NATIONAL BUREAU OF STANDARD;'' WW 9 W%

Proceedings of the Conference Held at the

National Bureau of Standards, Gaithersburg, Md.

March 22-24, 1972

Edited by

R. Mavrodineanu, J. I. Shultz, and O. Menis

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

U.S. DEPARTMENT OF COMMERCE, Frederick B. Dent, Secretary

NATIONAL BUREAU OF STANDARDS, Richard W. Roberts, Director

Issued May 1973
Library of Congress Catalog Card Number: 73-600066

National Bureau of Standards Special Publication 378

Nat. Bur. Stand. (U.S.), Spec. Publ. 378, 268 pages (May 1973) CODEN: XNBSAV

Reprinted from the National Bureau of Standards Journal of Research, Section A. Physics and Chemistry

Vol. 76A, Nos. 5 and 6, Sept.-Oct., and Nov.-Dec. 1972.

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402

(Order by SD Catalog No. C13. 10:378). Price: 84.85, domestic postpaid; $4.50, GPO Bookstore
Abstract

This volume contains the 18 papers presented at the Conference on Accuracy in Spectrophotometry and

Luminescence Measurements organized by the Analytical Chemistry Division of the National Bureau of Standards and held from March 22 to 24, 1972. These papers discuss the problems encountered in accurate spectrophotometry and luminescence measurements of materials. They also define the status of these competences from the standpoint of basic

principles, critical factors involved in the measurements, and the instrumental conditions which must be

fulfilled to assure accurate measurements. Particular attention is given to the selection, production, and

use of Standard Reference Materials in spectrophotometry and spectrofluorometry. Problems related to health are also covered in six of the papers, two of which include original contributions to the application

of luminescence techniques to specific biochemical problems.

Key words: Accuracy, critical parameters; instrumentation; linearity; quantum efficiency; spec- trofluorometry; spectrophotometry; standards.

HI
Foreword

"When you can measure what you are speaking about, and express it in numbers, you know something

about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meager and unsatisfactory kind ; it may be the beginning of knowledge but you have scarcely, in your

thoughts, advanced to the stag? of science," by William Thompson, Lord Kelvin, Popular Lectures and

Addresses 11891-18941.

This work presents the formal Proceedings of the Conference on Accuracy in Spectrophotometry

and Luminescence Measurements which was organized by the Analytical Chemistry Division of the NBS Institute for Materials Research. The Conference was held at NBS from March 22 through

March 24, 1972. The papers contained in this volume have also been published in two consecutive

issues of the Journal of Research of NBS, Volume 76A, Nos. 5 and 6, 1972.

This Conference brought together a number of leading authorities from the United States and from

overseas whose broad knowledge and experience provided valuable scientific contributions to the various topics of the Conference. The papers assembled in these Proceedings focus attention on the problems associated with accuracy in spectrophotometry and luminescence measurements of materials, and define the status of these competences from the standpoint of basic principles, in- strumental parameters, measurements, and Standard Reference Materials.

A major objective of the Conference was to provide the analytical chemist with the means to establish and maintain accuracy in spectrophotometric and luminescence measurements. The works

presented in these Proceedings should contribute significantly to the advancement of spec- trophotometric and luminescence measurements and constitute a solid ground for further progress.

W. Wayne Meinke, Chief

Analytical Chemistry Division

May 1973

IV
Preface

This volume is divided in two parts. The first part consists of six invited lectures and four invited

panel discussions on the subject of accuracy in spectrophotometry. The three invited lectures and five invited panel discussions, which constitute the second part of the volume, are devoted to the topic of accuracy in luminescence measurements.

These contributions are intended to interrelate theory and instrumentation with methodology for measuring "real samples" which are encountered in the general field of analytical chemistry, and more

specifically in the fields of health and environment. The roles of the basic principles and in- strumentation in the science of measurements are reflected in several papers. Three of these, by Clarke,

by Mavrodineanu, and by Mielenz, represent parallel approaches to the evaluation of accuracy of spectrophotometers and serve to augment the other contributions. At the same time, independent

measurements of a series of glass filters (NBS-SRM-930), serve to establish the magnitude of the bias

in this calibration. In this manner a better understanding of the needs and limitations of a material in a measurement situation has been established. Points of view on the philosophy of spectrophotometric

standards are expressed by Venable.

In the area of luminescence measurements, the three papers by Melhuish, by Crosby, Demas and

Callis, and by Winefordner survey the basic problems and instrumental criteria. Their descriptions

include a discussion of the state of the art and provide a clear prediction of the direction of future

efforts. The two papers describing the absolute calibration of light sources and photodetector linearity

by Rutgers, and by Sanders relate equally to both measurement problems. The three papers by Burke,

Deardorff and Menis, by Reisfeld, and by Velapoldi, interface the instrumental performance with real sample measurements through the development of suitable material standards in spectrophotometry

and spectrofluorimetry. On the other hand, the important areas of health and environment are ex- tensively covered in four papers by Penton, Widdowson, and Williams, by Burnett, by Rand, and by

Chen. They help to pinpoint the problems and progress associated with automation and clinical measurements. Finally, the papers by Udenfriend, and by Guilbault present insight into original

contributions to the application of luminescence techniques to specific health measurement

methodology.

This collection assembles in one volume valuable and original contributions, and constitutes a unique and important source of information which should serve equally well the workers in the field of research and applications of spectrophotometric and luminescence measurements.

The NBS Committee responsible for the organization of the Conference were Oscar Menis, general

chairman and panel discussion moderator; James I. Shultz, associate general chairman; Radu

Mavrodineanu, program chairman; Ranee A. Velapoldi, associate program chairman; Robert W.

Burke, arrangement chairman ; and Sara R. Torrence, arrangement liaison. The Conference had the encouragement of the National Institute of General Medical Sciences, who

also cooperated with the National Bureau of Standards in supporting part of the work of the

Analytical Chemistry Division. This work led to the design and development of instrumentation for high accuracy spectrophotometry; for the study, preparation and evaluation of liquid absorbance

standards, and in the investigations related to solid compounds and inorganic ion glasses as Standard

Reference Materials for spectrofluorometry. This support resulted in the issuance of a number of standards which assure that more meaningful measurements will be made in the field of biochemistry

and related areas. The undertaking of the Conference and publication of this book would not have been possible

without the cooperation and assistance of many persons. The important contributions of all speakers and the assistance of the session chairmen is acknowledged with deep appreciation.

V
The NBS Office of Technical Information and Publication, under the direction of W. R. Tilley,

gave invaluable assistance to the publication of this volume; we wish to acknowledge in particular the

assistance of Mrs. Betty L. Oberholtzer, Mrs. Bertha S. Darrow, and Mrs. Mary V. Betizel. Within the Analytical Chemistry Division, special thanks are given to Mrs. Mary Pantazis for her

tireless efforts in typing some of the manuscripts, and her diligent work in taking care of correspon- dence and program publications. Particular appreciation is expressed to Mrs. Rosemary Maddock for providing coordination and invaluable assistance in the many phases of preparing this volume. Special

thanks are also extended to R. A. Velapoldi for his assistance in reviewing the papers on luminescence

and for preparing the author index.

R. Mavrodineanu

J. I. Shultz

O. Menis

May 1973

VI
Contents

Page

Foreword IV

Preface V

Part I. Spectrophotometry

High Accuracy Spectrophotometry at the National Physical Laboratory

F. J. J. Clarke 1 An Accurate Spectrophotometer for Measuring the Transmittance of Solid and Liquid Materials R. Mavrodineanu 31

Absolute Spectroradiometric Measurements

G. A. W. Rutgers 53

Accurate Measurements of and Correction for Nonlinearities in Radiometers

C.L. Sanders 63

Physical Parameters in High-Accuracy Spectrophotometry

K.D.Mielenz 81

Liquid Absorbance Standards

R. W.Burke. E. R. Deardorff, and O. Menis 95

Accurate Measurement of Molar Absorptivities Robert W. Burnett 109 Problems Associated With the Need for Standardization in Clinical Spectrophotometric

and Fluorometric Measurements

James R. Penton, Graham M. Widdowson, and George Z. Williams 117 The Role of Spectrophotometric Standards in the Clinical Chemistry Laboratory

RoydenN. Rand 125

Spectrophotometric Standards W. H. Venable, Jr 135

Part II. Luminescence Measurements

Absolute Spectrofluorometry

W.H.Melhuish 137

Absolute Quantum Efficiencies G. A. Crosby, J. N. Demas, and J. B. Callis 151

Phosphorimetry

J. D. Winefordner 169 Measurements of Absolute Values in Biochemical Fluorescence Spectroscopy

Raymond F. Chen 183 Newer Fluorometric Methods for the Analysis of Biologically Important Compounds

G. G. Guilbault 197

Inorganic Ions in Glasses and Polycrystalline Pellets as Fluorescence Standard Reference Materials R. Reisfeld 203 Development of a New Fluorescent Reagent and its Application to the Automated Assay of Amino Acids and Peptides at the Picomole Level

Sidney Udenfriend 227

Considerations on Organic Compounds in Solution and Inorganic Ions in Glasses as Fluorescent Standard Reference Materials R. A. Velapoldi 231

Author Index 245

Subject Index 253

VII

PART I. SPECTROPHOTOMETRY

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedi

of a Conference Held at NBS, Gaithersburg, AAd., March 22-24, 1972 (Issued May 1973).

High Accuracy Spectrophotometry at the National

Physical Laboratory

F. J. J. Clarke

National Physical Laboratory, Teddington, Middlesex, UK.

(June 5, 1972)

The techniques and equipment used at the National Physical Laboratory (NPL) to achieve high accuracy spectrophotometric measurements are described and discussed. The emphasis at NPL has always been on the determination of systematic components of error and their elimination or correction rather than on the attainment of mere precision, which is largely a matter of variance and resolution. The scales of regular transmittance, diffuse transmittance, total transmittance of scattering

samples, regular reflectance, diffuse reflectance, total reflectance and radiance factor are determined,

maintained, and made available in practical form to industry by combined use of a reference NPL

manual spectrophotometer and commercial recording spectrophotometers.

The presentation will concentrate on transmittance measurements made with the reference in- strument, which is designed specifically to allow separate investigation of the various possible sources

of systematic error, processes that are not practicable with commercial spectrophotometers. The

investigation of the linearity of the complete photoelectronic system has always been a key factor, and

double-aperture devices have been used consistently at NPL for over 40 years to monitor the perform- ance of this instrument and its predecessors. Besides instrumentation, the procurement of material standards of suitable quality is a major limitation of the art, and the types in use at NPL are described,

including the recently developed Ceramic Colour Standards.

Key words: High accuracy spectrophotometry; photoelectronic linearity; reflectance; transmittance.

I. Introduction

Spectral measurements and techniques in the ultraviolet, visible and infrared regions of the electromagnetic spectrum fall into two general classes. The first involves work aimed at elucidating the physics and

chemistry of the electronic, atomic, molecular and

phase states of the materials involved, and this is the ream, of the spectroscopist. The second involves work

aimed at developing and using measurement tech- niques for accurate quantitative determinations of such properties as emissivity, radiance and radiant

intensity of sources, transmittance, reflectance and

radiance factor of passive materials, and responsivity

of detectors. This is the realm of the radiometrist,

photometrist and colorimetrist, but the techniques

used (spectroradiometry and spectrophotometry) are

of interest to scientists and technologists in many

fields and industries because of their general usefulness. Those who develop the art are a very small

minority compared with the users of the techniques

and instruments, and are found usually at standardising

laboratories and instrument manufacturers.

We may conveniently distinguish between spectroradiometry and spectrophotometry by means of an

operational definition: spectroradiometry is concerned

with the spectral properties of sources and detectors,

whereas spectrophotometry is concerned with the

spectral properties of material specimens. The work

in these fields at the National Physical Laboratory

(NPL) is currently organized in two Sections along

just these lines: spectroradiometry forms part of the

work of the Quantitative Spectroscopy Section under

E. J. Gillham in Quantum Metrology Division, while

ultraviolet, visible and near infrared spectrophotometry

forms part of the work of Colorimetry and Photometry

Section under myself in Metrology Centre. I regret

to say that at present there is no programme of work

at NPL on spectrofluorimetric standards, a subject

still in its infancy but likely to become increasingly

important. However fluorescent materials are measured for reflectance or diffuse transmittance as part

of our spectrophotometric work, using either monochromatic illumination with broad-band detection or

alternatively broad-band illumination with monochromatic detection, and the excitation and emission

spectra can be distinguished from the reflected or transmitted components. Historically, spectrophotometric standards work in the ultraviolet and

visible regions at NPL has always been closely in- tegrated with colorimetry, and there is a very sound

1
reason for this: until relatively recently, the only users

of spectrophotometers who really needed limit-of-theart accuracy are those who use the instruments for colorimetric purposes. It was no accident that the color- imetrist Hardy at Massachusetts Institute of Technology (M.I.T.) set to work to design and develop the

first commercially manufactured automatic recording

spectrophotometer (The General Electric 1 instru- ment, ref. L1J) immediately following the establishment of the CLE. System of Colorimetry in 1931 and

its widespread adoption by divers industries. That

instrument remained unsurpassed for a great many

years. High accuracy spectrophotometry is needed for colorimetry because the chromaticity discrimination of

the normal human observer is so fine that only very

few of the instruments on the market can guarantee a commensurate accuracy in the derived colour specification. It may be pertinent to note that the colour

industries generally use tristimulus filter colorimeters

for differential measurements on production samples

of known spectral profile, where speed and precision

is more critical than freedom from possible systematic

error, whereas spectrophotometers are used with tri- stimulus computation in cases where the spectral profile

is unknown or likely to be variable and in cases where

the highest absolute accuracy is required in colour

specification. The development in recent years of colorant formulation by instrumental colour matched

prediction systems [2, 3] 2 nearly always involves

spectrophotometers that are as accurate as are avail- able on the commercial market and can be afforded

bv the firm involved.

The largest class of usage of spectrophotometers is by chemists, and except where their interests have

been colorimetric, their demands have usually been

for convenience, reliability, reproducibility and precision rather than for accuracy, for their uses are often

diagnostic rather than critically quantitative. In cases where they have used spectrophotometric methods for determining concentrations, they have generally

worked relative to standardised solutions and have not worried too much about the absolute accuracy of the

optical density (absorbance) scales of their instru- ments. However, this situation is changing, and an in- creasing number of chemical and clinical applications

are arising where high absolute accuracy is needed.

Of the contributors to this Symposium, R. Mavrodineanu is probably best qualified to deal with these

matters, and I will not myself comment further.

II. The General Nature of Errors and Accuracy

in Spectrophotometry

Before considering in detail the methods of spec- trophotometry and the equipment used at NPL, it is

1 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does

it imply that the particular product or equipment is necessarily the best available for that purpose.

2 Figures in brackets indicate the literature references at the end of this paper.

necessary to explain just what the term "high ac- curacy" can mean in this subject. It can mean several

things: for instance one can distinguish between high

quality commercial instruments and lower quality ones, and can say that by normal industrial and uni- versity laboratory criteria, high accuracy spectrophotometry is achieved when careful and competent

staff are using a high quality instrument that is properly

serviced when needed by the manufacturer or his agent. This approach may be embellished by

ownership of several different designs of good

quality instrument and the taking of an average

of results from them or even some weighted mean,

where the weighting depends on inspiration, prejudice

or perhaps experience of their past history of diver- gences from the mean. However, such an approach is not acceptable for a standardising laboratory, or indeed

any laboratory that aspires to making a determination to a known absolute accuracy, for it smacks of the

"black box" approach which implicitly assumes

that the manufacturers have somehow managed to build absolute accuracy into the photometric scale of the instrument or instruments. As it is well known

from interlaboratory comparisons that different in- strument types (and even different specimens of the same type) give systematic discrepancies that are experimentally significant in terms of their reproducibility and precision [4, 5, 6, 7, 8, 9], it is clear that

it would be remarkable if even one instrument always

gave a correct result. Since certain types of systematic

errors are common to most instruments to some extent,

it would also be remarkable if a mean result (or a weighted mean) could exactly or even approximately

balance out the various sources of systematic error. For the purposes of this Symposium we will take a more rigorous meaning of the term "high accuracy

spectrophotometry." We will make no assumptions

whatever about the validity of commercially manufactured equipment or about any calibration procedure

which has been attempted by the manufacturer.

We will question and test every aspect of the function

of optical components or instruments which are used,

and we will seek out, determine and correct for every

systematic component of error which we can think

of and discover, either in these component parts or

in the complete reference instrument such as is de- scribed later. Random components of error make their presence

manifest in a rather obvious fashion, and most laboratories and text books are rather obsessed by them,

with repeated use of phrases such as "signal-to-noise

ratio" and similar concepts. Standardising laboratories

should not be overconcerned with these problems,

but rather with the systematic components of error. Random components of error are readily reduced by

suitable statistical pooling of data from repeated runs, whereas systematic errors are not. A standardising

laboratory can afford to and should make many

more observations than an industrial laboratory normally would, and it usually becomes obvious as to how

much reduction of variance by pooling is necessary. Again, I would point out that many laboratories are

2
unduly concerned with the statistical treatment of

data, but this is not adequate to deal with the sys- tematic errors that should be of most concern. Excessive precision, arising from a combination of

high resolution and good repeatability, is probably

a bad thing since it tends to beguile even experienced

staff into a false sense of security as to the real ac- curacy achieved. In the hands of inexpert staff the

situation is worse, because of the confusion between

the concepts of precision and accuracy that exists

in so many minds. Of course, we must bear in mind

that for many industrial purposes repeatability is more important than absolute accuracy. At this point we might note the difference between reproducibility and repeatability. Repeatability refers to the

ability of an instrument to duplicate a result with

a given sample presentation, and refers exclusively

to the properties of the instrument. Reproducibility

refers to the ability to duplicate a result when the

sample presentation is repeated from scratch on

different occasions i.e., when it is cleaned or prepared

afresh, repositioned and realigned in the instrument.

Reproducibility therefore refers to the whole measure- ment procedure and covers effects arising both from

the instrument and the sample

In general, components of errors are seldom purely

random in nature or purely systematic, but tend to be

intermediate in nature or rather closer to one aspect

than the other. To assist in making this clear in practical terms, I have set out in figure 1 a classi- fication of the more important types of error found

in spectrophotometers in terms of their systematic

or random nature, and it is instructive also to include

for comparison the errors found in analogous instruSPECTROPHOTOMETER

| FAULTS

stray light in monochromator waveband AX

X calibration (25°C)

pen servo lag

pen carriage alignment

chart drive

X calibration (A9°)

pen zero

chart irregularity

reading errors

(detector

l,neari, V (output device

reference standard

non-standard geometry

faulty geometry

sample presentation

dark correction (sample

stray light idetector

polarisation

thermochromism

drift

noise

TRISTIMULUS COLORIMETER

FAULTS .

filter design

source s.p.d. P (X)

detector S(X)

filter realisation

source P(X)changes

detector S (X) changes

filter changes

Figure 1. Classification of types of error found in spectropho- tometers and colorimeters in relation to their random or systematic

nature.

ments i.e., photometers, colorimeters, and abridged

(filter) spectrophotometers. Some of these will be

discussed in detail in section IV. Some of the errors which are partly systematic and partly random are very difficult to deal with, and these manifest themselves as errors which are systematically present for an

unpredictable period and which then change to a

different systematic error for another unpredictable

period and so on. Such errors tend to be found in

very complex systems, such as some of the more

sophisticated double-beam automatic recording

spectrophotometers, which may have several inter- linked servos and feedback loops. This is one of the

factors which has influenced us for decades at NPL

in having a reference high accuracy spectrophotometer

which is as simple as possible in function, with as

little interaction as possible between its component

parts, so that each aspect of its function can be separately investigated.

The term "high accuracy spectrophotometry"

refers to quite different magnitudes of probable error when considering different types of measurement.

The simplest case is measurement of direct transmit- tance i.e., with no scattered or diffused component of

flux included, and the lowest attainable error levels are found here. The next least complicated case is measurement of direct (or specular) reflectance, again

with no diffused or scattered component included, and

error levels almost as low can be achieved, the

additional errors arising from the necessary movement

of the optical system. Measurement of radiance factor

introduces additional errors in the conditions of il- lumination and view of the sample and uncertainties

in the reference white standard used. Measurement

of the total or of the diffused component of transmit- tance for scattering samples introduces the geometrical

errors of integrating spheres (see later) and rather

higher net error levels result, at least 0.2 percent in absolute value. In fact for this type of measurement and

the remaining ones described below, the geometrical

errors found in practice in the conditions of illumination and view are such that the monochromators and

photoelectronic systems of good quality commercial

spectrophotometers are often adequate to ensure the

overall error level is not significantly increased.

Measurement of total or of diffuse components of

reflectance introduces not only the integrating sphere

errors, but also the uncertainties in the reference

white standards against which the samples are measured. Attainment of an error level of 0.2 percent

in reflectance, while not impressive to a transmission

spectrophotometrist, nevertheless qualifies for the

term "high accuracy," because it cannot be attained

reliably by using even an expensive commercial

instrument in the usual way.

III. Spectrophotometric Equipment at NPL

Spectrophotometry standards at NPL are derived

from measurements made on a specially constructed

manual instrument (nonautomatic and nonrecording)

known as the High Accuracy Spectrophotometer. This

is the reference instrument, described in section III. A, and the performances of other instruments used

in standards work are checked relative to it. Until

about 25 years ago there were sometimes two and

sometimes three manual spectrophotometers at work,

these being used partly for establishing standards

and partly for providing a calibration service for outside organizations. From then on the bulk of the

outside calibration work was handled by two auto3
matic recording instruments, a General Electric

and a Cary Model 10, and there was eventually only one manual instrument remaining, which became the

reference instrument. In recent years the recording

spectrophotometers mentioned above have been re- placed by a Cary 14 and a Cary 14R with a number

of improvements and accessory units, some designed

and made at NPL, and these have now been interfaced

to a local computer in an advanced time-sharing data

handling system as described in section III. B. In the spectrophotometry standards work at NPL

the principle of substitution is strictly adhered to. This means that for double-beam instruments the

measurement on a sample is sandwiched between

measurements made on a reference standard ("Open"

reading for absolute transmittance), and the second

beam uses a comparison standard rather than a true reference standard. In addition to the instruments used in spectrophotometric standards work, there are nowadays

a number of other spectrophotometers at NPL, all regular commercial models and used as tools in a number of research and development projects, particularly

by chemists. These are not instruments of the highest

calibre, and are not used with any serious intention of obtaining accurate results, so we will not mention them

further. One interesting exception concerns the work

of S. C. Ellis and his colleagues in Radiation Science

Division. Here a Cary 16 and an Optica CF4 are used

to provide a calibration service in the dosimetry of hard radiation, the change in optical density in a photochemical actinometer being the measured indication

of the previous irradiation [10]. The absolute accuracy

of the optical density scale of these instruments at the appropriate UV and visible wavelength has been

determined using transfer standards measured

on our reference instrument described immediately

below. The transfer standards were in the form of neutral density glass filters and cuvettes of accurately

known pathlength containing known concentrations

of pure potassium nitrate solution, so that this work

also amounts to a new determination of the molar

absorptivity of this material.

A. The NPL High Accuracy Spectrophotometer

The reference instrument for spectrophotometric

standards at NPL is currently being completely

rebuilt. The installation has been developed and improved in a piece-wise fashion over a number of years,

first one portion being replaced or improved, then

another, as the art has advanced in different areas

at different times. In the earlier years much pioneer

work was done by Preston [11, 12], Donaldson [13,

14, 15] and Harding [16]. An important milestone was

in 1947 when a Hilger "Uvisir" (Miiller-Hilger) quartz

double-monochromator was introduced to supersede

the use of series pairs of Hilger constant deviation

prism monochromators, under the aegis of H. G. W.

Harding, and from then to the present day one of these Uvisir instruments has formed the "fixed"

portion of the optical system.

The principle of operation of the NPL reference

instrument is, and always has been, deliberately

simple. The layout is indicated in figure 2. A steady source (chosen for stability and output in the spectral

region of interest) illuminates via the double-monochromator (chosen for aperture, freedom from stray

light, wavelength adjustability and reproducibility) one of a pair of samples mounted on a two-position

kinematic carriage. The transmitted or reflected

light is collected over the required solid angle by suit- able adjustable optics and assessed by a photoemissive

detector (chosen for responsivity, stability and low

dark current) which is dc coupled to a dc electrometer-input measuring system (chosen for high input

impedance, low offset current and good zero and

scaling stabilities). The single-beam principle is used

throughout, both in optics and in electronics. No

chopping or phase sensitive devices are used in the

optical-photoelectronic system, though they may be

used within electronic units such as stabilised dc

supplies or digital voltmeters.

Operotor's

position Electronics Writing rock

ledge

Photometric

enclosure

Steel surface

plate

FIGURE 2. Layout of the NPL High Accuracy Spectrophotometer, 1966-1972. DM: double monochromator; WD: wavelength drum;

Si, S2, S3: entrance, intermediate and exit slits; CH: condenser

mirror housing; Li, L2, L3: alternative lamps for use in measure- ment or in wavelength calibration; SC: sample carriage; K:

operating knob for SC; T: baffle tube; S: integrating sphere with

central screen; DH: detector housing, containing a selected EMI 9558Q photomultiplier P.

This use of a dc single-beam technique for the

past 40 years at NPL may strike an outside observer

who has read the common text books in spectrophotometry and electronics as rather strange. The reason may

be summarised by saying that the various developments in double-beam, chopped or phase-sensitive

techniques have made their impact largely on the

problems of coping with source instability, and improving detectivity, signal-to-noise ratio and repeatability, so that more read-out resolution can be used

to obtain greater precision over a wider range of scan- speed, wavelength and waveband conditions. Signalto-noise problems are inherently more serious in automatic scanning instruments because of the greater

rate of information output required to reveal the

4
shape of a profile. However these techniques do

nothing for absolute accuracy, and their most sophisticated employment in an advanced commercial recording instrument actually hinders attempts to determine

systematic errors in the photometric values obtained

(see sec. IV). For standards work there is no point in precision greatly exceeding the final accuracy that can be achieved after correcting for known systematic

errors, whereas in ordinary commercial instruments

where the systematic errors are sometimes rather

serious, there is an advantage for many industrial

purposes in having a rather better level of precision

than can really be justified.

The basic measuring procedure is to take three

types of reading in a preferred time sequence of the

following character:

DRSRSRSRD

where D represents a dark reading, R a reference

reading (for absolute transmittance work an "open"

reading) and S a sample reading. The required photometric ratio is calculated initially as (S-D)I(R-D)

in terms of the mean values S, R and D found, and a

linearity correction is subsequently applied. This

last is determined at intervals, and always immediately

preceding and succeeding any limit-of-the-art deter- minations, as described in section IV. A. The use of a time-symmetrical sequence of observations is important in reducing the influence of "drift" on the

results. Apart from shot-noise fluctuations and other

short term instabilities, slow variations (drifts) of out- put are the main source of intrinsic photometric error

in this technique. The time-symmetrical sequence

will completely nullify the effect of drift, provided the

drift be at a consistent rate and in the same sense.

In practice the drift tends to be fairly consistent, and

approximate nullification of drift effects is what is achieved with the technique. Irregular drift cannot be

allowed for. As will be seen in the description of the

installation that follows, great attention has been paid

to reducing all possible sources of drift and instability.

The source most favoured for visible region work

from 350 nm to 800 nm is a gas-filled lamp with

single vertical coil, and a stock of a suitable design

of 12V 48W obsolete-pattern automobile headlamps

is used. The lamps are selected for freedom from

filament distortion and striae in the bulb, such as would

give rise to a seriously nonuniform irradiance in the

field aperture of the monochromator. They are also aged and tested for stability prior to use in measurement, and a small proportion of these lamps are found to give rhythmic variation in output due to slow thermal oscillations in the convection currents

in the gas filling: such lamps are rejected. The lamp

is not simply held in a commercial fitting (bayonet

or screw) but has soldered electrical connections to eliminate contact resistance. This type of source

has not been improved for decades, but its operating

stability has, due to advances in electronics. Whereas

an operator used to monitor the lamp current with

a precision vernier potentiometer and galvanometer, and vary a trimming rheostat accordingly (which

controlled the current supplied from massive banks

of lead-acid accumulators), nowadays the current through the lamp is automatically stabilised to typically a part in 105 over a 10 minute period by means of one from a range of custom-built variable dc-power

supplies developed specially for photometric work

at NPL. A light output stability of about ± 1 part in 104 is routinely achieved over a 10 minute period.

For near ultraviolet work in the region 280 nm up- wards, the preferred source is nowadays a tungstenhalogen lamp, but we have not yet discovered a type

which is ideal in all respects (but see section III. B).

Although the stability should ideally be the same as with the conventional gas-filled lamp when run from

the same stabilizer, we have not been able in practice

to achieve the same stability of light output during

measurements. For work at shorter wavelengths the

deuterium arc has replaced the hydrogen arc as the

preferred lamp, and although discharge lamps are inherently far less stable than inoandescent filament

lamps, the type we use gives a stability of rather better

than 1 part in 103 over a 10 minute period.

The double-monochromator used up to the present

is a Muller-Hilger instrument of aperture 60 mmX60

mm. In each component monochromator there is a double pass through each of two 30° natural quartz

prisms, and the dispersions at all eight passes through

such prisms are additive. The slit micrometers open

the jaws symmetrically in all three cases, and fishtail templets allow the slit height to be varied at the en- trance and exit slits. A prism instrument is preferred

to a grating instrument for reduction of heterochromatic stray light, and for both manual and recording

type instruments used in standards work a true doublemonochromator is considered mandatory.

The fight emerging from the exit slit enters a large

light-tight photometric enclosure with matt black

walls and measuring 1050 mm X 620 mm X 520 mm.

This size of enclosure not only allows great freedom of

optical configuration, with adequate room for baffles and light traps, but also reduces the influence of homochromatic stray light scattered or reflected from optical

components and reflected from and round the en- closure. An extensive range of optical bench fittings

and photometric components can be deployed to

suit almost any kind of spectrophotometric measurement: direct transmittance, diffuse transmittance,

total transmittance, direct reflectance at almost any

angle, diffuse reflectance, total reflectance, or radiance

factor at almost any angles of illumination and view.

A kinematically designed sample carriage enables the

sample or samples to be moved to accurately deter- mined positions by remote control, these positions

being located by stops that can be clamped at any

position on the guides. All components are mounted

on a 2100 mm X 900 mm steel surface plate conforming

to Grade B of BS817 for long term stability.

5
The photodetector in the enclosure is held in a lightproof housing with a sliding shutter covering a 50 mm

diameter port in a plate to which a 100 mm diameter

integrating sphere can be screwed. The integrating

sphere has two diametrically opposite portholes, the entrance port being the smaller, and light cannot pass

directly from one to the other because of the presence

of a central disc-shaped screen. This particular sphere

is not used for measurements invoking integrating

sphere theory (such as diffuse or total transmittance

or diffuse or total reflectance) but is employed in direct transmittance and direct reflectance measurements to ensure that any movement of the beam or change in

its size or distribution at the detector region will have

no influence on the readings. This is important in measuring samples that are of nonideal optical quality,

and the uniformity of the central screen is vital. Until 1961 the photodetector used was a vacuum

photoemissive cell of a type specially developed for accurate photometry, latterly Rank-Cintel Types VB39

or VB59 for good red response or VA39 with silica window for ultraviolet response. The VB59 cell is still currently the preferred detector used in white-light

photometry such as the determination of luminous

intensity or luminous flux of incandescent or discharge

lamps: present photometer circuits are based on

the impedance converter developed by Jones [17]. A succession of designs of dc electrometer amplifier

null-balance detector systems had been developed

over the years at NPL, especially by Preston [11], Harding [16] and Crawford [18]. In 1960 I started work

on improving the photoelectronic system of Harding

that was then in use, and found suitable operating

conditions that would allow the EMI 9558 type of

photomultiplier to be used in a linear fashion: until then photomultipliers were viewed askance by stand- ards photometrists as being unstable and nonlinear

as compared with single-stage vacuum photoemissive

cells. At the same time, I developed two new designs

of electrometer photocell amplifier, one of which

(fig. 3) served for two years on the High Accuracy

Spectrophotometer giving the best performance

achieved at NPL with the null-balance method, and

an accuracy of measurement as good as the digital volt- meter system which superseded it. The other design

shown in figure 4 was invaluable in the investigation on Time-Ratio Photometry (see sec. V). In the null-balance method traditional at NPL, the

photocell amplifier has the task of detecting and dis- playing via a galvanometer spot deflection any change

of potential difference between A and B in figures 3 or 4. Potential drop across the selected anode load

resistor R is then balanced manually by means of a

potential difference obtained from a precision potentiometer to give a null-output indication. As the po- tentiometer can easily have a stability and linearity

in its dial-output relationship of order 1 part in a

million, and as the null-detector circuit itself is not required to be very linear, there are no limitations on

systematic accuracy except for those imposed by the

intrinsic characteristics of the photodetector and its circuit. For the photocell amplifiers shown in figures

3 and 4 the relevant performance parameters were:

input resistance approximately 1014 fi, zero stability

about 2 /aV per 10 minutes, the open reading being

up to 1.9 V. The photodetector used nowadays is an EMI Type

9558Q photomultiplier with end-window S-20 (tri- alkali) cathode. The best tube used is one of a group

specially selected by myself in terms of certain characteristics whose importance became clear as a result of unpublished researches into the use of photomultipliers for high accuracy work. The EHT supply used

has only lmV of ripple, noise or transients, which is

Figure 3. Photocell electrometer amplifier system developed by Clarke, as used 1961-1963 on NPL High Accuracy Spectrophotometer.

6
Figure 4. Photocell electrometer amplifier system developed by Clarke, as used

in Time Ratio Photometry work on photomultipliers.

very important as the gain of an eleven-stage Venetian

blind type of photomultiplier is proportional to the 7.7 exponent of the EHT voltage. The low frequency noise

in the photomultiplier output relevant to our dc usage

conforms to the limiting value given by Schottky's

Equation for shot-noise (allowing for the appropriate

dynode enhancement factor of 1.3) while the dark

current is equivalent to a stray flux of about 3 X 10~ 13 lumens of 2856 K Planckian radiation. In 1963 digital voltmeters (DVM) became available

with electrometer input circuits with input resistance

of order 1011 fl, offset currents of order 10~ 12 A,

10 /xX sensitivity, scale lengths of 20,000 or more

per range, linearity and stability commensurate with

the resolution limit and a useful choice of sampling

modes. The advent of these instruments and their satisfactory performance under rigorous tests caused

me promptly to set aside the improved photoelectronic system I had introduced only 2 years before,

and the system has remained substantially unaltered

since. The present system is no more accurate than

its predecessor but is quicker and more convenient

to use and hence improves staff productivity.

The voltages developed across an anode load resistor

(selected by PTFE wafer switch from a set with values

1 megohm, 3 megohm, 10 megohm, 30 megohm and

100 megohm) are measured by a Dynamco type 2020

or 2022 digital voltmeter. The coupling circuit sometimes used, figure 5, is of some interest as it incorporates a special compound filter to reduce low frequency

noise while allowing a reasonable slewing rate to avoid

lengthy waits for the potential change to become complete following interchange of photocurrent. A partic- ular problem is that the potentiometric digital volt- meter has, and requires for common mode rejection, a twin-T mains frequency rejection filter with capacitors of value 0.1 fiF and 0.2 fiF in its input circuit, while we want to be able to use anode load resistors of value up to 100 megohms for certain high sensitivity

measurements. The system would obviously be too

Figure 5. Special filter circuit for coupling anode of photomultiplier

to a digital voltmeter sampling repetitively at mains frequency

(50 Hz). The photometric ranging using Swl involves values of

ft, to R 7 of 108ft to 105fi in steps of about 3, and f?C=0.02 s for

this first stage of low-pass filtering. Stage 2 is simple low-pass, with RC = 0.016 s for low values of load resistor, but tapered off for high values to compensate for stray capacitance in the photo- tube circuit. The time constant of this stage can also be augmented

using Sw2. Stage 3 is simple low-pass, of RC= 0.018 s. Stage 4

is a twin-T network set to reject 34 Hz. Stage 5 is simple low-pass,

of RC = 0.018 s. Stage 6 is a twin-T network set to reject 50 Hz.

sluggish if we used the normal filter provided, but as

it can be switched out of circuit, this gives us the

opportunity to provide our own external version.

As figure 5 shows, a combination of four cascaded

stages of R-C low-pass filtering and two stages of

twin-T rejection filters tuned to 34 Hz and 50 Hz

respectively are employed. Figure 6 shows the fre- quency response measured for such a filter, and indi- cates how successful the approach is in allowing low

attenuation below about 5 Hz but a steep average

roll-off above about 10 Hz. This special coupling circuit was developed for special investigations and allows sector discs to be used

validly over a range of speeds. A simpler coupling

circuit is used for everyday work when 108 fl resistors are not used. An alternative approach to the problem

has been devised by Samways [19], and this has been

used recently in spectroradiometric work at NPL.

502-591 O - 73 - 2
IO Voltage

transmlttana

(

E/e 0) . IO

log scale

A.B C

...

! \ \

3 \c

i

a\ \

5

6

Attenuation

of power 20

6 IO

f (Hz) log scale

120

FIGURE 6. Transmittance versus frequency characteristic of special

filter circuit (curve A). Note the 120 dB rejection at the sampling

frequency of the DVM. Curve B shows the performance of a simple low-pass filter whose RC is set to 0.108 s to give an equivalent effect at low frequencies (<5 Hz). Curve C shows the effect produced by the 4 cascaded stages of simple low-pass filter present

in the special filter circuit. Because of the large number of optical configurations

used to provide the different directions and solid angles

of illumination and view and give different sizes of illuminated patch on the sample, it is impractical to show diagrams of them. Figure 2 shows a simplified

indication of the optical arrangement used in measuring the direct transmittance of a small filter. However

some of the points raised in section IV will give guid- ance on devising the most suitable optical layout.

The NPL High Accuracy Spectrophotometer is currently being rebuilt, and the double-monochromator to be used is a Hilger D300 which uses a double

pass through a 60° Cornu prism of aperture about

55 mm X 55 mm in each monochromator section. A

yet larger photometric enclosure measuring roughly

1300 mm X 900 mm X 700 mm will be used, and the

sample carriage will employ a Schneeberger linear

bearing as suggested by R. Mavrodineanu of NBS. The

photoelectronic system is not being altered for ultra- violet and visible region work, but we are planning

to add a synchronised detector chopped system for near infrared work in the region 800 nm to 2600 nm.

This need arises because in 1967 Colorimetry and

Photometry Section took over the responsibility for near infrared spectrophotometric standards (at up

to 2600 nm) and until the present we have had no

independent means of verifying the accuracy of our Cary 14 instruments in this region.

A number of checks and determinations of various

kinds of systematic error are routinely made when

working at high accuracy. These are detailed and

discussed in section IV.

B. On-Line Data System for Automatic Recording

Spectrophotometry

The bulk of the work in spectrophotometric standards and measurement services for outside organisations is performed on good quality recording spectrophotometers, and Cary 14 and Cary 14R instruments

are used for these purposes. A detailed description

of the Cary 14 instrument would be out of place here

as it is a very well-known design and full details are obtainable elsewhere [20]. It is particularly note- worthy among commercial instrumentation for the

size, freedom from stray light, wavelength calibration

reproducibility and wide range of scan speeds of its prism-grating double-monochromator. In fact it is our

experience that a wavelength value for a peak, trough

or emission line can be determined more accurately

with one of these recording instruments than with our reference manual instrument, and this is undoubtedly due to the ability to scan and record continuously

and to the absence of stick-slip irregularities when

scanning at even the slowest speed (0.05 nm/s).

Accessories available include the direct reflection attachment and the Model 1411 sphere. The direct

reflection attachment uses the "V/W" optical arrange- ment after Strong [21] and invokes the use of a speci- men compartment that is unusually large by commercial standards. In view of my comments on the

importance of having a large compartment for the

sample and supplementary optics in section III. A,

it will come as no surprise to the reader to learn that we use this compartment unit in preference to the

normal one for all direct transmittance work as well,

and that we have built a duplicate ourselves for the

second instrument. In addition we have a box extension

above the lid which allows rather lengthy items to be

measured a reasonable distance from either end, a feature which has proved very useful when measuring

the envelope transmittances of photometric standard

lamps.

The Cary 1411 sphere attachment is noteworthy

among commercial instrumentation in having a pair

of screens inside, between the reflectance sample

and comparison ports and the detector port. This means that the largest source of systematic error found in the majority of commercial integrating spheres

is taken care of, for without such screens the component of flux diffusely reflected directly to the de- tector port is grossly overweighted relative to the

flux diffusely reflected in all other directions. The

unit is provided with supplementary deuterium and

tungsten sources for broad-band irradiation of samples,

and the result is that a total of 30 measuring modes

(combinations of source, detector and geometry of

illumination and view) are available with our Cary 14

instrument and 32 modes with our Cary 14R. These

are set out in table 1. At the present time it is not practicable to deter- mine the geometrical errors in integrating spheres by

calculation. This is because classical integrating

sphere theory depends on certain elegant simplifications which arise from the assumptions that a wall

coating is uniform over its whole surface and that it is also Lambertian i.e., a uniform diffuser. Other

common simplifications are to assume that samples

are small enough to have a negligible effect on the

8
Table 1. Modes of measurement available for automatic recording spectrophotometry at NPL

Measurement with monochromatic irradiation and

broad-band collection (geometry in brackets)

Source-Detector combination

Deuterium Tungsten Tungsten

PMT PMT PbS

Measurement with broad- band irradiation and monochromatic collection (geometry in brackets)

Source-Detector combination

Deuterium Tungsten Tungsten

PMT PMT PbS

Direct Transmittance t(0°/0°) 1 1 1* Direct Transmittance t(0°/0°) 0 1 1 Diffuse Transmittance t(0°/«0 It It 0 Diffuse Transmittance t(c?/0°) 1 1 1 Total Transmittance T(0°/i) It It 0 Total Transmittance t(j/0°) 1 1 1 Direct Reflectance p(8°/8°) 1 1 1* Direct Reflectance p(8°/8°) 0 1 1 Diffuse Reflectance p(Q°jd) It It 0 Diffuse Radiance Factor f3(d/(f) 1 1 1 Total Reflectance p(8°/0 It It 0 Total Radiance Factor /3(i/8°) 1 1 1 Radiance Factor /3(0°/45o

) 1 1 0

t Alternative version available with NPL Large Sphere Attachment.

* Available with Cary 14R instrument only. mean sphere wall reflectance, that the other portholes

are small enough to have a negligible influence on the

spatial sampling of the flux collected from the sample

and not to interfere with the interreflections needed

for complete integration, and that the necessary

screens are coated so as to be perfect reflecting diffus- ers i.e., with no absorption. The errors from these

last three assumptions can be allowed for by theoretical

analysis, but only by making use of the first two

assumptions about the wall coating: in any case no

analytical treatments to allow for real (nonideal)

wall coatings have yet appeared. Some work on this problem has been going on recently at NPL by Ed.

Fulton and myself using a numerical analysis method

rather than an algebraic analysis approach, but results have only so far been obtained for particular cases,

not the more intractable general cases. Because of these theoretical difficulties in deter- mining sphere errors, a practical approach has been

adopted. An alternative sphere attachment for the

Cary 14 instruments has been designed and constructed, and is known as the NPL Large Sphere Attachment

[22]. The idea is to have available for use on the same

instrument two comparable sphere reflectance attach- ments, both conforming as far as practicable with

ideal requirements and yet with geometrical designs

that differ as widely as possible. Any differences in measurements found with the two sphere attachments

must then be due to their own errors, and not in- fluenced by optical faults if any in the main instru- ment. To this end the sphere from a General Electric

recording spectrophotometer (GERS) was used and

modified, and a toroidal mirror system was used to couple the Cary 14 optics to it. The optical configuration of the two attachments are shown for comparison

in Plates I and II, and apart from the size difference

of the spheres (90 mm against 200 mm) the contrasted

features are: wall coatings, smoked MgO for Cary

From monochromatc

Plate 1. Layout of the optical configuration of the Cary 1411 sphere attachment.

9
Plate 2. Layout of the optical configuration of the NPL Large Sphere Attachment

for a Cary 14 instrument.

1411, Distillation Products special BaS04 paint for GERS sphere; optical axes, perpendicular for Cary

1411, nearly parallel for GERS; photocell port, at top

for Cary 1411, at bottom for GERS sphere; gloss

traps, entrance ports for Cary 1411, separate ports

for GERS sphere. The contrasted positions of the

photocell port are very significant, as sphere coatings

darken due to atmospheric dust and pollution in a systematically nonuniform way in the sense that the

dirtiest portion is at the bottom and the cleanest is at the top. Apart from this, some measurements we made

with a reflectance head on the freshly painted wall of aim integrating sphere show that even fresh sphere

coatings (judged visually to be uniform) are surprisingly

nonuniform, and variations of several percent are normal. Both the Cary 1411 and the NPL unit have the

necessary screens between the reflectance sample

ports and the photocell port, but the NPL unit also has screens between entrance ports and the photocell

port to make it fully valid for measurements of diffuse or total transmittance. The complete attachment is shown in Plate III. The two recording spectrophotometers are inter- faced in a time-sharing mode to a dedicated Micro

16P computer fitted with a core holding 16K words

of 16-bit length and with a backing store facility in the

form of a drum store holding 64K words. Two views

of the complete installation are shown in Plates IV

and V. As this spectrophotometer data handling system

is unique and one of the most advanced yet developed,

a description is worth including. The system was

evolved from ideas provided jointly by staff of In- strumental Colour Systems Limited, Digico Limited

and NPL, and a system schematic is shown in figure 7. The two spectrophotometers can be operated simultaneously and quite independently of each other, and

this also applies to the real-time routines and user

Plate 3. View from above of the NPL Large Sphere Attachment showing the toroidal mirrors used to couple the Cary 14 optics to the GERS sphere used.

10
Plate 4. View of the NPL on-line spectrophotometry data-system,

showing the Cary 14 and 14R instruments. The near one is fitted with the Cary 1411 small sphere attachment, and the far one is fitted with the large sample compartment with box-lid extension

for transmittance work on long samples.

Plate 5. View of the NPL on-line spectrophotometry data-system,

showing the dedicated Micro 16P computer with 16K of core to which the two spectrophotometers shown in the foreground are interfaced in a time-sharing mode for independent operation and

processing.

programs whereby the instrument outputs are subject

to digital noise filtering, normalising, referencing, dark

correction, and radiometric photometric or colori- metric calculation as the case might be. The user programs are in a high-level language

called MATHCHAT, not unlike BASIC, and because

the interpretive compiler is present in core under

all normal conditions, the system seems very like a public telephone-modem linked time-sharing system

based on a large processor, such as IBM's Call 360

or Honeywell-GE265. The avoidance of tedious doubleDrum ] Store h* 3 (64 K)

Micro 16 P

computer

(16 K core)

Hesitate Control Hardware

Mult-Div

Micro 16 P medium speed interface

X DVM

encoder , (A -D) X

encoder DVM(A D)

Cary 14 Spectrophotometer

ASR 33

Teletype

Channel 1

Cary 14R

Spectrophotometer

Channel 2

ASR 33

Teletype

Figure 7. System schematic for the NPL's unique on-line data system (see text).

pass or triple-pass loading and compiling/assembling

procedures, such as are currently common with small

computers, is a tremendous advantage, as is the

ability to instantly modify a user-program in a highlevel language. One special feature of the language

MATHCHAT, provided at our request, is that it allows

subscripted integer arrays in addition to the usual real variables, simple or subscripted. This permits very

efficient storage in core, particularly of spectral arrays for subsequent processing, as well as faster real-time operations, which can then use a hardware

multiply/divide unit which reduces the time for a

multiplication from about 1 ms to about 10 ^ts and

for a division slightly longer.

Associated with each user area in core i.e., each

"channel," are three peripherals, namely the photometric interface, the wavelength interface, and a ASR 33 teletype. "Reference" beam and "sample"

beam photometric signals are extracted from a Cary

14 instrument at the feed to the slide wire potentiometer on the reference side and at the output from the

multipot system on the sample side, so that the complete pen servo system is bypassed for the data system

thereby avoiding possible troubles with nonlinearity,

damage, dirty contact or displacement errors in the

slide wire potentiometer and lag, nonideal damping,

ill-defined temporal integration or mechanical troubles

in the pen drive system. Signals are also extracted

from the appropriate high-speed relays in the instru- ment to allow corresponding high-speed relays in the

interface to provide proper synchronisation of sampling. The sample and reference values are read

alternately each mains cycle by a digital voltmeter,

and the values transferred to the appropriate buffer

for real-time processing.

The wavelength value is also read into the same

buffer each mains cycle by an absolute wavelength

encoder coupled to the output shaft provided on the

spectrophotometer to give a resolution of 0.1 nm. An

11
absolute encoder is preferred to the incremental type more commonly used in such systems because it avoids troubles from the operator starting at the wrong

wavelength by mistake, problems with extraneous

pulses (perhaps from power thyristor operation in a

stabiliser in a neighbouring room) causing a gradual

lack of registration between the wavelength and the

record held in the register of an up-down counter, or perhaps momentary malfunction in the system. With

an absolute encoder and wavelength being read at least once per 0.1 nm, momentary malfunction can only

influence the current value, not future ones, and even

if intermittent malfunction occurred, only a very small

proportion of the data employed in the real-time

routine described below would be affected. The most advanced feature of our data system is the real-time procedure by which digital noise filtering

is applied to the collection and initial processing of

spectral data. Suppose that during a scan the wavelength encoder has just changed its indication to a new value 0.1 nm different from the last. During the

time that it indicates the same wavelength (rounded

to 0.1 nm) the sample and reference signals are col- lected and summed into separate registers, and the

number of photometric values of each type is also held, so that on the next change of indication of 0.1 nm in wavelength value the arithmetic mean of the

reference and sample beam values can be properly

computed and their ratio taken. For ultraviolet and

visible work the commonest scan speed used (1 nm/s)

gives on average 2\ reference and 2\ sample beam

readings per 0.1 nm interval. This process continues

during the scan, but the arithmetically meaned photometric ratios are also multiplied by a special weighting

factor or factors, depending on whether the wavelength

value is at the time within one or more spectral

"windows" within which piece-wise fitting of low order

polynomials is required. The user can command the

system to give at any required output wavelength

\0 the value which would be obtained by making a

least-squares fit of a cubic (or alternatively a quintic)

to (2n+l) photometric ratios, one for each 0.1 nm

interval, where a wavelength range of this "window"

extends from (Ao~ n) to (k0 + n) in units of 0.1 nm. The larger the value of (2n + 1 ) , the more effectively is noise and variance filtered out, but if (2n + 1) becomes too large the portion of spectral profile being

investigated within the "window" will not be properly

represented by a portion of a cubic function, hence

there is an optimum choice for (2n+l) to suit the

type of profile being measured. The largest value for (2n+l) permitted is 101 and this implies a spectral

"window" of ± 5 nm about the required value. However

this "window" does not lead to anything like the

amount of degradation in the spectral profile which

would occur if a simple "running mean" were being

evaluated over the same width of "window." When

dealing with very sharp profiles the minimum value of (2n+l) permitted is 3 for the cubic fit, which gives no data improvement over the initial photometric

ratio at the wavelength required, apart from that

arising in the first stage of data processing when a photometric ratio of arithmetic means of reference

and sample beam readings is produced at each 0.1 nm interval. Obviously in such cases one uses a slow scan speed to allow the simple arithmetic means to give adequate noise reduction, and if one used a speed

of 0.05 nm/s then the means of 50 reference and 50

sample beam readings are used to give the output data

at 0.1 nm intervals. The weighting factors used in executing the piecewise cubic fit are automatically evaluated by a special

algorithm as soon as the command choosing (2n+l)

is made prior to a spectral run, and the essential algebraic basis is given below.

For (2rc + l) equispaced ordinates extending from

(Ao — n) to (\o+rc) in units of 0.1 nm in this case, the photometric value can be written T(i), where

i = — n (l) + n. The quadratic fitted value for T(0)

is F(0) , where

F(0)= J W{i)-T{i)

i=-n

which is calculated during a scan. The weighting

factors W{i) are calculated beforehand, immediately

the value of n is typed in:

W{i) = (S4-S2 • i

2 )l[(2n+l) S4 -S22 ]

where

n S2 = 2 i2 = (2/i+l) (n+T) rc/3 i=-n

n S4 = 2 i 4 = S2 (3n2 + 3«-l)/5.

i=-n

It should be noted that the general algorithm given

here is that required for fitting a quadratic to any

number of equispaced ordinates: owing to the fact that the odd-order terms in a polynomial do not in- fluence the fitted value at the central ordinate of a symmetrical "window" (the only value we are using),

the result of fitting a quadratic is identical to that of

fitting a cubic, and that for a quartic is identical to that for a quintic, etc. [23]. As the algorithm becomes

more difficult to derive for successively higher order

polynomials, we obviously use the lower (even)

order case that gives the same result. We also have the

general algorithm for the piece-wise fitting of quartic

(and hence quintic) polynomials, but it is omitted for brevity.

One particularly useful feature arising from the

flexibility of the real-time procedure is that the spectral scan speed can be altered freely during a scan; for instance, a much slower scanned speed can be adopted

when passing through a water absorption band in the near infrared than for the rest of the scan, which avoids

certain well-known problems with slit or gain servos. Not only this, but the scan can be stopped and re12
started or even partly back-tracked and restarted

without corruption of the acceptable part of the scan

already produced.

The use of the backing store to hold an assortment

of user program texts and standard data blocks as well

as an image of the system software, compiler and

maths packages is a great convenience, but is quite

conventional and needs no further comment here. What have proved to be the main advances in NPL's

recording spectrophotometry given by the on-line

data system? The main objective has been amply

achieved, namely a major reduction in the staff time

involved in all stages of standards work and calibration services for outside organisations, from making

the measurements through various stages of data

reduction to the final tabulations in our records or the

despatch of a calibration report. The ease of producing

and comparing data has led to anomalous operating

conditions of the spectrophotometers being spotted more certainly. We have been gratified to find that the precision and repeatability of measurement has

also been significantly improved, which suggests

that the slide wire potentiometer and pen servo system

of the Gary 14 instrument imposes a major limitation on its performance when used with a pen recorder

output in the normal way. However we have not been

deceived into imagining that the data system has

necessarily reduced the systematic errors of measurement. Since we do not rely on commercial spectrophotometers to provide our scales of measurement,

this is no great worry to us. A considerable number of improvements have been

made to the Cary 14 instruments and their accessories

by NPL staff, but it would make a long and tedious

discourse to describe them all and the majority of

them are specific to the particular instrument. However one of the more minor improvements is worth

mentioning as it can be applied to a number of other

types of spectrophotometer as well as the Cary 14. This concerns the replacement of the RCA side- window photomultiplier type 1P28, used in the normal

transmittance configuration, with one of the new EMI

type 9670 tubes. We purchase these tubes specially

selected for high near-infrared response, and they can

be supplied with uniform sand-blasting of the silica side-window to give diffusion of light over the cathode.

The diffusion is needed to reduce effects due to any

displacement of the beam by the presence of the

sample, and such sand-blasting is present in the

special 1P28 tube supplied with the instrument.

The great difference lies in the relative spectral

responsivity of the S-10 type cathode as compared

with the S-5 response of the normal or selected

1P28 tube, see figure 8. The limitations imposed by

cathode responsivity only make their presence felt towards the ends of the spectral range that can be

covered, and the lower quantum efficiency of the

9670 tube at wavelengths below about 590 nm is no

practical problem. However at wavelengths above

600 nm the newer tube is clearly better, not merely

because of higher spectral responsivity (which ex10

Quantum

efficiency

(log scale)

-4

IO

S-2Q

S-5

\lS-20

s-io

\ s-io

S-5

200 300 400 500 600 700 600

Wavelength (nm)

Figure 8. Spectral responsivity characteristic for EMI 9670 tube (S-10 cathode), a plug-in replacement for RCA 1P28 (S-5 cathode). The most desirable cathode type (S-20) is not available in compatible side-window designs.3

tends its range by about 150 nm) but particularly

due to the much lower spectral gradient. Performance

is improved in two ways: heterochromatic stray light

reduction and a more gradual variation of the slit servo during a scan. Heterochromatic stray light

itself is benefited in two ways in the 600 nm to 900 nm

region: the lower spectral gradient would mean less shorter-wave stray light at a given slit opening, and the

proportion of stray light for a given spectral gradient

decreases markedly as slit width is reduced by the

slit servo due to the higher absolute spectral responsivity in this region. The EMI 9670 is convenient as it is a plug-in replacement for the 1P28 tube, so that no

modifications are necessary. While a tube with a S-20 response would be better still, figure 8, no

compatible type is known to us.

3 A new compatible

type has recently been announced by RCA with considerably extended red response, the C31025 series

with Ga As or Ga In As cathodes, but I have not yet

heard any satisfactory reports on these from other

photometrists.

Another of the more minor improvements which is of general application concerns the choice of tungsten

halogen lamp. Curiously in a double-beam system, we found that most of the types we tried gave rather

poorer repeatability of measurement than the conventional gas-filled lamp originally supplied, and we

attributed this to changes of filament position in rela- tion to the rather striated sihca envelopes used, which

caused alterations in the light distribution at both

slit and field aperture and which affected beam

balance. We have found that an Atlas 30 V 250 W

single-coil lamp type Pl/8, running at 3400 K on full rating, has a particularly good quality envelope and

gives both stable and efficient operation. The single

coil filament allows one to tilt it to get maximum

filling of the entrance slit using the Moire effect,

Subsequent to the Conference EMI have announced a compatible tube type 9785 with S-20 cathode. 13
and about 50 percent more throughput can be obtained

than with the 120 V 650 W tungsten halogen source which the manufacturer can supply, while heating

problems with the monochromator are eased.

IV. Determination and Correction of

Systematic Errors As described in section II, the spectrophotometrist

seeking after high accuracy should concern himself

much more with correction of systematic errors (and

indeed avoiding them as far as possible) than with

statistical exercises that relate to variance and random

types of error. In the subsections 4.A to 4.D that follow some guidance is given as to how to deal with the more

important types of systematic error. As the precise

procedure depends on the details of the installation used, any description must refer to some particular

type of configuration, and the NPL High Accuracy

Spectrophotometer has been used as the model. It will become apparent that the procedures can easily

be applied to any other instrument working on the same manual single-beam dc principle, but often not at all easily to a typical commercial instrument

working on the double-beam chopped synchronous

detection principle. While manufacturers have sometimes made drastic modifications so as to produce

special versions of their instrument to investigate

errors, this does not normally lie within the power of a user.

A. Linearity Correction, using the Double-Aperture

or Multi-Aperture Methods

At NPL the preferred method of determining linearity errors in the photoelectronic system is the DoubleAperture method, and it has continued in use in high

accuracy spectrophotometry for over 40 years. The

method tests the linearity of response of the complete

instrument system as it is set up for measurement of a sample, whereas other methods (sec. IV. B) often

involve drastic modifications or major additions to the

system or separate investigations on parts of the

system i.e., the photocell, or amplifiers or potentiometer and the pen recorder or the digital readout, or some combination thereof. Provided that the method

is sound, it therefore allows a single investigation of

linearity to settle the question for the complete

instrument at the time involved. The double-aperture

method is the most accurate, sound, simple and fool- proof technique that we know of for testing linearity in a manual single-beam dc-mode instrument.

Essentially, the only modification required to the

system is the introduction of a double-aperture device

into a suitable place in the optics. The advantage of

this is that no change need necessarily be made to the

system, and linearity testing can be commenced at any time. The most recent design in use at NPL since

1967 is shown in Plates VI and VII, and this was used

as a model by R. Mavrodineanu for the one made for

his new installation at NBS. We have devices with

both square and circular aperture formats to suit differPlate 6. NPL double-aperture device for linearity testing: general view of the side facing the incident radiation showing the bevelled edges.

Plate 7. NPL double-aperture device for linearity testing: detail of the side facing the detector system showing the mechanism and

use of bevelling where possible.

ent applications, and the sliding leaves are operated

independently by Bowden cables from a pair of pushpull knobs each with a detent to permit the leaf to remain open. Leaf and knob are each spring-loaded to ensure that if the knob is not caught in the detent the

leaf will always close fully. When closed all the edges

of the leaf adjacent to the edge of an aperture are enclosed in closefitting slots to ensure that no light can leak through, and this requirement explains why

a septum is used to separate the apertures. (Earlier

designs have often not had this septum). The edges of

14
the apertures are bevelled on both sides where possible, and the brass used in construction of the aperture

plate and leaves is chemically blackened. This blackening leaves a weak specular component of reflection

but very little diffuse component, and to prevent re- flections of light back to some preceding optical component and thence back through one or both of the

apertures, the whole device is slightly rotated about a

vertical axis from the perpendicular orientation when

being aligned.

The double-aperture device permits the departure

from linearity of the complete instrument to be measured over any 2 : 1 step within the range of the photometric scale, and the procedure is first to place and

align the device in a suitable part of the optical system

(see later) and adjust the vertical position until the

readings of each aperture (the other being shut) are approximately equal. If the readings from each aperture alone are A and B, those from both together are (A+ B), and those with both shut are D, then a minimal

sequence of readings is DAB (A + B) (A + B) BAD

and this would be called a single-volley set. This is of course a time-symmetrical sequence to minimise

drift and variance effects, and equal numbers of observations of each type are made to give equal statistical weight. For more accurate work, double-or-triple

volley sets of observations are made for any particular

2 : 1 step. If the mean values of each type of reading

are calculated then the linearity error e is calculated

as e=[A + B-(A +B)-D]l[(A + B)-Dl

The error e is normally expressed as a percentage,

and the correction to be applied to a measured photometric value of 50 percent over the corresponding part

of the scale is — e. But what if the photometric value to be corrected is not 50 percent, or is not measured over precisely the same part of the scale? To cope with the general case

the procedure is to cascade the linearity testing in binary steps, so that if the first step involves output

readings of full scale (f.s.) and 50 percent f.s., then

the second step involves outputs of 50 percent f.s. and 25 percent f.s., the third step 25 percent f.s. and

12£ percent f.s., and so on. Various methods can be

used to reduce the (A+B) reading of the nth step to the A reading of the (n— l)th step, such as reduction

of lamp power, slit widths or use of a supplementary

attenuator such as a filter. The settings of the successive steps to half the readings of the preceding

step need not be closer than 1 percent of the value. This, and the fact that the A and B readings need not be equal to closer than 1 percent of the value, arises because the input-output relationship of the photoelectronic system, if not exactly linear, is always at least a smooth continuous function and approximately

linear in any instrument that is in sound operating

condition.

The method of linking the errors from each 2 : 1 step to form a series of points on a continuous error function is illustrated in figure 9. On the NPL reference

instrument this function has always been found to be

smooth and regular, but if it were not a regular smooth

curve this fact might be missed due to the gaps between data points, especially at the upper end of the

curve. Hence intermediate steps forming a second

series of steps such as 70 percent f.s. and 35 percent

f.s., 35 percent f.s. and 17£ percent f.s., etc. should be

determined on any new or modified instrument. The

70 percent f.s. point is attached to the smooth curve drawn through the normal series 100 percent f.s., 50 percent f.s., 25 percent f.s., etc., and the points

corresponding to 35 percent f.s., 17£ percent f.s. etc., are then linked to the assumed position for 70 percent

f.s. as for the normal series. If the smooth regular curve assumed through the normal series of data points has

been drawn correctly to fit the true error function,

then the points at 35 percent f.s., 17i percent f.s., etc., will also be found to lie on the curve. In general

the second series will suggest some slight improvement

in shape to the error function as in figure 9. A really

sceptical standards man would then proceed to find a third series of value corresponding to some series

like 85 percent f.s., 42£ percent f.s., 2L| percent f.s., etc., and test the error function further.

Linear

error € C/o

of valu

Normal range of 2 = 1 steps k

1

1

1

1 -

1

1

1

1 1

1

1

1

t>U% l2''2°/o 25% 50°/o Output reading, percent of full scale

Curve obtained with normal steps

- Curve after improvement using supplementary range of steps

I007o

Figure 9. Upper diagram shows how the linearity error function

is built up from the errors ei, e 2 etc. found with each successive cascaded 2:1 step, starting with 100 percent full scale. Lower

diagram shows how the linearity error function found with the normal cascaded 2 : 1 steps (dotted) may need altering to accom- modate the errors ej,e'2 found with a supplementary range of 2 : 1 steps, starting in this case at 70 percent full scale.

The double-aperture method may be regarded as completely sound if subject to the following conditions.

(1) Any additional component of reflected light

introduced by the presence of a leaf blocking one

aperture must not be allowed to pass to the detector

15
by the other aperture or by any other stray light path.

This is met by keeping the double-aperture devices

as far from other optical components on the illuminated

side as practicable, by masking any bright parts of these components with black flock paper and by tilting

the device slightly as mentioned before.

(2) By the same argument, there must be no alter- ation of reading from one aperture due to changes in interreflected components on the detector side of the

device caused by alteration of the other aperture leaf. This is met by keeping the double-aperture device as

far from optical components on the detector side as possible.

(3) There must be no relative change of light distri- bution at the double-aperture during a volley of

observations. Apart from trying to prevent relative movement (not always completely possible, due to the

need for adjustability and the opening and closing

forces imposed), the light distribution should be as uniform as possible and slightly larger than the size of the double-aperture, so that to first order any rela- tive movement produces no variation of reading.

(4) There must be no change of fight distribution

at the actual detector surface during a volley of observations, since an ideal filter inserted into the beam for measurement will cause no such change. In the NPL

instrument the use of the integrating sphere as a

diffusing device ensures that this condition is met.

If the double-aperture device were focused onto the

photocathode, the test would not be valid since when

both apertures are open for (A+ B) there would be two

separate portions of the cathode acting like two photocells in parallel, and an intrinsic nonlinearity would

not be detected if two photoelectron streams remained

reasonably separate as in many types of photomultiplier.

(5) There must be no optical interference between

the fluxes transmitted through each aperture. According to K. G. Birch of Optical Metrology Division,

NPL this condition is met for all practical purposes in typically dimensioned equipment when conventional

tungsten or discharge sources are used, provided that

slit widths are not reduced below 0.002 mm during the

linearity testing.

A consequence of (1) and (2) above is that at NPL

we usually change the optical system within the photometric enclosure for linearity testing to allow the

double-aperture device to be as far from other optical

components as practicable.

The freedom from optical interference effects mentioned at (5) above arises because with slit widths

commonly met with and with conventional sources

the "coherence patch" at the double-aperture device

is not large enough to straddle the septum, which is of order 3 mm wide. However to make doubly sure

that interference effects cannot invalidate the test, the practice at NPL is to actually focus the doubleaperture onto the target screen in the integrating

sphere in front of the detector. Then even if the "co- herence patch" were to cover the whole doubleaperture, there would be no overlap of the fluxes from

each aperture at the screen where they receive their

first diffusion, so that no fringes could be formed there. The region of the slit image is the only place where

fringes could be formed, and we make sure that the

mirror or lens which images the double-aperture onto

the target screen is not in this region as such fringes

could interact with flaws, blemishes or dust particles

to give readings capriciously high or low according to the precise relative positioning.

For systems employing temporal or spatio-temporal

chopping of the fight, a further condition must be met.

(6) There must be no change in the temporal features

of the waveform reaching the detector between the

three types of reading, A, B, or (A + B), whether of shape or phase: only a strict change of amplitude is permitted. This cannot be met if the light distribution

of the beam alters at all during the sampling time,

due to actual movement or due to sector-mirrors or rocking mirrors having a non-uniform reflectance

due to poor coating, ageing, dust or pollution.

The requirement (6) arises because no ac-detector

circuit (tuned narrow-band, synchronous sine-wave,

synchronous square-wave, homodyne, heterodyne,

or any other type) is ideal in the sense that it functions exactly as supposed, and changes of waveform

shape or phase at a given amplitude will therefore

generally cause a spurious change of output. This

condition (6) can seldom be proved to be met in commercial instruments.

EHT

volts

Resistor R ohms Wavekngth (n m) 438 S50 740

75 O 3 I0 6 V 950 3 I0 6 * 750 lO' o + 950 lO' 0

Output reading (volts)

Figure 10. Linearity correction function of the NPL High Accuracy

Spectrophotometer, showing corrections found for the range of EHT voltages, load resistors and wavelengths covered in one

particular investigation. It happens that in this case the correction function is not sensitive to changes of operating conditions over

a certain range of values, and a common curve is representative.

The dotted curves show the probable error that would be quoted

for the accuracy of the correction.

The linearity correction function of the NPL High

Accuracy Spectrophotometer is shown in figure 10 in terms of results obtained before and af ter a particular

programme of filter standardisation. The curve drawn

for the normal sequence 50 percent f.s.,25 percent f.s., 12£ percent f.s., etc., has not been modified to suit the

16
sequence 70 percent f.s., 35 percent f.s., 17i percent

f.s., etc. Results are shown for the two values of

dynode load resistor used, for different values of

photomultiplier E.H.T., and for several wavelengths.

Strictly speaking one should not assume that the cor- rection required is the same when any of these key

parameters is varied; however it can be seen that the

correction function turned out to be not significantly different in any of the cases tested. In general, there

is usually a preferred range of output current —

E.H.T. combinations where an invariant and almost

linear characteristic is obtained for the photocell.

However departure from Ohm's Law behaviour of

the load resistor is often a larger source of linearity

failure than the photocell itself in well-designed

systems, so each load resistor needs a separate in- vestigation. As it has been found that the linearity

correction function has remained stable over a period

of several years, no attempt has been made to find load resistors that are more nearly ohmic in characteristic: a survey of resistors with thorough testing of

all available types and ratings of interest would

prove too costly to be justified in these circumstances.

It is not necessary to test linearity at different wavelengths unless it is thought possible that different mechanisms might then be operative in the photodetector. Some photocells do indeed have different characteristics in certain wavelength regions, usually

at the long wave end of their spectral response. So

when a new instrument or photocell is being tested

for linearity, several wavelengths covering the whole

range of spectral response should be used to check

this point.

The multi-aperture method, commonly employing

ten apertures, is but a variant of the double-aperture

method, and is equally sound in principle. It has been

used at NPL by lamp standards photometrists, whose

needs are not quite so exacting as spectrophotometrists.

In fact the multi-aperture method cannot be made as accurate as the double-aperture method with a given

number of observations on a particular aperture combination, because it is impracticable to use a time-symmetrical sequence of readings which allow the values

A, (A+B), (A+B+C), . . . (A+B+C+D+E+

F+G+H+I + J) to be determined with equal and

optimum accuracy relative to each other so as to pro- duce ten points on the error function of equal uncertainty. To get results of equivalent accuracy, the

demands on system stability are rather greater than

with the double-aperture approach.

The double-aperture method allows the linearity

correction required for any given 2 : 1 step to be deter- mined to a few parts or less in 104

, depending on the

number of volleys of readings used. The accumulation

of probable error for cascaded steps is illustrated in figure 10.

B. Linearity Correction, Using Other Methods

A great many approaches have been suggested

over the years to the problem of determining linearity

errors, and it is not proposed to review them critically

in this paper as each would need a section equivalent

to the preceding one to cover relevant points thor- oughly. In this section some indications will be given

as to why these other approaches are not preferred

at NPL. In essence they nearly all amount to employment of a device or equipment which varies the

irradiance at the photodetector in a predictable or calculable manner. Methods which involve the photocell being removed and tested away from the rest of

the spectrophotometer can tell us much about the

photocell but require supplementary investigations

to be carried out on the rest of the photoelectronic

system, for example on the Ohm's Law behaviour of the load resistors. Since even when this has been

done, assumptions have to be made as to how the

photocell might behave when in the instrument and

connected into the system circuitry, such an approach

cannot be regarded as foolproof. To give illustrations,

temperature rises, magnetic fields, earth loops or cross modulation effects within an instrument can give rise to real or apparent linearity departures which have

confused many an unwary investigator.

The double and multiple aperture methods are examples of the general principle of addition of fluxes,

and the other methods which use this principle are equally sound but are difficult to implement experimentally with sufficient rigour. The use of several sources is clumsy as demands on the stability of the

optics and the sources are great, and the apparatus

is bulky. The device of using one source but multibeam optics avoids severe source problems but leaves

the problem of optical stability. Where beam-splitters

are used for dividing or recombining the beams, the

problems of polarisation, scatter and multiple reflec- tions have to be faced, and where one source is used

the interference effects are more likely to be trouble- some than with the multi-aperture method because

optical superposition of related wave fronts is more

closely achieved. Beam-splitters are in general spectrally selective, and this makes their use for linearity

testing difficult over a wide spectral range. A pair of plane polarising devices used as polariser

and analyser offers a possibility of producing a net transmittance varying predictably as a function of the angle of rotation of the analyser. Most polarisers

(sheet polarisers, Brewster windows and electro- optic devices) are too poor in quality and too spectrally

selective to be taken seriously, but polarising prisms

of high quality have been used in attempts to obtain accurate linearity testing. The problem here is that the predicted law is only obeyed in practice over a

limited range of optical density (absorbance) for as the "crossed" position is approached the optical

density starts to change very rapidly making the

orientation and alignment very critical, and at densities above unity the slightest flaw, surface blemish,

or foreign particle on the intermediate surfaces causes

partial depolarisation and hence a breakdown of the

predicted law. Another problem is that the light passed

by the analyser is plane polarised and at varying

orientations, and most photodetectors are sensitive

to polarisation effects to some extent. Unfortunately

17
it is not possible fully to depolarise plane-polarised

light by means of a single diffusion, by reflection from

smoked or pressed magnesium oxide or from opal glass, or by transmission through opal glass when its trans- mittance is of reasonable magnitude.

The inverse square law has often been used as a means of controlling the flux incident on a photocell

in a predictable manner, but it is not good enough

for work of the highest accuracy due to the non- uniform radiation field emitted by practical sources.

If the source and detector apertures are finite, a cor- rection to the inverse-square law has to be calculated

using configuration integrals (a. process sometimes

called calculation of the mutual exchange coefficient),

but this assumes a Lambertian (Cosine Law) behaviour of source emission and detector response.

Practical sources and detectors never obey Lambert's

Law closely. I myself have used pot opal glass as a secondary source, carefully masked to give a known

aperture and with a tungsten ribbon source focused on the far side, but have not obtained good enough

obedience to Lambert's Law: the departure from

Lambert's Law is very difficult to measure by goniophotometry to sufficient accuracy to calculate an

adequate correction, and is wavelength dependent.

A "black body" source might prove acceptably

Lambertian, but its defining aperture presents severe

difficulties. Variable apertures are another old favourite for producing a controlled attenuation of flux, but the

method is generally unsound because of the impossibility of producing an optical beam in a spectrophotometer of uniform radiance cross-section. To over- come this the division of the aperture into numerous

small variable apertures has been tried using combs,

rotating grilles and Venetian blind devices. The problem then becomes a geometrical one of realising the

desired construction by precision engineering, and

high accuracy cannot be achieved reliably. An alternative approach is to use fixed meshes or perforated screens as attenuators, but they too require calibration

by some metrological procedure and this is unsound

because not only is transmitted light lost in an instru- ment by diffraction but also for small apertures the

effective area for transmission is less than the metrological area. This last point arises because radiant

energy is absorbed for a distance of the order of its wavelength away from any metal edge: this effect of physical optics is too complicated to permit of any

adequate calculations for a correction in any real

situation. Testing for obedience to Bouguer's Law or Beer's Law is another much used method of testing linearity,

especially among chemists. However it is fallacious,

for if these laws are verified for an instrument it only

establishes that the photoelectronic system obeys a simple power law, not that the power (exponent) is exactly unity. In fact some particular faults in photoelectronic systems lead to precisely this type of

nonlinearity, so that the method can be dangerously

reassuring. Apart from this, Beer's Law seems to be

exactly obeyed in remarkably few of the solutions

that have been rigorously examined. The usual form

of application involves cuvettes of varying path length

or solutions of varying concentration. A variant is the

technique suggested by Campbell in the 1930's but

resurrected by Slavin and Porro [24], of grinding and

polishing homogeneously absorbing glass to different thicknesses and verifying whether the density (absorbance) is proportional to thickness after allowing

for reflection losses. This is equally fallacious of course. Sector discs are an important method of accurate

attenuation, and they are spectrally nonselective, not

sensitive to polarisation and relatively easy to construct accurately and measure metrologically. If the

Talbot-Plateau Law is obeyed the transmittance is linearly proportional to the angular opening. The

problem is whether the detector really obeys the

Talbot-Plateau Law: while many solid-state types do

not, it happens that with suitable circuitry and choice

of operating conditions vacuum photoemissive devices

obey the law very closely indeed. This has led to the

development of the technique of Time Ratio Photometry

at NPL, as described in section V.

C. Heterochromatic Stray Light

The unqualified term "stray fight" in spectrophotometry usually means heterochromatic stray light i.e., light transmitted by the monochromator that is outside

the required or set waveband. There are in fact other

types of stray light, referred to in the following section

IV. D. Heterochromatic stray fight arises from three main causes: general small angle and wide angle

scatter from dust, blemishes and pollution of optical

surfaces; stray reflections, especially where lenses

or prisms are used; and unwanted orders of diffraction

in grating instruments. General scatter is usually the worst culprit, and because of this it nearly always

happens that the components of stray fight become

progressively smaller as their wavelengths depart

further from the set wavelength.

The obvious approach in reducing errors is to pur- chase a high quality double-monochromator, and for guidance we would rank the different types of system

in the following order for stray light: best is prismprism, then prism-grating and finally grating-grating.

However, even with such an instrument there are circumstances when significant heterochromatic stray

light errors can arise. This is most commonly met at the long wave limit of ultraviolet and visible instru- ments, where a photomultiplier is used and particularly

where a grating has been blazed for optimum per- formance in the awkward 280 nm to 340 nm region

where one changes from an incandescent lamp to a hydrogen or deuterium arc. That choice of blaze has

of course been dictated by the need to increase net response in this latter region and reduce heterochromatic stray fight there, for this region is the second

most suspect, apart from the region of short wave cutoff imposed by air and silica absorption at around 185 nm. For any source-detector combination the error

is maximal where the net response is very low compared with the average over the usable range and

18
where the spectral gradient of response is steep, and

this normally occurs near the limit of the usable range.

It is for this reason that we are prepared to sacrifice peak quantum efficiency if necessary in order to obtain

a less steep spectral gradient of response at the red

end of the working range. The general method of testing for errors is to use

selective band or cutoff filters, and is well enough

known to need no description here. It is important to check that introduction of the selective filter has no

adverse optical effect. A convenient place to introduce

such a filter into a common-path part of the system in a double-beam system is between the source and

entrance slit, but this may alter the light distribution

at both slit and field aperture and hence give a bias to the reading other than due to its absorption properties.

It is therefore particularly important to adhere to the

strict substitution principle in making the additional

measurements. The same requirement applies equally

when matched pairs of selective filters (even if of high

quality and closely matched) are introduced into the sample and comparison beams of double-beam

instruments.

D. Homochromatic Stray Light

Homochromatic stray light is any light which is present in addition to the light which has passed to the point in question along the optical system in the

desired manner, and which is of approximately the same spectral distribution. Thus it means in practice

all stray radiation except the heterochromatic stray

light from the monochromator or any leakage of

ambient light through chinks in the casing. (Leakage

of ambient light becomes obvious if one switches off or covers all fight sources in the room and blacks out the windows and one can then detect an alteration of output or of operating conditions, and this will not be

commented on further.) Homochromatic stray light

usually arises from specular or diffuse reflections, or from diffuse components of transmitted light in the case of transmitting elements of the system, and

consists in general of light which has departed from

its desired optical path and which somehow re-enters the system so as to be included with the required light

being measured. There are so many mechanisms by

which this can happen, and they are so particular to the precise optical configuration, that one cannot

discuss the various possibilities in any general way.

I will only mention therefore a few specific types of homochromatic stray light and how we deal with them.

Interreflections, involving specular reflections from

optical surfaces or from bright metal components like

slit jaws, and lens or mirror mounts, have two effects:

if they involve the reflection from the sample itself then they impose a direct error on the photometric

value being measured, whereas if they are independent of the sample itself they only impose an

indirect error due to the spatial or angular distributions

of illumination and collection not being quite as re- quired. Tilting the sample usually reveals the direct

type of error, but there is one circumstance (commonly

met with in commercial instruments) where this is not

necessarily so. I refer to the practice of placing a

filter or cuvette at a slit image to minimise beam

movements at a conjugate image formed at the

detector. If an optical surface at a slit image is tilted slightly the reflected beam is deviated to cover a slightly different part of the aperture of the mirror or lens which

has focussed the radiation, but a slit image will still be formed from the reflected light back at the exit

slit of the monochromator, and it will not move. As

slit jaws are bright-edged and either flat to the outside

world or bevelled (often at 45°), and as monochromators

often have plain windows there to prevent entry of the atmosphere or its pollutants, it is clear that there

is usually a reflection back once more down the optical

system towards sample and detector, and this will be focussed yet again to form an image at the specimen.

Direct errors from interreflections can also occur on

the detector side of the sample, especially if a bare

(diffuserless) end-window photomultiplier receives

light normally. In NPL standards work the sample

is therefore never placed at a slit image if this can be avoided. The presence of both types of inter- reflection (i.e., involving the sample or not) can be

checked most readily by eye using a dental mirror.

Both specular and diffuse reflections often occur at surfaces close to the beam and parallel to it, such as inner surfaces of lens or mirror mounts or tubes, and

the cure is to add suitably shaped and dimensioned

stops in the system. Stray specular reflections may

strike the instrument casing or other parts and be

diffusely or specularly reflected so as to "by-pass"

the sample. This provides a further argument for a

large photometric enclosure (sample compartment).

This type of effect can cause serious errors in very

dark samples, and a routine practice is to place a dummy (black) sample in the sample position and of identical dimensions to the sample and then take dark

readings, and compare them with the more normal

dark readings obtained by blocking the beam prior to entry into the photometric enclosure. The latter reading gives the photoelectronic system component of dark error (assuming there is no leakage of ambient light), whereas the difference in these readings gives the optical component of dark error. This

procedure is very important in total and diffuse re- flectance measurements, for small angle scatter in the sample beam optics preceding the integrating

sphere causes a halo of scattered light surrounding the

illuminating patch on the sample, both on the outer

parts of the sample and on the sphere wall. In this case

the "black" sample consists of a blackened cavity

brought up against the sample port, and the error is usually the largest component of systematic error found in reflectance measurements, representing a zero offset ranging from roughly 0.1 percent in the

infrared (2 /xm) to roughly 0.5 percent in the ultra- violet (0.3 (Am) in reflectance value but depending

on the condition of the preceding optical surfaces.

For transmittance measurements an opaque dummy is used, with a mirror or matt white surface attached in some cases to exaggerate particular types of stray

light effect.

19
Samples should always be masked so as to cover

their edges and have an opaque surround. This is to prevent by-passing of light, especially in small specimens, for it is not uncommon for stray reflections or small angle scatter to produce a weak flux distribution

alongside or surrounding the beam: again I emphasise

the use of the eye looking along the direction of the

beam. The reference sample (or the absence of a sample for an "open" reading in absolute transmittance) must also have precisely the same masking

present, to prevent systematic error. However these

local masks on the sample carriage are not sufficient:

it is also necessary to have a fixed mask as close as possible forming a locally limiting stop. This is to cover

the possibility that the sample and reference masks

do not assume the same position due to misalignment on the carriage or faulty adjustment of the stops on the

carriage guides, or that they are not precisely the same

size and shape. The fixed mask should either be slightly

larger than the beam cross-section there, with clear- ance all round, or it should be smaller than the nearly

uniform "core" of the beam cross-section. This is to obtain optimum photometric stability if one accepts

that some slight relative movement of optical components might occur. At NPL the direct interreflection error (involving

the sample itself) is avoided by tilting the filter by an

angle of order 1°, keeping the solid angle of illumination down to half-cone values of under 1°, avoiding

forming a slight image at or near the filter, and catching

the beam reflected back on to a piece of black velvet or flock paper placed just to the side of the exit slit. Even with such precautions, recent investigations with

highly reflecting filters have shown that great care needs to be taken with metal-film or thin-film filters, and errors are likely to be appreciably greater than

with normal absorbing filters. Phenomena which can give rise to systematic errors

in spectrophotometry are photochromism, thermochromism and fluorescence; but these could arguably

be regarded as defects in the sample rather than

defects in the measurement procedure. As with testing

for heterochromatic stray light, the principles of

testing for these phenomena are well known and

largely common sense once the nature of the problem

is understood, and further comment will not be made

here.

E. Polarisation Errors

If we confine our attention to the normal isotropic

materials used in standards and calibration work,

then Fresnel's Equations tell us that for light incident

or reflected at normal incidence polarisation has no

effect on the measurement, while for angles near to normal polarisation effects are negligible. This is fortunate, for all monochromators produce a partial

plane polarisation that is very significant, and it means

that transmittance measurements are usually not subject to a significant error due to polarisation

effects. For measurements of direct reflectance,

diffuse reflectance or of radiance factor involving

inclinations above 10° from normal however, certain

precautions must always be taken as explained below.

The proportion of polarisation varies appreciably

from monochromator to monochromator, so it is never

satisfactory to quote a measurement which refers to the particular polarisation conditions (often not known

reliably) in one particular instrument only. So we must

specify "standard" conditions: but what are these?

There are 3 obvious candidates: 100 percent s-polarised

light, 100 percent p-polarised light, or randomly

polarised light. The most generally useful value for a photometric quantity is the one which refers to randomly polarised light. iJnfortunately randomly

polarised incident irradiances and collector/detector

systems not selective for polarisation are almost impossible to produce in instruments without sacrificing light

by the use of heavy diffusion, and one therefore measures the quantity with a plane polariser present in either the irradiating or the collecting system or both,

depending on which of these are inclined at appreciable

angles to normal. This results in two (or sometimes

four) measurements being made, and the required

result for randomly polarised light is easily calculated

from these as it is the arithmetic mean of the measurements. V. Time Ratio Photometry

A few years ago O. C. Jones and I invented a new

technique of photometry [25, 26] aimed at equalling

or perhaps surpassing the accuracy achievable by the

dc single-beam method, which relies as we have seen

earlier on the close approximation to true linear

behaviour of certain preferred types of photoemissive

detector and on the fact that any slight non-linearity

found in such systems is very stable and therefore

accurately correctable by use of the cascaded doubleaperture method of linearity testing. Our aim was to develop an independent scale for determining photometric ratios by which we could verify the validity of the existing method. At the same time we wanted

the technique to be self-calibrating and not relying on

corrections determined on another occasion, and also more suited to exploitation^ of modern developments

in digital data techniques.

A. Talbot's Law and the Principle of Time

Ratio Photometry

Talbot's Law, sometimes called the Talbot-Plateau

Law, states that light fluctuating periodically above

the critical fusion frequency of vision is exactly

equivalent to steady radiation of the same average

value, and the law was of course discovered (in 1834)

in connection with the response of the human eye. Mathematically, if F{t) is the periodically fluctuating

light and R is the response evoked,

R{F(t)} =R{F}

where

20
In 1913 Marx and Lichtenecker found that Talbot's

Law applied also to vacuum photoemissive cells, as

far as could be judged with the accuracy possible at that time. For a photocell that obeys Talbot's Law the mean photocurrent observed with a periodically

fluctuating illumination is the same as that obtained

if the incident flux is distributed uniformly throughout

the duration of the period. For this to be true in practice, the associated electrical circuit must be linear

and with an appropriate overall time constant. The

linearity is necessary to prevent rectification, whereby

energy can be transferred from the oscillatory terms

to the constant term in the Fourier series for the

chopped waveform as a function of time. In passing I would point out that linearity of the circuit elements

is also necessary in the cognate case of tuned or phase sensitive detector systems, to prevent errors due to cross modulation.

In 1929 Carruthers and Harrison working at NPL

[27], found strict obedience to Talbot's Law for a

variety of photoemissive cells, including some that were markedly nonlinear as judged by their steadystate behaviour. It would not be out of place to emphasise that if a sector disc, whose opening is say 10

percent of its circumference, is inserted in a beam of

light falling on a photocell obeying Talbot's Law, it is not correct to assume that the photocurrent falls to 10 percent of its former value. That will only be

true if the photocell happens to have a linear characteristic as well as obeying Talbot's Law. What Talbot's

Law does say is that the photocurrent falls to the same

value that obtains if a 10 percent filter is inserted in the beam.

To consider the essence of the time ratio method, let us assume that one is trying to measure the trans- mittance of a filter. The method is, however, readily

applicable to other types of photometric measurement.

Imagine a steady beam of fight illuminating a photoemissive cell with a suitable dc-measuring circuit. One needs a repetitive shutter, which opens and

closes fully, and whose open-to-shut time ratio can be

varied. The photocurrent obtained with the shutter

open and the filter attenuating the beam is noted,

then the filter is removed and the shutter sequence is started. The fraction of the total time during which the

shutter is open is varied until the same photocurrent is obtained, then some accurate timing technique is employed to measure this time-ratio. The transmittance

of the filter is then equal to the fraction of time for

which the shutter was open, provided that the photometric detector obeys Talbot's Law. It is as simple as

that. The beauty of the method is that the shutter can be

calibrated absolutely, in terms of time, in situ, and even

simultaneously with the measurement as described

later. Thus the shutter need not possess long term

stability of characteristics: an adjusted value need only

be held for a few seconds at a time.

It would be appropriate to break off for a moment to consider what is really happening, that a vacuum

photoemissive device can adhere exactly to Talbot's

Law yet give a slightly nonlinear response to steady

radiation. The three important factors in this are:

Firstly, the cell has an intrinsic response time of order 10-9 second, which is much shorter than any

chopping period that would be employed. Secondly,

the cell has an instantaneous intrinsic response that is normally strictly linear, if it could be measured a few

fj,s after a change of illumination with some suitable

fast circuit, this instantaneous linearity not being

affected by previous illumination. Thirdly, the cell possesses a relatively slow fatigue or drift characteristic whereby the observed photosensitivity changes

progressively under illumination. In our technique, if the cell obeys Talbot's Law,

drift or fatigue do not affect the measurement, because

the output produced by chopped radiation must suffer exactly the same drift or fatigue as that arising with

steady radiation of the same average value. Otherwise

the cell would not obey Talbot's Law.

B. Experimental Verification

We can now look at the experimental work carried

out at NPL to see a selection of the evidence relating

to the accuracy and the generality of Talbot's Law

obedience in actual specimens of photoemissive detectors. The finer details of experimental technique

and methodology will be omitted for brevity in this description of the work. Figure 11 shows the optical

and electronic arrangement used in the initial studies. Later work has used modified systems of the same

general kind. The variable shutter chops a slit image

to provide a full on-off modulation with a fast rise time

of a few microseconds, and for all our accurate fundamental work we have used a variety of sector discs. We have favoured mechanical or electro-mechanical

shutters throughout, for such devices really do provide

complete and stable modulation irrespective of wavelength, polarisation or angle of incidence.

A fraction of the light passed by the shutter is re- ceived via a beam-splitter at a supplementary gating

photocell, which was a spare photomultiplier of no

great quality. This had the function of simply opening

and closing electronic gates rapidly to permit pulses

from a stable 100 KHz oscillator to pass to counters

as appropriate. Most of the light in the beam reached

photodetector A, which in these first experiments was

connected to one of several types of dc-electrometer

current-amplifier via adjustable smoothing filters, the

dc-null-balance technique being employed. The elec- trometer null-balance system shown in figure 4 was

used for much of the work on photomultipliers. In some

of the later experiments, a digital voltmeter was used

to indicate directly the residual difference between

the signal and a backing-off voltage: the backing-off

technique proved very helpful in respect of the compromise needed between smoothing and slewing rate. We have emphasised small transmission factors of order 1 percent in most of the work, due to the fact that the differences in waveform and instantaneous values

between the test and comparison fluxes are then most

pronounced, and impose the most difficult sampling

conditions. Work on a vacuum photo-diode was carried

21
SOURCE *^

VARIABLE SHUTTER

MICROSCOPE

OBJECTIVES

FILTER

•F

1

| LIGHT , ELECTRONIC IOO KC/S ICOUNT SWITCHING DARK , UNIT OSCILLATOR COUNT

FIGURE 11. Optical and electronic system schematic for experiments on Time Ratio Photometry.

out by O. C. Jones and assistants, while work on

photomultipliers was carried out by myself and

assistants.

If Talbot's Law applies to some particular experimental configuration, then it follows that variation of

flash frequency at constant mark to space ratio should

have no effect on the output unless the frequency

becomes so low that a sufficiently smoothed and

readable output becomes impossible. The test is a necessary but not sufficient condition for Talbot's Law

obedience. It was found that a simple vacuum photodiode, with capacitive smoothing of the anode circuit gives no significant change of reading as frequency is dropped to 2 Hz. With photomultipliers, it was found

that discrepancies of up to a few percent of the value were obtained for a 1 percent sector if there was

inadequate capacitance loading of either the anode

or the last four dynodes. The reason for discrepancies

with inadequate capacitance in the anode circuit

is clear enough: with a reading of say 1 V dc, a 1 per- cent sector can give pulsed reduction of up to 100 V

on the final collecting potential difference, leading

to synchronised collapse of collection efficiency. The

reason for Talbot's Law failure when the dynodes

are not heavily loaded with capacitance is rather more

interesting. For any one stage of a venetian-blind type

of photomultiplier, there is a 0.7 power law relating

stage-gain to applied volts. Since most of the output

current is drawn from the last dynode, and most of the penultimate output current is drawn from the

penultimate dynode, and so on, it is clear that there

must be some voltage pulsing of the dynodes, which

are usually fed from a chain of resistors. The voltage

pulsing of the dynodes produces cross-modulation of the powers in the various terms of the Fourier series representing the square wave light modulation, and in this particular case some spurious rectification. By

analogy with the old term "anode-bend demodulation," we can call this effect "dynode-bend demodulation."

Our dynode resistors are made as low in value as practicable, namely 100K, and we found that with

25/xF per stage (giving 2£ s time constant) we could

eliminate the effect of dynode-bend demodulation.

In fact recent measurements show no significant

change for frequencies reduced to as low as 0.5 Hz,

provided that the currents are kept down to the range

where Talbot's Law is known to hold at frequencies

above, say, 20 Hz. In fact with a 0.4 percent sector and peak anode currents of up to 4 X 10_4 A (far above

the limit for linear behaviour under dc-conditions) no change was detectable to within ±2 in 10000 of the

reading for 1 Hz, or ±5 in 10 000 for 0.5 Hz. Larger

output currents give progressively larger departures

from Talbot's Law for a given gain or for a given flux. For a given output current, increasing the incident flux and reducing the gain to compensate will produce an

increase in Talbot's Law error. The most fundamental test for Talbot's Law obedi- ence is to measure a sector transmittance by ratio of photo-currents, to correct for nonlinearities and compare the result with the true value. But what is the

true value? This may be given by the time ratio technique:

T(o) T(o) T(t) - T(c)

T(t) ' °

r T(o) + T(c) ' °r T(t)

where T(o) is the total of "open" times, T(c) is the

total of "closed" times, and T(t) is the total elapsed

time during a counting period. However these ratios are only valid if T(t) is either an exact number of chopping periods or else so large that the error due to the usual residual incomplete period is negligible.

Further, the gating circuits must trigger at exactly

50 percent up the leading and trailing edges of the chopped waveform. These three versions of the sector transmission seldom agreed exactly, of course, and

these divergences indicated our probable error. Some results on this are shown in table 2. The

linearity correction for the vacuum photo-diode and

its circuit was negligible, whereas that for the photomultiplier and its circuit was finite. The linearity

corrections were obtained from supplementary

measurements by successive application of the

double-aperture technique of absolute linearity cor- rection. As explained in section IV. A this linearity

correction allows properly for nonohmic behaviours

of the load resistor as well as for true linearity de- 22
Table 2. Tests of Talbot's Law

METHOD OF °/o Transmission Effect of speed MEASUREMENT change from 30f/s

Singje Stoqe Cell °/o

Ratio of Photocurrents 1-473 3 126 <0 OOI

for speeds

Time - Ratio 1 474 3 129 down to 2 f/s

Computed from

dimensions 1 477 3 127

(uniform motor

speed)

Photomultiplier Ratio of 1 470

Photocurrcnts <0 003

Corrected

(for non-linearity)

1 473 for speeds down to IO f/s Time -Ratio 1 475

parture of the photocell. A further estimate of the

sector's transmission is given by metrology, difficulties arising here from uncertainties of the position of the true centres of rotation of these demountable

sectors. The sectors were directly mounted on the

shaft of a large dc compound-wound motor, and there was only inertial smoothing of any commutator pulsing.

The data shown here indicate Talbot's Law obedience

to within two or three parts in 10ft 000 of the

"open" reading, and the discrepancies are of doubtful

significance.

We now proceeded to apply time ratio photometry to measurement of a rather neutral absorbing glass filter of transmittance about 1 percent, and results are shown in table 3. To obtain reliable data a narrow- band green filter combination was used, so as to minimise effects due to nonneutrality of the test

filter and changes in colour temperature of the lamp.

Table 3. Comparison of time ratio method with conventional photometry

METHOD OF MEASUREMENT RESULT

(a) Transmission factor of filter measured directly by ratio of photocurrents in d c. photometry

(b) The same, corrected for linearity departure

1185% - O OOI 7o

1187% - O 002 lo

(c) Transmission factor of sector by

timing

(d) Transmission of filter relative to

sector, by ratio of photocurrent

(e) Transmission of filter deduced

from (c) and (d) i.e. by the time - ratio method

1475% i O OOI%

0-806 ± O OOI

I 189% ±0 002%

Only photomultipliers gave adequate data here, and

in fact all the later work was done with photomultipliers. We measured the transmittance of the filter by direct ratio of photocurrents, and then corrected

for linearity departure, giving the most accurate answer possible by conventional means. We then

measured the transmittance of the sector then avail- able that was closest in value to the filter, by the

time ratio method, and followed this up by determining

the transmittance of the filter relative to the sector by ratio of photocurrents. Ideally this last would have

given a value close to unity had we had a closely

matched sector-filter pair. As it was, the value of 0.806

required only a minimal linearity correction. The

product of these last two measurements gives the

transmittance of the filter by a simple form of time

ratio photometry, and it will be seen that the residual

discrepancy of 2 parts in 100 000 in "open" reading

is not significant.

C. Generality of Talbot's Law With Various Operating

Conditions in a Photomultiplier

A consequence of Talbot's Law is the invariance of

the measured ratio of the transmittance of a sector disc

to that of a filter. We later made up a special multicomponent filter to match a certain 1 percent sector rather closely under conditions where Talbot's Law

was known to hold, that is at low illuminations and low

output currents. Table 4 shows results obtained when

the flux and cathode current were fixed, and the gain

and output current was varied over a wide range. The

values S/F give the readings obtained with the 1 per- cent sector relative to the readings on the filter. Clearly,

increasing the output current above a certain threshold

level introduces progressively greater departures

from Talbot's Law. Measurements of linearity errors under steady flux conditions showed not only no

correlation in magnitude but these were of opposite

sense. The Talbot's Law errors indicate some instantaneous saturation at the peak current levels, whereas

the linearity checks show over-additivity due to dynode

heating. When output current is held constant, it is found that as the flux is increased and the gain de- creased to compensate, progressively larger errors

arise. The results are general for all tubes tested

Table 4.

TALBOT'S LAW ERRORS, e, FOR VARIOUS ANODE CURRENTS

Gain la (mean) la (peak) S/F e% A lin.(st)°/o

2 6 x IO

3 19 x IO"

8 19 x IO"6 O 984 o o O-OO

2 6x IO

4

1-9 x IO" 7

1 9 x IO~

5 0984 oo o oo

2 6x IO

5 19 x IO

-6

1-9 x IO

-4 0-972 -12 ooo

2-6 x IO6 19 x IO~

5 19 x IO~3 0-869 -117 + O 62

2-6 x IO

7 19 x IO

-4

1-9 x IO~

2 O 550 -44-

1

4- 3-3

-12 O Fixed cathode current = 7xlO A. Sector of I O /o Tube No. 6622

23
which were of the EMI 9558 type. Peak currents of

up to about 2 X 10-5 A produce no significant error, if the gain is adequate.

The intercomparison of fatigue-curves of photomultipliers with equivalent chopped and steady fluxes provided an independent and convincing check on

these facts. Such curves should be identical for de- vices that obey Talbot's Law, and this was indeed

found to be the case. Yet another consequence of Talbot's Law is that if a linearity test is made on equivalent steady and

chopped radiation levels, the same result should be

obtained. This powerful technique of measuring the

difference between chopped and steady radiation

linearity errors has also been applied to a number of

photomultipliers using the sound double-aperture

method, and once again it has been found that up to a certain threshold value of output current, no error occurs; above that level progressively increasing

failures of Talbot's Law occur in the sense of "in- stantaneous saturation" of peak currents above

about 2 X 10~3 A, confirming results mentioned

earlier. This technique was also applied to investi- gating what happens as we vary the mark-to-space

ratio or sector opening. Table 5 shows results from

only 3 sectors out of a whole set with progressively

varied transmittances that were used. This shows a very important result: it is not the mean value of cur- rent that determines the threshold for Talbot's Law

failure: it is the peak current. This means that the

presence of a specimen in a time-ratio spectrophotometer does not alter the optimum operating conditions, for the optimum operating conditions involve

using currents slightly below the critical threshold

for Talbot's Law failure.Table 5.

TALBOT'S LAW ERRORS, €, FOR VARIOUS SECTORS

Sector r la (mean) la (peak) Alin.(ch) A lin.(st) e x io

4

O 40% 9 3 x lo" 8 2 3 x IO"

5

(x IO"4

) - IO

(xlO~4

) - 4 -6 ±3

3 20°/o 6-4 x lO"

7 2 Ox IO

_S -H + 1 ±3

75 04°/o 17 x IO~

5 2-3 x IO"5 40 40 O ±5

O 40°/o 1 Ox IO~

6 2-5 x IO~

4 -40 - 1 -39 ±8

3 20°/o 8 1 x IO~

6 2-5 x IO"" -15*5 14 ^2 -30 ±7

75-04% 1 9x IO"

4 2-5 x IO"

4 477 542 -65 ±9

Tube No. 5564 Net gain = 2 x IO

I would like to mention two facts which reinforce our claims as to knowing the absolute uncertainties.

Firstly, T. Quinn has used our time ratio techniques

to determine the transmittance of certain sectors used

in establishing the temperature scale at NPL and found

consistency over long periods and with alternative

timing and optical equipment to 1 or 2 parts in a million

of the open reading, for sectors of 10 percent and 1

percent transmittance. Secondly, O. C. Jones designed

a super-quality 0.4 percent sector which allowed

metrology to 2 parts in a million. I measured this by

ratio of photo-currents, corrected for linearity and

produced a value that agreed to 1 part in a million of open reading, even though my probable error was 3

parts in a million of open reading.

D. Modes of Application

Three ways of applying Time Ratio Photometry to a double-beam automatic spectrophotometer will be

briefly described below. There are in fact many other

ways of using the principle. Figure 12 shows a system

which uses fixed gating intervals from timer 28 and

Figure 12. Schematic of one possible mode of application of Time

Ratio Photometry to a double-beam spectrophotometer (see

text). gates 32 during which the null detector 18 looks at fixed amplitude, fixed duration pulses delivered by

shutters 30 and 31. During the test phase pulses at fixed frequency are triggered by oscillator 34, and are attenuated by the specimen 42 to be equivalent to unattenuated pulses of lower frequency triggered by

variable oscillator 35, which is controlled by the off- balance signal from null detector 18. A ratio of fre- quencies from counters 36, 44 energised by supplementary photocell 38 gives the transmittance.

Figure 13 shows a system in which a high frequency

shutter 130 modulates a common portion of the optical

train. A timer 128 controls the beam-switching and

allows a fixed test phase interval. A shutter 131 allows

the reference phase duration to be reduced so that the

24
I?8

Figure 13. Schematic of another possible mode of application of Time Ratio Photometry to a double-beam spectrophotometer

(see text).

reference flux is equivalent to the fixed duration but

amplitude-attenuated test phase. A supplementary

detector and two counters 136 and 144 allow the high

frequency pulses from shutter 130 to be used to time

the open durations that give a balance from null detector 118, with and without specimen 142 in place.

Figure 14 shows a system in which the gating

intervals from timer 228 are fixed for test and reference

phases. An intermediate-frequency variable mark-tospace shutter 216, such as a variable sector, is controlled by the off-balance signal from null detector

218 to achieve equivalence with the unchopped but

attenuated flux passed by specimen 242. Timing is achieved by means of a supplementary detector,

fixed frequency oscillator 248, gate 246 and counters as

before. Owing to the staff shortage in this field at NPL

during the past few years, these ideas above have not been implemented in the sense that no Time Ratio

Spectrophotometer for practical use has been built. The fundamental research described in sections V. A

to V. C above was only performed on installations that could be described at best as abridged (filter) spectrophotometers. It remains for an instrument manufacturer to judge when the market would be ready for an automatic recording instrument that could achieve

the accuracy which only a few of the world's standardising laboratories can at present achieve in a staff- intensive way with manual instruments of their own

P36 244 222

220

Figure 14. Schematic of another possible mode of application of Time Ratio Photometry to a double-beam spectrophotometer

(see text).

design. What is certain is that Time Ratio Spectrophotometry is ideally suited to on-line data systems

with dedicated computers, since the technique is inherently digital.

VI. Material Standards for

Spectrophotometry

A. Direct Transmittance

Absorbing glass filters form the most satisfactory

transmittance standards in general, but they have

their limitations, especially in respect of the spectral

range available. It is desirable to separate out the

effects of nonlinearity, heterochromatic stray light,

wavelength calibration and wave band errors for diagnostic reasons, and this means that a neutral portion

of the spectral profile must be used for holding or

testing the photometric value. There are two classes

of glass suitable for this: very neutral glasses such as Chance ON28, ON29, ON30 or ON31 or Schott NG3,

NG4, NG5, NGll, or pseudo-neutral glasses such as Chance ON10, ON11A, ON31A or ON32A which

have several broad smooth maxima and minima at different levels but are not selective enough to impose

serious risk of heterochromatic stray light errors if measurements are made at the maxima or minima.

These latter have the advantage that a single filter gives calibrations at several densities. Both classes

of filter are used at NPL, and optical tolerances used

in fabrication are that the flatness of the surface

must be better than 1 fringe per cm and the parallelism

25
must be better than 15 s of arc. Such filters require

several years to stabilise in transmittance because

the surface reflection loss reduces progressively from

the initial value (given roughly by Fresnel's Equations) due to the effect of the atmosphere and even modest cleaning procedures in leaching out metal

oxide components from the surface of the complex

glass, which lowers its superficial refractive index.

Because absorbing glasses cannot be used in the

ultraviolet region satisfactorily, thin-film metal coat- ings on a good quality silica substrate are also used.

These can be made very neutral, but it is difficult to avoid pin-holes or nonuniformity in manufacture, so

a large illuminated patch must be used in measurement

in order to obtain a representative result. They also require special care in measurement because of the

high reflectance, and this is no trivial problem.

At NPL we prefer large material standards to small

ones, so that a large enough illuminated area can be

employed to ensure that blemishes or nonuniformities

do not unduly affect the results, which can then be

held to be representative of the sample. This provides

a further reason for avoiding a slit image in the vicinity

of the sample (see also sec. IV. D). The accuracy with

which the dimensions and position of the illuminated

patch on the sample need to be specified is obviously

less critical with a large patch than with a slit image.

At NPL the preferred size of sample is 50 mm x 50 mm,

and the preferred patch size is about 15 mm x 10 mm.

These dimensions give one considerable freedom in the optical configuration and so help with the problems

of preventing homochromatic stray light.

Standard solutions, such as acid potassium dichromate, alkaline potassium chromate or potassium

nitrate, are not used for holding our photometric

scale in the ultraviolet or visible regions because of

the additional uncertainties which are introduced in the preparation and handling of solutions. Inter- laboratory comparisons have always shown greater

discrepancies between measurements on solutions

than on solid filters [6, 28, 29]. It should also be

pointed out that the determination of the optical path

length of a cuvette is a far from trivial problem in metrology, due to the presence of the outer pair of

faces in addition to the inner pair being measured. It is possible to measure a 10 mm cuvette to an uncertainty of ±0.002 mm, so that if a nominal density

of 1 were being measured the uncertainty due to calibration error in the path length would be ±0.0002.

The method used at NPL to achieve this involves

the use of a laser for alignment and a mercury source

with interference microscope and standardised pre- cision micrometer for measurement.

B. Direct Reflectance

Material standards of direct reflectance are very

useful in allowing calibration by substitution with a fixed optical system, and a particularly stable type is kept at NPL. This was developed by myself some years ago and consists of a piece of Spectrosil grade fused

silica which has had both surfaces ground and polished

flat but wedged at an angle of 7° to each other. The

front surface forms the standard of reflectance

(roughly Z\% in value) so that the reflection from the

back surface has to be eliminated as otherwise it would give a second and displaced reflection as well

as uncertainties due to interreflections. This elimination is done in two ways: its angle of reflection is 14° away from the required front surface reflection so that

it can be excluded from the viewing system almost

completely, and its magnitude is reduced to about

0.01 percent reflectance by means of a special coating

applied to the back face. The performance of this coating is shown in figure 15, and the diffuse component of reflectance is even lower than the direct

component. This means that with a near-flawless front surface, the standard has a negligible diffuse component of reflectance. Studies of the polished surfaces of various glass

types at NPL have shown that only pure silica is unaffected by the atmosphere and mild cleaning

procedures. Our own spectrophotometric measure- ments show that the direct reflectance of these silica standards has not apparently altered over a period

of years by an experimentally significant amount

1. e., about ± 0.01 percent.

C. Diffuse Reflectance and Radiance Factor A very extensive program of work has been going on for several years at NPL on these topics, and

only an indication of this work can be given here. The

process of determining the absolute scales of reflect- ance and radiance factor is fraught with difficulty,

and has not yet reached a satisfactory conclusion.

Claimed uncertainty levels of ±0.5 percent quoted

recently by several laboratories seem a little dubious

in view of the discrepancies between them which

extend over a range of more than 1 percent. The earlier determinations of Preston at NPL [30, 31] have not

yet been surpassed for absence of systematic error by other laboratories, and the current investigation at NPL is far from complete. The primary standard is the

perfect reflecting diffuser by definition, a theoretical

abstraction. Secondary standards are pressings of

analytical magnesium oxide or barium sulphate, which

have replaced smoked magnesium oxide as being more

reproducible. In fact a reproducibility of ±0.1 per- cent in reflectance can be obtained with pressings,

compared with up to ±1 percent for the smoked

preparation, and these pressings form our interim

scale pending a satisfactorily accurate absolute scale. In the visible region the radiance factor for pressings

of analytical grades for both barium sulphate and

magnesium oxide is about 100 percent while the

reflectance is about 98i percent.

Practical working standards in the form of ceramic

materials are used in two forms. One of these is a special photometric opal glass MS-14 (replaced

later by MS-20) which is only obtainable from

U.S.S.R. This material is exceptionally white and

neutral in the visible and near ultraviolet region, and

26
REFLECTION FACTOR OF PIGMENT LOADED H.T. CEMENT ON POLISHED FUSED SILICA

polishes well. It forms an excellent reference white

standard for a reflection spectrophotometer, but of course needs calibrating against a secondary standard.

The other form of working standard used consists

of sets of Ceramic Colour Standards. These were

developed jointly by the British ceramics industry

(especially British Ceramic Research Association)

and NPL to provide durable, stable and uniform

working standards in various nonwhite colours. They

were developed mainly to provide a convenient means

of testing the consistency of operation and accuracy

of industrial colorimeters and spectrophotometers.

Each set has a light grey of about 65 percent reflec- tance, a medium grey of about 30 percent reflectance

and a dark grey of about 10 percent reflectance, these

being used for testing linearity. In addition there are nine spectrally selective standards which are useful

for testing wavelength calibration, waveband and

stray light errors. The ultraviolet, visible and infrared

diffuse reflectance profiles are shown in figures 16 to 20. Ceramic Colour Standards are more fully de- scribed in [32, 33, 34]. A thousand standards were manufactured by an

automatic continuous process in a single batch for each colour type to give a statistically homogeneous

population, and from these some sixty sets were

purchased by NPL for its standardisation services.

The remainder are available in uncalibrated form, and

over half the stock has now been sold. The uncalibrated sets are very useful for transferring to them

the calibration of an NPL-standardised set, especially in the case of a large company with several

manufacturing plants at different sites: as the goniophotometric and spectrophotometric characteristics are closely similar, such a transfer is a purely differ- ential measurement and can safely be done with a commercial instrument without systematic errors

arising. NPL-standardised Ceramic Colour Standards

are in use all over the world i.e., in Great Britain,

The Netherlands, Norway, Germany, Italy, Switzerland, Hungary, U.S.S.R., Australia, Panama, Canada

and, of course, the United States of America.

ioo90

SO

5

s 70^

c 60-

o c | 50^

u 40r

0*3 05 07 09 I I 13 15 17 19

9 862

11616 MOUNTAIN GREY - Type LT illumination

IOO 90

SO

|

ft 60

'

5040

03 0 7 0 9 II 13

Wavelength micrometers FIGURE 16. Ultraviolet, visible and near infrared diffuse reflectance

profiles for the prototypes of the Light Grey, Medium Grey and Dark Grey Ceramic Colour Standards. The specular component

was excluded in these measurements. 27
0-3 0 5 0 7

07 09 II 1-3 1-5 17 Wavelength micrometers —

Figure 17. As in figure 16 for the prototypes of the Light Green and Dark Green Ceramic Colour Standards.

07 09 II 13 1-5 Wavelength micrometers FIGURE 20. As in figure 16 for the prototypes of the Greenish Blue and Pink Ceramic Colour Standards.

IOO 03 05 07 09 I 3 I 5 17 19i 1 1 r

SIBERIAN BLUE COBALT BLUE

Type II illumination

03 05 07 09 II 13 1-5 Wavelength micrometersI 7 1-9

IOO

80 | 70.0

o 60 S

co 50 u 40 2 30 £

20 5

10

Figure 18. As in figure 16 for the prototypes of the Medium Blue and Dark Blue Ceramic Colour Standards.

03

07 0-9 II 1-3 1-5 Wavelength micrometers FIGURE 19. As in figure 16 for the prototypes of the Maroon, 1 and Yellow Ceramic Colour Standards.

The author wishes to express his thanks to the Institute of Materials Research, National Bureau of

Standards for their generous assistance to facilitate his contribution to the Conference. The work de- scribed forms part of the programme of research of the National Physical Laboratory.

VII. References

[1] Hardy, A. C, Recording spectrophotometer, J. Opt. Soc. Amer. 25, 305-311 (1935).

[2] Billmeyer, F. W., Precision and Accuracy of Industrial Color Measurement, Procs. Int. Colour Meeting, Lucerne, 445-456

(1965).

[3] Gailey, I., Visual versus instrumental colour matching, J. Soc. Dyers and Colorists 83 , 481-492 (1967).

[4] Edisbury, J. R., COLLABORATIVE TEST: Relative readings on twenty-eight Beckman spectrophotometers, Photoelec. Spectrom. Grp. BuU. 1 , 10-14 (1949).

[5] Gridgeman, N. T., Statistical analysis: the accuracy and pre- cision of photoelectric spectrometry, Photoelec. Spectrom.

Grp. Bull. 4, 67-79 (1951).

[6] Edisbury, J. R., Some musings on errors in spectrophotometry,

Photoelec. Spectrom. Grp. Bull. 5, 109-114 (1952).

[7] Ketelaar, J. A. A., Fahrenfort, J., Haas, C, and Brinkman,

G. A., The accuracy and precision of photoelectric spectrometers, Photoelec. Spectrom. Grp. Bull. 8, 176-9 (1955).

[8] PSG Collaborative Test of 94 Recording Spectrometers, Photoelec. Spectrom. Grp. Bull. 16, 443-467 (1965).

[9] Robertson, A. R. and Wright, W. D., International comparison

of working standards for colorimetry, J. Opt. Soc. Amer. 55 , 694-706 (1965).

[10] Ellis, S. C, Problems in Spectrophotometry and their influence

in Radiation Measurements, Symposium: Radiation Dose

and Dose-Rate Measurements in the Megarad Range, NPL,

18—23 (UK Panel on Gamma and Electron Irradiation,

1970).

[11] Preston, J. S. and Cuckow, F. W., A photoelectric spectro- photometer of high accuracy, Proc. Phys. Soc. 48 , 869-880

(1936).

[12] Preston, J. S., Errors due to stray light in spectrophotometry,

J. Sci. Instrs. 13, 368-370 (1936).

[13] Donaldson, R., Spectrophotometry of reflecting materials, J. Sci. Instrs. 16, 114-117 (1939).

[14] Donaldson, R., Stray light in monochromators, J. Sci. Instrs. 29, 150-153 (1952).

[15] Donaldson, R., Spectrophotometry of fluorescent pigments,

Brit. J. Appl. Phys. 5, 210-214 (1954).

[16] Harding, H. G. W., Precautions necessary for accurate meas- urements of optical density standards, Photoelec. Spectrom.

Grp. Bull. 4, 79-86 (1951). 28
[17] Jones, O. C, An impedance converter for use with digital voltmeters, J. Sci. Instrs. 40, 196-197 (1963).

[18] Crawford, B. H., Physical photometry, Notes on Applied Sci- ence No. 29, H.M.S.O., London, 1962.

[19] Samways, P. R., Optimum noise filter for dc measurements,

J. Phys. E 1, 142-144.

[20] Bauman, R. P., Absorption Spectroscopy, p. 89 (Wiley; New

York, 1962).

[21] Strong, J., Procedures in Experimental Physics, p. 376 (PrenticeHall, New York, 1938).

[22] Clarke, F. J. J., Fulton, E. R., and Samways, P. R., An inte- grating sphere reflectance and transmittance attachment for use with a Cary 14 spectrophotometer, to be published.

[23] Hildebrand, F. B., Introduction to Numerical Analysis, p. 301 (McGraw-Hill, New York, 1956).

[24] Slavin, W. and Porro, T. J., Measurement of photometric accuracy in UV-VIS spectrophotometry, Procs. Pittsburgh

Conf. on An. Chem. and App. Spect. (1960).

[25] Jones, O. C, and Clarke, F. J. J., A new photometric technique

using a variable shutter device, Nature, 191, 1290 (1961).

[26] Jones, O. C. and Clarke, F. J. J., Attenuation of Radiation,

British Patent No. 1031781 (1962); US Patent No 3256444

(1966).

[27] Carruthers, G. H., and Harrison, T. H., Application of Talbot's Law to photoelectric cells with a non-linear illumination- current characteristic, Phil. Mag. S, 7, 7 , 792-811 (1929).

[28] Vandenbelt. J. M., A collaborative study of Cary spectro- photometers, J. Opt. Soc. Amer. 44,641 (1954).

[29] Vandenbelt, J. M., Collaborating readings with the Cary 14 spectrophotometer, J. Opt. Soc. Amer. 50, 24 (1960).

[30] Preston, J. S., The reflection factor of magnesium oxide. Trans.

Opt. Soc. 31, 15-35 (1930).

[31] Preston, J. S., A new determination of the luminance factor of magnesium oxide, Procs. Phys. Soc, B, 65 , 76-80 (1952).

[32] Clarke, F. J. J., and Samways, P. R., The spectrophotometric

properties of a selection of ceramic tiles, Report No. MC2,

NPL (1968).

[33] Clarke, F. J. J., Ceramic colour standards, Procs. AIC Symposium, Color 69, Stockholm, 445-452 (Miisterschmidt,

Gottingen, 1970).

[34] Clarke, F. J. J., Ceramic colour standards^ an aid to industrial

control, Printing Technology, 13, 101-113 (1970).

(Paper 76A5-728)

29

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md„ March 22-24, 1972 (Issued May 1973). An Accurate Spectrophotometer for Measuring the

Transmittance of Solid and Liquid Materials

R. Mavrodineanu

Analytical Chemistry Division, National Bureau of Standards, Washington, D.C. 20234

(May 31, 1972)

The optical transmittance of solids and liquids as well as the molar absorptivity of various chemical

species are parameters of fundamental significance in characterizing these materials. Meaningful trans- mittance data can be obtained only when the measurements are performed with well-known accuracy and precision. To perform such measurements, a high accuracy spectrophotometer was designed and

assembled at NBS, Analytical Chemistry Division, and will be described in this paper. This single- beam instrument is composed of a constant radiation source, a monochromator, a sample carriage, an

integrating sphere-photomultiplier assembly followed by appropriate electronics, and a read out system

consisting of a digital voltmeter and a computer data acquisition and handling provision. The accuracy

of transmittance measurements is determined by the light-addition principle used in conjunction with a two-aperture arrangement. Thz spectrophotometer can be used in manual or automatic modes of operation. A detailed discussion of the data obtained with this instrument, used in both modes, will be pre- sented together with its application to the certification of solid and liquid Standard Reference Materials

for checking the photometric scales of conventional spectrophotometers.

Key words: Absorbance; automation of accurate spectrophotometer; instrumentation, spectrophotometric; spectrophotometry, high accuracy; standard reference material in spectrophotometry;

transmittance.

Transmittance is the ratio of two radiation flux in- tensities. It is therefore necessary that the photometric

scale of the spectrophotometer used to perform the

measurements be accurate. The transmittance of a particular material is also a function of wavelength; hence

the wavelength scale of the monochromator should also be accurate, and appropriate spectral bandpasses

should be used. The measurements should be made

using collimated radiations. Such radiations define un- ambiguously the actual path length through the trans- mitting medium, the reflection losses, and eliminate

the effects of polarized radiations that are produced at the surface of the sample. Other important factors which must be considered are: homogeneity and stability of the sample, radiation scatter inside the sample,

interference phenomena, stray radiation, polarization,

fluorescence, temperature, particulate matter, and sur- face conditions. Since transmittance measurements

depend on a diversity of factors, meaningful values can

be obtained only by defining the experimental conditions for obtaining transmittance data [1, 2].

2 Spectrophotometers are used to perform two types of measurements:

(1) Quantitative determination of chemical species

using the relation between optical transmission of the

material, and the concentration as a measuring parameter. Under these circumstances, the photometric scale

2 Figures in brackets indicate the literature references at the end of this paper.

I. Introduction

Optical transmittance is due to an intrinsic property

of matter and characterizes a particular transparent

material. Since this parameter is not known a priori, it must be determined by experimental procedures.

True transmittance values can be obtained only by

using accurate measuring techniques and by taking

into consideration all factors which can affect and distort the data. 1

1 The optical transmittance of a solid material includes the reflection losses which occur

at the air-solid interface. The internal transmittance is defined as the transmittance of the material corrected for reflection losses (2). This internal transmittance can be calculated in principle from the transmittance by using the well known Fresnel equations (1, pp. 98 to 100). For collimated radiation the reflectance R, for a material with an index of refraction, n, and an absorptivity, a, at wavelength, A, is given through: _ (n,-l)' + ni oj

4 (n» + l) 2 + nj al For a nonabsorbing material and collimated radiation: («>-!)' (nx+1) 2 For glass, n is approximately 1.5 in the visible region of the spectrum, and R will be about 4 percent at every air-glass interface. When noncolUmated radiation is used:

sin2 (q-fl)>

sin2 (a + 0)»

for perpendicular polarized radiation, and

tan2 (a-/9U

Rll =

k tanMa + /3)»

for parallel polarized radiation, where a and /3 are the angles of incidence and refraction, respectively. In collimated radiation and in air, a = fj = 0 and RL =Jl" = R- 31
of the spectrophotometer is calibrated in meaningful

units, using a series of reference solutions having

known concentrations of the species to be determined,

rather than values of optical transmittance.

The accuracy of the measurements is related solely

to the accuracy with which the concentration of the

reference solutions is known and to the precision (stability, sensitivity, reproducibility) of the spectrophotometric method and instrument used. The accuracy of the photometric scale per se, is not a critical factor in such measurements.

The precision, stability, and reproducibility of the

instrument can be checked before each series of meas- urements by careful use of solid or liquid reference

filters having well established transmittance values.

(2) Determination of the optical transmission characteristics of solid or liquid materials, and the determination of molar absorptivities of chemical compounds.

In both cases the accuracy of the photometric scale of

the measuring instrument, among other things, is essential to provide true values. Ways to establish

and check this important parameter are critically

needed.

Since conventional spectrophotometers do not pro- vide means to check photometric accuracy or to evaluate the possible sources of systematic errors, it was decided in 1969 to design and construct a re- search spectrophotometer on which transmittance

measurements could be performed with well defined accuracy. Such an instrument would be used to deter- mine optical transmittance of selected solids and

liquids at various wavelengths. These materials can be

used as standard reference materials (SRM's) to check

the accuracy of the photometric scale of conventional

spectrophotometers. The same certified SRM's could

likewise be used to monitor the precision, stability,

and reproducibility of those instruments [3, 4]. After a comprehensive examination of the literature

in this field [5 to 34] arranged in chronological order, an instrument was developed which is similar in principle to the instrument at the National Physical

Laboratory (NPL), Teddington, England, where a long

tradition of high accuracy spectrophotometry exists. The instrument described in this work performs'

measurements of radiant energy in the visible and

ultraviolet region of the spectrum, with well established

and high photometric accuracy. Transmittance meas- urements on solids and liquids can be made with this instrument using collimated as well as noncollimated

beam geometry. The wavelength accuracy and spectral

bandpass achievable are adequate to avoid degradation of photometric accuracy, and the other inter- ferences mentioned have been given careful consideration, and, in most cases, have been assessed quantitatively.

The transmittance measurements on the optically

neutral glass filters discussed in this work have been

made with a noncollimated beam geometry corresponding to an aperture of about f:10. The image of the exit

slit of the monochromator (8 mm x 0.5 mm) was

produced at the center of the entrance face of the

filter. All measurements have been made against air for the nonattenuated radiation flux, and no correction for reflection losses was made. Transmittance

measurements made with noncollimated radiation by

projecting the image of the exit slit of the monochromator on the entrance face of the sample using an opening of f:10 (total angle of about 7° or 8°), may

differ by several parts in 104 of the value when compared with similar measurements made with colli- mated radiations, as indicated in this Journal by

K. Mielenz.

Noncollimated beam geometry was applied in this work to approach the measuring conditions used in most of the conventional spectrophotometers which

are available today. A brief description of this instru- ment was given earlier in reference [3].

II. Description of the Instrument3

The high accuracy spectrophotometer, completed

and tested in 1970, is a single beam instrument which

contains the following components: (a) a constant

radiation source, (b) a monochromator, (c) a sample

holder, (d) a system to check the accuracy of the

photometric measurements, (e) an integrating sphere

attached to a photomultiplier-digital voltmeter unit,

and (f) the data presentation system. Figure 1 illus- trates schematically the arrangement of these various

components. A circular neutral wedge is placed after the light source to select various levels of radiation

intensities required for measurements. A description

of the components is presented in the following

sections.

a. The Radiation Source. Since the instrument is a single-beam type, it is essential that the radiation source be constant and homogeneous. Additional

desirable conditions are: capability of monitoring the current supplied to the source and radiation similar

to that from a Planckian radiator. The source is similar in design to that developed and used at NBS

by H. J. Kostkowski and R. D. Lee of the Institute for Basic Standards. This source was duplicated in our

instrument with the kind assistance of its developers.

The source is used in the spectral range 360 nm to 800 nm and consists of a tungsten incandescent fila- ment lamp with a tungsten ribbon 8 mm long by 2 mm

wide. The connections to the lamp terminals are soldered to minimize contact problems (see fig. 6). The direct current required to operate this lamp at approximately 3000 K is 18 A across a 6 V drop; our source is operated at 5 V and 15 A. The d.c. power supply is capable of delivering 15 V and 50 A,

and can be operated in constant current or constant

voltage modes. To achieve the constant current mode

an external sensing resistor of 0.1 Cl and 50 A and

a current control circuit are placed in series with the

power supply. A feedback voltage across this resistor

is connected to the sensing system. The character

3 The commercial instruments and parts used in the construction of the spectrophotometer are identified in the addendum.

32
Precision Optical Bench m suns mi

BIBI-iCeUBACT SPSETROPH0T0HBTBR

Mirror

Circular|Neutral r|N< Wedge

NPL Aperture for Linearity Control

(After F.J.J. Clarke)

Sample -Br Carriage 1m Grating Monochromator (Czerny -Turner)

Precision Optical Bench in Lightproof Enclosure l

Blank

r* Constant

| Light Power

1 Source Supply

Integrating Sphere

Figure 1. Principle of the single beam high accuracy spectrophotometer. The aperture unit is placed on the optical bench only when linearity measurements are performed.

istic of this function is the ability to automatically

change its output voltage to maintain a constant

current to the load resistor, which, in our case, is the lamp source. The nominal current regulation

obtained is better than 0.01 percent, and the stability over an 8 hour period, at constant load temperature,

is better than 0.02 percent. The stability of the current

delivered to the lamp is monitored with a high accuracy

potentiometer used in conjunction with a null meter. This meter is sensitive to variations in the current supplied to the lamp from 1 part in 1000 to 1 part in 1,000,000 per division (fig. 1 and fig. 14). The po- tentiometer is connected to the current source across

a resistor (O.Olfl and 100 A) placed in series with the

lamp.

The demagnified (2 to 1) image of the ribbon fila- ment is projected on the entrance slit of the pre- disperser by a fused quartz (nonfluorescent Si02)

lens whose focal distance is 254 mm and diameter is 44 mm. This and the other lenses used in the optical

system, were calculated by K. Mielenz of the Institute for Basic Standards at NBS. The lenses are mounted

in carriers which permit orientation in any position.

A circular neutral wedge is placed between the fight source and the predisperser. This wedge, evaporated

inconel on a fused quartz disc (150 mm diam), is linear

in density and provides a light attenuation of 100 to 1. The wedge is motor driven (1 rev. per s) to select proper radiation intensity levels as required by the

measurements (figs. 2, 3, and 4). The radiation source used for measurements in the ultraviolet region to 275 nm is a single coil tungsten-bromine incandescent

lamp (fig. 5) supplied by an adequate power source; below 275 nm, a deuterium discharge lamp is con- templated.

b. The Monochromator. The monochromator is a 1-m Czerny-Turner type grating instrument with a dispersion of 0.8 nm/mm. The flat grating has 1200

grooves per mm covering a surface of 100 x 100 mm.

The monochromator is provided with a predispersing

attachment to reduce the stray light (fig. 3). This preFlGURE 2. General views of the spectrophotometer. Rear: optical bench carrying the tungsten-halogen radiation source used for checking the alignment of optical components, followed by a quartz

lens, the circular quartz neutral wedge, and a flat mirror. Left: the 1-m Czerny-Turner grating monochromator (the predisperser

is not illustrated here). Front: optical bench carrying a quartz

lens, the single-sample and blank carriage, a second quartz lens, and the integrating sphere with the photomultiplier housing.

33
Figure 3. Close view of the tungsten ribbon filament lamp on its adjustable holder, followed by the quartz lens — circular neutral wedge assembly, and by the flat mirror in its adjustable holder. The 30° quartz prism Littrow-type predisperser is located at the entrance slit of the 1-m grating monochromator. Extreme left: neon gas laser used to check the optical alignment, and mercury

discharge lamp for wavelength calibration. When in use, the tungsten ribbon lamp is surrounded by an enclosure with black

walls (50 cm x 50 cm x 70 cm high). Rear: enclosure containing the

optical units illustrated in figure 2.

disperser is a small quartz prism monochromator

connected to the scanning system of the 1-m instru- ment. A wavelength counter permits readings to 0.1 nm and the scanning speed can be varied from 0.05 nm to 200 nm/min by a 12 speed synchronous electric motor. The optical components are placed on precision lathe bed type optical benches which are 160 and 120 cm

long, and are equipped with appropriate carriers

provided with x-y-z adjustments.

c. Sample Carrying Systems. The spectrophotometer is provided with two sample carrying sys- tems. One system measures one sample and its blank,

while the other system permits sequential measure- ments for seven samples and eight reference reading

positions against air, and can be operated manually

or automatically through a computer interfaced

with the instrument.

The single sample carrying unit consists of a platform provided with two vertical holders which can

accept f-in (14 mm) rods and a variety of sample

supports (fig. 2). These holders can be moved laterally

through a rack and pinion arrangement. The platform

is mounted on 4 ball bushings which ride on two

horizontal rods and can be moved pneumatically

across the optical axis. The pneumatic operation was recommended by G. E. Moore and J. T. Sterling of the Institute for Materials Research at NBS and

by L. Owen, a guest worker at NBS. The travel dis- tance is 8 in (20 cm) and the linear movement is smooth; the position of the platform and the sample

in and out of the optical beam, can be reproduced

within 0.025 mm. This unit is illustrated in figure 2

Figure 4. Close view of the circular, neutral wedge. The front plate which carries the fused silica lens was removed to show the fused

silica disc with the evaporated metal layer.

and is located between the two quartz lenses. The

sample holder is designed to accept conventional

solid or liquid filter holders which fit most spectrophotometers. These holders are provided with a thermostating jacket, and can be rotated in the

horizontal plane through a 10 cm diameter rotating

table. A filter holder which permits the rotation and

scanning of the sample in the x—y direction is also available (fig. 7). It is provided with micrometer

screws having a total linear motion of 25 mm with

0.01 mm per division. The seven-sample carrying

unit is illustrated in figures 8 and 9 and consists

of a semicircular aluminum-alloy plate placed hori- zontally on an appropriate carrier on the optical bench

along the optical axis. This plate, which is 32 cm in diameter and 2.5 cm thick, can be rotated clockwise

through a pneumatically operated precision ratchet

system in increments of 12°. The stepwise rotation

utilizes a solenoid valve which is operated electrically

by a switch located outside the enclosure. This

switch can be operated manually or automatically by

computer (fig. 14). 34
Figure 5. Single coil tungsten halogen lamp in the adjustable

holder.

The semicircular plate carries seven sample holders

similar to those used for the single sample system

described earlier. The holders are placed at 24°

intervals and are separated by blank spacings. About

1 atm of air pressure is used to operate the plate and

the rotation is set at 2 s per 12° step when the auto- matic computer operating mode is used.

d. System to Check the Accuracy of the

Photometric Reading. Since the high accuracy

spectrophotometer is single beam, accurate photometric data are obtained when there is a linear relation

between the measured radiation flux and the corre- sponding response of the photodetector.

Linearity of photodetectors can be measured by

several means: the inverse square law [7, 15]; the use of

optical elements having a known transmittance which

can be determined by other means [17] and the light

addition principle of Elster and Geitel using a plurality

of light sources [5, 6, 8, 9, 10, 13, 18, 19, 20, 28, 31, 33,

34] or multiple apertures [11, 12, 14, 16, 21, 23, 25, 26,

27, 30]. A novel approach to the problem of accurate

Figure 6. The tungsten ribbon filament lamp in the newly designed

adjustable holder. The platform which carries the lamp is similar

to that described in figure 5 and can be oriented in the horizontal plane through the six screws spaced around the edges of the platform at 60° intervals. Three screws push the platform while

the other three pull. The current-supplying wires are soldered directly to the lamp

terminals to eliminate contact problems.

photometric measurements was described by O. C. Jones and F. J. J. Clarke [24, 29] and by F. Desvignes

and J. Ohnet [32]. A critical discussion of some

aspects of accurate spectrophotometry will be found

in an NBS manuscript by Gibson and associates [22]. The radiation addition principle, using two apertures

with one source of radiation, was chosen for our work.

The aperture method for checking the linearity of

photometric data was in use at the National Physical

Laboratory from about 1930 onwards, and one form of it was described by Preston and Cuckow [11] in conjunction with a single beam spectrophotometer, using a

five aperture screen. One year later, Buchmiiller and

Konig [12] described and used a two aperture unit. At

NBS, Barbrow [14] used a 10 aperture arrangement,

while Harding [16] and Cordle and Habell [25] at NPL

described a two aperture system. Multiapertures were

used by Hoppmann [21], Bischoff [23], Sanders [26] and Nonaka and Kashima [27]. Finally, Clarke T30] 35
FIGURE 7. Same as figure 2 except tor the sample holder which

in this case is capable of rotating the sample 360° and to displace

it in the x-y direction through the micrometer screws.

Figure 8. Circular platform carrying seven filter holders. The lb

position switches (7 sample positions and 8 blank positions) are

visible along with the two quartz lenses. The exit slit of the monochromator is at left.

discussed in detail the use of a two aperture system to check the accuracy of photometric data obtained on the

spectrophotometer at NPL. It is this two aperture

system which is used at NBS.

The two aperture unit consists of a metal plate

(130 mm by 100 mm) containing two rectangular

Figure 9. Same as figure 8. The pneumatic cylinder which rotates the circular platform through a ratchet mechanism is visible at the rear of the platform. The integrating sphere with its pneumatic

shutter is seen at right.

Figure 10. Same as figure 2. In this case the dual-aperture unit for linearity control is located on the optical bench after the exit

slit of the monochromator.

windows, A and B, (20 mm by 8 mm) located one above

the other (figs. 10, 11, 12). Each aperture can be closed

by a light-tight shutter which is operated pneumatically

by remote control (fig. 14). The aperture plate is placed

in the optical path after the exit slit of the monochromator and within the optical solid angle of the

instrument. The image of the apertures is then pro- jected on the target of the integrating sphere. A fused

quartz lens with a focal distance of 190 mm and a diameter of 60 mm is used for this purpose. The arrange- ment is illustrated in figure 10. No optical element

36
Figure 11. Detail of the dual-aperture unit showing its construction and the pneumatic system which operates the two shutters. One aperture is open, the other is closed.

should be placed between the aperture plate and the

monochromator. The linearity check consists of

measuring the photocurrem produced when aperture

A is open then closed, and then aperture B is open and

then closed. The value of (A) + {B) is compared with the

values obtained with both apertures (A + B) open. If the

system is linear these two values should be identical:

(A) + (B) = (A + B).

If this is not the case, the system shows nonlinearity

which is proportional to the amount by which the sum of (A) + (B) differs from (A + B). This difference is then used to correct the transmittance values measured

on the solid or liquid filters.

e. Integrating Sphere and Photomultiplier

Arrangement. The radiations emitted from the exit

slit of the monochromator and passing through the

aperture or the filter are received on the target of the

integrating sphere. This sphere is illustrated in figures 2, 7, 9, and 10. A block of aluminum made

from identical halves was cut to produce a half sphere

in each block. The halves were joined together to form

a hollow sphere. Its diameter is 125 mm and a target,

made from a circular plate, 35 mm in diameter, is located at the center of the sphere. The front surface

of the sphere has a 20 mm diameter opening. This

FIGURE 12. Front view of the dual-aperture unit, with both

apertures open.

opening can be closed by a shutter which is operated

remotely by a pneumatic system. A 50 mm diameter

opening is at the opposite end to which the housing of the photomultiplier is attached by an "O" ring to pro- vide a light-tight joint. The inside of the sphere is coated using a suspension of BaSd; the outside is painted black.

Under these circumstances the sensitive surface of the photodetector receives the radiations originating

from the exit slit of the monochromator only after these radiations have undergone at least two diffuse

reflections. The photomultiplier is a 50 mm flat-faced, silica end window tube with a 44 mm cathode and 11 Venetian blind dynodes having CsSb secondary emitting

surfaces. The cathode is an S-20 or tri-alkali type.

The spectral range of this tube is from below 200.0 nm

to 850.0 nm. The operating voltage used is 850 V. The

photomultiplier output is supplied to a current-to- voltage converter consisting of an operational amplifier with high precision feedback resistors with

values of 106

, 3 X 106

, 107

, 3 X 107, and 10 8a Dark

current compensation is also available. This electronic

system, described in figure 13 was designed and

assembled by K. W. Yee of the NBS Electronic In- strumentation Section. The output from the currentto-voltage unit is connected to a digital voltmeter,

illustrated in figure 14, with one microvolt resolution on the 1 V full scale range. 37
r

CLOSED SHIELDED 801 r

9.lifi Ukfl

I

| RtNCt |

| ADJUST |

LOW NOISE CABLE IHPHEHOL 21-5!? C

| MPUT >—

0

INSULATED /

FRO"

CHASSIS ^

I

1

j lo«a,.i»|

ZOOpF «

IMUfl IOOTJD POLTSTTBCiE

tOOpF

4.VF —

tun

0

niimn

I

OUTPUT

IV FOR INDICATED FULL SCALE (»»»«)

NOTES

ALL NONELECTROLTTIC CAPACITORS

ARE KETALBED HTLAR UNLESS OTHERWISE NOTED

•° SELECTED FOR 2 Hi I RATIO TO IITHIN 0 1%

ALL RESISTORS BETTER THAN i\ TOLERANCE HAVE TC

OR LESS

2 J A S 6 T I 9 10

SEMICONDUCTOR

CIRCUITS 2-15 - 50

Figure 13. Schematic of the current-to-voltage circuitry. Courtesy of K. W. Yee.

The optical components located after the exit slit of the monochromator, including the photomultiplier

tube, are enclosed in a light -tight box 200 cm long, 70

cm wide and 76 cm deep (fig. 3). The removable

front panel is provided with a sliding door to permit

rapid access to the filter-holder system. The box

contains outlets for the compressed air which operates

the apertures, sample carriage and integrating sphere

shutter, and for the electrical connection from the photomultiplier. The inside walls are lined with thermal

insulation painted black. When in use, all nonblack

metal parts are covered with a black cloth to reduce

stray light. The entire equipment is placed on a vi- bration isolation table 3.66 m by 1.52 m. The optical

benches and the monochromator are secured by stops

which are attached to the table surface. The alignment of the optical parts is made and checked periodically with a low-power laser shown in figure 3 (CW gas

laser, output power 2 mW, X 6328 A) and with a high

intensity tungsten-halogen lamp shown in figure 5.

f. Data Collection and Presentation Systems.

The data output from the digital voltmeter (DVM),

corresponding to the current generated at the photomultiplier tube by the radiations passing through the

aperture system (A, 5, A + B) or the samples (/) and

blanks (h), can be obtained by visual means or computer operation. Both methods have been used in this work with good results. In the visual mode, the

operator examines the digital voltmeter display and

takes a mental average of the data. The display rate

is adjusted to about one reading per second.

When measurements are taken by computer, the

display of the digital voltmeter is adjusted to a faster

rate; for instance, 10 to 20 data per second, depending

on the capabilities of the instrument and measurement

requirements. In our work, we use 10 data per second

and collect 50 individual data for each measurement.

This information is fed to the computer which calculates and prints the results as averages with the cor- responding standard deviation, relative standard

38
Figure 14. Console containing the power supply for the photomultiplier tube, the dc null detector, the current-to-voltage converter, the digital voltmeter, the command panel for computer

operation, and the command panel for the pneumatic operation

of the shutter, aperture system, and single sample carriage. The

electric switches for operating the circular neutral wedge are also located on this panel. Middle right: potentiometer for monitoring the dc current supplied to the tungsten ribbon filament lamp. At bottom left: teletype for data presentation.

Right: light panel which indicates the position of the automatic seven sample holder.

deviation, and sample position number to identify

the measurement. When transmittance measurements

are made on individual samples or when linearity

checks are performed, the readings are initiated manually for every position. When the seven sample

holder is used for sequential measurements, the

operation is performed automatically by the computer. It is programmed to take a predetermined

number of individual DVM readings (50), print the

arithmetic average, followed by the standard deviation, relative standard deviation, percent transmit- tance and sample position (fig. 15). At the conclusion

of each measurement, the computer initiates a signal

which rotates the holder to the next position. This is followed by the data taking and sample changing

sequence until the measurements are stopped manually or automatically by a provision made in the

computer program.

CALIBRATION OF SRM930 AT 440 NM 2-2 -72 SAMPLE DUMBER AND POSIT I ON

1-70 IN 2; 1-79 IN 4; 2- 79 IN 6; REFERENCE IN 8; 3-79 IN io; 1-91 IN 12; 3-91 IN 14

pos AV s S/AV PERCENT T

i •2001700E 01 •4388E-03 •2192E-03

2 .6413441E 00 .3256E-03 .5077E-03

3 •2001230E 01 •4320E-03 .2 1 59E-03 32 .044

4 •6S28346E 00 •4804E-03 •73S9E-03

5 •2001040E 01 .4254E-03 .2126E-03 32 .623

6 .4221 1 10E 00 •2293E-03 . S432E-03

7 .2000995F 01 .304 1E-03 . 1520E-03 21 .095

8 •6560142E 00 •3189E-03 .4861E-03

9 .2001402E 01 .4476E-03 •2236E-03 32 .781

10 .2358762E 00 . 1425E-03 .6039E-03

1 1 •2001539E 01 .41 14E-03 •2055E-03 1 1 .785

12 .6739426E 00 .2848E-03 .4286E-03

13 •2001205E 01 .4678E-03 .2338E-03 33 .674

14 •2386761E 00 . 1064E-03 .4456E-03

15 .2001 101E 01 •4150E-03 .2074E-03 1 1 .927

1 .2001622E 01 .3187E-03 . 1 592E-03

2 •6412983E 00 . 1321E-03 •2060E-03

3 .2002043E 01 •6139E-03 .3067E-03 32 .036

4 •6526462E 00 •2474E-03 •3790E-03

5 .2001S51E 01 •4756E-03 •2376E-03 32 .603

6 •4222282E 00 .2174E-03 •5149E-03

7 .2001S07E 01 •4355E-03 .2176F-03 21 .095

8 .6562004E 00 .3344E-03 .509SE-03

9 .8001 529E 01 •5337E-03 .2667F.-03 32 .785

10 •2359933E 00 . 1886E-03 .7992E-03

1 1 •2001634E 0 1 •4532E-03 .2264E-03 1 1 .790

12 .674 1099E 00 .3418E-03 .5070E-03

13 .2001589E 01 .6368E-03 .3182E-03 33 .678

14 •2386828E 00 . 1604E-03 •6720E-03

IS •2001829E 01 .3388E-03 . 1693E-03 1

1

.924

1 .20022 14E 01 •2826E-03 . 1412E-03

2 •6413472E 00 -3518E-03 •5486E-03

3 •2001882E 01 .3882E-03 . 1939E-03 32 .035

4 .65261 77E 00 •2513E-03 .3851E-03

5 .2001741E 01 . 384 1E-03 . 1919E-03 32 .601

6 •4223418E 00 .2957E-03 . 7000E-03

7 •200207SE 01 •4892E-03 •2443E-03 21 .097

8 .6562083E 00 •1365E-03 .2081E-03

9 •2001871E 01 • 3708E-03 . 18S2F-03 32 .7 78

10 •2358786E 00 . 1 596E-03 •6767E-03

1 1 •2002130E 01 . 5036E-03 .2515E-03 I 1 .782

12 .6740563E 00 .3022E-03 .4484E-03

13 .2001997E 01 •4545E-03 .2270E-03 33 .668

14 .23883S4E 00 •1704E-03 .7133E-03

1 5 • 2.002478F 0 1 .297SE-03 . 1485E-03 1 1 .928

Figure 15. computer data presentation.

The programming of the entire computer operation was developed by J. Aronson, R. Freemire, and J. Wing. The computer-instrument interfacing was

performed by F. Ruegg and R. Shideler of the NBS

Analytical Chemistry Division, Technical Service

Group, under the supervision of J. DeVoe.

III. Stability of the Electronic System

As a rule, before taking measurements with the

spectrophotometer, a warmup period of one hour is required. The room temperature is kept at 24 ±1 °C,

and the relative humidity is 35 percent. The particulate matter is controlled through special filters which

rates the room in the 100,000 class. The dark current of the photomultiplier tube was

measured by taking 15 replications each consisting of

the average of 50 individual digital voltmeter readings.

These measurements were made using 850 V at the

anode. The average dark current under these cir- cumstances produced 0.000682 V with a relative standard deviation of 0.71 percent.

In all of our work, a dark current buck-out arrange- ment was used. A series of measurements were per- formed to determine the stability of this dark current compensation. To this effect, 15 consecutive measurements, each representing the average of 50 indi- 39

502-591 O - 73
vidual digital voltmeter readings, were made and the

average dark current value was 0.000024 V with a

relative standard deviation of 23.1 percent.

Four tests were made to determine the stability of the electronic system and the radiation source using

the computer data acquisition mode.

a. Stability of the Current-to-Voltage Converter. A constant voltage was supplied to the converter using the dark current compensation provided on the unit. Fifty individual measurements were taken

every 5 seconds and the average value was printed

along with its percent standard deviation. The meas- urements were then repeated 15 times and an average

of the 15 values was calculated along with the cor- responding percent standard deviation. These meas- urements were then repeated three times. The results are summarized in table 1. This table also presents

the values for the first group and the average values

and corresponding percent standard deviation for the

two consecutive groups. It can be seen from the

stability of the current-to-voltage unit that measure- ments can be performed with a reproducibility of

about 0.0012 (at the 67% confidence level) expressed

as percent standard deviation for a single determination. The time interval between the first and last group of measurements was 15 min.

Table 1. Stability of the current-to-voltage converter alone measured in three groups of 15 replications each

Average of 50 individual Percent standard

ilication measurements; volts deviation

1 1.003494 0.00136 2 1.003496 0.00129 3 1.003482 0.00146 4 1.003507 0.0011, 5 1.003515 0.00134 6 1.003508 0.00136 7 1.003497 O.OOllg

8 1.003498 0.00133 9 1.003505 0.001

1

0 10 1.003510 0.00142 11 1.003518 0.00133 12 1.003521 0.00120 13 1.003522 0.0013, 14 1.003507 0.00126 15 1.003527 0.00137

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

1.003507

0.0012

1.003535

0.0015

1.003545

0.0010

First group

Second group

Third group

b. Stability of the Current-to-Voltage Converter Plus the Photomultiplier Tube Supplied

with 850 V and in Total Darkness. The measure- ments were made as previously described and the

results are presented in table 2.

c. Stability of the Current-to-Voltage Converter and the Photomultiplier Tube Supplied

with 850 V and Exposed to the Radiation of a Tritium Activated Fluorescence Source. A constant radiation source consisting of a tritium activated

phosphor was placed before the integrating sphere

and a series of measurements were taken following

the technique described above. Table 3 shows the

results.

d. Stability of the Current-to-Voltage Converter, the Photomultiplier Tube Supplied

with 850 V, and the Tungsten Ribbon Filament

Lamp. The same measuring procedure as mentioned

in a, b, and c was used here. In this case, however,

the incandescent tungsten lamp was used as the source of radiation. Table 4 summarizes the results of four groups of measurements over a period of 20

min. This last series of measurements indicate that the single-beam spectrophotometer is capable of

producing measurements of radiation fluxes with a percent standard deviation of about 0.0225 for single

measurements with 2.00 V at the photomultiplier tube

anode.

40
Table 3. Stability of the current-to-voltage converter and the photomultiplier tube at 850 V and exposed to the

radiation of a tritium activated fluorescent source

Average of 50 individual Percent standard

lication measurements; volts deviation

1 1.536345 0.0275 2 1.536326 0.0302 3 1.536196 0.0228 4 1.536289 0.0235 5 1.536106 0.0309 6 1.536117 0.0314

7 1.535916 0.025, 8 1.536065 0.023s 9 1.536179 0.0293 10 1.536003 0.0233 11 1.536083 0.021s

12 1.535961 O.O263

13 1.536052 0.031 3 14 1.536095 0.0266 15 1.536092 O.O262

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

1.536122

0.0082

1.535768

0.0095

1.535522

0.0054

First group

Second group

Third group

accuracy with a low pressure mercury discharge

lamp placed before the entrance slit of the monochromator. The following wavelengths were used

for calibration: 3650.2 A; 4046.6 A; 4077.8 A; 4339.2

A; 4347.5 A; 4358.4 A; 4916.0 A; 5460.7 A; 5769.6 A;

and 5790.7 A. If additional reference wavelengths

are needed, a Cd-Hg or a He-discharge lamp could be

used for calibration. The wavelength counter was

then checked using the procedure recommended by

Gibson [2], and a slit of 0.1 mm which is equivalent to an effective spectral bandpass of 0.08 nm. The deviation of the wavelength counter from the true value was found to be less than ±0.1 nm; hence no wavelength correction was applied to the measurements

discussed here.

V. Stray Radiation

Tests were made to determine the stray radiant

energy (SRE) in the monochromator proper, as

well as in the photometric arrangement. The measure- ment of stray radiation in the monochromator, that is, the radiation energy at wavelengths different from

those of the nominal spectral bandpass transmitted

through the instrument, is not easy or infallible. A

detailed discussion of this instrumental parameter was

given in an ASTM Tentative Method [35] and the procedure recommended in this work was used to deter- mine SRE in the blue and yellow spectral range. In this procedure, a solution of methylene blue, which has a strong absorption in the range from X 600 to 660 nm is used. The SRE using a slit of 1 mm (0.8 nm) was equal

to or less than five parts in 105. The SRE generated inside the photometric system is defined as the radiant energy which falls on the

photosensitive detector without passing through the

absorbing sample. This SRE is usually produced by

reflections and scattering of radiations on the optical

and mechanical parts located between the exit slit of

the monochromator and the integrating sphere. The

measurements were performed using a slit of 1 mm by

placing a front surface mirror at the sample position,

which reflects to the instrument all radiations received

from the exit slit imaged at the mirror surface. The size of this image was about 8 mm high and 1 mm wide. In

this way, a maximum SRE was generated in the

spectrophotometer. The measurements were then

performed at A. 577.3 nm, using a radiation flux intensity

five times greater than that used in routine trans- mittance measurements, by determining the dark cur- rent of the photomultiplier with the shutter in the

closed position at the integration sphere. An average

dark current of 0.040 mV was observed. The mirror was then placed at the sample position, the shutter

41
Table 2. Stability of the current-to-voltage converter and the photomultiplier tube at 850 V in total darkness

Average of 50 individual Percent standard

Replication measurements; volts deviation

1 1.012329 0.027,

2 1.012342 0.0488 3 1.012322 0.0272 4 1.012320 0.0338 5 1.012394 0.0435 6 1.012421 0.0153 7 1.012404 0.0184 8 1.012406 0.0296 9 1.012365 0.0196 10 1.012402 0.019,

11 1.012465 0.0252 12 1.012412 O.O6I5 13 1.012451 0.0235 14 1.012417 0.0298 15 1.012481 0.0248

Average of replications Percent standard

deviation

1.012395

0.0050

First group

Average of replications Percent standard

deviation

1.012467

0.0033 Second group

Average of replications Percent standard

deviation

1.012510

0.0035 Third group

In these measurements the stability of the direct current (nominal 5 V; 14 A) supplied to the tungsten

ribbon lamp was monitored with the potentiometer,

and the variation of this current was less than one

part in 105 during a series of 15 consecutive meas- urements (5 min).

Following the four stability tests discussed earlier, a consecutive series of six measurements were made

to determine the reproducibility of transmittance

measurements. To this effect seven Schott NG-4

neutral glass filters were placed in the automatic

sample carrying system and the data acquisition and

sample changing operations were performed auto- matically through the computer unit. As mentioned

previously, the sample carrying system can accept seven samples in positions 2; 4; 6; 8; 10; 12; 14, and

eight intermediate positions 1; 3; 5; 7; 9; 11; 13; 15. The odd numbers correspond to measurements of the nonattenuated radiation beam passing through

air and are marked 7o, while the even numbers cor- respond to measurements of the attenuated radiations

after passing through the absorbing material and are marked 7. The uncorrected transmittance, T, is then

lo The radiation flux from the tungsten ribbon filament

lamp was attenuated with the circular neutral wedge

until a photocurrent corresponding to about 2.0020 V

was obtained for the nonattenuated beam Io. The photomultiplier tube was supplied with 850 V and the 30

MO resistor was used at the current-to-voltage converter. For every position, 50 digital voltmeter readings were taken by the computer at a rate of 10 to 15 per

second. The average value was printed along with

the sample position, the standard deviation, the relative standard deviation, and the transmittance values

for the glass filters 2; 4; 6; 8; 10; 12; and 14:

7° + 7°

1 3 7° + 7°3 5 7° + 7°'

5 7

etc.

until the seven glass filters were measured. This

sequence was repeated six times and the results are given in table 5. As can be seen from these data, the reproducibility

of sequential transmittance measurements can be

performed with an average standard deviation of 0.010 percent for a single determination.

IV. Wavelength Calibration

The wavelength scale of the monochromator is provided with a counter which indicates wavelength

directly in angstroms. This counter is checked for

42
Table 4. Stability of the current-to- voltage converter, the photomultiplier tube at 850 V, and the tungsten ribbon

filament lamp

Replication

12

34

56

78

9

10

111213

14

15

Average of 50 individual measurements; volts

2.002395

2.001356

2.002145

2.000975 2.001944

2.000925

2.001832

2.000825

2.001551 2.000960 2.001739

2.000851 2.001729

2.000825

2.001557

Percent standard

deviation

0.038o 0.0226 0.0247 0.0262 0.0207 0.028!

0.026! 0.0235 O.O263 0.021 2 0.023, 0.0244 0.0282 0.0230 0.0244

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

Average of replications Percent standard

deviation

2.001441

0.026

2.001517

0.012

2.000826

0.025

2.001268

0.027

First group

Second group

Third group

Fourth group

Table 5. Reproducibility of transmittance measurements on seven Schott NG-4 glass filters No. 2; 4; 6; 8; 10;

12; and 14

Percent transmittance

Replication No.

12

34

5

6

Average

Percent cr

Average percent a was opened and measurements were made again.

The average value found was 0.037 mV. This indicated

that no SRE could be detected under the experimental

circumstances.

2 4 6 8 10 12 14

33.327 21.711 12.236 50.990 33.377 20.906 13.473 33.325 21.710 12.237 50.983 33.377 20.903 13.471 33.321 21.711 12.241 50.992 33.383 20.900 13.474 33.320 21.708 12.240 50.988 33.375 20.901 13.470 33.323 21.710 12.239 50.983 33.379 20.901 13.474 33.325 21.710 12.238 50.986 33.377 20.904 13.470

33.32 21.710 12.238 50.987 33.378 20.902 13.472

0.0080 0.0051 0.0150 0.0072 0.0083 0.0108 0.0141

0.010

VI. Linearity Control

The single-beam static optical system described in

this work permits the unequivocal use of the radiation

43
addition principle by means of the double-aperture

method for determining departure from linearity of the

entire optical, photometric, and electronic system, and

thus of the photometric accuracy of transmittance

measurements.

The double-aperture and its positioning on the

optical bench was described earlier. Its use will now

be illustrated, and follows the procedure developed

and used at the National Physical Laboratory.

Since the linearity of photometric data for a given

photomultiplier tube depends on the anode voltage,

the values at the current-to-voltage converter, and

the ambient temperature, all measurements were made

using identical experimental conditions. These same

conditions were maintained when transmittance meas- urements were performed. Since the linearity is, within 1 part in 104

, not usually a function of wavelength [36], all measurements were performed at A. 565.0 nm. A recent study of this parameter at NBS by

Mielenz and Eckerle indicates that there may be a

relation between wavelength and linearity at the level of 1 part in 105 [38]. The intensity of the radiation flux produced by the

tungsten ribbon lamp was attenuated with the circular

neutral wedge until a photocurrent equivalent to 2.0020 V was obtained when both apertures, A and

B, were open. A setting of 850 V was used at the photomultiplier tube with a 30 MCI resistor at the currentto-voltage converter. Fifty individual DVM readings were taken and the average value for (A + B) was

printed. Aperture B was then closed, and 50 DVM

readings were taken. The average value for aperture

A was printed. The average value for aperture B was

then obtained in a similar manner by closing aperture

A and opening aperture B. This sequence was repeated

three times, ending with an {A + B) value. Identical measurements were made over a range of attenuation corresponding to 4 cascaded steps of 2

to 1 as illustrated in the actual example which follows:

Step 1

Av.

Diff. % Corr.

Step 2

Av.

Diff. % Corr.

Step 3

(A + B)

2.00147 2.00156 2.00206 2.0021!

2.00181

2.00233- = -0.026

(A + B)

1.00049 1.00078 1.00066 1.0009,,

1.00071

1.00112 —

= -0.041

(A + B)

0.50062

B

0.9864!

0.98640 0.98625

1.01596 1.01599 1.01609 1.01598 + 0.98635 = 2.00233

2.00181 =+0.00052

A

0.51020 0.50996 0.51004 0.51007 + 0.49105=1.00112

B

0.49108 0.4909i

0.491

1

5

1.00071 =+0.00041

A

0.25657 B

0.24430

0.50056 0.5007o

0.50062

0.25673 0.2566s

0.24436 0.24432 0.25665 + 0.24433 = 0.50098

Av.

Diff. % Corr.

Step 4

Av.

Diff. % Corr.

0.50063

0. 50098 - 0. 50063 = + 0. 00035 = -0.069

(A + B)

0.25020 0.25028 0.25019

0.25023 0.25023

0.25031 - = -0.031

A

0.12872 0.12856 0.12854

B

0.1217s

0.12168 0.12166 0.12861 + 0.12170 = 0.25031

0.25023 = + 0.00008

The correction curve is established from these data

by plotting voltages on the abscissa and the corre- sponding additive correction value on the ordinate.

'These are tabulated below and illustrated in figure 16.

FIGURE 16. Linearity correction curve.

Voltage %T % Correction of % T

2.00 100 0.0 1.00 50 0.026

0.50 25 0.067

0.25 12.5 0.14 0.125 6.25 0.167 When transmittance measurements are performed,

the h reading is initially set with the circular neutral

wedge to a value near 2.0020 V. The I value is then

measured. If the initial /0 = 2.00214 V and final /o = 2.00228 V and 7=0.54220 V, then percent T is:

0.54220

2.00214 + 2.00228

X 100 = 27.081

which is the noncorrected value. To correct this value, one takes from the ordinate of figure 16 the value

corresponding to 0.54220 on the abscissa which, in

this case, is 0.072. The corrected percent T value is then:

27.081 -f

7- 081^4072

) =27.061.

44
Mielenz and Eckerle have studied recently the

double aperture method for testing photometric

linearity, and have used a curve-fitting procedure

for finding the nonlinearity correction rather than

the method described earlier (38).

VII. Sample Position

A series of measurements were performed to deter- mine the magnitude of error which could occur when

the sample is oriented with its entrance face at an

angle to the incoming radiation beam. The single

sample holder provided with the rotating table, as described in section II, paragraph c, was used. Trans- mittance measurements were performed by producing

the image of the exit slit of the monochromator at the entrance face of the sample (aperture f:10). The data

are shown in table 6. The consequence of this condition on transmittance measurements is discussed by

Mielenz in this Journal.

Table 6. Percent transmittance (%T) , measured on

three neutral glass filters 1.0; 1.5; and 2.0 mm

thick at a = 440 nm, at three angles of

incidence

Angle of Filter Filter Filter incidence 1.0 mm 1.5 mm 2.0 mm

Normal incidence 32.91 5 19.83 8 11.606 1° 32.899 19.833 11.604 2° 32.897 19.81 9 11.599 3° 32.88, 19.81 2 11.589

Similar measurements were made to determine the

identity of positions on the seven-sample automatic

changer described in section II, paragraph c. For

this experiment, seven neutral glass filters A; B; C;

D; E; F; and G were used and were positioned in holders 2; 4; 6; 8; 10; 12; and 14 in three different

arrangements as described by Garfinkel, Mann and

Youden [39].

Table 7. Evaluation of the identity of the seven stations of the automatic sample changer

Percent Transmittance (%T) at station number (St. No.) 2; 4; 6; 8; 10; 12; and

14 for niters A, B, C, D, E, F, and G at A = 465.0 nm

Run No. A B C D E F G

%T 27.08 16.44 46.39 26.12 15.22 37.34 23.23

I St. No. 2 4 6 8 10 12' 14 %T 27.07 16.40 46.39 26.11 15.21 37.34 23.23

II St. No. 14 2 4 6 8 10 12 %T 27.08 16.43 46.39 HI St. No. 10 12 14

Transmittance measurements were then performed

on all filters for the three different arrangements and

the results are given in table 7. From these data it can

be concluded that the seven stations are interchangeable and will produce measurements which will not

differ by more than one part in one thousand.

VIII. Influence of Polarized Radiations on

Transmittance Measurements

This effect was determined by measuring the trans- mittance of a Schott NG-4 neutral glass filter at four

wavelengths using radiations emerging from the pre- disperser-monochromator unit, and by projecting the

image of the exit slit (8 by 0.5 mm) at the entrance

face of the filter with a convergent beam geometry

corresponding to an f : 10 opening. The glass filter was

checked prior to measurements with a polariscope for freedom of internal tensions. Column one of table 8

shows the results obtained when transmittance meas- urements were made using the radiations produced by

the spectrophotometer. Column two shows the results

26.11 15.21 37.33 23.23

2 4 6 8

obtained when a polarizing sheet, with the vibration

plane horizontal, was placed in front of the glass filter. The measurements obtained with the vibration plane

in vertical position, are given in column three. These measurements show that polarized radiations :an affect transmittance measurements of solid glass filters when noncollimated beam geometry is used. This effect is predicted by the Fresnel equations

mentioned in the introduction and should disappear

when collimated radiations are used (1, pg. 100).

XI. Comparison of Transmittance

Measurements

Two sets of solid filters were used in a comparative

test to determine the reproducibility of transmittance

measurements between two laboratories. One set was made from three neutral glass Schott NG-4 filters having nominal percent transmittances of 10; 20; and 30. The second set was made as described elsewhere [4]. Three evaporated metal (Inconel) on fused quartz

(nonfluorescent) plates having nominal percent

45
Table 8. Effect of polarization on percent transmittance (%T) measured at four wavelengths on a Schott NG-4

glass filter %T

Wavelength nm

Produced by

spectro- photometer

Polarizer, plane of vibration horizontalPolarizer, plane of

vibration

vertical

% Difference

%7\ - %T3

440.0

465.0

590.0

635.0

19.81« 22.597 19.17s 20.61,

2

19.800 22.600 19.170 20.60219.788 22.569 19.099 20.54 7

- 0.15 - 0.12 - 0.41 - 0.31

transmittances of 25; 50; and 75 were used. The trans- mittance measurements were performed on two sets of

filters at the National Physical Laboratory (NPL) in England using their high accuracy spectrophotometer, and at NBS on the instrument described in this paper. The measurements at NBS were carried out before and after the measurements at NPL. All measurements were made with noncollimated conver- gent beam geometry. A rectangular surface of the

filter about 3 mm by 8 mm was used at NPL and the

beam was only slightly convergent. At NBS an area about 8 mm by 0.5 mm was used for the transmittance

measurements.

The results given in table 9 indicate that an average

difference of —0.19 percent of the values was obtained

between the measurements carried out at NPL and at NBS. An average difference of —0.30 percent of the

value was found when similar measurements were

performed on the inconel-on-quartz filters, as shown in table 10.

X. Standard Reference Materials for

Spectrophotometry

The need for providing means and materials to check the proper functioning of a spectrophotometer was discussed in some detail in previous publications [3, 4]. At that time it was established that the

accuracy of the photometric scale is a critical and most

demanding parameter in spectrophotometry. Hence,

particular attention was given to a number of ways for checking this parameter. Investigations showed that

solid colored glass filters, exhibiting optical neutrality over the spectral range from 400.0 nm to 700.0 nm,

would constitute an acceptable Standard Reference

Material (SRM). From the various colored glass

Table 9. Comparison between the percent transmittances (%T) measured on three Schott NG-4 glass filters

at NPL and NBS

ivelength NBS, %T NBS, %T NBS, %T NPL, %T % Diff. nm March 12, May 18, average February NBS to N

1971 1971 1971

440.0 12.92 12.91 12.91s 12.93 - 0.11 465.0 14.965 14.98 14.97.3 15.01 - 0.25 590.0 11.70 11.64 11.67 11.67 0.0 635.0 12.72 12.68 12.70 12.72 - 0.16

440.0 19.62 5 19.58 19.6O3 19.62 - 0.09 465.0 22.385 22.35 22.367 22.43 - 0.28 590.0 19.06 18.95 19.00s 19.01 - 0.03 635.0 20.455 20.37 20.41 3 20.47 - 0.28

440.0 32.89 32.86 32.875 32.98 - 0.32 465.0 35.52 35.54 35.53 35.66 - 0.36 590.0 31.16s 31.10 31.13a 31.21 - 0.25 635.0 32.565 32.52 32.54:! 32.62 - 0.24

Average difference between NBS and NPL percent T values = — 0.19 percent.

filters available, Schott NG-4 "neutral glass" was

selected, prepared and characterized. It is now offered by NBS as a means to check the photometric scale of spectrophotometers.

46
Table 10. Comparison between the percent transmittances (%T) measured on three inconel-on-silica filters at NPL and NBS

Wavelength NBS, %T NBS, %T % Diff. nm 1 2 average NPL, %T NBS to NPL

450.0 24.87 550.0 23.78 650.0 23.38

450.0 49.35 550.0 47.60

650.0 46.85

24.88 24.875 23.82 23.80

23.39 23.385

49.33 49.34 47.60 47.60

46.85 46.85

24.93 - 0.22 23.86 - 0.25 23.46 - 0.32

49.56 - 0.44*

47.81 - 0.44 47.14 - 0.62

450.0 72.17 72.20 550.0 72.05 72.11 650.0 72.20 72.34

72.185 72.30 - 0.16 72.08 72.20 - 0.17 72.27 72.33 - 0.08

Average difference between NBS and NPL percent T values = — 0.30 percent.

*This filter had a flaw in the form of a crack which was sometimes visible and other times invisible. The larger differences found in the measurements of this filter may be due to this flaw.

SRM 930, developed in the Analytical Chemistry

Division and available since March 1971 consists of

three glass filters. Each filter bears an identification number, and the upper left corner has been removed

to indicate correct orientation in the metal holder

(fig. 17). The transmittance measurements were made with

the high accuracy spectrophotometer described in

this paper, and are certified with an uncertainty of

±0.5 percent of the value. This uncertainty is the sum of the random errors of ±0.1 percent (2SD

limit) and of estimated biases which are ±0.4 per- cent. These biases are due to possible systematic

errors originating principally from the inherent

inhomogeneity and instability of the glass as well

as from positioning of the filter. Measurements were made at 24 °C, and variations within several de- grees Celsius of this temperature will not significantly affect the calibration of the filters. The neutral

NG-4 glass for the filters was provided by Schott

of Mainz, Germany and is designated as "Jena Colored

and Filter Glass." Nominal transmittance for a

filter 1.5 mm thick is 20 percent at 400.0 nm wavelength and 32 percent at 700.0 nm wavelength. Be- tween these limits the transmittance varies in a monotonic manner. The filter is held in a frame and the size and shape

of the filters and frames were selected, for practical

considerations, to conform to the dimensions of the

standardized cuvettes for which holders are supplied

in most conventional spectrophotometers. The filters are approximately 1.0, 1.5, and 2.0 mm thick. Cor- responding to these thicknesses are nominal trans- mittances of 30, 20, and 10 percent, respectively.

These thicknesses were selected to provide a means for calibrating the photometric scale at three different

levels.

The effective spectral bandpasses used to determine

the certified values were equal to or smaller than 2.2 nm

at 440.0 nm; 2.7 nm at 465.0 nm; 5.4 nm at 590.0 nm;

and 6.0 nm at 635.0 nm. The transmittance measure- ments are made by producing the image of the slit (about 8 mm by 0.5 mm) using a convergent beam

geometry with an opening of f:10 corresponding

to an angle of 7° to 8° in the middle of the entrance

face of the filter. This beam geometry was used to reproduce the average experimental conditions

found in most of the conventional spectrophotometers available today. Prior to the certification, each

filter is examined for surface defects and thoroughly

cleaned. If, through handling, the surface of the

filter becomes contaminated, it may be cleaned

with a small soft brush attached to a rubber tube

connected to a vacuum source [40]. If contamination

results from fingerprints, they must be removed

before making measurements. This may be accomplished by removing the filter from its holder, breathing lightly on it, and rubbing the surface gently

with optical lens tissue. The clean filter is then properly

positioned in its holder. To remove and replace the

filter in the holder, the spring-loaded plate should

be lifted with care to prevent damage to the filter. As little handling as possible is recommended. SRM

930 should be used according to the directions on

the certificate; consult the manufacturer of the

instrument if differences are obtained that exceed

those specified by the manufacturer.

Under no circumstances should other cleaning

procedures which make use of detergent solutions,

organic solvents, etc. be applied.

When a filter has become contaminated beyond

cleaning by the procedure described in the certifi- cate, it should be forwarded to NBS. After proper

cleaning, the filters will be checked and, if needed,

47
Figure 17. NBS Standard Reference Material 930 and calibration certificate.

recalibrated using the high accuracy spectrophotometer described in this work.

It was already stated that the accuracy of photometric scales defines only one of the parameters re- quired for obtaining accurate transmittance values

and molar absorptivities. Other factors must also be

established. These are wavelength accuracy, adequate

spectral bandpass, stray light, cell parameters (when

solutions are measured), fluorescence, polarization,

reflection, and temperature coefficient. Some of these

variables were discussed in NBS Technical Notes 544

and 584 and are also examined in this paper. The transmittance data given in the certificate which accompanies each SRM 930 depend not only on the intrinsic properties of the glass and the ex- perimental measurement conditions, but on the surface

state of the glass. This parameter varies with time

and exposure conditions. When glass is exposed to normal room atmosphere and temperature, its surface

is corroded to an extent depending on the composition, time of exposure, concentration, temperature

and nature of the glass surface acting agents. This

action produces a change in the reflecting and trans- mitting properties of the material [41]. For instance,

a well-known phenomenon called "blooming" of the

glass is due to the formation of an SiO layer at the

surface of the glass. This layer, which increases the

transmittance, acts as an antireflection coating. The

speed with which such a layer is formed varies with

the composition of the glass, the atmosphere and time.

Generally speaking, several years are needed for a

fresh surface to reach equilibrium. This, and similar

48
phenomena are presently being studied, along with means to stabilize the surface state of glass filters. Until more information is acquired in this field, we

recommend that the colored glass filters issued as SRM 930 be rechecked annually to determine whether

any physicochemical changes, which might affect the

transmittance values, have occurred.

Another important factor is the need for defining

and producing a clean glass surface. Until now the

final cleaning of the NG-4 filters was made with re- distilled ethyl alcohol and pure water (thermally

distilled and deionized). Other cleaning procedures

are under consideration. The use of isopropyl alcohol

in vapor or liquid form associated with mild ultra- sonic action is being investigated [41]. The transmittance characteristics of the SRM 930

limit the use of this material to the visible region of

the spectrum from about 400 nm to the near infrared.

Since the ultraviolet region, from about 200 nm is also important to the analyst who uses spectrophotometric

methods, exploratory work is underway to select and

certify solid material for checking the photometric

scale in this spectral region. Optical filters exhibiting

small transmittance-wavelength dependence in the

spectral range 200 nm to near infrared can be obtained

by evaporating thin semitransparent layer of a metal on a suitable transparent substrate [42, 4], and such

filters have been considered in this work. The metals

selected were inconel and chromium which exhibit

adequate transmission characteristics and good ad- hesion to the substrate. The substrate was nonfluo- rescent fused quartz. A series of filters were prepared

by the optical shop at NBS according to the following

specifications: a number of nonfluorescent optical

quality fused quartz plates, 10 mm X 25 mm and 1 mm

thick, were cut and polished, Inconel or chromium

metal was evaporated on the surface to produce

nominal transmittances of 25, 50, and 75 percent.

The surface bearing the evaporated metal was coated

with a layer of optical cement which was transparent

to the visible and ultraviolet radiations down to 230 nm. A clear plate of the same material was used to cover and protect the evaporated metal layer.

The filter assembly was then marked at one corner

to insure its proper positioning and the finished filter was placed in a metal holder of conventional size (approximate o.d. 13 x 13 x 57 mm) fitting the cuvette holder found in most spectrophotometers. The metal

holder was also marked at one side to permit positioning of the filter in a reproducible manner.

In addition to the evaporated metal filters, a number

of units were prepared using only the clear uncoated

fused quartz plates and assembled with the same

optical cement. When desired, these clear filter assemblies could be used as reference samples in the blank compartment.

Before submitting the evaporated metal filters to transmittance measurements, a study was made of the

effect of radiations on their transmittances. A filter was exposed to an accelerated test in which radiations

had the same spectral distribution as the fluorescent

lighting of the laboratory, except that they were 1000

times more intense. The filter was exposed for an

equivalent of 36,000 hours of continuous irradiation. This test was made on a radiation accelerator made

available by the Building Research Division of NBS.

The percent transmittance was measured before and

after the exposure and gave the following results:

Transmittance, percent

Wavelength, nm 250 380 500 650

Before exposure 44.48 51.35 48.90 47.41

After exposure 44.11 51.34 48.92 47.47

The relative standard deviation for a single deter- mination of these measurements was 0.01 percent.

As can be seen, the only significant relative change in transmittance of about 0.84 percent of the value

occurred at 250 nm.

Several sets of these filters were calibrated at five selected wavelengths, 250 nm; 350 nm; 450 nm; 550 nm; and 650 nm, using the cleaning and measuring

procedures outlined for Schott NG-4 colored glass

neutral filters. The results indicated that the reproducibility of transmittance measurements is good

(percent standard deviation 0.009 to 0.024) and is comparable to those obtained for the colored glass

filters at all wavelengths except 250 nm. From the

experimental data, it is evident that the transmittance

of the evaporated metal filter at 250 nm is critical and, at present, no satisfactory explanation for this phenomenon can be given. A limitation of the evaporated metal filters is that they attenuate the intensity

of radiation by reflecting a part of it, rather than ab- sorbing. This can produce, in certain circumstances,

undesirable stray fight in the instrument and make the

transmission measurements dependent on the geometry of the optical beam. However, since these filters are closer to optical neutrality than the colored glass

filters, and since they can be used in the ultraviolet region as well, they were included in this work.

This limitation was apparent from the data ob- tained in a cooperative study conducted at C. Zeiss

by A. Reule using conventional spectrophotometers.

On the other hand, a similar comparative test, made

on the same filters by F. J. J. Clarke at NPL has

produced the results presented in table 10. One can

observe that, in spite of the limitations mentioned

above, an agreement within —0.30 percent of the

value was obtained between NBS and NPL measure- ments at the indicated wavelength.

Further studies will be needed to assess unambiguously the transmittance characteristics of evaporated

metal-on-quartz filters, with or without a protective

quartz plate, and to assess their suitability as Standard

Reference Materials to check the photometric scale

of spectrophotometers in the ultraviolet and visible part of the spectrum.

49
XI. Addendum

The identification of commercial instruments and

products, is given in the Addendum only to permit

reproduction of the work described in this paper.

In no instances does such identification imply recom- mendation or endorsement by the National Bureau of Standards, nor does it imply that the particular equipment or product is necessarily the best available for the

purpose.

Radiation source for visible — Microscope lamps,

type 18A/T10/1P-6V: General Electric Co., Lamp

Division, Nela Park, Cleveland, Ohio 44112. For ultraviolet: Atlas single coil halogen (Bromine) lamp,

type Pl/8, 30V, 250W: GTE Sylvania, Inc., 6610

Electronic Drive, Springfield, Virginia 22151.

Power supply for microscope lamp, Kepco, Model

JQE 15-50-M-VP: Kepco, Inc., 131-38 Sanford

Avenue, Flushing, New York 11352. For tungstenhalogen single filament lamp: same manufacturer,

Model JQE-36-30 Mt-VP.

Potentiometer: Leeds and Northrup Model K3 with

null meter and power supply. Resistors: Leeds and

Northrup 0.1, 50 A and 0.01, 100 A: Leeds and

Northrup, Sumneytown Pike, North Wales, Pa.

19454.

Nonfluorescent fused silica: Dynasil Corporation of

America, Berlin, New Jersey 08009.

Neutral Density Attenuator and BaS04 white paint:

Eastman Kodak Co., Special Products Sales, Kodak

Apparatus Division, Elmgrove Plant, Rochester, New

York 14650.

Monochromator with predisperser: McPherson In- strument Corp., 530 Main Street, Acton, Massachusetts 01720.

Optical benches with carriers and x — y sample

holder with micrometer control: Gaertner Scientific Corp., 1201 Wrightwood Ave., Chicago, Illinois 60614.

Lens holders: Ardel Instrument Co., Inc., P. O.

Box 992, Jamaica, New York 11431.

Ball bushing and rails: Thompson Industries, Inc., Manhasset, New York 11030.

Pneumatic cylinders and accessories: Clippard

Instrument Laboratory, Inc., Cincinnati, Ohio 45239.

Rotating table: Ealing Optics Division, 2225 Massachusetts Avenue, Cambridge, Massachusetts 02140.

Thermostating holders for glass cells and glass

filters: Cary Instruments, 2724 South Peck Road,

Monrovia, California 91016.

Pneumatic ratchet system: Allenair Corp., P. O.

Box 350, 255 East 2nd Street, Mineola, New York

11501.

Black paint -Nextel 101-c 10 Black: Reflective

Products Division 3M, 2501 Hudson Road, St. Paul,

Minnesota 55101.

Photomultiplier tube EMI-9558QA: Gencom Division, 80 Express Street, Plainview, New York 11803.

Power supply for photomultiplier tube: Model 415B

and digital voltmeter 8400A: John Fluke Manufacturing

Co., P.O. 7428, Seattle, Washington 98133.

Thermal insulation: Photoshroud, Shumway

Optical Instruments Corp., 2118 Beechgrove Place,

Utica, New York 13501.

Vibration isolation table: Lansing Research Co.,

705 Willow Avenue, Ithaca, New York 14850.

Low power laser; Model 195 cw gas laser, output

power 2 mw: Optics Technology, Inc., 901 California

Avenue, Palo Alto, California 94304.

Tritium activated fluorescent source, Beta light

Marker HM-110: Canrad Precision Industries, Inc., 630 Fifth Avenue, New York, New York 10020.

Colored glass neutral filters, Schott NG-4: FishSchurmann Corp., 70 Portman Road, New Rochelle,

New York 10802.

Spectral lamp: Oriel Optics Corp., 1 Market Street,

Stamford, Connecticut 06902.

Polarization filters: Polaroid Corp., 119 Windsor

Street, Cambridge, Massachusetts 02139.

Computer: 24K memory and 16 bit words. EMR

computer, Division of Weston Instruments, Inc., Schlumberger Co., 8001 Bloomington Freeway,

Minneapolis, Minnesota 55420.

The author gratefully acknowledges the assistance

of the following persons, in addition to those already

mentioned in the text: F. J. J. Clarke, for the considerable assistance given

through personal discussions on the philosophy and

principles of high accuracy spectrophotometry at NPL.

Anne Compton, for advice and help during the ex- perimental work carried-out at the high accuracy

spectrophotometer at NPL.

G. E. V. Lambert, for information concerning glass

filters (NPL).

K. D. Mielenz (NBS), for constructive discussions on

the principles of spectrophotometry and for critically

reviewing the manuscript.

W. H. Venable (NBS), for critically reviewing the

manuscript.

K. L. Eckerle (NBS), for advice in measuring stray

radiations and polarization produced in the monochromator.

C. R. Yokley (NBS), for advice on the radiation

stability of incandescent tungsten filament lamps.

W. K. Haller (NBS), for valuable information on the

properties of glass surfaces and optical glass filters. H. H. Ku (NBS), for advice on interpretation of the

data discussed in this paper. NBS Instrument Shop, for constructing the mechanical parts described in this work.

NBS Optical Shop, for grinding the quartz lens used

in this work.

D. S. Bright (NBS guest worker), for the programs

written at various times during the initial phase of this work.

V. E. Gray (NBS), for assistance in performing the

accelerated radiation tests. H. J. Hofert and A. Reule (C. Zeiss, Oberkochen,

Germany), for valuable information on linearity of

50
photodetectors and for demonstration of the radiation

addition method.

R. J. Carpenter (NBS), for constructive discussions on electronic circuitry and photodetectors.

L. Marzetta (NBS), for advice and assistance in assessing various functioning parameters of photomultiplier tubes. O. Menis, Section Chief, Analytical Coordination

Chemistry (NBS), for approving and encouraging the

work reported in this paper.

J. I. Shultz, Assistant Section Chief, Analytical

Coordination Chemistry (NBS), for critically reviewing

the manuscript.

XII. References

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[2] Gibson, K. S., Spectrophotometry, Nat. Bur. Stand. (U.S.). Circ. 484,48 pages (1949).

[3] Mavrodineanu, R., Spectrophotometry, instrumental develop- ment, O. Menis and J. I. Shultz, Eds., Nat. Bur. Stand.

(U.S.), Tech. Note 584, pp. 9-21 (1971).

[4] Mavrodineanu, R., Solid materials to check the photometric

scale of spectrophotometers, O. Menis and J. I. Shultz, Eds., Nat. Bur. Stand. (U.S.), Tech. Note 544, pp. 6-17 (1970).

[5] Elster, J. and Geitel, M., Un tne comparison of light intensities by photoelectric methods, Ann. Physik, Chemie 48, 625-635

(1893).

[6] Elster, j. and Geitel, H., The photoelectric effect on potassium

at low light levels, Phys. Ztsch. 13, 468-976 (1912).

[7] Koller, L. R., and Breeding, H. A., Characteristics of photoelectric tubes, General Electric Rev. 31, 476-479 (1928).

[8] Campbell, N. R., The experimental proof of a fundamental

photoelectric law, Trans. Opt. Soc. (London) 32, 61-65

(1930-1931).

[9] Fleury, P., An addition method for the precise study of the

variation of the current output of a photoelectric cell as a function of the incident luminous flux, Compt. Rend. Acad.

Sci. (France) 199, 195-197 (1934).

[10] Preston, J. S., and McDermott, L. H., The illumination re- sponse characteristics of vacuum photoelectric cells of the

Elster-Geitel Type, Proc. Phys. Soc. (London) 46, 256-272

(1934).

[11] Preston, J. S., and Cuckow, F. W., A photoelectric spectro- photometer of high accuracy, Proc. Phys. Soc. (London)

48,869-880 (1936).

[12] Buchmiiller, F., and Konig, H., Precision measurements on

incandescent lamps using selenium photocells, Assoc.

Suisse Electriciens, Bulletin 28, 89-99 ( 1937)

.

[13] Atkinson, J. R., Campbell, N. R., Palmer, E. H., and Winch,

G. T., The accuracy of rectifier-photoelectric cells, Proc. Phys. Soc. (London) 50, 934-946 (1938).

[14] Barbrow, L., A photometric procedure using barrier-layer photocells, J. Res. Nat. Bur. Stand. (U.S.), 25, 703-710

(1940) RP1348.

[15] Kaiser, H., Photographic-photometric calibration of step

filters, Spectrochimica Acta 3, 518-537 (1947-1949).

[16] Harding, H. G. W., Precautions necessary for accurate measure- ments of optical density standards, Photoelectric Spectrometry Group Bulletin 4, 79-86 (1951).

[17] Kortiim, G., and Maier, H., Dependence of photocurrent and

illuminating intensity in photocells and photomultipliers, Z. Naturforschung8A, 235-245 (1953).

[18] Hansen, G., Check of photometric function of spectrophotometers, Mikrochimica Acta 1955, 410-415.

[19] Hermann, W., Noise and linearity check on photomultipliers, Z. Naturforschung 12A, 1006-1013 (1957).

[20] Reule, A., Checking the photometer scale of absorption instru- ments, Zeiss-Mitt. 1, 283-299 (1959).

[21] Hoppmann, H., An instrument for checking the proportionality of physical radiation receivers, Technisch-Wissenschaftliche Abhandlungen Der Osram-Gesellschaft 7, 306-312 (1958).

[22] Gibson, G. L., Hammond, H. K., Ill, Holford, W. L., and

NimerofT, L, Calibration of photometers, NBS Manuscript, Nov. 1, 1960.

[23] Bischoff, K, Measurement of proportionality of radiation re- ceivers over a large radiation intensity range, Z. Instr. 69,

143-147 (1961).

[24] Jones, O. C, and Clarke, F. J. J., A new photometric technique

using a variable shutter device, Nature 191, 1290 (1961).

[25] Cordle, L. C, and Habell, K. J., Photometry of telescopes and

binoculars. National Physical Laboratory, Notes on Applied

Science No. 14, 18 pp. (1962).

[26] Sanders, C. L., A photocell linearity tester, Appl. Optics 1,

207-271, (1962).

[27] Nonaka, M., and Kashima, T., Linearity characteristics of multiplier phototubes, Japanese Journal Applied Physics 2, 785-

791 (1963).

[28] Hofert, H. J., and Loof, H., Calibration of photometric scales of a reflection photometer, Z. Instrumentenkunde 72, 139-143

(1964).

[29] Clarke, F. J. J., Time ratio photometry, Lecture to the Optical

Group of the Institute of Physics and the Physical Society (June 9, 1966).

[30] Clarke, F. J. J., High accuracy spectrophotometry of three filters, NPL; Report No. 3042, 14 pp. (Nov. 4, 1968).

[31] Reule, A., Testing spectrophotometer linearity, Appl. Optics 7, 1023-1028(1968).

[32] Desvignes, F., and Ohnet, J., Characteristics and measurement

of properties of radiation receivers, Techniques Philips N.6,

1-18 (1968).

[33] Kunz, H., Representation of the temperature scale above 1337.58 K with photoelectric direct current pyrometers, Metrologja 5, 88-102 (1969).

[34] Kostkowski, H. J., Personal communication (NBS 1970).

[35] ASTM Tentative Method of Estimating Stray Radiant Energy,

ASTM:E 387-69T.

[36] Clarke, F. J. J., Personal communication (NPL, August 1970).

[37] Mielenz, K. D.. and Eckerle, K.. Accuracy of oolarization attenuators (to be published), Appl. Optics (1972).

[38] Mielenz, K. D., and Eckerle, K., Spectrophotometer linearity testing using the double-aperture method, Appl. Opt. 11,

2294 (1972).

[39] Garfinkel, S. B., Mann, W. B., and Youden, W. J., Design and

statistical procedures for the evaluation of an automatic gamma-ray point-source calibrator, J. Res. Nat. Bur. Stand. (U.S.),70C (Eng. and Instr.), No. 2.53-63 (1966).

[40] Edisbury, J. R., Practical Hints on Absorption Spectrophotometry (Plenum Press, New Y'ork, 1967).

[41] Holland, L., The Properties of Glass Surfaces (Chapman and

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51

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Absolute Spectroradiometric Measurements

G. A. W. Rutgers*

Physical Laboratory Rijksuniversiteit Utrecht, Netherlands

(June 7, 1972)

There are two general methods for measuring a quantity of radiation emitted by a source. One

can compare it with the radiation emitted by a standard source or one can measure the radiation with a detector calibrated in absolute units. When using the latter method, one must know the spectral transmittance factor of the optical components between source and detector. In the present paper, a survey will be given of the standard sources available for spectroradiometry:

cavity radiator, tungsten strip lamp, anode of the carbon arc, xenon arc and cascade arc. Several

types of detectors such as the absolute bolometer and thermopile, with their properties, will be

discussed.

Key words: Absolute spectroradiometry; absolute standard source of radiation; calibrated photodetector.

I. Introduction

If a physicist delivers a lecture for analytical chemists, he should take care that his language cannot lead

to misunderstandings. From the Summary of Activities

1970/71 of the Analytical Coordination Chemistry

Section (NBS Technical Note 584) it appears that the

vocabulary used by physicists and chemists is not always the same. The main difference is in the use of the term photometry. For a physicist, particularly if he is engaged in the activities of the Commission Inter- nationale de l'Eclairage (CLE.), photometry is the

measurement of quantities referring to radiation evaluated according to the visual effect which it produces,

as based on certain conventions [l]. 1 These conventions

require the evaluation to be done according to the

international spectral luminous efficiency curve V(\),

defined by CLE. in 1923. In spectral measurements of

radiation, the V( \) -curve does not play a part. Therefore, in my opinion, it is better to speak of spectroradiometry instead of photometry. In this terminology

the main apparatus is a spectroradiometer instead of a photometer. Then the term photometer can be re- served for an apparatus, used to measure light (= visi- ble radiation evaluated according to the V(\) -curve)

and the pleonasm in the expression "photometric

scale in the visible region" can be avoided.

The following quantities will be used:

Spectral radiant intensity I\ (in a given direction):

Power emitted in that direction per unit of solid angle

per unit of wavelength. Unit: W/m-sr,

*We have learned with sorrow during the publication of this paper of the death of Professor Rutgers on October 4, 1972.

1 Figures in brackets indicate the literature references at the end of this paper.

Spectral radiance L\ (of a source in a given direction): Power emitted in that direction per unit of

projected area per unit of wavelength. Unit: W/m3 -sr, Spectral irradiance E\: Radiant power received by

a surface per unit area per unit of wavelength. Unit:

W/m3

, Spectral exitance M\: Quotient of the spectral

radiant flux leaving an element of a surface by the area of that element. Unit: W/m3

. It should be noted that the same definitions, without

the adjective "spectral," can be used; then the units

should be multiplied by m. Another point to be mentioned in the introduction is the usefulness of absolute

measurements. Again referring to NBS Technical

Note 584, the whole chapter on spectroradiometry is devoted to relative measurements, viz., the study of

different color filters on behalf of radiometry. In these

measurements, a constant radiation source and detector, reproducable samples and standard filters, and

linearity of the detector in a wide range, are the most

important requirements for the apparatus. However,

there are other fields in which absolute measurements

can give more information. For example, in line-re- versal experiments, knowledge of the radiance of the

background can give information on the radiance of a spectral line and therefore the number of transitions

belonging to the line. Another example is the measure- ment of the spectral distribution of a radiation,

emitted by an arbitrary source with either a continuous

or a discontinuous spectrum. An example is the

measurement of the radiance of a fluorescent surface. By comparing the spectral distribution of the radiation with that of a standard source, an absolute

measurement can be obtained if the geometry of the

53
measuring equipment is known.

From these examples, it will be clear that for absolute

measurements sometimes a comparison is made with an absolutely calibrated source; in other cases, an

absolute receiver will be used. In the latter case, the

influence of the optical components between source and detector upon the measured quantities are often

required. In the following sections a survey will be given of:

(1) the standard sources available for spectroradiometry and their properties with respect to spectral

radiance, accuracy and reproducibility,

(2) some detectors, used for absolute measurements,

their calibration, reproducibility, accuracy and other

properties, and

(3) some additional procedures used in absolute

measurements.

Literature references will be far from complete;

usually, references are given only to review articles and to special devices.

II. Spectroradiometric Standards

A. Black-Body Radiator

A radiator of which the spectral properties can be

derived completely from theory is the full radiator,

also called the black-body radiator. At a given temperature, T, the spectral distribution of the radiation

emitted is only a function of the wavelength k and is described by Planck's law. According to this law, the

spectral radiance, L\, of a black-body as a function

of k is given by the relation:

2hc2 1

he e UT,

(1)

1 where h is the Planck's constant, c the velocity of light

and k Boltzmann's constant. With ci = 27rhc2 and

c<i = hc\k (first and second radiation constant) eq (1) can be written2

Cl 7r\5 e

hclKkT -l (la)

ci = 3.7415 X 10-16 W • m2

; c2 = 1.4388 X 10-2 m K.

The spectral distribution (on the A -scale) has a maximum at X m , given by the equation

k m T= 2.8978 x 10-3 m K. (2)

For temperatures between 1200 and 3000 K, k m is situated between 2.4 and 1 fim, respectively (infrared

region). The ratio of the radiances at e.g., 300 nm to those in the top increases from 2.4 X 10^5 at 1000 K

to 0.0055 at 3000 K. Generally, the black -body radiator

is more practicable for use in the infrared region than

in the ultraviolet.

I a b

Figure 1. Simplified black-body radiators. The most simple device is a hollow tube with a small

hole in its (thin) wall, through which the radiation

emerges. Another type is a rod with a hole drilled in

its axis or a cavity of arbitrary shape (Hohlraumstrahler).

Deviations from ideal black-body conditions have

been calculated, by Gouffe [2], De Vos [3] and recently

by C. L. Sanders [4]. De Vos has shown that for a hole

in a cylindrical rod (fig. la), the emissivity e is between

0.978 and 0.997 if l/d ~ 10 and the inner wall has a

reflection factor of 0.60. The lowest value of e was

found for a material with a specular reflection characteristic, the highest value for a material with perfectly

diffuse reflection. For a cone-shape radiator, we

measured a somewhat higher e for a specularly redw

2 The same expression without n in the denominator holds for the exitance My

FIGURE 2. Cavity radiator to calculate the radiation loss through

the hole dO.

fleeting wall. The first order approximation of e in the

general case (fig. 2) is, according to calculations by

De Vos [3]:

e = 1 - rlWw

where r°° is the reflection factor of the element, dw,

in the direction dO dO and dQ.%, the "loss" cone. In order to avoid specular reflection (large r°°) , the ele- ment observed should not be perpendicular to the

direction of observation (fig. lb). The temperature of the radiator must be measured

according- to the procedures described in the text of the International Practical Temperature Scale (IPTS

1968). For temperatures below the melting point of gold (1337.58 K= 1064.43 °C) down to 630.74 °C a thermocouple calibrated on three fixed points is used.

Above the gold point, the temperature measurement

is based on Planck's law. Here, the optical pyrometer

can be used as a measuring instrument. Then, the

radiation should be measured in a small wavelength

region, usually at 650 nm, and the temperature is cal- culated from the ratio of the radiant intensities at the

unknown temperature and at the gold point. The accu54
racy of radiation measurements with a black-body

standard is related to the accuracy with which the

temperature of the radiator can be measured. In order

to obtain an accuracy of 1 percent in L\, the accuracy

in the temperature required at some wavelengths and

temperatures is mentioned in table 1.

Table 1

Accuracy needed in K for an error of 1 percent in L\

KIT 1337 2000 2500 3800 K

300 nm 0.4 0.8 1.3 3.0 650 nm 0.8 1.8 2.8 6.5 2000 nm 2.5 5.4 8.2 17

The accuracy with which the gold point can be

transferred to a visual pyrometer (650 nm) can be

estimated to be 0.5 K and with a photoelectric pyrometer to 0.05 to 0.1 K (apart from inaccuracies in the

gold point itself). A visual pyrometer calibrated at 650 nm with an accuracy of 0.5 K, has an inaccuracy

of at least 3 K at 2500 K (ratio of radiances to be

bridged about 2500). An accuracy of 1 percent in the

visible region does not seem an excessive requirement

for radiation measurements, but the figures in table 1 show that it is difficult to obtain this accuracy with a black-body radiator calibrated with a visual pyrometer.

According to Gray and Finch [5] , the accuracy of

calibration of a visual optical pyrometer at 2500 K is about 7 K; for an automatic photoelectric pyrometer the

accuracy is 0.6 K at 1337 K and 3 K at 2500 K. These

authors conclude that "the accuracy attainable outside

the standards lab is, at best, only moderate, even with

the most careful work and frequent recalibration." In conclusion, it can be stated that (1) the blackbody radiator is, from a physical point of view, the

best defined radiator, because its radiation does not depend upon the physical properties of the material

of which it is made, (2) its calibration does not permit a higher accuracy than 1 to 2 percent in its radiance at T ~ 2500 K and \ ~ 650 nm. The use of the blackbody radiator is restricted because of the auxiliary

apparatus, such as a furnace of accurately adjustable

temperature or a tube of high melting point. For

laboratory purposes the tungsten strip lamp is perhaps

less accurate but more practical. Nevertheless,

black-body furnaces up to 2500 K have been con- structed and successfully used for radiation measure- ments [4a].

B. Tungsten Strip Lamp

This section deals only with the tungsten strip lamp

as a standard source for spectral radiance. In pyrometry, this source is also used as a secondary temperature standard, for instance for the calibration of

commercial pyrometers.

The principal part of the strip lamp is a tungsten

ribbon with a width of some millimeters and a length

which varies for different types of lamp. The ribbon can be heated by an electric current up to a temperature of

~ 2360 K if the strip is mounted in an evacuated tube

and to ~ 2800 K if the tube is filled with a noble gas. The spectral radiance L\ is determined by the temperature of the strip and can be derived from Planck's

law, multiplied by the spectral emissivity e(\, T)

(Kirchhoff's law):

LX (K T)=e(k, T)Lb , x (\, T). (3)

In this formula L6) \(X, T) is the spectral radiance of a black-body at the same temperature, given by eq (1). e(A, T) as a function of k and T has been the subject

of many investigations. A discussion of the measure- ments has been given elsewhere by Schurer [6] and

the present author [7]. Since then, further results have

been published [8]. For the visible region (at 660 nm)

the new measurements are in agreement with the

measurements by De Vos ([9] fig. 3), and the conclusion

^according to Schurer [6]: "The e-values of De Vos,

after correction with —0.5 percent (Ae ~ 0.002) for possible influences of stray light and diffraction offer

in the u.v. and the visible up to 800 nm a set of data

with the required accuracy and reliability" can still be maintained.

In the infrared at 2.6 fim (the largest wavelength in De Vos' measurements), the e-values of Kovalev and

Muchnik are about 5 percent higher than those of

De Vos. According to Schurer [6], all previous measure- ments in the infrared at 2.6 fxm have results which are from 5 to 10 percent lower than those of De Vos. It is difficult to explain why the differences between the

results of different authors in the infrared are so much

larger than in the visible region. They can be caused

partly by different properties of the glass envelope

of the lamps, partly by different properties of the

tungsten strips themselves. More investigations in

this spectral region seem to be needed to solve this problem.

At the shorter wavelengths, Buckley [10] has shown

that a tungsten ribbon filament lamp with a sapphire

window can also be used as a calibration source in the

wavelength range 150 to 270 nm (ultraviolet). The

e-values, derived by Buckley from his measurements

(at wavelengths between 150 and 200 nm), show a

rather large uncertainty, but the relation to other

measurements at 200 nm is very satisfactory.

The calibration of the lamp is usually performed by means of a pyrometer at one (650 nm) or two wavelengths. In this way the luminance temperature Tl %

at that wavelength is measured and all uncertainties

in the pyrometer calibration are transmitted to the

ribbon calibration. The true temperature T is calculated from the relation (in Wien's approximation):

t(X) • e(\, T)e-C^T = e -c2ixTL

4)

or

1 1 _ Xlne(\, T)-t(X)

T TL c2 {b}

3 Luminance temperature = temperature of the full radiator for which the spectra) radiance at a specified wavelength is the same as for the radiator considered.

502-591 O - 73 - 5 55
Figure 3. Spectral emissivity of tungsten as a function of wavelength and temperature after De Vos [9].

t(\) is the spectral transmission factor of the window.

For X = 650 nm and c2 = 1.4388 X lfj-2 m • K (5) can be

written: - 1.041 X 10-4 lg{e(X, T) -t(X)} (5a)

Furthermore it should be kept in mind that there is a temperature gradient across and along the strip. The

latter is smaller in the center part of the strip if the

strip is longer and the end sections are folded back to the mounts. Detailed information for the use of the

tungsten strip lamp is given, a.o. by Barber [11] and

Kunz [12]. Usually, the current through the strip is supplied

by a stabilized dc supply. A dc current can be measured with a great accuracy and electronically stabilized

power supplies are commercially available. However,

if a dc current is passed through the strip during a long time in the same direction, changes in the surface

of the strip can be observed (De Vos [9], Quinn [13]). The grooves which are produced on the strip will have

an influence upon its emissivity and therefore on the

difference between true temperature and luminance

temperature (Cf. eq (5a)). De Vos therefore recom- mended to reverse the current at regular time intervals. One could also think on supplying the lamp with ac. Because of the finite heat capacity of the strip, it

can be expected that the temperature of the strip

changes periodically with the current and that a phase shift occurs between temperature and current. Measurements by Bezemer of our laboratory have

shown that the temperature during a half-cycle of the 50 Hz supply changes between two values, Tmax and rmln , which differ about 30 K at r=2000 K for a tungsten strip of 20 x 2 mm and a thickness of 0.02 mm. If the temperature is measured with a visual

pyrometer, no differences were found in the pyrometer

readings for the same dc and ac (effective) current,

because the eye is too slow to follow the 50-Hz fluctuations. If a chopping system is used, care should be

taken that measurements are not made at a fixed point

of the sine-wave.

The main reasons for nonreproducibilities in a

particular strip lamp are:

(1) variations in the temperature because of changes

in the position of the lamp (with respect to the vertical),

typical for gas filled lamps [11],

(2) changes in the structure of the filament because

of the direction of the current [9, 13],

(3) influence of the ambient temperature upon the

calibration, particularly at low ribbon temperatures

(T< 1500 K), and

(4) changes in the transmission factor of the window

because of tungsten deposits. Evaporation of the

tungsten has also an influence upon the ribbon itself.

56
Notwithstanding all these reasons for nonreproducibility, measurements of the spectral radiance of a number of lamps from different manufacture by

Schurer [6] gave very satisfactory results. If all lamps were adjusted to the same luminance temperature at 650 nm, the spectral radiances at 280 nm did

not deviate more than ±3 percent and at 2500 nm not more than ±2 percent.

For the user, the factors mentioned above mean

that the lamp has to be recalibrated regularly. For

absolute measurements, the accuracy is determined

both by the accuracy of the calibration of the lumi- nance temperature as a function of the lamp current and by the knowledge of the spectral emissivity of tungsten. In a review article such as this, the author

cannot do much more than draw the user's attention

to all the effects mentioned.

C. The Carbon Arc as a Standard for'Spectroradiometry

A disadvantage of the tungsten strip lamp, inherent

to the generation of the radiation, is the strong de- crease in radiance with decreasing wavelength. At a temperature of 2800 K the maximum of the spectral

radiance is at 950 nm; at 700 nm the radiance is still 74 percent of its maximum value, but at 300 nm it is only 0.3 percent of the maximum value. For a blackbody radiator, the relative slope of the radiancewavelength curve in the visible and ultraviolet range

of the spectrum decreases if the temperature increases..

If e(A, T) does not change much with wavelength, a

similar variation of radiance is found for other radiators. Therefore, one must look for a nearly grey

radiator with a temperature as high as possible. In

this respect, the anode of the carbon arc has favorable

properties. Since the first measurements by Waidner

and Burgess in 1904, the temperature and emissivity

of the carbon and graphite anode have been subject

of many investigations. A survey of these investigations has recently been given by Schurer [14]. The

author drew attention to the remarkable differences in spectral reflectance of carbon, found by different investigators; they range from 1 to 30 percent, cor- responding to emissivities of 99 to 70 percent.

Table 2. Some data of the anodes, used by Schurer [14]

Manufacturer Code Kind of carbon Density Resistivity • Grain size Overload (KFkg/m3

) (10-6nm) A/mm2

Ringsdorff RWO Graphite 1.79 7.7 Fine 1.12 RWI Graphite 1.59 7.5 Fine 1.07 RWII Lampblack 1.56 61 Fine 0.71 RWIV Graphite 1.78 7.2 Coarse 1.20 Ultra Carbon Ul Graphite 1.75 7.2 Coarse U2 Graphite 1.99 12.1 Fine 1.15 National Carbon AGKSP Graphite 1.66 6.4 Coarse 1.04 SPK Graphite 1.87 11.8 Fine 1.11 L113SP Lampblack 1.50 53 Fine 0.68 Conradty Noris Lampblack 1.20 96 Fine 0.6 Noris vacuum Coke 1.32 79 Coarse 0.75

57

First some words about the use of the carbon arc in practice. Generally, the arc is drawn between a hori- zontal anode (used as the radiance standard) and a

vertical cathode. The electrodes must be mounted in a

casing, which protects the arc from draught and the

surroundings from radiation. Because of the rapid

burning-off of the electrodes, their distance should be

held constant, either manually or with an automatic

device. It is often useful to make an enlarged image of

the arc on a screen, on which the required position can

be indicated. The arc is powered from a dc source, which should supply a current of about 10 A (for a low- current arc). To stabilize the arc, a ballast resistance

is needed, which is usually chosen such that the

voltage across the resistor is at least equal to that across the arc. Dependent on the quality of the supply,

a ripple filter can be added to suppress a possible

50-Hz ripple of the supply source. Schurer did measure- ments on several electrodes from different manufacturers: 4 Ringsdorff Werke G.m.b.H. (FRG), 6 different types,

Ultra Carbon Corp. (USA), 5 different types, National

Carbon Company (USA), 3 different types, Conradty

(FRG), 3 different types. The data of these electrodes

are collected in table 2. It can be seen that great varia- tions in density and specific resistance exist in the

different species. Much less variation was found in the current density at the overload point (current at which

the arc starts hissing). Therefore, the variations in the

temperature of the anode crater (the radiation source

proper) are rather small. The lowest (true) temperature was found for Conradty-electrodes (3775 to 3785 K,

± 15 K), the other temperatures range from 3790 K

to 3865 K. The values of the spectral emissivity, found

by Schurer for RWI anodes at arc temperature ranged

from 96 percent in the ultraviolet, via 99 percent at 500 nm to about 96 percent at 1.5 /xm. In some parts

of the spectrum, the radiation from the arc itself can

affect the observed anode radiance, particularly at

4 In order to adequately describe materials and experimental procedures, it was oc- casionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the

best available for that purpose.
those wavelengths where strong cyanogen bands are found and at wavelengths below 250 nm. Nevertheless,

Schurer concluded "that the anode of a carbon arc in

air offers a highly reproducible standard of spectral

and total radiance." The total radiance, found for a RWO anode, appeared to be 3.84 W mm-2 sr

_1 ±l

percent, of which 0.054 W • mm-2 sr

_1 has its origin

in the arc.

D. Some Other Standard Sources

Besides the standards, described in the foregoing

sections, there are some radiation sources which, with

certain restrictions, can also be used for absolute

spectroradiometric measurements. Alternatives for the

black -body radiator are the tungsten lamp, described

by Magdenburg and Wende [15] and the black-body

radiator after Quinn and Barber [16]. In the first case, a cylinder of tungsten foil is used

with a slit in the surface. It can be used as a tungsten

ribbon filament lamp, but with the possibility to measure e(\, T) of the tube by comparing the radiance of

the tube and the slit. In the second case, a tungsten

tube is used, which is observed end-on. Inside the

tube is a bundle of tungsten wire, which is the proper

source of radiation (fig. 4). It can be considered as a grey body with an emissivity as high as 0.95. The argonfilled lamp needs a current of 56 A at 12 V for a temperature of 2700 °C. Measurements by Jones [17] of the reproducibility of this lamp are promising and the

Comite Consultatif de Photometrie of the Bureau Inter- national des Poids et Mesures, in its meeting of September 1971 has recommended to continue the study of

this radiator.

a b

FIGURE 4. Quinn-Barber black-body radiator [16]. a, tungsten tube

with tungsten wires; b,exit diaphragm, <f> = 1 mm; dick diaphragms

for eliminating stray light. For some spectroradiometric measurements, it would be worthwhile to have a background with a radiance still higher than that of the anode of the

carbon arc. Attempts have been made to use the highpressure xenon arc lamp for this purpose. Most investi- gations have been done for XBO-lamps of various

wattage, made by Osram. For its use as a radiance

standard one should take in mind that the discharge

consists of two parts, the so-called "Plasmakugel," a small spot of high radiance situated just above the

cathode and the column with a radiance which is about one quarter of that of the sphere. The spectral radiance

of the sphere of XBO-900 lamp at 400 nm is about

1 W/cm2 -sr-nm, that of the arc~0.3 W/cm2 • sr • nm. The Tatter value is by a factor of 3 to 4 larger than the

radiance of the anode of the carbon arc and by a

factor of 200 larger than the radiance of the strip lamp

at 2800 K, both at the same wavelength. In our laboratory ter Heerdt has measured the spectral distri- bution of the radiation emitted by different parts of the

arc and investigates whether the reproducibility of the xenon-arc lamp is good enough for the use as standard

source. Another stable source of high spectral radiance is the

cascade-arc of Maecker [18], a stabilized high cur- rent arc with a current of about 1000 A and a temperature of about 12000 K (fig. 5).

FIGURE 5. Maecker's cascade arc [18] between a carbon anode and a tungsten cathode. Around the arc the cooling discs. Argon

is used here as protecting gas and SF6 as the gas the properties

of which are investigated. The arc column burns within the bore of a stack of copper discs and has a diameter of about 5 mm. The

spectrum consists of a strong continuous background

on which spectral lines of the arc gas are superimposed. Most measurements up to now are related to the properties of the arc gas or gases at high temperatures, but it seems that this arc has also good properties

for being used as radiation standard.

58
E. Irradiance Standards

All the standards mentioned in the foregoing

sections are primarily meant for radiance standards.

For absolute measurements of radiation, an irradiance

standard can be required, that is to say a standard

which causes a known spectral irradiance at the sur- face of a detector. If a black-body radiator is avail- able, this can also be used as an irradiance standard

if a diaphragm of known diameter is used. For the

tungsten strip lamp this is more difficult to achieve,

because the radiance is not constant along and across

the strip. This makes it difficult to calculate the ir- radiance at a given distance from the lamp. More

suitable standards for this purpose are the special

tungsten filament lamps and quartz-iodine lamps,

developed at the National Bureau of Standards [19]. An irradiance standard, which can also be used in the ultraviolet, is the UV-Normal by Krefft, Rossler

and Riittenauer [20], which consists of a high pres- sure mercury vapor lamp, run at dc, 2A, 200 W. An

alternative is the Standard 75 lamp, 0.95 A, 80 W [21]. The spectrum consists of a weak continuum on which

the mercury spectrum is superimposed. The spectral

distribution has been measured a.o. by Rossler, van

Stekelenburg [22], Coolidge and recently by Kok [21]. According to van Stekelenburg's measurements the

differences between various lamps of a series are* about 10 percent, the relative spectral distributions

from different lamps are nearly equal. The UV-Normal

gives an irradiance of 0.70 W/m2 at a distance of 1 m

at a wavelength of 365 nm (strongest line); at 296.7 nm

this value is 0.10 W/m2. The irradiance by the con- tinuum at the same distance is 0.48 mW/m2

• nm at 500 nm, 3.0 mW/m2 • nm at 300 nm.

III. Absolute Radiometers

In the preceding section, standard sources have been

described which can be used if absolute radiation

measurements are required and a comparison of the

unknown source with a standard source is possible.

Another procedure for absolute measurements is to measure the radiation emitted by the unknown source

directly with an absolute receiver, called also an

absolute radiometer.

Most detectors used in optical measurements, such

as the photomultiplier tube, the photoelectric cell

etc., have a spectral responsivity which depends on

the wavelength. Such a detector can be calibrated,

either relatively or absolutely, with the help of a standard source, combined with a spectral apparatus

or a series of interference filters. If the transmission

characteristics of the spectral apparatus or the filters are known, the calibration can be made in absolute

values. However, in many cases a relative spectral

response curve is sufficient. We will come back to this procedure later on.

First we will pay attention to devices which offer the possibility of making an absolute measurement

directly; that is to say, without using a standard

source. In such a device the radiant energy or power

absorbed must be compared with a quantity of energy

or power of another form, usually a quantity of electric power. The apparatus, best suitable for this purpose

and most thoroughly investigated is the absolute radiometer, belonging to the class of thermal receptors.

Essentially, this type of receptor is independent of

the wavelength of the incident radiation in the spectral

region where the absorption factor (black surface) is independent of wavelength. The incident radiation is absorbed in, e.g., a metal foil which consequently

increases in temperature. The increase in temperature can be measured in different ways, for instance with a thermopile or as a change in the resistance of the

receiver, a bolometer.5 Afterwards the metal foil can be heated by an electric current, which must be

adjusted in such a way that an equal change in the

detector element is caused.

One of the older instruments, based on this principle, is Wouda's bolometer [23] , developed in the

thirties in our laboratory. The radiation absorbing

element in Wouda's bolometer is a manganin foil of 50 x 15 x 0.03 mm, blackened with carbon black on

the front. On the back, a copper wire 50 urn in diameter is spread across the whole surface of the foil, as shown in figure 6.

TZZJT

7T

Figure 6. Wouda's absolute bolometer [23]. a,a= bolometer wire,

tf>= 50 /nm; b,b, copper clamps on which the metal foil is soldered

(at c,c); d,d, potential wires. A change in temperature of the receiver element by

absorption of the incident radiation causes a change in the resistance of the copper wire, which can be

measured with a Wheatstone bridge. Local differ- ences in the temperature are eliminated by the shape

of the wire which operates as an integrating device.

Afterwards the foil can be heated with a known electric current; its resistance (nearly independent of temperature) is also measured in a bridge circuit. A similar

element in the opposite arm of the bridge compensates

for a possible influence of the surroundings. The heat

balance equation is:

3 According to [1], a bolometer is a thermal receptor in which the heating of the part thai absorbs the radiation gives rise to a change in its electrical resistance. Here, bolometer is also used as a device where the receiver heats a resistor which resistance changes because

of the heating of the radiation receiver (see Wouda bolometer). 59
aEA = PR (6) 4<:m -

where E is the irradiance on the plane of the foil (W/m2

), a the absorption factor of the foil, A its surface (m2

), i the current which causes the same

effect in the detector as the radiation and R the

resistance of the foil. The accuracy is determined by

the accuracy with which a, A, i, and R can be deter- mined, the time constant by the heat capacity of the

foil.There is a difference between the heating of the

copper wire by radiation or by the electric current through the foil, since in the first case the heat ab- sorbed in the black layer has to pass this layer before

it reaches the metal foil; in the second case the heat

is produced inside the foil. Wouda has shown that the

procentual error caused by this effect is ~ ad/k where a is the heat loss per mm2 and per °C of the frontside

by radiation, convection and conduction of heat, d

the thickness of the black layer and k its heat conductivity. In the bolometer, used by Wouda, this correction was only 0.12 percent and was taken into account in the results of the measurements. Another

point to be mentioned is the absorptance of the black

layer. Carbon black has a reflection factor of 3-4 per- cent in the visible region; in the infrared beyond 4 \x the

layer becomes more and more transparent. This can be avoided by the use of other layers, developed since

then. Ultimately, Wouda claims an accuracy of 0.1 percent of his apparatus. Some years ago, we made a comparison between this bolometer and a thermopile,

calibrated at the National Physical Laboratory, and

found a difference of less than 1 percent. At that time, a more accurate comparison was not possible.

An alternative device was designed by this author

[24]. In this device the copper wire was replaced by a

thin aluminum foil made by evaporation on the back

of a mica foil. The receiver was a layer of evaporated

manganin on the frontside of the mica foil. The

accuracy was estimated to be better than 0.5 percent.

Another alternative has been described by Bischoff of the Physikalisch-Technische Bundesanstalt [251 and

by Gillham of the National Physical Laboratory [26]. In fact, Gillham developed three radiometers, of which the first one — an improvement of the older

Guild radiometer — consisted of a circular metal disc,

thermally insulated from its surroundings and provided

with a number of thermocouples to measure its average

temperature. The disc is heated either by the incident

radiation or by an electrical heating element. Gillham

made a new disc radiometer with a much smaller heat

capacity and added a complete second receiver

"identical to the first receiver, except that it looks in the opposite direction." The receiver and heating

element are shown in figure 7. In his third radiometer,

the black-body radiometer, the receiver is mounted in a

cavity with an absorption factor sensibly equal to unity

(fig. 8). This instrument has a sensitivity of 0.012

V • W_1 cm-2

, an area of the limiting aperture of 3.00

mm2 and a time constant of 14 s. A comparison of

the new instruments has shown that they agreed to within 0.2 percent.

receiver disk (aluminium)

I hiraluinin botlv

thermopile

mica disk

Figure 7. Gillham absolute radiometer [26].

« 2-4 cm

receiver tube

(aluminium)

radial thermopile

outer shell (Duralumin)

FIGURE 8. Gillham radiometer in a cavity [26]. At the National Standards Laboratory (Australia) a radiometer of the Gillham type has been used by

Blevin et al. [27] for a new determination of the

Stefan-Boltzmann constant. Preston [28] described a

similar device, also intended for measuring the energy

of radiation pulses. It had a rise time of i s and a cooling time constant of 8 s. An advantage of the black-body radiometer is that it is independent of the spectral characteristics of the

black surface layer, used in the devices described

above. It is often difficult to measure this absorption

factor over a wide spectral region. Previously, Miiller used a hemispherical mirror with the receiver in its center; the radiation is admitted through a hole in the

mirror. The radiation which is reflected by the re- ceiver impinges upon the mirror and is reflected back

to the receiver. In this way the effective absorbance

of the receiver can be increased.

Two other cavity-type radiometers have been devel- oped and described in detail by Kendall and Ber- dahl [29]. One type is used in vacuum, the other

either in vacuum or air. They are designed for measuring irradiances from about 0.10 mW/mm2 to 8 W/mm2 with an accuracy of 0.3 percent.

60
With all precautions taken, it seems possible to determine the total radiation on a certain plane with an accuracy of some tenths of a percent. For absolute

spectral measurements the accuracy is much less. Intercomparisons by National Standardizing Laboratories of absolute spectral irradiance scales are carried

out under the auspices of a committee on spectroradiometry of the Commission Internationale de

l'Eclairage with C. L. Sanders as the chairman. All devices described above have a rather large area and

time constant, and are generally not suited for measure- ments behind the exit slit of a monochromator. Therefore, the devices described must be considered as primary standard instruments, against which other

radiation receivers, either nonselective or selective, can be calibrated.

IV. Additional Remarks Concerning

Absolute Measurements

In the foregoing sections we have seen that for absolute measurements both absolute radiation

standards and absolute receivers are available. For a proper use of these devices, several precautions have

to be taken depending upon the aim of the measurements. The main problems in absolute radiometry are:

(1) the calibration of an arbitrary source in absolute

units,

(2) the calibration of a selective receiver in absolute

units. In both cases the user is generally interested in the

dependence of the quantities measured on the wavelength of the radiation emitted or received.

A. The Calibration of an Arbitrary Source

If an absolute standard source is available, both

sources can be compared with the aid of a monochromator and an arbitrary receiver behind the exit

slit. If the sources are comparable in size and spectral

distribution, no special problems will be encountered.

Both can sucessively be focused upon the entrance slit of the spectral apparatus and, if the receiver is a linear

instrument, the ratio of the signals is a measure for the

ratio in the radiances or radiant intensities. If the

sizes or intensities of the sources differ considerably,

a MgO- or BaS04 -screen can be irradiated successively

by the sources. Then, the radiances of the screen are compared. If the source to be calibrated has a spectrum

composed of a continuous background and spectral

lines superimposed upon the background (contrary to most standards described before, which have a rather

smooth spectrum), the signal has to be separated into

its component parts. This can be done in different ways:

(1) by variation of slit width. If for equal slit widths

the width of the slits is changed, the intensity of the

continuum leaving the monochromator is another function of this width than the intensity of the line radiation (intensity (:)(A\)2 and (AA.) respectively under

certain precautions [30]).

(2) with fixed (narrow) slits and continuous recording

of the signal as a function of wavelength. The area between the total response and the interpolated

continuum in the region of the line provides the radiant

flux in the line. The slit width should be chosen in accordance with the dispersion and the slope of the

spectral distribution curve. More about a similar

method can be found in a paper by Bauer and Erb [31]. If an absolute receiver is available, one can either measure the radiant flux leaving the exit slit of the

monochromator directly with the calibrated receiver

or, if this receiver is not suited for this purpose (e.g.,

because his surface is too large and must be filled homogeneously), calibrate another receiver against the

standard and use this one behind the exit slit (Cf. IV. B). In order to relate the signal of the receiver to the radiant intensity of the source to be calibrated, one must furthermore know the absorptance of the

optical components between source and detector and

the width of the spectral region transmitted by the

monochromator. This last quantity is needed for the

reduction of the measured signal to a quantity per

unit of wavelength.

A method, which gives all the information needed, is schematically given in figure 9 (Krijgsman [32]). Two monochromators are required in crossed position.

The source to be calibrated is focused on the entrance

slit Si of the first monochromator (i.e., a double monochromator), which in its turn is successively focused on S2 and S3. S3 is focused with lens L3 in P. The

spectral irradiance in P is measured with the absolutely

calibrated receiver. Afterwards the image in P is re- placed by the source K. The radiant intensity of K

and its image (both in P) are compared with a second

monochromator with arbitrary receiver or spectrograph but with a direction of dispersion which is perpendicular to that of the first one (S4 perpendicular to S3). The spectral region transmitted by the

first monochromator can be derived from the shape of the region transmitted by the second monochromator.

For detailed information on this method, the reader is referred to the literature.

Figure 9. Krijgsman's arrangement [32] for the absolute calibration of an arbitrary source K. D, double monochromator with

slits Si, S2, S3 ; P, image of K; Sp, spectrograph with horizontal

slit S4.

61
B. Calibration of An Arbitrary Receiver

Here, also, the method depends upon the standard

available, either a standard source or an absolute

receiver. If a standard source is available, one can use

either a series of interference filters or a monochromator to separate the radiation into individual

wavelengths. In both cases the spectral transmittance

of the wavelength-selecting device should be known.

If a standard receiver is available, an arbitrary source can be used and the receiver to be calibrated can be

compared to the standard receiver in an arrangement

similar to that shown in the first part of figure 9 (both

receivers in P). If the receiver has a spectral response

curve with steep slope (or slopes), it might be necessary to calculate the effective wavelength of the radiation incident upon the receiver.

Factors, like nonlinearity, fatigue effects and area

sensitivity of the receiver are not discussed here. But the user should not forget them in the evaluation

of his measurements.

The author is much indebted to J. A. ter Heerdt

for carefully reading the manuscript.

V. References

[1] Vocabulaire International de l'Eclairage. 3rd Ed. Publ. CLE.

N° 17 (E-l.l) 1970.

[2] Gouffe, A., Aperture corrections for artificial black-bodies

taking account of multiple internal reflections. Rev. d'Optique

24, 1-10 (1945).

[3] De Vos, J. C, Evaluation of the quality of a black -body, Physica

20, 669-689 (1954).

[4] Sanders, C. L., Thermodynamic considerations in realizing the primary standard of light. Metrologia 3, 4, 119-29

(1967).

[4a] Suzuki, M., Nanjo, M., and Yoshie, K., Standards of spectral radiance and irradiance for the wavelength region of 0.25

to 2.5 ixm. P-71.21, XVII- Session CLE., Barcelona 1971.

[5] Gray, W. T., and Finch, D. I., How accurately can temperature be measured? Physics Today 24, 9, 32-40 (1971).

[6] Schurer, K., The tungsten striplamp and the anode of the carbon arc as radiometric standards. Thesis Utrecht 1969; The tungsten strip lamp as a standard of spectral radiance, Optik 28, 400-406 (1968/69); Discussion of some uncertainties in the spectral emissivity of tungsten, Optik 28, 44-53

(1968/69).

[7] Rutgers, G. A. W., Standards for photometry. Appl. Optics

10, 12, 2595-99 (1971).

[8] Kovalev, I. I., and Muchnik, G. F., Normal spectral emissive power of tungsten etc.. High Temperature 8, 5, 983-88

(1970).

[9] De Vos, J. C, The emissivity of tungsten ribbon. Thesis Vrije Universiteit Amsterdam 1953; A new determination of the emissivity of tungsten ribbon, Physica 20, 690-714 (1954).

[10] Buckley, J. L., Use of a tungsten filament lamp as a calibration standard in the vacuum ultraviolet. Appl. Opt. 10,5, 1114-18

(1971).

[11] Barber, C. R., Factors affecting the reproducibility of brightness

of tungsten strip lamps for pyrometer standardization, J. Sci. Inst. 23 , 238-42 (1946).

[12] Kunz, H., Priifen technischer Strahlungspyrometer, V.D.I.- Berichte Nr 112, 37-46 (1966).

[13] Quinn, T. J., The effect of thermal etching on the emissivity of tungsten, Brit. J. Appl. Phys. 16, 973-80 (1965).

[14] Schurer, K., The spectral emissivity of the anode of a carbon

arc, Appl. Opt. 7, 3, 461-5 (1968).

[15] Magdenburg, H., and Wende, B., Uber die Erzeugung Schwarzer

Strahlung mit einer Wolfram-Lampe, PTB-Mitt. 74, 431

(1964).

[16] Quinn, T. J., and Barber, C. R., A lamp as a reproducible source of near black-body radiation for precise pyrometry up to 2700 °C, Metrologia 3, 1, 19-23 (1967).

[17] Jones, O. C, and Gordon-Smith, G. W., Utilisation d'une lampe a corps noir comme etalon de temperature de couleur

et de luminance, Comite Consultatif de Photometrie 71-14

(Reunion Sept. 1971).

[18] Maecker, H., Fortschritte in der Bogenphysik. Proc. 5th Int. Conference on Ionization Phenomena in Gases, Munich

1961, 1793-1811.

[19] Stair, R., Schneider, W. E., and Jackson, J. K., A new standard

of spectral irradiance. Appl. Opt. 2, 11, 1151-1154 (1963).

Stair, R., Schneider, W. E., and Fussell, W. B., The new

tungsten-filament lamp standards of total irradiance, Appl.

Optics 6, 1, 101-105 (1967).

[20] Krefft, H., Rbssler, F., and Riittenauer, A., Ein neues Strah- lungsnormal, Z. techn. Phys. 18, 20-25 (19371.

[21] Kok, C J., Measurement of spectral irradiance of UV Normal

and Standard 75 Lamps. Paper Committee E 1.2 — CLE.,

Barcelona 1971.

[22] Proceedings 12th Congress CLE., Stockholm 1951, Committee

41 (Ultraviolet Radiation).

[23] Wouda, J., Nauwkeurige absolute intensiteitsmeting, Thesis

Utrecht 1935.

[24] Rutgers, G. A. W., A new absolute radiometer, Physica 17,

129-36 (1951).

[25] Bischoff, K., Ein einfacher Absolutempfanger hoher Genauigkeit, Optik 28, 183-89 (1968).

[26] Gilham, E. J., Recent investigations in absolute radiometry,

Proc. Roy. Soc. (London) A 2 69, 249-76 (1962).

[27] Blevin, W. R., and Brown, W. J., A precise measurement of the Stefan-Boltzmann constant, Metrologia 7, 1, 15-29

(1971).

[28] Preston, J. S., A radiation thermopile for cw and laser pulse

measurement, J. Physics E: Sci. Inst. 4, 969-72 (1971).

[29] Kendall, J. M. and Berdahl, C M., Two black-body radiometers

of high accuracy, Appl. Opt. 9, 5, 1082-91 (1970).

[30] Morren, L., Une methode de separation des raies et du fond

continu (etc.), 17th Session CLE. Barcelona 1971, Paper

71.08.

[31] Bauer, C, and Erb, W., Auswerteverfahren beim Vergleich

eines Linienstrahlers mit einem Kontinuumstrahler, Optik

30, 371-381 (1970).

[32] Krijgsman, C, De anode van de koolboog in lucht als standaardlichtbron, Thesis Utrecht 1938.

(Paper 76A5-730)

62
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Accurate Measurements of and Corrections for Nonlinearities

in Radiometers

C. L. Sanders

Division of Physics, National Research Council of Canada, Ottawa, Canada Kl A OS1

(June 7, 1972)

The methods described in the literature for accurately measuring photocell linearity are surveyed and assessed. The effect of not measuring photocell linearity under the conditions used in the final apparatus are considered. Some of the conditions necessary for accurate assessment of the nonlinearity under working conditions are specified. The use of the NRC "Photocell Linearity Tester"

to measure and correct for the nonlinearity of various receivers is described. Key words: Nonlinearity; photocell linearity; photometric accuracy; radiation addition method.

I. Introduction

A radiometer is linear if at time, to, the response,

N(to), indicated by the radiometer is exactly proportional to the incident radiant flux, 4>(to), i.e., N(t0) = a(f)(to), where a is a constant. This is the ideal and

is never exactly realized. Our problem is to measure one radiant flux, <\>\, relative to another, $2. Thus, we want to know (\>\l4>-i from the measurements N\, and N2 . In general, 4>i and

$2 are functions of time since we must compare the

two fluxes and thus cannot irradiate the receptor

indefinitely with either flux. In general, the response, N(t0), depends on the flux which fell on the receptor from the time the receiver was made up to time, t 0. It also depends on the elec- tronic circuit associated with the receptor and on all of the things which control its behavior, such as component temperatures, power supply voltages, charges on its capacitors, magnetic fields, etc. Again, in the

ideal case the response of the electronic circuit should

depend only on (f)(t 0 ) , but in practice it will depend on

<j>{t) where t goes from —00 to t 0 . For accurate measurements, we must apply the

two fluxes to be compared in a way which will, for the receptor and electronics used, produce a ratio which is precisely reproducible. We also must devise

a method to measure and correct for the nonlinearity

which is present at the time of the measurement.

The purpose of this paper is to describe the principal

methods of reducing and measuring nonlinearity, to indicate the accuracies attained by each, and to point out a few of the merits or drawbacks in each. Hopefully this will aid in deciding which method is appropriate for a particular task, and also in deciding how

many precautions are required to attain a specified accuracy. The reference list given is not intended to be complete, but rather to indicate a sufficient variety

of methods.

II. List of Methods of Measuring and Correcting

for Nonlinearity

1. Superposition (Additive) method. The superposition method is a basic physical method and is useful in all radiometric applications. It may be di- vided into two classes: (a) Multiple sources, (b) Orte source providing several independently interceptible

beams.

2. Bouguer's Law.

3. Beer's Law.

4. A combination of the superposition method and

Bouguer's Law as described by Hawes [l]. 1

5. Inverse Square Law.

6. Standard absorbing filters of either liquid or glass.

7. Standard reflecting materials.

8. Rotating sectors. WARNING: This does not measure all types of linearity because in the extreme

case the flux is either <f> or zero and nothing can be

deduced about linearity.

9. Measurement of amplitude of harmonics or beat

frequencies. Increases precision of measurements

and speed of measuring nonlinearities.

10. Measurement of known radiant fluxes. Erminy [2] has shown how to provide the known radiant fluxes by

the superposition method using three sources.

1 Figures in brackets indicate the literature references at the end of this paper. 63
11. NuD method eliminates nonlinearity. Lee and

others use it in optical pyrometry by providing standard

sources to match the test source.

12. Use of a linear receptor to calibrate a test receptor.

III. Summary of Methods of Reducing Nonlinearities and Increasing Accuracy

1. Kunz [3] has shown that one can attenuate the

flux by a known amount using a sector and thus improve the results by keeping the average anode current

in a photomultiplier low enough to make negligible the

drift of dark current.

2. Jung [4] has demonstrated improved linearity by

attenuating the larger flux with a sector. This is effective if the chopping rate is much faster than the

time constant of the nonlinearity.

3. Jung [5] has demonstrated improved linearity by

chopping the flux to be measured and then adding a steady flux, Po, to the chopped flux to keep the average

flux constant. The mixture of chopped and steady flux

is then passed to a phase sensitive rectifier and the

output caused by the chopped radiation is shown to be

very linear provided the rectifier is linear. Po need not be measured.

4. Special circuits in photomultipliers to keep the

dynode voltage independent of anode current have

been developed. The linearity is improved by using an adequate cathode to first dynode voltage and maintaining a constant last dynode to anode voltage.

5. Low or zero load resistance for selenium (barrier

layer) cells and silicon diodes improve the linearity.

6. Jones and Clarke [6] used a photocell obeying

Talbot's Law and a variable sector measured by time

ratio photometry to reduce nonlinearities in photometric measurements.

7. Potentiometric or feedback system to keep constant the input to electrometers increases the stability

and precision of measurements. Vacuum photoemission diodes with a cylindrical anode are extremely

linear.

A. Detailed Descriptions of Some Methods

for Measuring Nonlinearities

1. Superposition (Additive) Method— In the superposition method with two sources fluxes </>i and 4>2 produce the responses iVi and N2 - The combined flux,

<£i + $2, produces the response Nu. If iVi + /V2 = ./Vi 2 then the photometer is linear. If Ni + N2 ^ Ni2 the

nonlinearity may be given by the factor Ni2 l{Ni + N2 ), as described by Sanders [7], where this factor may be

used to correct the response at the scale position

(Ni + N2 )l2. If Ni^Nz the measured correction is only an average correction factor, which will have an

error dependent on how the nonlinearity changes with response. Zero correction is assumed necessary at /v,+/v2 . Rotter [8] preferred to find the nonlinearity as a difference correction to the response. He assumed that the correction was zero at the value N\ = N2 , and then

defined the correction at by

k2 =N1 + N2 -N12 .

With Rotter's method, it is possible to make hand

computations more easily. a. Multiple Sources — Many descriptions have

been given of the use of this method. Preston and

McDermott [9] in 1934 reviewed some earlier papers

and then described their linearity tests using six incandescent lamps inside an integrating sphere. The lamps

which were screened from the diffuse viewing window

could be switched on or off independently of one

another. They were each adjusted to have a similar

luminous flux. Each lamp was measured independently

with the test cell and then in combinations of 2, 3, 4,

5, and 6. In this experiment, each lamp had to be

switched on and stabilized before being used.

In most cases the photo cells tested decreased in sensitivity with the illumination on the receiver.

They used blue, green, and red filters and found that the nonlinearity was worst for blue light. In some cases, increased anode to cathode voltage improved the

linearity. Nonlinearities of up to 15 percent were

measured. The best cell was an Osram KMV62 with

a nonlinearity of 0.1 percent, at a ratio of 1 :6 in flux. All KMV6 cells were not as linear.

It is possible that the nonlinearity was caused by

decreased cathode to anode potential at the higher

flux.This type of nonlinearity can be avoided by using

a cathode which is mounted at the end of a long cylin- drical anode in the manner described by Boutry and

Gillod [10]. The VB59 photodiode from Rank Cintel,

England, which is very linear has the cathode mounted

along the axis of the cylindrical tube with a small

window in the metallized tube wall. The nonlinearity can also be avoided by using an auxiliary potential to cancel out the potential developed across the anode

resistor, or by using the connections to an operational

amplifier as described by Witherell and Faulhaber

[11]. The operational amplifier decreases the anode

load resistance by the factor \\A where A is the open

loop gain of the amplifier.

Other users of multiple sources have mounted the

lamps in individual compartments and left the lamps

lit and selected the flux by shutters. Erminy [2], Kunz [3], Jung [4], and Reule [12] used two or more

sources which could be isolated by baffles or rotatable

mirrors. See items below for more details on these

applications.

Reule [12] described some linearity measurements

on a Carl Zeiss DMR21 Recording Spectrophotometer.

He used a method of testing the linearity of a single

beam spectrophotometer which had earlier been

described by Hansen [13]. This step source or supplementary light method uses the superposition principle.

A supplementary source, Ss, provides an adjustable

2 In order to adequately describe materials and experimental procedures, it is occasionally necessary to identify commercial products by manufacturer's name or label- In no

instances does such identification imply endorsement bv the National Bureau of Standards,

nor does it imply that the particular product or equipment is necessarily the best available

for that purpose. 64
amount of flux which is measured as N by the spectrophotometer in the single beam mode. Ss is blocked off and the internal source, Si, in the spectrophotometer is adjusted to provide an equal reading. Both shutters are opened and the sum is noted. Ss is again blocked off and Si is adjusted to give a reading equal to the sum.

This stepping procedure is repeated until the top of the spectrophotometric scale is reached. Reule used

steps of 20 percent as a compromise between good

scale coverage and drifting errors. The analysis of the measurements is complicated if there is drift of the zero, or drift of the readings caused

by lamp drift. It is not possible to read below zero or above 100 percent.

Reule also described how to use a similar method

for double beam operation. The agreement between the

corrections for double and single beam operation was

within 0.1 percent of full scale. The corrections necessary to correct for nonlinearity depended on wavelength,

but were always less than 0.1 percent when a photomultiplier was used as the detector. Reule was able to use identical geometry for the two

sources so the measurements should be applicable to the measurements made with the spectrophotometer.

The two sources are independent, so no interference can be caused by partial coherence.

b. One Source Providing Several Fluxes —

This is a very convenient method, since it means that only one power supply is required to provide the two

or more fluxes which are required, Since the source is usually a tungsten lamp, the voltage must be controlled

with four times the stability which is required in the

flux. The current should be controlled with about seven times more stability than that required in the

flux. Thus, the power supply will be expensive and a considerable saving will result from using only one

supply.

There is, however, a danger in deriving the two fluxes

for the superposition method from a single source. The two fluxes may be coherent to some extent and

this may cause errors in measuring the nonlinearity. In fact, Mallick [14] describes a method in which the

departure of additivity of two intensities indicates the

degree of coherence of the light vibrations at the two

points. Since one cannot use the same measurement

to determine both nonlinearity and coherence, it is necessary to be sure which or what combination of these phenomena one is measuring in a given experi- ment. The equation given by Mallick is

I{Q) =h + h+2 {hh) 1/2

1 7i2 (t) I cos [<* 12 (t) - 8]

.

One can see that if yi 2 — 1 and the phase is correct, we can obtain a value of I(Q) = 2{h +I2 ) in the case where h — h. This is twice the value expected for in- coherent sources. Similarly, I(Q) could be zero if the

phase was shifted by 180°. Admittedly these are the

extreme cases. For some ideas on how to evaluate the possible errors in linearity measurements due to coherence, see Born and Wolf [15], Mielenz and

Eckerle [16], and Bures and Delisle [17]. The coherence will depend on the wavelength,

wavelength range, area of source, coherence in original source, angular separation of apertures, size of

apertures, area of receiver, path lengths, difference in path lengths, and diffusion in the system. Thus, it would be advisable to use two different parts of the

strip filament which acted as the original source. This may be accomplished in Sanders' [7] linearity tester by placing a thin prism in front of one-half of

Figure 1. Circular glass plate with a 3 degree wedge removed from one half. This half deflects rays from 1 aperture of each pair of apertures in the linearity tester of figure 2.

the pairs of holes. As shown in figure 1, this may be

easily constructed by removing a 3° wedge from onehalf of a circular glass plate with the thin edge of the

wedge on a diameter. The plate may be positioned in front of the lens in the linearity tester with the diameter positioned relative to the master plate, shown in figure 2, so all the rays passing the upper half of the

holes are deflected downward. A strip filament lamp

has replaced the lamp with diffusing bulb. A photocell

placed at the image of the lamp filament, will receive

images from two separate parts of the vertical strip

filament lamp. There may be some slight residual co- herence due to interreflections in the lamp or by

diffraction or stray fight at the apertures, but it will be considerably reduced. If it is desirable to measure

with monochromatic fight or to extremely high ac- curacy, it would be advisable to use a diffuser before

the receiver to remove even the residual coherence.

The master plate in figure 2 has 18 apertures related

in area as 1:1:2:2:4:4:8:8:16: . . . 64:128:128.

This master plate is placed between a lens anc

the image of a lamp. Thus, each aperture as viewed

from the image plane has a luminance equal to the luminance, L, of the source times, T, the trans- mittance of the lens. Thus, the irradiance from

each aperture at the image is proportional to the area of the aperture. A rotatable disk with suitable

holes can be positioned to transmit light through one

hole or a pair of holes from the master plate. Thus, a range of illuminances of 1:256 are obtained at the

image position. A receiver is placed at this position

and the response measured for each aperture or

pair of apertures, as shown in table 1, which is taken

from Sanders [7]. For the whole series of measure- ments we must select one position which requires no

correction. Sanders in 1962 used normalization at

/,, the maximum value. It is now believed that normalization at some fixed position on a scale would be more

reasonable and advantageous in comparing nonlinearity measurements obtained at two different

65
FROSTED 60W LAMP

INDEXING DIMPLES f| a CLAMP

MASTER PLATE

POINTER FOR

°/ APERTURE SELECTION

ROTARY DISC

RELEASE LEVER FOR BALL a DIMPLE CLAMP

Figure 2. Sanders' Photocell Linearity Tester. (Courtesy, Applied Optics.)

times. With normalization at the maximum, one over the whole range of 256:1. At the upper readings

obtains the correction factors as shown in column 4, the correction required was less than 0.05 percent

which are applicable to the responses shown in column

5. Using this apparatus, Sanders obtained correction

factors for two detectors, the best ot which was a vacuum diode photocell of Boutry and Gillod [10] which required a maximum correction factor of 0.997

for a range of 30:1. The larger corrections with the

smaller apertures may be caused by errors in measuring the smaller fluxes, by stray light, or by interfer- ence between partially coherent beams. The ad- vantage of Sanders arrangement is that only one

Table 1. Determination of factors to correct readings to a linear scale

Net

reading Sum Ratio

Aa A +

Aa A + a

Correction factor"

Correction

applies to reading

Ii Hh Gg Ff Ee Dd Cc Bb Aa A + a I+i' H+h' G+g' F+f ' E+e' D+d' C+c' B+b' A+a 2

Bb

bC

Cc

cD

Dd

d

E

B + b

C+c

D+d

Bb

B + b

Cc C+c

Dd

D+d

Ii Hh Gg Ff Ee Dd Cc Bb

I+i ' H+h ' G + g

' F+f ' W+~e ' D+d ' C + c' B + b

Ii Hh Gg Ff Ee Dd Cc I+i' H+h' G+g' F+f ' E+e' D+d' C+c

Ii Hh Gg Ff Ee Dd

B + b

I+i H+h G+g F+f E+e D+d

Ii Hh Gg Ff Ee I+i' H+h' G+g ' F+f ' E+e

66

C+<

2

D + d

E + e 2
Ee E+,

Ee E + ,

eF

Ff

f

G

Gg

F+f

G + g

Ff

F+f

Gg

G+g

li Hh Gg Ff

1+i ' H+h ' G+g ' F+f

Hh Gg G + g I+i H+h G+,

H

Hh

h

I

li

H + h

I+i

Hh

H + h

li I+i

li Hh

1+i ' H+h

H + h

li I+i

1.000

I+i

2

° Assuming no correction to reading of li. moving part is required.

We have now modified it by cutting gear teeth on

the edge of the large disk and driving the disk with a stepping motor with 500 steps per revolution. The

positioning accuracy is adequate and it is no longer

necessary to use the ball in the conical indentations to locate the disk exactly. A Slo Syn [18] Tape Control

System operates the linearity tester. The Slo-Syn sys- tem was available for use since it was incorporated in 1968 into the spectroradiometer described earlier by

Sanders and Gaw [19]. The apertures may be selected

by punched paper tape in the order described in table 1 or in any other desirable way. It takes about 5 s to ro- tate the disk 180° so it is faster to proceed following

approximately the order of table 1. One program used is to go through the table to the bottom and then to repeat

starting from the bottom up. Nine zero readings are spaced fairly uniformly through this measurement

cycle. The results are recorded on punched cards by means of a digital voltmeter. The measurements are analyzed by computer and a table such as table 2 is produced for each half-cycle.

The top line gives the description of the test. The

next fine shows the zero readings in volts which were

recorded. The next line shows the eight averages of the

successive pairs of adjacent zeros. The data in this table were obtained between the first and fifth zero.

The table then follows the pattern set out in table 1. The data in table 2 are for a silicon diode connected to an operational amplifier to produce an effective O.lfl load resistance. The diode was a Pin 10 from United

Detector Technology and was connected in the photovoltaic mode to an operational amplifier as described

by Witherell and Faulhaber [11]. The silicon diode had

a flashed opal glass in front of its V(\) correction filter. Each measurement made after the cell had been

illuminated for about 11 s. A 1 s reading was taken by

the integrating DVM. The correction ratios in column

4 for a ratio of 1 : 2 in flux show a maximum deviation

of 0.0026 from 1.0 at the response of 0.00567 V. This

corresponds to a photocurrent of 0.00567 /xA. The

correction factor based on zero correction at the

maximum reading is given in colmn 5. Here the cor- rection is farthest from unity at the middle of the range, with the maximum departure of 0.0014 at 0.011 V. There is considerable variation from one measure- ment series to another and a number of repeat meas- urements were made.

Table 3 shows the correction factors for various

selected voltage outputs for twelve repeat measure- ments on the same silicon diode. The first six fines are

for a 1.5 s delay and the next six for an 11 s delay. The

first line shows the response voltages at which linear

67
Table 2. Computer Output for Nonlinearity Measurements on a silicon diode

11 2102720006 TEST DIODE XI 12.45 AMPS 10 S, 1 S COUNT

0.000100 0.000100 0.000100 0.000120 0.000150 0.000120 0.000130 0.000110 0.000110 0.000100 0.000100 0.000110 0.000135 0.000135 0.000125 0.000120 0.000110 READING NET CORRECTION READING AT WHICH

UvIE READING SUM RATIO FACTOR FACTOR APPLIES

Al 0.002500 1.0000 1.0013 0.002880 A12 0.005760 0.005760 A2 0.003260 Bl 0.005060 1.0026 1.0013 0.005670 B12 0.011370 0.011340 B2 0.006280 CI 0.010360 0.9996 0.9986 0.011300 C12 0.022590 0.022600 C2 0.012240 Dl 0.024660 0.9994 0.9991 0.024600 D12 0.049170 0.049200 D2 0.024540 El 0.051370 0.9997 0.9997 0.047885 E12 0.095740 0.095770 E2 0.044400 Fl 0.103610 1.0007 1.0000 0.087310 F12 0.174750 0.174620 F2 0.071010 Gl 0.207140 0.9997 0.9992 0.179910 G12 0.359710 0.359820 G2 0.152680 HI 0.309115 1.0001 0.9995 0.359185 H12 0.718455 0.718370 H2 0.409255

11 0.779645 0.9994 0.9994 0.786410

112 1.571915 1.572820

12 0.793175

Table 3. Correction factors at selected voltages for successive measurements on a silicon diode. Measurements

1-6 with a 1.5 s exposure before the reading. Measurements 7-12 with an 11 s exposure before the reading

Meas. No. Volts 0.003 0.006 0.01 0 .05 0.10 0.30 0.50 0.70 1.00 1.30 1 .50

1 1.0038 0.9986 1.0006 ii.0005 1.0002 0.9993 0.9993 0.9994 0.9995 0.9998 0 .9999

2 1.0057 1.0022 1.0008 i..0000 0.9998 0.9992 0.9993 0.9994 0.9996 0.9998 1 .0000

3 0.9930 0.9982 0.9989 0 .9998 0.9994 0.9990 0.9993 0.9993 0.9995 0.9998 0 .9999 4 0.9979 0.9962 0.9989 0 .9999 0.9997 0.9993 0.9994 0.9994 0.9996 0.9998 0 .9999 5 0.9985 1.0035 0.9967 0 .9999 0.9997 0.9992 0.9993 0.9994 0.9996 0.9998 1 .0000 6 0.9943 0.9960 0.9974 1 .0003 1.0003 0.9996 0.9995 0.9995 0.9997 0.9998 1..0000

7 0.9951 0.9968 0.9988 0 .9993 0.9994 0.9992 0.9993 0.9994 0.9996 0.9998 1 .0000 8 1.0004 0.9987 0.9994 1 .0006 1.0010 0.9996 0.9993 0.9995 0.9997 0.9999 1 .0000 9 1.0025 1.0043 1.0022 1..0013 1.0013 1.0002 0.9999 0.9998 0.9998 0.9999 1 .0000 10 1.0038 1.0003 0.9990 0 .9994 0.9998 0.9991 0.9994 0.9996 0.9998 0.9999 1 .0000

11 1.0013 1.0013 0.9992 0 9997 0.9999 0.9994 0.9995 0.9994 0.9996 0.9998 0 .9999 12 0.9977 0.9943 0.9936 1..0012 1.0012 0.9998 0.9995 0.9996 0.9998 0.9999 1..0000

Ave. 1-6 0.9989 0.9981 0.9989 1. 0000 0.9999 0.9993 0.9994 0.9994 0.9996 0.9998 1. 0000

Ave. 7-12 1.0013 0.9990 0.9987 1. 0002 1.0004 0.9995 0.9995 0.9996 0.9997 0.9999 1. 0000

interpolations were made to find the applicable cor- for each voltage. The correction factors are quite

rection factor. The next line shows the measurement consistent from one run to the next at voltages from

number in column 1, followed by the correction factors 0.3 to 2.0 V. The average correction factors are 68
shown in the next to last line of table 3. The correction

factor of 0.9995 is the farthest from unity. For this set of measurements the lamp used was a vacuum strip

filament lamp.

The irradiance due to one aperture must not change

when another aperture is opened. Interaction between

one aperture and another may result from interreflec- tions which depend on whether another aperture is open or closed. The precautions required to reduce

these effects to the minimum possible have not been

taken to date in the National Research Council (NRC)

linearity tester. It seemed, previously, that the instability of the source or the receiver and its attendant

electronic circuits, were the dominant sources of fluctuation and systematic error. Present developments suggest that some improvements would be

warranted as follows. One could put antireflection

coatings on all the optical surfaces, put light traps to collect major stray fluxes, make the aperture covers

into light traps to prevent back reflected light when an

aperture is closed and in general tighten up the experiment to ensure that all these effects produce negligible

errors. Nonaka and Kashima [20] made a series of measure- ments on several RCA photomultipliers of the types

1P21, 1P22, and 1P28. They used ten equal sized

apertures in parallel light between two lenses which

focussed the source on the receptor. Their measurements, made with a precision of about 0.1 percent,

showed that the nonlinearity depended on the color of the light; on the position of radiation on the cathode; on the voltages (a) from the cathode to the anode, (b) from cathode to first dynode, and (c) from the last dynode to the anode. Nonaka and Kashima adjusted the

radiation to obtain an anode current of 0.3 jiiA with all ten apertures open. They did not compensate for the

voltage drop in the anode resistor and, hence, some

nonlinearity will be caused by anode load feedback

which results in a lower voltage from the last dynode

to the anode as the anode current increases. At 500 V

overall, the gain decreased with anode current, but at 1000 V overall, the gain usually increased with anode

current. With 1000 V overall, the collection by the

anode is complete, but the higher voltage produces a higher gain which with increasing incident flux causes enough voltage redistribution in the dynode chain to cause an increase in overall gain. This agrees with the

analysis of Moatti [21] and Lush [22]. The nonlinearity was greater near the edge of cathode.

These measurements by Nonaka and Kashima, show

that it is essential to measure the nonlinearities of a photomultiplier under the same optical and electrical conditions which will be used in obtaining the measure- ments to be corrected for nonlinearity. The nonlinearities can vary in the range ±1.0 percent when

these conditions are changed.

Rotter [8] analyzed the possibilities which existed

for selecting and providing the irradiances to be

measured in testing the nonlinearity of receptors by

the superposition method. His treatment assumed

that the irradiances were provided by a single stable source. A lens system with apertures graduated in

size provided irradiances related in several different ways. Rotter analyzed the system with respect to the

work involved and the accuracy produced.

He divided the measuring systems into two main

classes:

1. Method in which more than two irradiances may

be received at the same time.

1.1. Method with, n, nearly equal sized openings,

i.e., 1:1:1:1:....

1.2. Method with gradation of the openings by a

factor of two, i.e., 1 : 1 : 2 : 4 : 8 : 16 : 32 : . . 2. Method in which, at most, two irradiances are received at one time.

2.1. Method with steps of pairs of equal irradiances

increasing in size from one pair to the next by a factor of two, i.e., 1:1:2:2:4:4: . . ..

2.2. Method of increasing the size by steps of 62

percent which was first suggested and used by

Rotter. These steps are arranged in size, ac- cording to the equation

Ai + Ai+i = Aj+2

except that the first two irradiances are equal

in size, i.e., A0 — Ai=l. The series then be- comes 1 : 1 : 2 : 3 : 5 : 8 : 13 : 21 : 34 : 55 : 89 : 144. For each method, Rotter gave the order of measurements, and the order of calculating the additive

correction. As an example of this, the measurement

program for Method 2.2 follows:

Reading Irradiance Correction

Nv 1' k, Nv i VI h

K i *i Nlt2 1 2 A:3 N2 2 k2 N2 , 3 2 3 k5

It is noted that each aperture is used in three con- secutive readings, except aperture 1', and the largest

apertures are used only twice. The corrections ki are found from

k\ = 0 (arbitrarily chosen)

k2 = Nv + N1 + 2k-Nv,i

k3 = Ni + N2 + ^ + h - , 2

k5 = N2 + N3 + k2 + k3 -N2>3

k8 = N3 + N5 + k3 + k5 -N3>5 The calculations to find the corrections, A:;, are thus

easily determined by additions and subtractions in

this method, whereas that presented by Sanders re- quires extensive mulplication.

Rotter's analysis showed that the Method 1.1 using n equal sized apertures was very expensive (in the

number of measurements required) compared to the

other three methods, if the range was greater than 1 :4

69
Thus, for the range 1:8, Method 1.1 required twice

as many readings. For the range 1:64, it required

about ten times as many. However, Method 1.1 is more likely to be used with eight sources and will cover a range of 1:8. Then the source will be raised

in intensity by a factor of eight to cover the next

factor of eight on the scale. With this procedure, the

equal sized aperture method would only require twice

as many readings as the other three. In Rotter's analysis of the errors in the corrections

relative to the errors caused by the source variations,

he found that Method 1.1 produced correction values

with errors between two and three times lower than

those produced by the other methods. Thus, at least four repetitions of the measurements in the last three

methods would be required to produce measurements

with the same error as those obtained by a single set of measurements by Method 1.1.

It thus seems that the choice of the best method

must be made based on other criteria, such as cost of the equipment, ease of use, or minimization of drifts. Rotter described three physical arrangements of

0°

Figure 3. Rotter's aperture arrangement using 13 apertures with a

sliding shutter on each. The angular position and relative diameter

of each aperture is indicated. The outer circle indicates the out- side diameter of the lens. (Courtesy, Messtechnik.)

apertures. One, reproduced in figure 3, has 13 apertures increasing in size by a factor of 62 percent,

except for apertures 1 and 1', which are equal. Each

aperture is covered as required by a sliding shutter. The outer circle shows the size of the lens. This is a

variation of BischofFs [23] arrangement and makes

good use of the lens area. The range covered is 1 : 377. The 13 sliding shutters make the method more complicated to use than that of Sanders or the one shown in figure 4 which was also given by Rotter. This apparatus has two moving disks, B and C, with

six apertures in each. The lens is outlined by the dotted

circles enclosing both the smaller dotted circles D and

E. These are apertures in a plate fixed in front of the

lens. The numbers near the circles shown on the disks

FIGURE 4. Rotter's aperture arrangement using two rotatable discs B and C with 6 apertures in each. Apertures D and E are in a disc before lens L with its outer diameter indicated by a dashed circle. (Courtesy, Messtechnik.)

indicate the relative area of these apertures. The disk B

may be rotated to place any of the six apertures

inside D or to cover D as required. The same applies

to disk C relative to aperture E. The lens may be more

effectively utilized if the holes D and E are oblong in shape and the largest holes in the disks are similarly

distorted. This method of using two disks eliminates

the requirement for a large number of moving parts. At

the same time it requires less space and less accurate machining than Sanders linearity tester. Each disk

remains stationary for three measurements as dis- cussed above. The large disk in Sanders apparatus

must be moved between the reading on A alone and the

reading on A in combination with a. Thus there is a

possibility of inexact reproduction of the flux through

A or through a. Also, by using different disks in Rotter's apparatus, a different scaling of sizes may

easily be selected. Also, disks B and C may be inter- changed, which could be useful in eliminating certain

errors.

2. Bouguer's Law— This law states that if filters a and

b have transmittance Ta and 7& respectively, then the

transmittance of the two filters placed one after the

other in an optical beam, will be Ta X 7&. Thus, if one 70
knows the transmittance of each of a set of filters, one

can combine them to produce various transmittances

to be used to test a photometric system.

The validity of the method is not always certain. Hawes [1] noted difficulties caused by transmittances

changing with change of temperature, with age, with

cleaning techniques, with interreflections, with non- uniformity of the filters, and with bandwidth of the

transmitted beam. He found the system useful if one

did not depend on the long term stability of the filter transmittance and took the proper precautions to control temperature, interreflections, uniformity and

bandwidth.

Hawes, Bischoff [23] and others have noted that Bouguer's Law does not check for nonlinearity unless

the transmittance of at least one filter is known by an independent method. See below under A(4) for Hawes'

method which combines superposition and Bouguer's

Law. Hawes found it necessary to select filters suf- ficiently uniform that a 1-mm aperture moving over

the filter detected no changes larger than 0.01 in ab- sorbance. He described an apparatus to use with a Cary spectrophotometer in making these measurements.

3. Beer's Law — This law states that log (1/7) is pro- portional to the concentration of the solute in the

solution, where T is the transmittance of a constant thickness of the solution. This is only true for monochromatic light unless Tis independent of wavelength

over the full extent of the measured radiation. This

method is used in chemical analysis where the tech- niques are available to make the solutions properly and

where the scientists may depend on the law in their analyses and know when it will be valid. Problems have

been noted with particles growing in the solutions.

Interreflections may change with concentration and

the law may not be quite exact in all cases.

4. Combination of the Superposition Method With

Bouguer's Law to Test a Spectrophotometer— Hawes

[1] gave a useful set of criteria which should be met in selecting a method for calibrating photometric linearity

in spectrophotometers. Hawes' method for spectrophotometry cannot be transferred to broad band

photometry because the absorbing glass filters which

are available are not exactly neutral. He used three

Chance ON 10 filters about 1 mm thick. Two had trans- mittances of about 0.49 and the third about 0.57. He

made the apparatus shown in figure 5. The solid block

of metal with the two large holes is screwed firmly in place in the test beam of the Cary. Any one or any

combination of the three filters may be placed in slots in the metal block, B. The possible selection of holes

is: both open, a open, b open, or both closed, re- spectively, when no slide, slide a, slide b, or slide cis in the slot. The three filters cover both beams and are

oriented, by the retainers shown at R, to prevent inter- reflections from reentering the optical beam. The test compartment is optically remote from the cathode of the photomultiplier in the Cary, so the beams from the

two apertures fall on the same area of the cathode. It was necessary to insert and remove the slides! by means

FIGURE 5. Dual aperture with filter assembly used by Hawes to determine and correct for nonlinearities. Fixed block B. Slides a, b, and c. Retaining rings R for three filters. (Courtesy, Applied

Optics.)

of a flexible material in order not to disturb the posi- tion of the metal block. This procedure may also prevent temperature changes from being introduced

by the fingers. It is not clear how the filters could be

inserted and removed in the Bouguer Law tests without

disturbing the position of the limiting aperture.

The slides produced a measurement at 100 percent on the scale and at two positions near 0.50 and at zero. Thus, using the superposition method, one could find the correction required near the middle of the scale

and could use this to find corrected transmittances of

filters 1 and 2. Hawes assumed that in the instrument

the following relationship held over the whole scale:

7r//o=7Xl+c) =f

where e is the amount by which the exponent differs from unity, T is the true transmittance, 7r is the measured current with the filter in place, and I0 is the current for 100 percent transmittance. Thus,

log (7r/7o) = (l + e) log 7= log 7"

and, from the measurements on filters 1 and 2 and

those with the superposition method, e can be de- termined. Hawes calculated in absorbance rather than

transmittance and applied a correction, eA, to each

absorbance A. Using two different photomultiplier tubes he found a difference of 0.07 percent in the transmittance when

the two photomultipliers were used in the same

instrument to measure the three component filter with

a transmittance of 13.59 percent. The use of the value

of e determined by the superposition method did not reduce the discrepancy. Hawes felt that the trans- mittance of one filter probably changed between the

two sets of measurements. It thus remains to be shown

whether the assumption of a constant value for e is valid and useful. If no sufficiently stable filters can be

found, then it is questionable whether the corrections

are necessary.

5. Inverse Square Law— the accuracies attainable

502-591 O - 73 - 71
with the inverse square law depend on the accuracy

available in measuring: the distance from the source

to the receiver, the uniformity with angle of the in- tensity of the source in the direction of the receiver,

the size of the receiver, and the range of intensities required. It should be possible to refine the inverse

square law method by measuring the angular distri- bution of intensity of the source over the largest solid angle which the receiver will subtend at the source. With this distribution and the cosine law to allow for the angle of incidence of the receiver surface, the flux incident on the receiver could be calculated for a number of distances. Changing the voltage on the

lamp or inserting a filter would extend the range. Stray light due to reflections and due to diffraction caused by the baffles (see Blevin [24] would need to be

given careful consideration.

6. Standard Absorbing Filters — See comments under

Bouguer's Law. NBS filter sets described by Keegan,

Schleter and Judd [25] are useful for detecting errors

in a spectrophotometer and in keeping two or more

spectrophotometers of similar geometry in agreement

within a few tenths of a percent provided that the same filter set is used on both instruments.

7. Standard Reflecting Materials—Robertson and

Wright [26] reported the results of measurements on

grey ceramic tiles in a number of different laboratories.

Standardized ceramic tiles [27], now available from

National Physical Laboratory (NPL) or from the

British Ceramic Tile Council, may be used for checking photometric linearity if the geometry of the

spectrophotometer being used matches the geometry

provided during the calibration.

8. Rotating Sectors — In some cases, sectors are used to reduce the average flux incident on a re- ceptor or to measure the nonlinearity of a receptor.

If the response produced by the intermittent flux is identical with that produced by a steady flux whose

magnitude equals the mean magnitude of the inter- mittent flux taken over one period then the receptor

is said to obey Talbot's Law. This does not necessarily mean that the receptor is linear. It just means that the

nonlinearity is independent of whether the flux is chopped or steady. The faster the sector rotates the more likely it is that Talbot's Law will be valid. See

parts B(l) and B(2) for applications of sectors to improve linearity of measurements. Kunz [3] describes

methods used in accurate construction and calibration of sectors.

9. Measurement of Amplitude of a Harmonic or the Beat Frequency— Jung [4] described a very interesting,

useful and rapid method of measuring the nonlinearity

of receivers. In this method, two chopped beams of

slightly different frequencies are combined on the

receptor and the output of the receptor is examined

at the difference frequency. Jung showed that the

amplitude of this difference frequency is proportional

to the nonlinearity if the nonlinearity is proportional

to the flux. He also demonstrated that one could determine the time constant of the nonlinearity even if it was as small as a few milliseconds.

Jung assumed that

i = BP+CP2 + DP3 (1)

with | CP2

1 and \DP*\ <§ BP where B, C and D are constants, and i is the photocurrent and P is the in- cident flux. He defined NLG as the nonlinearity measur- ed by the superposition method at U + i2 , where

NLG (i1+2 )=2 tl+2 - {il + i2) (2)

Using eq (1) to find the current, t'i and t 2 , for two fluxes Pi — P2 = 0.5 P and placing the currents in eq (2), we get

NLc(ii +2 )~j(CP + j-DP*) (3)

In the dynamic method, the flux, Pi, is modulated at frequency w, and P2 at 0*2, where a>i > a)2 . Jung pro- ceeded to use a Fourier expansion to show that A (oji — (o2 ), the amplitude of the frequency wi — 0)2 is given by

A( (t>i-(1>2 )=—(CP* + ^-DPz) (4)

7T4 Z

where the basic frequency o>i, had the amplitude A

(q)i) = BPItt. Jung then defined NLD (i) the nonlinearity at the maximum current, i = i\ +2 , by

NLD (i) =77v4(a>i — (o2 )/A(o)i) = (1/B) (CP+ (3/2) DP2 (5)

under this assumption NLD = NLG but the assumption

of eq (1) is too simple because the photocurrent

does not instantaneously change when P changes.

Thus, Jung found it necessary to postulate

i = BP+CP* (l-e-( "T)

) (6)

where P is a function of time, t is the time constant of the nonlinearity, and t is the time from commencement of the steady signal. By use of suitable expressions for i and P as a function of time and applying a Laplace transformation, Jung found that NLD (i,(oT) = CPIB (l+(o>0 2

) Thus by eq (3) with D = 0 and assuming that in the

superposition method t > t, the relationship between

NLG and/VLois

NLD = NLGl(l + ((or) 2

) (7)

If the frequency of the chopper is 200 Hz and t=1

72
ms, then NLD/NLG = OA. Thus one can find t from

the ratio oiNLolNLn.

The experimental arrangement used by Jung is

Figure 6. Jung's optical apparatus for dynamic measurement of

nonlinearities. The semireflecting plate SP combines radiation from sources L, and L^. The radiation is chopped by shutters

Si and S2 . (Courtesy, Z. Angew. Physik.)

illustrated in figure 6. The oscillating shutters, Si

and S2, operate at frequencies of 213.5 Hz and 200

Hz, respectively. The semitransparent plate, SP,

combines the beams from the lamps, Li and L2, so they illuminate the receiver, R, identically. An

interference filter, IF, could be used to isolate a narrow band of wavelengths.

The measuring circuit Jung used is shown in 1=

IWHz JT RA£

HT

Al MIXER A2

FIGURE 7. Jung's electronic apparatus for dynamic measurement of nonlinearities. Al, A2, and A3 are amplifiers. Si and S2 are shutters. PS is a phase shifter and PSR a phase sensitive rectifier. (Courtesy, Z. Angew. Physik.)

figure 7. The oscillating shutters, Si and S2, were

driven by independent generators, Gi and G2, through

power amplifiers, Ai and A2. The signals from d and

G2 were fed to a mixer to produce the beat frequency

at 13.5 Hz which was filtered by a filter, RC, to at- tentuate the base frequencies by 80 db. A phase

shifter, PS, was required before the phase sensitive

rectifier, PSR, and was adjusted to give maximum

signal at the digital voltmeter, DVM. Provision was

made for using either a photomultiplier, PMT, or a photodiode, Si. They could be connected at points

A and B. In addition, B could be connected to either

terminal 1 or 2. The first of these connections produces

a voltage in the impedance transformer which keeps

the voltage constant from the anode of the photomultiplier to the last dynode. The input impedance of

the transformer is greater than 10 11 CI. This connection keeps the collection efficiency of the anode constant. The nonlinearity obtained by Jung using con10

3 NL

103 NL

D

"3

Figure 8. Jung's measurements on an EMI 9558 photomultiplier. Measurements without anode voltage compensation by the dc and dynamic method are shown respectively by circles and crosses. Those by the dynamic method with anode voltage compensation

are shown by triangles. (Courtesy, Z. Angew. Physik.)

nection 1 for B is as shown by the triangles in figure 8. With B connected to position 2, there is a voltage drop

produced in RA by the anode current which causes the

collection efficiency of the anode to decrease and

causes nonlinearities, as shown by the crosses in figure 8. Jung's measurements by the dc superposition method with the point A connected to position 2 gave the results shown by circles. There is much more scatter in the dc measurements

than in the dynamic measurements. This instability

may be partly caused by instability in the source, but

also by drifting dark current caused by the large

changes in average flux incident on the photomultiplier. Kunz [3] describes this effect in detail and his findings will be discussed further in B(l). Since the

two measurements made without compensation of

anode drop by Jung agree within the scatter, it is not

possible to estimate a value of t from eq (7). It only

suggests that o>t =£ 1. As shown by the triangles in figure 8, the nonlinearity is much less when the change in the voltage across the anode resistor is compensated by connecting

B to position 1. The sign of the nonlinearity is re- versed as one would expect from Moatti [21] (see B4

below).

Jung's measurements on a silicon photodiode with

24 V bias and connected to give compensation of the

voltage drop showed good agreement between the

dynamic and dc method. The nonlinearity in this case was about 0.8 percent at 1 [jlA photocurrent.

Bressani, Brovetto and Rucci [28] described a method of testing nonlinearity of a photomultiplier

by modulating the flux on a slit in front of a photomultiplier. The method depends on the modulation

being exactly a sine function. Any nonlinearities in the

receiver will produce the second harmonic which is

73
selected by a lock -in amplifier whose reference channel

is driven by a frequency doubler from the oscillator,

which also drives the oscillating mirror which modulates the beam. The 1 percent error in the linearity

measurements was said to be due to the lock-in

amplifier. This does not compare very well to the

error of the phase sensitive rectifier used by Jung

which Jung claimed was responsible for 0.05 percent

of the error. The illumination at the slit must be uni- form in Bressani's method and nonuniformity of

sensitivity over the cathode must not be able to inter- fere, if the nonlinearity is to be measured correctly.

This makes the potential of the method much lower

than that of Jung's method using the beat frequency.

10. Measurement of Radiant Fluxes to Test Linearity—To the series of steps of irradiance which

Rotter described, we should add that described by

Erminy [2]. Erminy used three strip filament lamps,

EMERGING BEAMS

s3

FIGURE 9. Erminy's arrangement using 3 sources for producing

integral and fractional multiples of a radiance. (Courtesy, J. Opt.

Soc. Amer.)

as shown in figure 9. The current on each lamp could

be adjusted to provide selected levels of radiance.

Each source could be blocked off with a shutter. Starting with No, Erminy showed how to obtain the

radiances mNol2i where

m= 1, 2, 3 ... and i= 1, 2, 3 ... .

Erminy used the device to obtain radiances which

could be matched to the radiance of another lamp by

a null detector. He produced in this other lamp a radiance scale obtained by additive means. The detector was not required to be linear or to have a good

stability over a long period of time. Thirty seconds were required for each measurement.

In testing the nonlinearity of photocells, we are assuming their stability. The adjustment routine used

by Erminy to provide a variety of radiances can also be

used to provide irradiances with the same multiples,

m/Vo/2', and thus may be used to check the nonlinearity of photocells at a much greater variety of

irradiances than can be obtained by the use of two

sources. In producing an irradiance scale, one could,

by appropriate optical means, adjust the irradiance

from each source without changing the lamp current on the three lamps. An iris diaphragm, distance varia- tion neutral wedge, etc. could be used with each

source. The two-step method can only produce irradiances

differing by a factor of two, so one is very dependent

on having a device which changes smoothly in nonlinearity as the irradiance increases. Erminy's arrange- ment can help to avoid such an assumption.

11. Null Method-Lee [29], in establishing the NBS

pyrometric temperature scale, used Erminy's method

with three strip filament lamps to calibrate a fourth

lamp at 14 currents, so the radiances were in the

proportion 0.007812 : 0.015625 : 0.03125 : 0.0625 : 0.125 : 0.25:0.5:0.67:1.0:1.5:2.0:3.0:4.0:6.0. Using / =

P{Tr ), where P (Tr ) is a power function of Tr , he

found seven coefficients to fit the 14 radiance temperatures Tr corresponding to the 14 currents. Tables were made using the equation so current could be re- lated to temperature. A similar process could produce

a (completely adjustable) radiance standard so the

detector would need only serve as a null detector. It takes seconds for the lamp to stabilize at each temperature, but there may be some circumstances where

this method should be used. The paper by Lee [29] describes the behaviour of strip filament lamps.

Vacuum lamps are more stable than gas filled lamps,

provided the temperature is kept low.

12. Use of a Linear Receptor to Calibrate a Test Receptor— Edwards and Jeffries [30] used a linear

planar photodiode as a reference detector to measure

the linearity of silicon diodes using a cathode ray

oscilloscope as the display element. They were able to detect nonlinearities of 3 percent over a range of 105

: 1. Their applications involved measurements of pulses

down to 3 ns duration. For slower tests, it should be

possible to use, as a linear reference device, a vacuum

photoemissive diode with an anode subtending almost

all the solid angle surrounding the cathode.

B. Method of Increasing Linearity and Accuracy

1. Selection and Operation of Photomultipliers for

Measurement of High Flux Ratios -Kunz [3] in a paper

describing the development of a temperature scale above the gold point, listed the following 12 points to consider in the selection and use of photomultipliers:

(1) Photomultipliers with nonfocusing dynode sys- tems are more suitable than other types.

(2) Photomultipliers with glass bases show smaller

leakage currents than those with plastic bases.

(3) One should seek a photomultiplier with the

smallest possible dark current leakage at the anode

and dynode, e.g., 2.5 X 10~12 A at step voltages of 30 V. The insulation test should take place several hours

after washing the base with very clean solvents and

distilled water.

(4) Photomultipliers should be selected for the most

constant possible sensitivity under changing illumina74
tion. In a check of the drift situation, particular attention should be paid to the drift following a decrease in illumination.

(5) For measuring uncertainties below 0.05 percent

the EMI 9558 A photomultiplier must be operated only

at photocurrents below 3 X 10~ 8 A.

In B2 and B3 below, Jung has shown that operation

at considerably higher currents under special conditions can produce results of similar accuracy.

(6) With the EMI 9558 photomultiplier tube, operation with five (or eight) dynodes is possible, and, especially at small gains of 10 to 1,000 (or 30 to 10,000), is more advantageous if an initial drift causes interference

when all stages are in use.

(7) The photomultiplier and its anode resistance

must be installed in a housing with high thermal inertia or in a thermostatted housing.

(8) The anode resistance must have small voltage

and small temperature coefficients.

(9) Anode feedback should be avoided by compensation or the provision of proper resistances.

(10) The capacitor in parallel with the anode resist- ance must have very low dielectric absorption.

(11) Interferences due to dielectric absorption in the electrometer input and its connecting cable must

be avoided by suitable selecting or handling.

(12) In addition to the recommended application of rotating sectored disks for attenuation of the radiation, it may be necessary to incorporate simple sup- plementary filters in vibrating capacitor electrometers.

Kunz followed these rules and showed that an EMI

9558 was linear to about ± 0.005 percent at final elec- trode currents from 0.03 to 20 nA with an overall gain

of either 30 or 300 and using the fifth dynode as the

final electrode. Kunz's paper should certainly be consulted by any- one wishing to improve their radiometric techniques.

2. Improving Linearity Using a Sector — After de- veloping the dynamic (beat frequency) method of measuring nonlinearity and finding the relationship

between NLo and NLq, Jung [5] proceeded to describe

two methods of increasing the linearity of radiation

measurements. The first method is to attenuate the

strongest, P2 , of the two radiations which are being

compared, with a sector with an opening which is such that the open time, Ds, relative to the period, T,

is given by Ds = P1T/P2. With this arrangement, the

average irradiance from Pi which reaches the receiver

is equal to PJ)s\T=P\. With the assumption of eq

(6) for the type of nonlinearity and using a Fourier

treatment, Jung showed that ec/e = 0 for g>t—» 00 and

€z)/e=l for (or—> 0. In this case, (o is 2-7T times the

chopping frequency, and t is the time constant of the nonlinearity. The errors, eo and e, are respectively,

the nonlinearity errors in the measurement with and

without the attenuating sector. Thus if the nonlinearity

appears much faster than the chopping period, the

sector will not reduce the error at all, but if the nonlinearity appears slowly relative to the chopping period,

the sector will reduce the nonlinearity. Note that the

nonlinearity assumed in eq (6) is a nonlinearity proportional to the flux.

3. Improving Linearity by Adding a Steady Flux to the Weakest Flux of Two Chopped Radiations —

Since some receivers, such as the silicon diode measured in figure 14, have a nonlinearity which is not proportional to the irradiance, Jung developed another

method of increasing linearity in a receiver which has

a nonlinearity that appears very quickly. The ap- paratus for comparing the radiant flux from Pi and

FIGURE 10. Optical arrangement for photometry of two radiances

P, and P2 selected by mirror RM. P, and P2 are chopped. An

adjustable radiation P0 is added to the weaker radiation. (Courtesy,

Z. Angew. Physik.)

P2 by this method is illustrated in figure 10, taken

from Jung. The rotatable mirror, RM, directs the radiation from either Li to L2 to the concave mirror CM. The

radiation is then chopped by LM, a 50 percent sector. During the time when radiation, Pi, from source, L*,

is falling on the receiver, R, radiation P0 from L0 is also incident on the receiver. Po is adjusted so the aver- age chopped flux on the receiver from Pi plus Po is equal to the average flux from P2 . The variation with

Pit)

P2"(t)

_!

2

P,"(t) 7

Pn + R Pn R"(t) = P,"(t)

Figure 11. Time dependency of radiation on receptor. Radiations

Po, Pi and P2 are indicated. After chopping and recombination

either P"(t) or P£(t) is incident on receptor. (Courtesy, Z. Angew.

Physik.)

time of the incident flux on R is shown in figure 11. Po, Pi, and P2 indicate the steady fluxes. P"(t) and

P2 '(0 indicate the respective incident flux depending

on whether the mirror selects Pi or P2 . The shutter on Po is also opened in the first case. A phase sensitive

rectifier permits the chopped signal P[' (t) to be separ- ated from the steady signal resulting from Po. Again

75
Jung provided a theoretical treatment and showed that the nonlinearity should be eliminated completely if the nonlinearity is proportional to the flux. If the photocurrent obeys eq (1), Jung showed that the quadratic part of the nonlinearity should be re- duced by a factor of four. Jung tested an EMI 9558 QB photomultiplier with

the voltage divider network shown at the top of figure

12. This circuit provides a positive anode current- proportional nonlinearity of about 2 X 10" 3 • /xA -1 with

a rise time of a little more than 1.3 ms. The rise time is caused by the capacitors across the last three stages.

He used three methods of measurement:

(1) the usual dc superposition method,

(2) the superposition method where the combined

beam was attenuated with a 50 percent sector,

(3) the dynamic method with the average frequency

206.5 Hz of the two signals.

The nonlinearity was measured over a wide range of

anode currents. The respective results for the three

— i i iii i i iii i iii

- Photomultiplier 9558 QB \= 650 nm

Figure 12. Jung's measurements of the nonlinearity of an EMI 9558

photomultiplier using 3 methods. (Courtesy, Z. Angew. Physik.)

methods are shown by NLX , NL2 , and NL3 in figure 12. The largest nonlinearity was measured by the first method. The sector attentuation of method 2 reduces

the nonlinearity by a factor of 2. The dynamic method

measures a nonlinearity less by a further factor of

nearly 5. Jung showed that these results were consistent with the theory and indicated a rise time for the

nonlinearity of 2.1 ms.

It remained for him to demonstrate experimentally

that the method of providing equal average photocurrent by adding a steady flux to the weakest signal would

produce improved linearity. For this purpose, he built the apparatus shown in figure 13. The equalization of

average signal is accomplished by a voltmeter measuring the average voltage after IC, the impedance conFlGURE 13. Apparatus for confirming the method of figure 10 bv

superposition. Radiation from sources L! and L2 may be recombined or measured separately. Radiation from adjustable source Lo

is added until the voltmeter at the output of impedance converter IC reads a constant value. Phase sensitive rectifier PSR

provides a signal to digital voltmeter DVM. Detector RD provides

synchronization from chopper LM. (Courtesy, Z. Angew. Physik.)

verter. This is followed by PSR, the phase sensitive

rectifier (controlled by a signal picked off RD, a detector, which is actuated by LM, the modulator) and a digital voltmeter. The results obtained by Jung are given

0

_ UB = 24V \=948nm

-Hp2

-

f

/ />T ^

y

-7 |0.5 1.0 1.5 M 1 - ^1 +

1 . .-NL6

3 0 p

2 SB

Figure 14. Jung's measurements of the nonlinearity of a silicon diode. NL for measurements by the dynamic method. NLG for measurements with equalization of average current on the re- ceptor. (Courtesy, Z. Angew. Physik.)

in table 4; column 1 shows the maximum anode cur- rent for each ratio of 2:1. Column 2 shows the nonlinearity for the method of figure 6, and column 3 the

nonlinearity for the usual dc superposition method.

The values in column 2 fluctuate in the range ± 0.2 X

10-3

, while the values in column 3 increase with anode

current to 30 X 10-3 at 19 juA. We can see that Jung

achieved a really vast improvement in linearity by

maintaining the average light constant by adding a steady fight during the presentation of the weaker flux. After these measurements for a ratio of 1 :2, Jung

made measurements of P\ relative to P2 for ratios of

1 : 30 and 1 : 100. To obtain the true ratios, he measured i for each source without a modulator and cor- rected the i values using the values obtained from curve NLi of figure 12. The corrected ratio is given by

Pl =n =

iid-NLl (i 1 )) P2 V= kil-NInfa))

76
The results for Q= are shown in column 4 of table 5. The ratio Q~ from the use of Po to create constant aver- age flux is formed without a correction of any kind. The

values are given in column 6 of table 5. Column 7 shows

the fractional difference between the two methods for the two flux ratios. These are respectively equivalent

to agreeing in the measurement of transmittance of

0.03 to within 0.04 percent of the transmittance and

agreeing in measuring a transmittance of 0.01 to within

0.06 percent of the transmittance.

Table 4

Max. Anode

Current Nonlinearity

i fiA NLo (i — const) NL, (i ^ const)

0.20 -0.18- 10-3 3.35 • 10-3

.40 .00 .70

.80 -.02 1.35 1.00 -.48 1.70

1.46 -.20 2.50

1.72 -.09 2.90

1.98 .21 3.30

2.78 .07 4.6 4.96 -.09 8.2 19.05 .10 30.0

pliers in photometry.

Finally, Jung applied the constant average light

method to a silicon diode with a bias voltage of 24 V

and obtained the points marked by crosses and the curve labelled NLG in figure 13. The measurements

for a ratio of 1:2 and using the usual dc superposition

method are shown in figure 14 by circles and a curve marked NL. The values, NLG , with a maximum of

about LOXlO-3 at 1 juA are much less in absolute

value than the values NL. This shows that the equalization method also increases the linearity of receivers

with curved nonlinearity characteristics. The nonlinearity may be high for a diode with a bias voltage

applied. The bias makes the response fast and Jung

may have used it biased for that reason.

4. Moatti [21] showed that in using a voltage divider

network of equal resistors, to provide the voltages to the dynodes, one should expect a decrease of gain

with anode current. Moatti suggested fixing the volt- age from last dynode to anode. Lush [22] showed that

this would produce an increase of gain with anode

current and suggested introducing some resistance,

in series with this fixed voltage, in the circuit from

last dynode to anode. He showed that the optimum

value of this resistance depended on the maximum

anode current which would be drawn relative to the current in the voltage divider network. Land [31] experimented with the straight resistance network

Table 5

D.C. method Chopping method

(i const) (j= const)

i /iA NL(i) <?=

1 10.0909 16.2 10-3 32.295

2 0.30756 0.53 • 10-3

1 12.2923 20.0 • 10-3 96.524

2 0.12483 0.22 • 10-3

This is indeed a remarkable achievement, especially

since one method requires no corrections for nonlinearity and the other requires a series of measure- ments of nonlinearity which must be combined to pro- duce the final correction. It should be noted that Jung

used vacuum tungsten strip lamps as sources with

currents stabilized so their irradiance have a stability

with time of 2 X 10~5. The measurements were made

with a photomultiplier at maximum currents up to 12

fiA. Jung felt that the maximum measurement uncertainty was ±0.7 X 10"3 and resulted from noise and

the nonlinearity of the phase-sensitive rectifiers.

If the procedure had been to add steady light to the

weakest of two unchopped signals, in order to make

a null reading, the added flux would need to be known

to 0.0006 percent to achieve the same accuracy, but,

in this constant average light method, Po need not be

known. With the useful detail given by Jung we

should all be able to make better use of photomulti10.0710 32.306 0.34 • 10"3 0.31174

12.2048 96.580 0.58 • 10"3 0.12637 and found that the changes in gain were about 1 per- cent if the ratio of the anode current to the current in the resistance network was less than 0.1. He sug- gested that this was good enough since instabilities would be this large.

W. Van de Stadt [32] in a short letter noted that "after exposure to a relatively high light flux (say, up- wards of 103 times the NEP) the tubes exhibit 'memory',

i.e. the anode current does not decrease to its previous

dark current level after removal of the incident light

flux. This memory effect may take several seconds to decay and is dependent in magnitude and duration on the light flux that generated it. The tube does re- cover to its original NEP given enough time."

Kunz [3] gave a detailed report on his experiences

with dark current. His measurements on EMI 9558

photomultipliers gave results which indicate a time

constant, of change in sensitivity after changing the

incident flux, of between 8 and 16 min depending to

77
some extent on the anode current. The change of

sensitivity was — 0.1 percent at 80 nA, — 0.4 percent

at 330 nA and - 0.7 percent at 860 nA. Kunz measured the dark current at the anode of an EMI 9558 photomultiplier tube with a gain of 300 and

found that the dark current increased rapidly as the

anode to last dynode voltage was increased. The anode

current at 30 V was 1 X 10-12 A increasing to 5 x

10 12 A at 50 V. He found hysteresis with the current remaining higher, as the voltage was reduced, than it had been at the same voltage as the voltage was in- creased. When he measured the dark current at any of the dynodes from 5 to 10 he found that the hysteresis was reversed. The sign may be reversed because the

dynodes emit photoelectrons, but the anode does not. However, on the dynodes the increase of dark current was fairly linear with last stage voltage. Because of the

nonlinearity of dark current on the anode and because

he found abrupt changes of anode dark current,

Kunz preferred to use the measurements of current

at one of the dynodes, rather than at the anode. He

also wanted to use a low gain in most cases because he

found it desirable to keep the current at the final stage below 30 nA.

5. Witherell and Faulhaber [11] discussed the

operation of silicon diodes for photometric applications

and gave data for silicon diode nonlinearity as a function of load resistance and cell current. They use the

cell in the photovoltaic mode, i.e. without the bias

used by Jung. They showed that the linearity improved

by a factor of almost 15 for every decade decrease in load resistance. The nonlinearity also increased

proportionally with cell current. With a load resistance

of 1 fl they found that the maximum nonlinearity was

0.1 percent for cell currents ranging from 50,000 nA

to 0.5 nA.

For the short circuit condition the device is in- sensitive to temperature, about —0.1 percent/°C

at 550 nm. They give the connections of the operational amplifier and also the properties of the amplifier

and the cell which must be considered in using the

cell in photometric applications. The bias voltage

speeds up the response time so this will make

Jung's measurements on the response time of the

biased silicon diode inapplicable to the circuit used by

Witherell and Faulhaber. Jung [4] measured at several

wavelengths and found the nonlinearity of silicon diodes much more severe at long wavelengths. It would be interesting to know whether the same applies

in the photovoltaic mode of operation.

6. Jones and Clarke [6] showed that it was possible to use a photomultiplier as a null device and thus to compare a larger flux, after transmission by a sector,

with a weaker fight which was unattenuated. They

measured the opening in a adjustable sector by a photoelectric timing device. This method takes advantage of the fact that some receivers obey Talbot's Law,

although they may be nonlinear when used to compare

two steady irradiances of different size. The Jones and

Clarke method can produce digital data directly from

the timing procedure.

7. Jones and Sanders [33] showed that keeping the

input voltage to an electrometer constant would improve the precision of measurements. This potentiometric method used an electrometer amplifier as a null device. It served at the same time to keep the voltage constant from cathode to anode of the vacuum photocell. This increased its linearity. The vacuum cell used had a cylindrical anode with the cathode at one end of the cylinder. The resultant device was precise

and linear, but inconvenient to use since some knowledge of the signal was required in advance. Jones [34] later described a circuit which avoided this requirement and made it possible to measure accurately

using a digital voltmeter. Similar techniques now use

operational amplifiers.

8. Sauerbrey [35], in a private communication of his

latest extension of Jung's method of constant average

anode current, was able to show that with an average

anode current of 10 fiA on an EMI 9558 photomultiplier

tube, it can be used with a nonlinearity of less than

0.1 percent. He investigated the effect of cathode-first

dynode voltage, last dynode-anode voltage and second

last dynode-last dynode voltage.

9. Schanda and Szigeti [36] showed that 50-W

tungsten-halogen lamps with a single coil made by

Tungsram were more stable in short term use than a 200-W lamp with a coiled-coil filament. The use of the

former type could, therefore, improve results of spectrophotometry or spectroradiometry.

10. Davies [37] has recently shown that photomultipliers cleaned thoroughly, painted with silver paint which is connected to the cathode by a 10 MCI

resistor, and the use of the tube with no socket will reduce the dark current by as much as 1000 times.

The dark current is more stable and is not increased

after operation with anode currents up to 10 /xA.

IV. Conclusions

The fundamental method of testing linearity is the

superposition method. Several methods have been

described and used to produce accuracies of 0.05 percent or better. Jung [4] has shown that nonlinearities can be measured quickly and precisely by a beat

frequency method. His measurements do not always

apply to the receptor as actually used, but his measure- ments suggested a method [5] of using receptors so they behave linearly or very nearly so. This method

involves keeping the average anode current constant. The flux to be measured is chopped, a steady flux is added to the weaker and the alternating response is separated by a phase sensitive rectifier. The results are linear to 0.05 percent without correction for nonlinearity.

The accuracy of the phase sensitive rectifier may

limit the accuracy of Jung's method. The accuracy

and nonlinearities of the rectifier system would need

78
to be less than 0.01 percent if the error from this source is to be negligible.

Jung's method can only be used to measure steady

radiation. For discharge lamps operated by ac, one

might make a null comparison between a steady

source and the periodic test source using one photometer. The steady source could then be measured using

Jung's system. These measurements would need to be

corrected for the residual nonlinearity as measured

by the superposition method. This residual nonlinearity

would be caused mainly by the increased space charges

at higher photocurrents.

Sauerbrey has recently shown that with appropriate

methods, a photomultiplier can be made to behave

linearly, within 0.1 percent, at anode currents up to

10/jlA. This is in contrast to Kunz who used dc measure- ments and found that he needed to limit the maximum

current to 0.03 /jlA on the last electrode to obtain

results accurate to ±0.005 percent.

Sauerbrey indicated that a photomultiplier tube

which he tested did not obey the 1+e power function

assumed by Hawes. It would, therefore, be advisable

to check Hawes' assumption by the superposition

method at several levels before using it in the Hawes'

combination method.

For accurate measurements, tungsten strip fila- ment lamps operating in a vacuum have been found

necessary because of their better stability compared

to gas filled lamps.

The receptor and its associated electronic circuit should be tested and used under the same conditions,

if the measured nonlinearities are to be applicable.

The nonlinearities are considerably altered by changing

the resistance circuit in the dynode chain of the photomultiplier. Different resistance ratios are optimum

for different types of tubes.

V. References

[1] Hawes, R. C, Technique for measuring photometric accuracy,

Appl. Opt. 10, No. 6, 1246-1253 (1971).

[2] Erminy, D. E., Scheme for obtaining integral and fractional multiples of a given radiance. J. Opt. Soc. Amer. 53, 1448-

1449 (1963).

[3] Kunz, H., Representation of the temperature scale above

1337.58 K with photoelectric direct current pyrometers,

Metrologia 5, No. 3 , 88-102 (1969).

[4] Jung, H. J., Eine Dynamische Methode zur Messung der Nichtlinearitaten Fotoelektrischer Strahlungsempfanger. Z. Angew.

Physik 30, 338-341 (1971).

[5] Jung, H. J., Kompensation von Nichtlinearitaten bei Photoelektrischen Strahlungsmessungen. Z. Angew. Physik. 31,

170-176 (1971).

[6] Jones, O. C. and Clarke, F. J. J., A new photometric technique

using a variable shutter device, Nature 191, 1290 (1961).

[7] Sanders, C. L., A photocell linearity tester. Appl. Opt. 1, 207-

211 (1962).

[8] Rotter, F., Die Prufung der Linearitat von Strahlungsempfangern, Z. Instr. 73, 66-71 (1965).

[9] Preston, J. G., McDermott, L. H., The illumination-response

characteristics of vacuum photoelectric cells of the Elster- Geitel Type, Proc. Phys. Soc. (London) 46, Part 2, No. 253, 256-272 (1934).

[10] Boutry, G. A., and Gillod, P., Studies in Photoelectric Photometry. A Photoemissive Cell Specially for High Precision Measurements. (Laboratoire d'Essais Publications), No. 43, 32

(1939).

[11] Witherell, P. G., and Faulhaber, M. E., The Silicon Solar Cell

as a Photometric Detector, Appl. Opt. 9 , 73 (1970).

[12] Reule, A., Testing Spectrophotometer Linearity. Appl. Opt.

7, 1023-1028 (1968).

[13] Hansen, G., Prufung der Fotometrischen Funktion von Spectralphotometern. Mikrochimica Acta, Heft 2/3, 410-415, (1955).

[14] Mallick, E., Measurement of the Degree of Coherence in Non- monochromatic Light. Appl. Opt. 8, 2501-2504 (1969).

[15] Born, M., and Wolf, E., Partially Coherent Light, Sec. 10.4.3

in Principles of Optics, p. 513, 4th Edition (Pergamon Press, New York, 1970).

[16] Mielenz, Klaus D., and Eckerle, Ken L., Spectrophotometer

linearity testing using double-aperture method, Appl. Opt. 11, 2294 (1972).

[17] Bures, J., and Delisle, C, Comptage des Photoelectrons Provenant de Points Statistiquement Independents et d'une Surface Etundue de la Photocathode. Can. J. Phys. 49, 1255-1262

(1971).

[18] Slo-Syn Numerical Tape Control, Superior Electric Co., Bristol, Connecticut.

[19] Sanders, C. L. and Gaw, W., A versatile spectroradiometer and

its applications, Appl. Opt. 6, 1639-1647 (1967).

[20] Nonaka, M. and Kashima, T., Linearity characteristics of multiplier phototubes, Japan. J. of Appl. Phys. 2, 785-791 (December 1963).

[21] Moatti, P., Linearite des Photomultiplicateurs. Effet des Courants de Dynodes. L'Onde Electrique XLIII, 787-793,

July- August (1963).

[22] Lush, H. J., Photomultiplier linearity, J. Sci. Instr. 42, 597-

602 (1965).

[23] Bischoff, K., Die Messung des Proportionalitatsverhaltens von Strahlungsempfangern uber gro/3e Bestrahlungsstar- kebereiche. Z. Instrum 69, 143-147 (1961).

[24] Blevin, W. R., Diffraction losses in radiometry and photometry.

Metrologia 6, 39-44 (1970).

[25] Keegan, H. J., Schleter, J. C, and Judd, D. B., J. Res. Nat. Bur. Stand. (U.S.) A66, 203 (1962).

[26] Robertson, A. R. and Wright, W. D., International Comparison

of Working Standards for Colorimetry, J. Opt. Soc. Amer.

55,685-706(1965).

[27] Clarke, F. J. J., Ceramic Color Standards — An aid for industrial

control, Printing Technology Magazine 13, 101-113 (1969).

[28] Bressani, T., Brovetto, P., Rucci, A., A method for testing the

linearity of a photomultiplier, Nucl. Instr. and Methods 53,

41-44 (1967).

[29] Lee, R. D., The NBS photoelectric pyrometer and its use in realizing the International Practical Temperature Scale above 1063 °C, Metrologia 2, 150-162 (1966).

[30] Edwards, J., and Jeffries, R. J., Automatic Linearity Tester

for Photodetectors, J. Phys. E, Sci. Instr. 3, 518-520 (1970).

[31] Land, P. S., A discussion of the region of linear operation of photomultipliers, Review of Scientific Instruments 42, 420-

425 (1971).

[32] Van de Stadt, W., Some remarks on photomultiplier tube

behavior. Elec. Eng. 41, 61 (1969).

[33] Jones, O. C, and Sanders, C. L. A High Precision Photoelectric Photometer. J. Opt. Soc. of Amer. 51, 105-108 (1961).

[34] Jones, O. C, An impedance converter for use with digital voltmeters, J. Sci. Instr. 40, 196-197 (1963).

[35] Sauerbrey, G., Private communication from PTB, Braunschweig,

Germany.

[36] Schanda, J., and Szigeti, G., Stability of High Color Temperature Etalon Lamps, Preprint Paper No. 71.27 of Proceedings

of CIE Session, Barcelona (1971).

[37] Davies, W. E. R., Reduction of dark current in photomultiplier

tubes, Rev. Sci. Instrum. 43 , 556 (1972). (Paper 76A5-731)

79

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Physical Parameters in High-Accuracy

Spectrophotometry

K. D. Mietenz

Optical Physics Division, Institute for Basic Standards, National Bureau of Standards,

Washington, D.C. 20234

(May 31, 1972)

The measured apparent transmittance Ta of a filter or liquid sample depends on the beam geometry in the spectrophotometer. For focused light incident upon the sample, Ta is different for systems

having different f-numbers, and also depends on the state of polarization of the light. These effects are eliminated when the incident light is collimated; in this case Ta approaches the "true" transmit- tance t of the sample. Both modes of operation suffer from stray light and interference effects. The

former may be reduced significantly by using mirror rather than lens optics, and the latter by suitable choice of the monochromator slit width. A new spectrophotometer based upon the above-mentioned

design principles is described. The photometric precision of this instrument is shot-noise limited, permitting measurements to better than 10 ~4 transmittance units. The double-aperture method of testing detector linearity to this level of precision is discussed. The conventional method of finding the nonlinearity correction can be replaced by a curve-fitting procedure giving bettor precision. Data on detector nonlinearity, and its dependence on wavelength,

are presented.

Key words: High accuracy spectrophotometry, physical parameters; linearity test of photodetector;

spectrophotometry, high accuracy.

I. Introduction

When the same sample is measured with two spec- trophotometers the results often disagree by amounts

several times greater than the precision of either

instrument. An example of this is given in table 1,

summarizing a recent NBS in-house intercomparison

of two spectrophotometers both of which have pre- cisions well below 10"4 transmittance units. When the

measurements were repeated, it was found that the

Table 1. Results of comparative filter measurements. The high-accuracy spectrophotometers described in reference [1] and in this paper were used for this test

Measured transmittance Range of nonuniformity 440 nm 465 nm 590 nm 635 nm of filter (635 nm)

10% Filter Labi 0.1159 0.1356 0.1037 0.1136 Lab 2 , 0.1145 0.1339 0.1024 0.1122 0.0005

Diff. +0.0014 +0.0017 +0.0013 +0.0014 20% Filter Lab 1 Lab 2 0.1980

0.1980

0.2259

0.2259

0.1916

0.1916 0.20600.2061 0.0004

Diff. 0.0000 0.0000 0.0000 0.0001 30% Filter Lab 1 Lab 2 0.3287 0.3289

0.3553 0.35570.3113 0.31150.3255 0.3258 0.0001

Diff. - 0.0002 0.0004 0.0002 - 0.0003 81
filters were slightly inhomogeneous. This inhomogeneity is too small to account for the relatively large

discrepancies for the 10 percent filter, but large enough

to dismiss the agreement on the 20 percent filter as

fortuitous, since no attempt had been made to measure the same area of these filters. Furthermore, the

filters were found to be slightly dichroic in one laboratory, but not in the other. A similar interlaboratory

test, which led to a small residual bias of roughly the same magnitude, is reported in reference [l]. 1 For the purposes of routine spectrophotometry,

the level of agreement of these measurements is quite

satisfactory, but not from the point of view of highaccuracy spectrophotometry. With well-designed

modern spectrophotometers, it is possible to achieve

repeatabilities of a few 10~5 transmittance units. Anyone using such advanced instrumentation cannot

ignore discrepancies as large as those mentioned. As

other cooperative tests have shown before [2], the

above-mentioned results indicate that a part of the

discrepancies must be attributed to the samples

used. In view of this difficulty, spectrophotometric

accuracy cannot be assessed by interlaboratory tests alone. In order to separate the effects of sample and

instrument, it is necessary to identify the various

possible sources of systematic errors for each of the

spectrophotometers involved, and to take them into account before comparing results. The main emphasis of this paper is on systematic

errors due to beam geometry. It will be shown that

different spectrophotometers are, indeed, likely to give different results. Conversely, it is entirely possible

to obtain agreement between two instruments of similar

design, but what was measured may not have been a meaningful material property of the sample.

Some of the beam-geometry errors are calculable by

straightforward application of optical physics. These

may be eliminated by numerical correction of the

measured results. In other cases, the magnitude of the

error must be determined experimentally. Ideally, a high-accuracy spectrophotometer should have off-axis mirror optics, and all measurements should be made in parallel light. Nevertheless, it is possible, although

significantly more difficult, to perform accurate measurements using a conventional focused-beam

spectrophotometer with lenses. Both types of instru- ments may suffer from systematic errors due to interference.

The particular samples discussed are glass filters, because these appear to be the most commonly used

transfer standards for assessing spectrophotometric

accuracy. A definition is given of what might be con- sidered the 'true' transmittance of an ideal filter, followed by a discussion of some of the pitfalls of real

filters. The necessity to eliminate the systematic error due

to nonlinear detector response is well recognized. It will be shown that the double -aperture [3] method can be adapted to yield the additive nonlinearity correction to ± 1 X 10"5 transmittance units.

II. A New Spectrophotometer

Most of the measurements reported in this paper were performed with a new single-beam spectrophotometer designed as a research tool for spectrophotometric measurements with an accuracy exceeding that of conventional systems. The instrument is described elsewhere [4, 5J in greater detail; its design

is shown schematically in figure 1.

COLLIMATING LENSFOCUSING LENS

(

o n n o

REGULATED POWER

SUPPLY

1 LINENS*' SOURCE

D

0

0 He - Ne

LASER

i

1

r

SHUNT

'TUNGSTEN

SOURCE

t t GLAN-THOMPSON

PRISMS

COMPUTER

DOUBLE APERTURE

CURRENT TO

FREQUENCY CONVERTER

AND COUNTER

OFF-AXIS

PARABOLIC MIRROR

*

SAMPLE

HOLDER

4

REGULATED POWER

SUPPLY

CZERNY-TURNER

I METER MONOCHROMATOR

LIMITING APERTURE

OFF-AXIS PARABOLIC MIRROR

SHUTTER Figure i. High-accuracy spectrophotometer.

Figures in brackets indicate the literature references at the end of this paper. 82
The monochromator is an f/8.7 Czerny-Turner

system with 1-m off-axis parabolic mirrors and a 1200-lines/mm plane grating blazed for 500 nm. The

entrance and exit 'slits' are interchangeable circular

apertures with diameters ranging between 0.25 and

1 mm, so that the exit aperture approximates a point source having a spectral band pass between 0.2 and

0.8 nm when the grating is used in the first order. This

light is collimated by a 195-mm off-axis parabolic

mirror, and focused into the detector by another.

These two mirrors are mounted at the ends of a 120-cm

precision optical bench; the space between them

constitutes the sample compartment of the spectrophotometer. It will become apparent in this paper

that this design; namely, the exclusive use of off-axis mirror optics, and the placement of the sample in a collimated and linearly polarized beam, constitutes

the optimal beam geometry for high-accuracy

spectrophotometry.

A large sample space was provided in this spectrophotometer, so that the beam geometries of other

instruments can be simulated by means of lenses

placed in the collimated beam. Several of the meas- urements reported in this paper were performed in

this manner. The light source is a tungsten ribbon lamp, rated

for 6V and 18A, and connected to a regulated power

supply using external sensing on a 0.1 fl shunt rated

for 50 A. Except for a possible linear drift in time,

the radiant-flux output of this source is constant to better than 0.01 percent for periods of the order of

20 min. The flux into the monochromator is varied

by variation of the lamp current or rotation of the first of the two Glan-Thompson prisms shown in figure 1. The second Glan-Thompson prism defines the state of polarization of the fight. The source is focused on

the entrance slit of the Ebert prism predisperser which

precedes the grating monochromator.

A 2-mW helium-neon laser was installed in the sys- tem for alignment purposes, and spectral lamps are provided for calibration of the wavelength scale to a 0.02-nm accuracy.

The detector is an end-on photomultiplier tube

attached to a 15-cm averaging sphere with an estimated

efficiency of 20 percent. The photomultiplier tube has an S~20 cathode and 11 stages; the dynode chain is linear with a Zener diode between cathode and first dynode. Its power supply is voltage-regulated to 0.001 percent, and the anode current is measured by

a current-to-frequency converter similar to that de- scribed by Taylor [6]. This converter and its associated high-precision counter were found to be linear

to better than one part in 10 4

, and to have a full-scale repeatability less than two parts in 10 5

. The counter

integrates over a 10-s interval. The counter signals are recorded automatically on punched paper tape by means of a data acquisition

and control system, which is also used as a computer

terminal to process the recorded data. Upon construction of the spectrophotometer, a

series of tests was undertaken to determine its photometric precision. The signal currents generally

drifted in time and exhibited random fluctuations

superimposed upon this drift. Since all quantities

measured with a spectrophotometer are ratios, the

drift is of no concern provided it is linear in time,

and provided that all readings are taken at fixed time

intervals. Under these conditions the quantity affect- ing the precision of transmittance measurements is the standard deviation,

dl = I{vt)YI(n-\)

1/2 (1)

where Ii, h, . . . In are n successive readings of the same signal current /, and I{vt) is their expectation

value, obtained from a least-square fit of the measured data as a linear function of time, t.

.003

~1 1 1—I I I M |

,1,1 J I

I _l 'I

3X10"'

Figure 2. Photometric precision dill of spectrophotometer versus

signal current /. When plotted as shown in figure 2, the random

error, dl, so defined, was found to be primarily a function of the signal current, /, itself independent

of the particular combinations of lamp current, flux attenuation, wavelength, and photomultiplier dynode

voltage used to produce any particular value of /. As

indicated by the solid line, the relationship between

dl and / is, approximately,

dlll =

10/7 X10-7 if/<10-7 A, (2a)

io- if/>10-7 A, (2b)

The low-current branch (2a) of this curve is due to photomultiplier shot noise. The limiting value (2b)

for large signal currents is attributed to lamp noise.

Equations (2a, b) can be used to predict the random

error, dT= m1/2 (3)

with which this spectrophotometer will perform

transmittance measurements. The result is that the

standard deviation of a single measurement of trans- mittance is of the order of 10~4 transmittance units or

less, provided that the signal current, /, for the 100

83
percent point of the transmittance scale is chosen to be equal or greater than 10 -7 A. Measurements

based on a series of repeated readings will then be

reproducible to within a few 0.00001 transmittance

units. This expectation was verified directly, when

separate transmission measurements of neutral- density glass filters with nominal transmittances of

0.1, 0.2, and 0.3 were found to be repeatable to ±0.00004.

III. The True' Transmittance of a Filter

The optical transmittance of a given sample,

transmitted radiant flux T=

incident radiant flux

(4)

is not a well-defined property of matter. It depends on

the form of the sample, on the path which the fight

travels, and is also a function of the polarization and

degree of coherence of the light. Thus, it cannot serve

a useful purpose in spectrophotometry unless constraints are imposed on sample and light beam so that the above ratio of fluxes is reduced to a meaningful

material property. In the following, this limiting form

of the optical transmittance, T, will be referred to as the 'true' transmittance, t, of the sample.

As an example, consider an ideal glass filter; that

is, a plane-parallel slab of homogeneous material of complex refractive index h = rc(l + Ik) and thickness,

Figure 3. Notation used in calculating the transmittance of a filter.

t, bordered by identical media with refractive index,

n', and illuminated by a linearly polarized plane wave

of wavelength X and angle of incidence 9' (fig. 3). Its optical transmittance is [7]

T(d')

(l-p2 )e^

l_ p

2 e

2i/3 (5a)

where p is the appropriate Fresnel coefficient of amplitude reflection at each of the two boundary surfaces,

given by

n' cos 9' — n cos 9

n' cos 9' + n cos 9

for S-polarization and

Pm=-

n cos 9 — n cos 6'

n cos 6+ n cos 9'

for P-polarization. Furthermore,

/3= (27r/A.)fracos 9,

with 9 defined by Snell's law,

n sin 9=n sin 9'

.

(5b)

(5c)

(5d)

(5e)

These equations indicate that T is a function of

the angle of incidence and state of polarization of the

light. Therefore, different transmittances may be

measured for the same sample with spectropho- tometers for which the angles of light incidence are

different. In addition, the measured transmittance

may vary when the polarization of the light is varied, so that a nonpolarizing filter may exhibit an instru- ment-dependent, apparent dichroism.

Equation (5a), which is based on the assumption of

perfectly coherent light, also leads to an interference term. In practice, the degree of coherence of the

light is different for different spectrophotometers, so

.that the actually observed interference effect will be more or less subdued. This may lead to further in- strumental discrepancies of measured transmittances.

These sources of error are eliminated when trans- mittance is measured in collimated light at normal inci- dence, and under conditions such that interference is negligible. Then, eq (5a) is reduced to

no) (6a)

where r is the energy reflectance at normal incidence,

given by

2 r=|pj 2 =|p„| 2 =

n —n

n' + n

for either state of polarization, and where

j.= g-4irnK</A

(6b)

(6c)

is the internal transmittance of the filter for normal

incidence.

Equation (6a) is a function of the filter parameters, n and t, only [8], and may therefore be identified as the

above-mentioned 'true' transmittance, t, of the filter. Any departures of measured transmittance from this value of t are to be considered errors.

84
IV. Requirements for Standard Filters

A few simple conclusions from eqs (6a, b, c) may

serve here to demonstrate some of the difficulties in assessing spectrophotometric accuracy by measuring

the same sample in different laboratories.

Differentiation of eq (6c) shows that the variation of

Ti due to a variation of sample thickness, t, is

ATi = Ti(ln Tt )Mt, (7) which has an extreme value, ATj =—0.37At/t, if Ti = 0.37. Hence, it may be seen that t must be constant to at least 0.5 fJiin if the transmittance of a 2-mm thick filter

is to be constant to 10 ~4 transmittance units. The filter must be plane-parallel to 0.1 milliradians if this requirement is to be satisfied over a 5-mm area. Equally strin- gent requirements must, of course, be imposed on the

homogeneity of the filter material.

The importance of proper sample handling and

cleaning may be illustrated by assuming the presence

of a thin contaminating layer of thickness, t\, and

refractive index, ni, on the filter surfaces. In this case, the Fresnel reflectance, r, in eq (6a) must be replaced

by [9] _ n

2 (n'-n) 2 + (n2 -

n' 2

) (n2 -

n

2

) sin2 jx/2)

r'~ n\{n' + n) 2 +(n\-n'2 ){n\-n2

) sin2 (jc/2 )

(n'-n) 2 P *n'n{n2 -n'2 )(n2 -n2

) . 2

1 (n + re) I nf(n — n)'s (ra + n) A

J

(8a)

x = 4>7rn 1 t 1/k, (8b)

and where it was assumed that k < 1 and that {n\ — n' 2

) {n\ — n

2 )ln\(n' + n) 2 is a small number [10]. In practice, the film may be water (ni ~ 1.33), oil («i ~ 1.47), or some similar contaminant, so that ni ~ 1.4 may be

taken as an average representative number. If n ~ 1 and n ~ 1.5, then n ~ r — 12 sin2 (x/2), with r as the

Fresnel reflectance (6b) of the uncontaminated sur- faces. Hence, it is seen that the film acts as an anti- reflection coating; its effect on transmittance will be

less than 10-4 transmittance units only if 2(r— n)

=£ 10 -4

. According to (8b), the corresponding tolerance on the film thickness is ti 2 10~3 X, so that a layer

only a few molecules thick may be troublesome. Repeated cleaning is likely to leave residual layers of at least this magnitude on the filter surfaces. A similar

difficulty arises from the fact that the surfaces may

gradually become leached by certain cleansing agents.

The additional problem of dust is too well known to de- serve more than passing mention at this point.

These examples show the large effect that inadequate samples and sample handling techniques may

have on interlaboratory comparisons. Efforts are being

made to develop more homogeneous filter materials,

and to grind and polish them with greater precision,

[11]. In the meantime some improvement may be

achieved by more elaborate calibration procedures;

for example, the calibration of a standard filter should

be done by mapping. The filters must be cleaned with

properly selected chemicals. They should also be

recalibrated periodically, so that the user is made

aware of any surface damage or accumulation of

residual layers.

85
V. Beam-Geometry Effects gated toward the detector is, according to figure 5a,

The sample compartment of many spectrophotometers is designed as shown in figure 4, where the monochromator exit slit is focused into the sample by a

lens, and is refocused into the detector by another. This

beam geometry is preferred by most designers for intensity reasons (all of the flux from the slit is passed

through the sample), but it will not yield the 'true' transmittance t of eq (6a). The major systematic

errors inherent in this design are due to beam displacement, interre flections, and oblique light incidence.

These errors will be discussed now, in order to establish the conditions for which they are either negligibly

small or may be eliminated by numerical correction.

A. Beam-Displacement Errors The solid lines in figures 4a, b, show the path of

fight in a focused-beam spectrophotometer without the

sample. As indicated by the broken lines, the sample

shortens the effective distance between the inter- mediate slit image, S', and the second lens by an amount t(l — 1/n), so that the final slit image, S", is shifted towards the detector. If the sample is not normal to the optic axis, S" will also be shifted laterally.

This causes different areas of the detector surface to be illuminated before and after the sample was in- serted and, therefore, leads to errors because most

detectors show a variation of sensitivity with illuminated area. This error must be avoided by passing the

light through an averaging sphere before it reaches the

detector. While this is a well-recognized fact, it is surprising that even some of the more elaborate commercial spectrophotometers are not equipped with

averaging spheres. The inefficiency of spheres in some

portions of the spectrum is no adequate reason not to use them at all. Another error arises when the second lens is the

limiting aperture of the beam, as in figure 4b. In this case, the insertion of the sample changes the effective aperture of the first lens, such that it collects less light

than without the sample, resulting in too low a measured transmittance. It is easy to show, by measure- ments with either lens as the limiting aperture, that

this error may be quite large. The results of such

meaurements, listed in table 2, demonstrate the

necessity to underfill the second lens. Table 2. Measured transmittance of three neutral- density filters in focused light with (a) the first lens,

and (b) the second lens as the limiting aperture (f/5)

(a)

0.10399

.31362

.58981

(b)

0.10303

.31211 .58588

(a)-(b)

0.00096

.00151 .00393

B. I nterreflection Errors

Before the sample is inserted into the beam of a spectrophotometer with lenses, the radiant flux propa-

(a) j

fh = T,T„(] 4- R< R„ -4- •),\ (9a)

(b)

T,T,

T, RjR, T2

T,TT 2 T|TR 2 TR|TT2 T,TR 2 RT 2

T, RR i TT 2

Figure 5. Reflected beams in a spectrophotometer with lenses.

where 7\, T%, Ri, and R2 are the transmittances and

reflectances of the two lenses, and where any contri- butions due to four or more reflections are considered

negligibly small. When the sample is placed in the

beam, the transmitted flux is, as indicated in figure 5b,

4>' = TJT2 {\ + R,R 2 T2 + RR 2 + R,R + . . .), (9b)

with T and R as the transmittance and reflectance of the sample.

As may easily be seen by measuring a sample with

and without two clear glass plates on either side of it, the discrepancy between the ratio of these two fluxes and the transmittance, T, of the sample may be quite

large. The results of such a measurement are plotted

in figure 6, according to which the error is a monotonic

function of transmittance and approaches 0.01 transmittance units for large values of T. This experiment represents a particularly bad case, but nevertheless, illustrates the seriousness of interreflection

errors.

3-10"

ERROR AT

TRANSMITTANCE

Figure 6. Difference A^in transmittance for three filters, measured

between two clear glass plates and without them. 86
The main cause of the error is the fact that the

insertion of the sample creates the two additional

beams shown at the bottom of figure 5b. As is well

known, these two beams may be prevented from

reaching the detector by tilting the sample. This is indicated in figure 7 for the component reflected from

the front surface of the sample and returned by the

first lens. If this lens is biconvex, the light is focused

into a real image, Si, by the first surface of the lens,

and into a virtual image, S2 , by the second. The

interreflection effect is large. A semireflecting non- absorbing sample may be used. In this case the error,

(<j)'l4>)-T, is largest when R=(l — T) =0.5. According to figure 6, a clear glass or quartz plate

also gives a large effect. In both cases, the dependence

of reflectance on angle of incidence and polarization

must be taken into account; otherwise a plateau

may not be found. This is illustrated in figure 8. A

2-mm quartz plate was used, and data were taken for both, S and P polarization in the tilt plane of the

APERTURE

Figure 7. Elimination of interreflections by sample tilting.

right-hand side of the figure shows the corresponding

images, S[ and S'2

, formed by the second lens, as well as the cones of light that must not pass through

the detector entrance aperture. Figure 7 deals with

only two of the eight ghost images thus formed, but

it is clear that they all lie on the straight line shown,

intersecting the optic axis at the tilt angle 6T- Thus,

it is always possible to eliminate the two bottom

beams in figure 5b by sufficiently tilting the sample.

Just how much tilt is required is a very complicated

function of beam geometry, and is best decided

experimentally by measuring transmittance versus

tilt angle until a plateau is reached. Such tests should

be done under well-defined conditions for which the

0.00

-0.01

AT

/S POLARIZATION

Figure 8. Difference A71 in measured transmittance versus tilt angle for clear glass sample between lenses.

sample [12]. According to section 5.3, below, the

average of the S and P data is theoretically independent of angle for small values of dr. Thus, the

figure indicates that a tilt angle of approximately 3°

is necessary for this particular instrument.

When enough tilt was introduced, the last two terms

in eq (9b) may be omitted. Then the ratio of the two

fluxes received by the detector becomes

1 +RiR2 T2 + . . . 4>'l<t>-T

1 + jRii?2+ ; ; ;

T[\ - RiRzO- - T2

) +...]. (10)

The residual interreflection error given by this equation, is due to the fact that the direct beam in figure

5b has passed the sample once, whereas the remaining interreflected beam has passed it three times.

This error cannot be eliminated by tilting.

Representing amounts of light which actually reach

the detector, the quantities R\ and R2 in eq (10) are rather complicated functions of beam geometry.

Nevertheless, it is clear that the residual error has a maximum, T — = 0.38/? iR2 for T= 0.577, and,

therefore, remains below 10~ 4 transmittance units as long as VrJT2 =s 0.016. In spite of the difficulty to assign numerical values to Ri and R2 , it is likely that well-coated lenses will meet this requirement. Whether

or not this is the case can be ascertained by means of a standard sample of about 60 percent transmittance

which was calibrated on a lensless spectrophotometer.

C. Obliquity Errors When defocusing and interreflection effects are eliminated, the remaining beam-geometry error of

focused-beam spectrophotometers is due to oblique

light incidence.

502-591 O - 73 - 7 87
Since 0' and 0 are small angles, and since the

extinction coefficient, jik, is also small, we have

P± = Vr^[l+(»'M)#2

]. x n t ai

P,=-V-T[l-(n7n)fl'»],

(Ha)

(lib)

as obtained by Taylor expansion of eqs (5b, c) to second order in 0' and 0, using Snell's law (5e), and

letting^ ~ n. Similarly, eq (5d) leads to

p = ('2nl\)nt {[1 - H2(n'd'ln) 2 ] + ik[l + 1/2 (n 6' In) 2 ]}, (11c)

when second-order terms in k are neglected.

With these approximations, the 'true' transmit- tance of eq (6a) becomes

(1 -r) 2r;

1 - r

2Ti 2

with Ti as given by (6c) and

\n' + n)

(12a)

(12b)

Similarly, eq (5a) is reduced to

T[d') =t(1 + y; 0' 2 ±yr0' 2 ), (13a)

y,= \l2(n'ln) 2 In r ; , (13b)

yr = 4>(n'ln)r, (13c)

where the upper and lower signs in (13a) pertain to S and P polarization, respectively, and where the

interference term was again averaged. All terms in

r

2 appearing in (13b, c) were neglected.

The two sources of error; namely, a decrease of internal transmittance due to increased path lengths

at oblique incidence, and dependence of Fresnel

reflectance on angle of incidence, appear as the

separate terms involving y, and yr in (13a). These

will be discussed in the following two sections.

1. Path-Length Error—In a focused-beam spectrophotometer, the average path of light through the

sample is a function of the sizes and shapes of the

monochromator exit slit and of the effective aperture

of the focusing lens. In the following, it will be as- sumed that the slit is sufficiently short to be treated

as a point source, and that the lens is underfilled so

that the beam cross-section is rectangular because of the rectangular grating or prism aperture. For simplicity, the aperture will be assumed to be square, subtending the same angle 20o in both the horizontal

and vertical planes. Furthermore, it will be assumed

that the sample is tilted only in one of these planes.

As before, the tilt angle will be denoted by 0r- Under these conditions, the average value of the

squared angle of incidence 6' 2 in the tilt plane is, according to figure 4a,

1 f0r+<>o (0' 2 )av=l^ O'W

= l/3(02 + 302 ). (14a)

In the remaining plane there is no tilt, so that

(0' 2

) av =l/302

. (14b)

The effective value of 6' 2 to be used in eq (13a) is the sum of these two. Therefore, the expected pathlength error is <r-r = -Tyi 2/3(02 + 1.502) = -l/3(n7n) 2T(lnri )(02o + 1-502

). (15)

If the Fresnel reflectance is small, this function

has a maximum at Ti = 0.37. For this particular value

of internal transmittance, and assuming n=1.5, n = 1, its magnitude is r-r= 0.05 (02 + 1.502 ),

2-10 '

I - 10

T-T

FRESNEL- REFLECTANCE ERROR

Figure 9. Maximal path-length error (for Tf = 0.37 and 0O = O.O5

rad) and maximal Fresnel-reflectance error (for t, = 1.00 and P

polarization) versus tilt angle dj, as computed from eqs (15) and

(16) with n = 1.5, n' = 1.

which is plotted in figure 9 as a function of tilt angle

6T for an f/10 cone of light (0O — 0.05 rad). The graph

shows that the effect of the cone angle, 0o, is small,

but that errors of the order of 10~ 3 transmittance

units may be caused when the tilt angle is appreciable.

2. Fresnel-Reflectance Error—According to eq

(13a) the effects of S and P polarization are approximately equal in magnitude, but opposite in sign.

Therefore, they cancel when unpolarized light is used.

For a symmetrical cone of light incident upon an

untilted filter they cancel as well, because the roles 88
of S and P polarization are interchanged in any two

perpendicular cross sections of the cone. Hence,

Fresnel reflectance effects are observable only when

polarized light is used, and when the filter is tilted. Because of the opposing effects of S and P polarization in two mutually orthogonal planes, the effective value of 6' 2 to be used in this case, in the last term

of eq (13a), is the difference of the two values in (14a, b). Therefore,

T-T=±Tyr di = ±4r(n'ln)r0*, (16)

where the two signs pertain to the two cases in which

the light is S or P polarized in the tilt plane.

This error is largest for large transmittances. If n— 1.5, n' = 1, r= 0.04, and r = 0.92, its magnitude

is t T — 0.1 6t2

, which is also plotted in figure 7. The figure shows that the Fresnel-reflectance error is of the same order of magnitude as the path-length

error. According to figure 10 the magnitude of this effect remains the same when a thin surface layer is present on the sample. The solid lines show the Fresnel

reflectances for 5 and P polarization and for n = 1.5, n = 1. As indicated by the broken lines, a thin layer of index rii = 1.4 and thickness X/100 merely shifts these curves by a small amount. Thus, the angular

dependence predicted by eq (16) remains unchanged,

only the factor, r, on the right-hand side is changed

insignificantly.

D. Elimination of Beam-Geometry Errors The obliquity errors discussed in the previous

section cannot be eliminated experimentally. In fact,

0.041 -

0.040

0.039 -

Figure 10. Computed reflectance of dielectric boundary (n=1.5,

n' — l) with and without thin surface layer of thickness k/100 and

index 1.4.

they are increased by the necessity of tilting the sample

in order to reduce the interreflection error. However,

these obliquity errors can be predicted quantitatively

and may, therefore, be removed by numerical cor- rection of measured transmittances.

Thus, it should be possible to perform accurate measurements of 'true' transmittance with a conventional focused-beam spectrophotometer by eliminating defocusing and interreflection errors as indicated, measuring the tilt angle, and then applying

a correction term, such as given by eqs (15) and

(16) [13]. The same goal may be accomplished, in a much

simpler fashion, by measuring in collimated rather than

focused fight. However, this cannot be done by

simply repositioning the two lenses to make the

light between them parallel. Such a beam geometry

would still suffer from interreflection effects, so that the necessity to tilt the sample would reintroduce

beam displacement and obliquity errors. In order to avoid interreflections, it is necessary to use off-axis mirrors to collimate the light, and re- focus it into the detector, as shown in figure 11. In

this case, no significant portion of the light reflected from the sample will reach the detector, provided that the sample is normal to the beam and that off-axis mirrors are used in the monochromator as well. Each successive optical element will then reflect this

light further toward the source, until some of it is returned by some lens on the source side of the monochromator. The final amount reaching the detector

will be negligibly small. To ensure that these conditions are realized, the

imaging properties of the system must be quite

good. Since a well-collimated beam cannot be derived

from an extended source, it is necessary to equip the

monochromator with small circular entrance and exit apertures rather than slits. Off-axis parabolas must be

used rather than spherical mirrors to provide good

imaging, so that the reflected light will still "fit" the

monochromator apertures. In addition, it is necessary to reduce the effects of

spurious reflections by baffling, and by blackening of all component parts. The system must be aligned very

carefully. This may be accomplished by means of an

alignment laser and precision leveling devices for all components, including the sample holder.

The new spectrophotometer mentioned in section II, above, was designed and constructed in accordance

with these guide lines. Returning the reflected light

through the monochromator apertures was found to be

a relatively easy alignment which, in addition to eliminating interreflections, also ensures that the samples

are normal to the beam to within 1 milliradian. Therefore, no measurable obliquity errors are encountered

and beam displacements will not occur, either. Unlike

a focused-beam spectrophotometer, this instrument measures "true" transmittance without the necessity

for elaborate precautions and numerical corrections.

The fact that this spectrophotometer is indeed

effectively free from interreflection errors is demonstrated in figure 12. The figure shows the results of a 89
REFLECTED X

LIGHT MONOCHROMATOR

EXIT APERTURE

DETECTOR ENTRANCE APERTURE

Figure 11. Elimination of interreflections by using collimated light at normal inci- dence and off-axis mirrors.

Figure 12. Measured transmittance T versus tilt angle for clear quartz sample between off-axis mirrors.

tilting experiment similar to that of figure 8. There is no

'interreflection hump' near normal incidence as in the

previous case, and the average of the measured trans- mittances for S and P polarization is the same for all

tilt angles. The particular sample used (a clear quartz

plate) was found to be slightly birefringent. This was

verified by checking it between crossed polaroids.

VI. Interference Effects

Figure 13 demonstrates the possibility of interfer- ence effects in spectrophotometry. A recording spec- trophotometer was used to measure the transmittance

of a very thin glass plate (nt ~ 0.24 mm) at near- infrared wavelengths (A — 1.05 fim). The observed

modulation of transmittance of roughly 1 percent, due to interference fringes spaced 2.3 nm apart, is significantly smaller than would be expected from eq (5a), which predicts a fringe visibility of about 8 percent for

this particular case. As already mentioned, this dis- crepancy arises from the partial coherence of the

illuminating light.

w\wyvwA/VV> YW\ WWWWWWvw> WV" ~*

1.0

0.9

0.8

Figure 13.

WAVELENGTH (nm)

Measured interference effect for thin sample at near- infrared wavelengths.

Due to various 'smearing factors', such as localiza- tion of fringes, unequal amplitudes of interfering

beams, and imperfect temporal or spatial coherence

of the spectrophotometer beam, the effect is likely to be small in most practical situations. The particular

type of interference observed and its magnitude are sample-dependent, and are also a function of the beam

geometry of the spectrophotometer.

Unless the sample is plane-parallel by interfero- metric standards, the interference pattern will be of

the Fizeau type, and will be localized in the sample.

These fringes will be 'seen' to a greater extent by the

detector of a focused-beam spectrophotometer (fig. 14a)

than by that of a parallel-beam instrument. On the other

hand, the latter is more susceptible to the Haidinger

rings localized at infinity (fig. 14b) which occur when

the sample is plane-parallel and is normal to the

beam. Both types of fringes may result in a modulation

of measured transmittance, such as shown in figure 13,

when the spectrophotometer wavelength is scanned.

The Fizeau fringe pattern may also cause a similar

modulation of transmittance when the sample is moved

across the beam at a fixed wavelength setting.

For most samples the reflectance r at the sample

boundaries is small. Thus, two-beam rather than

multiple-beam interference will be observed, and the

fringe contrast will be small because of the large ratio 90
1/r of the amplitudes of the two interfering beams. A

further reduction of contrast may be attributed to the

FIZEAU . FRINGES

MONOCHR0MAT0R

EXIT SLIT

(b)

HAIDINGER RINGS

Figure 14. Localization of (a) Fizeau fringes, and (b), Haidinger

rings in focused-beam and collimated-beam spectrophotometers.

is the complex degree of coherence of the interfering

beams. The theory of partial coherence permits the

computation of | 712 | as a function of the beam parameters [15]. For this particular spectrophotometer such

computations show, for example, that a variation of

measured transmittance as large as 3 X 10~ 3 transmit- tance units is possible for a sample of optical thick- ness 3mm measured at 500 nm and 0.8 nm bandwidth.

This result, although a worst-case estimate, indicates

that interference must be reckoned with in highaccuracy spectrophotometry. The presence or absence

of an effect should be ascertained experimentally

whenever indicated. If present, the interference effect should be measured, and then averaged to obtain the

'true' transmittance of the sample.

Generally, the effect is largest for thin samples,

small bandwidths, and long wavelengths. Thus, only

sufficiently thick filters should be used as transfer

standards for the assessment of spectrophotometric

accuracy. Neutral-density filters are preferable since

they permit the use of large bandwidths.

finite degree of coherence of the interfering beams. The

effect of imperfect temporal coherence is that the

spectral fringe spacing, 8k, is generally significantly

smaller than the bandpass AX of the monochromator,

so that the remaining net effect will be a much subdued

average over several fringes. For example, 8A = 0.04 nm

for a 2-mm filter of refractive index n=1.5 at k =

500 nm, whereas the normally used bandpass of the

spectrophotometer described in section 2 is AA =

0.8 nm. In this case, the detector averages over twenty

fringes and the reduction of contrast is at least twentyfold. Lack of perfect spatial coherence may similarly

lead to an averaging of several spatially separated

fringes comprised within the spot size seen by the

detector. The over-all effect of these various factors is difficult to assess. Within the context of this paper it is only

possible to estimate an upper bound of the effect on

transmittance measurements with the spectrophotometer described here. Assuming the case of a perfectly

plane-parallel, clear sample of optical thickness nt, arranged perfectly normal to the beam, the measured

transmittance is [14]

r= T [l + 2r| yi

/4-7T s I —nt + avz (17)

VII. Nonlinearity Correction

All spectrophotometers are subject to nonlinearity

errors which must be eliminated by applying a correction, A71

, converting the measured transmittance, T, into the true value

r=r+A7\ (19)

A simple and yet accurate method to determine this correction is the method of light addition, based upon

the fact that a linear system which gives readings

1(A) and 1(B) for radiant fluxes A and B will give a reading I(A + B) = 1(A) + 1(B) when the two fluxes are added incoherently. The system is nonlinear and a correction must be applied if the ratio

1 + (t(A, B)

I(A + B)

1 1(A) + 1(B)

(20)

is not equal to unity. The most practical implementation is the double-aperture method due to Clarke [3], in which the two fluxes are those transmitted by a

pair of apertures which may be opened and closed

separately or in combination. This method was used

in the present work, and is discussed in detail in references [16] and [17]. The nonlinear detector response may be expressed

in the form

where t is the average transmittance, r is the Fresnel

reflectance of the sample boundaries, and

/(</>) = 7,</>[l + e(</>)], (21)

where

712= I 712 1 e* (18) . /(<£) = signal current minus dark current,

91
t) = nominal detector sensitivity,

(j> = radiant flux,

e(</>) = departure from linearity.

If the two apertures are equal in size, and if <j> = A + B

is the combined flux through both apertures, then, eq

(20) may be written as

l + <r(<f>) /(<M ! + €(</>) 2/(<£/2) l + €(<A/2)

(22)

The measured apparent transmittance for a sample

with true transmittance. t, is

T

_I(T<t>) _ 1 + 6(T0)

1 — — 1 /(<£) 1 + €(<£)

(23)

so that the required additive correction Ar of eq (19)

is given by

r i + c(t0)

] (24)

These equations show that there is no straighforward

functional relationship between the double-aperture

datum, a, and the nonlinearity correction Ar. The

former samples the detector response at the two points

<f> and 1/2 <f>, but for the latter, we require the knowledge of €(<£) at a different pair of points, </> and T<f). In the usual application of the double-aperture method,

this difficulty is circumvented by using the fact that the multiplier of t on the right-hand side of (23) happens

to be the reciprocal of the right-hand side of (22), if t—1/2. Thus, [l + cr(0)] is the multiplicative correction to be applied when t= 1/2. Similarly, the correction factor for t= 1/4 is [1 + a(<t>)] [1 + cr(l/2 <£)] , and

for t= 1/2" it is

T/r=fj [l + o-(<A/2»- 1 )]. (25)

Thus, one can measure <x((/>), o-(l/2 (f>) , . . . calculate the corresponding multiplicative corrections for T=l/2, 1/4, . . ., and then draw a curve from which

the correction may be read for arbitrary values of t. This method is unsatisfactory because:

(a) The actually measured corrections are unevenly

distributed along the T-axis, with wide gaps between

them in the regions of most interest.

(b) Most measurements are made at low light levels,

where the correction is small and the experimental

error is large.

(c) Each measured point depends on the previous

ones, so that the errors accumulate as t is decreased.

(d) The necessity to read the correction from a graph

is awkward, especially if all other data are processed

by computer.

These disadvantages can be overcome since, according to (22) and (24), M is linked to a through €{(f>).

Hence, it is possible to measure a{T<t>) for evenly

distributed values of t, process these data to And

e(<t>), and then calculate AT directly from (24). In order to do so, a lamp current is chosen so that the

unattenuated flux, <f>, through both apertures corre- sponds to the 100 percent point of the transmittance

scale. After cr((f>) has been measured, the incident flux

is attenuated in four 20 percent steps, and ct(t<£) is measured for t = 0.8, 0.6, 0.4, and 0.2. This series of

measurements is repeated four times.

An example of the data so obtained is shown in figure 15. In all cases, the dependence of a on T could

TW—

Figure 15. Measured dependence of the nonlinearity datum ct(t<£) on Tfor7(<£)=3XlO-M and \ = 525 nm.

be represented satisfactorily in the form of a parabola,

o-(T<t>) = aT + br2

, (26)

a = a(</)),6-6(</.), (27)

where the coefficients, a and b, are determined from

the measured data by least-square fitting, using the

inverse variances of the measured values of ofn/>) as weight factors. The use of a second-degree equation was justified by a statistical analysis with the residuals

showing no patterns suggesting higher order terms. Hence, we can calculate AT

1 as follows. Letting

e(<f>) = aT</> + /8(r<^) 2 (28a)

92
and then using eq (22), we find

a(T<t>) = 1/2 ar<]>+ 1/4(3/8 -a2

) (t0) 2 + . . . (28b)

to second order in </>, so that

a = 1/2 a<f>, b = 1/4(3)8 - a

2)02

, (28c)

according to (26, 27). Solving for a and fi , and substitut- ing the result into (28a),

e((/)) = 2ar + 4/3(a2 + 6)r2 +. .

and, therefore,

2ar(l - T) +4/3(a2 + b)T(l

A71 Ty

1 + 2a+ 4/3(a2 + b)

(28d)

(29)

upon substitution of (28d) into (24) for t — T and

T=l.

Thus the coefficients, a and b, can be stored in the

computer after ct(t<£) was measured as described,

and then Ar may be calculated from eq (29) as re- quired for arbitrary values of the measured apparent

transmittance T. As an indication of the precision of this method,

table 3 lists the results obtained in two independent

determinations of the nonlinearity correction. The

two sets of final results for <x are in far better mutual

agreement than the individual raw. data of figure 15. The residual discrepancies of the additive correction,

A7\ never exceed 10-5 transmittance units. For the particular spectrophotometer described in section II the nonlinearity correction was found to be

independent of source polarization, free from inter- ference effects, but slightly dependent on wavelength.

This variation with wavelength (about 5 X 10-5 trans- mittance units or less between 400 and 750 nm) is taken into account in all measurements by means of a numerical interpolation formula derived from the

measured values of Arfor three wavelengths.

Table 3. Repeated measurements of cr and

A7\A = 525 nm, /((/>) = 3 X 10~ 7 A)

T

First measurement Second measurement

o- X 104 AT x 10" a X 104 AT x 104

0.1 0.07 0.72 0.19 0.77

.2 .25 1.38 .45 1.46

.3 .52 1.96 .79 2.02

.4 .91 2.40 1.21 2.43

.5 1.39 2.67 1.70 2.66

.6 1.97 2.73 2.27 2.68

.7 2.66 2.53 2.92 2.45

.8 3.45 2.04 3.65 1.95

.9 4.34 1.21 4.45 1.14

1.0 5.34 0.00 5.33 0.00

The cooperation of K. L. Eckerle throughout this work is acknowledged with great appreciation. The

design of the spectrophotometer was initiated by R. P. Madden, J. Reader, and J. Schleter. Various suggestions were made by C. E. Kuyatt, R. Mavrodineanu,

and W. H. Venable.

VIII. References and Notes

[1] Mavrodineanu, R. J. Res. Nat. Bur. Stand. (U.S.), 76A (Phys. and Chem.), No. 5, 405 (Sept.-Oct. 1972).

[2] Hawes, R. C, Appl. Optics 10, 1246 (1971).

[3] Clarke, F. J. J., J. Res. Nat. Bur. Stand. (U.S.), 76A (Phys. and

Chem.), No. 5, 375 (Sept.-Oct. 1972).

[4] Mielenz, K. D., Eckerle, K. L., Madden, R. P., Reader, J., Appl. Optics (to be published).

[5] Mielenz, K. D., and Eckerle, K. L., Nat. Bur. Stand. (U.S.), Tech Note 729, 60 pages (June 1972).

[6] Taylor, D. J., Rev. Sci. Instr. 40, 559 (1969).

[7] Born, M., and Wolf, E., Principles of Optics (Third Edition, Pergamon Press, 1965), Chapters 1 and 13.

[8] In all practical applications the surrounding medium is air. Most refractometers measure refractive indices with respect

to air. In this case, the dependence of t on n' is taken into account.

[9] Vasicek, A., Optics of Thin Films (North-Holland Publishing

Co., 1960), Chapter 3. [10] For n' = l, n=1.5, and «i = 1.4, the numerical value of this factor is — 0.02. In addition, sin 2 (jc/2) is small for a very thin

film. Further computations on this subject are presented in figure 10.

[11] Mavrodineanu, R., private communication.

[12] These measurements were performed by R. J. Bruening, NBS,

on a goniophotometer with lenses for k = 546.1 nm.

[13] It should be noted that the simplifying assumptions which led

to eqs (13) and (14) may not apply. The correction terms must

be derived for each spectrophotometer individually. A detailed knowledge of beam geometry is necessary for this purpose.

[14] Born, M., and Wolf. E., Principles of Optics (Third Edition, Pergamon Press, 1965), Chapter 10.

[15] Equation (19) was derived from the general expressions for

| -y , 2 1 for small quasimonochromatic sources derived in K. D. Mielenz, Gas Lasers and Conventional Sources in Inter- ferometry (Electron Beam and Laser Beam Technology,

Academic Press, N.Y., 1968). For a uniformly illuminated

circular aperture of angular radius a = a/2/, the degree of

spatial coherence is given by

Irlspatiai = \(sin A)IA\

where A = koa2 t/2, i0 = 27r/\. The degree of temporal coherence is the normalized Fourier transform of the spectral

profile transmitted by the aperture, which in this case is

F(k = k a ) 0

x

2 if*2 g-a2 4

otherwise,

where x = (k — k0 )dx/dk is the linear coordinate in the aperture plane, and where the aperture diameter is assumed

to be much larger than the Rayleigh width of the grating. This leads to |y|temporal=|2/1 (B)/B|, where B = atdk/dx. Expressing |-yi 2 | as the product of the degrees of spatial and temporal coherence, and substituting the result into (17), we obtain the numerical results quoted

in section VI.

[16] Sanders, C. L.,J. Res. Nat. Bur. Stand. (U.S.) 76A(Phys. and

Chem.), No. 5, 437 (Sept.-Oct. 1972;.

[17] Mielenz, K. D., and Eckerle, K. L., Appl. Optics, 11, 2294

(1972). (Paper 76A5-732)

93

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Liquid Absorbance Standards

R. W. Burke, E. R. Deardorff, and O. Menis

Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234

(June 14, 1972)

Errors in the measurement of the absorbances of liquid filters result from instrumental and chemical uncertainties. This paper presents a systematic study of these variables on the absorbances of selected filters. Three types of liquid filters are discussed. These are (1) individual solutions of high

purity compounds, (2) composite mixtures and (3) aqueous solutions of organic dyes. The accuracy of the absorptivity data is established using NBS-calibrated glass filters. The magnitude of the errors

arising from spectral bandpass, beam geometry, stray light, internal multiple reflections, and refractive index are delineated. Finally, as a practical outgrowth of this study, the development and issuance

of NBS Standard Reference Material 931, Liquid Absorbance Standards for Ultraviolet and Visible Spectrophotometry, is described.

Key words: Absorptivity data; accuracy; liquid absorbance standards; Standard Reference Materials.

I. Introduction

In the use of filters for checking the accuracy of

the photometric scale of spectrophotometers, one

needs materials which exhibit absolute spectral neutrality. Such ideal filters are not available at present.

The use of the materials which have been suggested is limited by instrumental and sample variables. For this reason, one must carefully specify conditions which

define the other sources of errors. The first part of this paper reviews several of the more important instrumental parameters and dis- cusses their effects on absorbance measurements.

Examples from the literature are given to illustrate the magnitude of the errors arising from spectral

bandpass, stray light, nonparallel radiation, and

multiple reflections. The second part presents some

experimental studies of several materials which may

serve as calibration "filters" or standards.

II. Instrumental Parameters

A. Wavelength Accuracy

The absorbances of most samples are sufficiently

wavelength dependent that even in the most favorable

regions of maxima and minima a wavelength error of 1—2 nm can produce absorbance errors of a few

tenths of a percent. When performing measurements

on the slopes of absorption peaks, the wavelength

setting is obviously much more critical and errors are

typically several percent per nanometer. Wavelength accuracy, therefore, becomes especially

important when making absorbance measurements

at, for example, an isosbestic point.

A fine source provides the most definitive means for establishing the accuracy of the wavelength scale. The best single source is a mercury lamp which may

be used throughout the range of 200-1000 nm. For

calibration in the visible region, helium lines are

also useful. Gibson [l] 1 has discussed the use of mercury and

helium sources and has listed those lines best suited

for wavelength calibration. Other sources which have

been used include neon, cadmium, cesium, and sodium.

The M.I.T. Wavelength Tables [2] summarizes the most

prominent emission lines of these elements. Not all wavelengths are given and care must be taken in using any of these lines in order not to confuse closely

adjacent lines with the one being checked. For those

spectrophotometers having a hydrogen or deuterium

source, the emission lines at 486.13 and 656.28 nm

(H) or 485.99 and 656.10 nm (D) may provide con- venient checks at these wavelengths.

Calibrated holmium oxide and didymium glasses may

be useful secondary standards, particularly for checking recording spectrophotometers in which a dynamic

check of the instrument is often desirable. The

apparent absorption maxima of these filters, however,

may vary with spectral bandpass. Therefore, for the

highest accuracy, they should be calibrated for the

spectral bandpass at which they are to be used. In general, prism instruments require more frequent

calibration than the grating type because their dis- persion is temperature dependent. Corrections in the

visible region are of the order of 0.1 nm per degree

Celsius. Hysteresis effects must also be considered

1 Fieurea in brackets indicate the literature references at the end of this paper. 95
and the wavelength settings should always be ap- proached from the same direction.

B. Finite Bandwidth

Spectral bandpass differences undoubtedly account

for a significant portion of the discrepancies in molar

absorptivity values reported in the literature. Some

have resulted from the employment of inadequate in- strumentation; others have resulted from improper use

of these instruments or complications arising from

solution equilibria.

Increasing the spectral bandpass at an absorption

maximum has two effects on apparent peak heights:

(1) the observed values are always less than the

true values and (2) the differences between the two

are proportionally greater at higher absorbances.

Thus, for a finite bandpass, a plot of absorbance

versus concentration or path length will always have

a smaller slope than it does in monochromatic radiation and, in addition, will be concave to the concentration axis. Figure 1 illustrates these effects. The reverse behavior will be observed for measure- ments at an absorption minimum. Meehan [3] has

given a simple example which verifies these two

effects mathematically.

CONCENTRATION (PATH LENGTH)

Figure 1. Effect of spectral bandpass on absorbance: (a) Monochromatic radiation (b) Finite bandwidth. Consider that an absorbing system is illuminated

by two monochromatic radiations of wavelengths

\i and A.2 and that the Lambert-Beer law is obeyed

at each wavelength with absorptivities a\ and ai.

If the effective intensities of the two wavelengths are

l\ and h, respectively, the transmittance, T, is T = ih-lO-"^ +h • \0- a * bc )l(h + h) (1)

where 6 = path length and c— concentration. If A.i is the wavelength of maximum absorption so that

ai > a,2, then

T =

1

(1 + r) ,10-«l6c[l + r .10<ai-a2>&c]

(2)

where r = /2 //i. The absorbance, A, defined as — log

Tis

A = aibc + log (1 + r) - log [1 + r-10(a i- a 2>&c]

(3)

which may be differentiated with respect to be to give

dAld(bc) = ai - [r(ai - a2 )10< a i- a 2>&c]/

[1 + r- 10<a i- a 2»>c]

(4)

At low absorbances

dA/d(bc)bc~o

ai + ra2 1 + r (5) whereas when be becomes large, 10ai*c ^>10a26c and

the limiting slope is

dAld(bc)bc^oo=a2. (6)

Thus, the limiting absorptivity at low absorbances

is a weighted average of a\ and a2 while, at higher

absorbances, it is equal to a2 , the smaller of the

individual absorptivities.

The theory for the correction of spectral bandpass

errors has been developed by Hardy and Young [4], Eberhardt [5], and Broderson [6]. Rigorous calculation

requires integration of the relation

^obs = log

hS^dX

hS)J0-A dK

(7)

where I\ is the incident intensity and S\ is the spectral

sensitivity of the detector. Equation (7) emphasizes

the fact that the observed absorbance depends not only on the shape of the absorption curve, but also on

the wavelength distribution of the source and the

detector response. If I\S\ is constant over the wavelength interval used, approximate corrections for spectral bandpass may be calculated by assuming a Gaussian shape for the absorption peak and a tri- angular slit function for the instrument. Some calculations are given in table 1 based on these assumptions.

The tabulated values of AobJA agree closely with

those observed experimentally for A up to 1. As

shown by Broderson [6] however, for values of RBW

larger than 0.5, AobJA also depends on A and the

above treatment is no longer applicable.

C. Stray Light

Stray light is defined as any fight outside the spectral

region isolated by the monochromator that reaches the

detector. It is produced by scatter from the optics and

96
Table 1. Dependence of Aobs on spectral bandpass

at an absorption maximum under idealized conditions (see text)

RBWa RBWa ^obs/^

0.0100 0.99995 0.0800 0.9970

.0200 .9998 .0900 .9962 .0300 .9995 .1000 .9954 .0400 .9992 .2000 .9819 .0500 .9988 .3000 .9604 .0600 .9983 .4000 .9321 .0700 .9977 .5000 .8987

a Relative bandwidth: Ratio of spectral bandwidth to half bandwidth of fully resolved peak.

walls of the monochromator and is present in varying

amounts in all spectrophotometers.

Stray light can lead to varied problems in spectrophotometry. Spurious absorption bands may arise in some cases. More frequently, however, deviations

from the Lambert-Beer law are produced. These

deviations are positive if the stray light is absorbed and

negative if it is not. Extensive literature exists on the detection, measure- ment and minimization of stray light. Several of the more useful papers are those by Hogness, Zscheile

and Sidwell [7], Perry [8], Slavin [9], and Poulson [10] in which additional references may be found.

The amount of stray light present is proportionally

large in those wavelength regions where the trans- mission of the monochromator, the source intensity or the detector sensitivity are relatively low. These

regions, which should first be checked, are 200-230

nm and 600-700 nm. If a tungsten lamp must be used

in the range 350-400 nm, a visible cutoff filter should

always be employed.

The quantitative assessment of stray light requires

the use of extremely sharp cutoff filters. Slavin [9] and Poulson [10] have described a number of liquid

and solid filters that may be used. Aqueous solutions of

the alkali halides, for example, are extremely useful

in the ultraviolet. Care must be taken, however, that

light leaks in the cuvet compartment are not contributing to the observed results. The apparent stray light

will not vary appreciably with slitwidth if only instru- mental stray fight is present. In a quick test for light

leaks, the apparent stray light will decrease rapidly as the slits are opened.

The stray light error encountered most frequently in spectrophotometry produces a decrease in an absorption peak and leads to negative deviations from the

Lambert-Beer law. If it is assumed that none of the

stray light is absorbed by the sample, the measured

absorbance is related to the true transmittance by the

expression [9]

^obs =iog [(i-r)s+r]-> (8)

where S is the instrumental stray light expressed as a

fraction. The effects of several levels of unabsorbed

stray light on absorbance are given in table 2. Most

samples absorb sufficiently in other regions of the spec- trum to filter out a proportion of the stray light. Thus,

indirect estimates of instrumental stray light based on

deviations from linearity are generally low.

Table 2. Effect of unabsorbed stray light on absorbance

A ^obs S = 0.0001 S = 0.001 S = 0.01

0.1 0.1000 0.0999 0.0989

.5 .4999 .4990 .4907

1.0 .9996 .9961 .9626

1.5 1.499 1.487 1.384

2.0 1.996 1.959 1.701

D. Nonparallel Incident Radiation

A perfectly parallel beam of radiation must come

from a point source and can carry only an infini- tesimal amount of energy. The light beam in a spectrophotometer always has some finite angular size. As a

result, the average light path is greater than the per- pendicular distance between the cuvet faces. Meehan

[3] has considered the case in which the incident

radiation is convergent or divergent in one dimension

such as corresponds, respectively, to radiation focused

by a cylindrical lens or leaving a narrow slit. If 0 and

R are the angles of incidence and refraction, re- spectively, the path length of the extreme ray (fig. 2)

is 6/cos R and the absorbance of this ray is A/cos R. Other rays enter at smaller angles and the observed

absorbance is given by

k b H

FIGURE 2. Nonparallel incident radiation [3]. Reprinted by permission of John Wiley & Sons, Incorporated.

f"max

^obs = ^/flmax 1/cOS RdR. (9) J 0

For this relation to hold, the incident beam must be

uniform over its cross section. Evaluation of this integral for Rmax of 2-10° (Omax = 2.7°-13.4°) gives

the following percent errors in A (table 3). Thus, for angles of incidence up to 5 degrees, the absorbance

error is less than 0.1 percent.

E. Multiple Reflections

On passage of light through a cuvet containing

solvent or sample solution, some radiation is reflected

97
Table 3. Dependence of absorbance error on nonparallelism of incident radiation

/!ma„ degrees 0max , degrees Percent error in A

24

68

10

2.7

5.3 8.0

10.7 13.4

0.020

.081

.18

.33

.51

at each of the two air-glass and the two glass-liquid surfaces. For perpendicular incidence, the fraction

/ reflected on passing from a medium of refractive

index n\ to a second having retractive index n2 is given by the Fresnel expression

/= n\— n2 «i+ n2 (10)

For an air-glass surface, / = 0.04 and for a glassliquid surface, /= 0.0035. When more than one surface

is involved, the effects of multiple reflections must be

considered. The essential question when dealing with

liquid filters is whether the solvent completely compensates for such reflections. According to Goldring

et al. [11], it does not. They consider the case where the

reflections from all surfaces perpendicular to the

fight beam on the two sides of the sample are grouped

together to form two effective reflection coefficients, n

and r2 , as shown in figure 3. Considering only first and

second order reflections, the observed absorbance is

=t* Byr, r,

PnT-PoTr,

*%Tr,-T-TrJ

SOLVENT SAMPLE

FIGURE 3. Internal multiple reflections [11]. Reprinted by permission of the American Chemical Society.

^obs = log-5-=log-—- — — (11) f2 /V (1 — ri + r^l 2

)

which, after transformation and series expansion, can be reduced to the following expression:

Aobs = A + 0.4343 (1-T2

) nr2 1-r,

(12)

Considering only the reflections from the two cuvet

faces and that ri = r2 = 0.05, the variation of ^ obs with

A isA 0.1 0.2 0.5 1.0 2.0

Aobs 0.1004 0.2007 0.5010 1.0011 2.0011

Thus, because of internal multiple reflections, the

measured absorbance should always be larger than the

true absorbance with the percentage difference being

greatest at low absorbances. Verification of these

deviations is difficult experimentally. Goldring et al. [11], however, have suggested ways of minimizing

reflection effects. These include positioning the cuvet

at a small angle to the beam and stopping down the

detector surface, immersing the cuvet in a fluid con- tained in a larger rectangular cuvet turned at an

angle to the beam, or by using a cuvet with prismatic

windows. In each case, the effective path length must

be determined separately. Other alternatives consist

t of using cuvets constructed of absorbing materials or immersion of a glass filter into the sample. For a

limited wavelength range, reflection errors can also be

reduced by coating the external window surfaces

with anti-reflection layers. In addition to reflections from the cuvets, reflections

from other surfaces must also be considered. Gibson

[1] has pointed out that reflections may occur from the

surfaces of the slit and detector and has suggested a means for checking this source of error. A thin glass

plate is placed in its normal position at right angles

to the beam and then at a small angle from this position. In the second position the reflected energy is directed out of the beam while scarcely affecting the

true absorbance.

F. Cuvets

The majority of absorbance measurements are performed on solutions and, in such instances, the cuvet becomes an integral part of the measurement

system. Presently, the uncertainty in the length of the

light path can be a limiting factor in the determination

of molar absorptivity. Nonparallelism of the end

windows can be of even greater consequence. However, the effect can be minimized by using the same

cuvet orientation for all measurements. The uncertainty in path length is nevertheless greater.

Cuvets are available in a variety of shapes and

sizes and may be made of glass or silica. Construction remains largely an art and cuvets may vary considerably in their quality. Two methods of assembly

are commonly used. These are: (1) fusion using only

heat and (2) the use of intermediate, low-melting

glasses. At present, each has its disadvantage. The

first technique is the more desirable, but our ex- perience, primarily with rectangular cuvets, indicates

that the edges are not always completely fused. These surfaces exhibit capillarity and may become

serious sources of contamination. Under such conditions, the cleaning of these cuvets is difficult, if not

impossible. The use of low-melting fluxes on the other

hand can produce strains because of differences in coefficients of expansion and cuvets so constructed

are generally more fragile.

Cuvets are frequently offered in matched sets. This

may be considered more of a convenience than a necessity since this terminology is normally used only

to describe the transmission of the window material.

98
Path length and parallelism of end plates are of more

fundamental importance. Unfortunately, no generally

accepted tolerances have been established in this country for the construction of cuvets. The British

Standards Institution, however, has published a set of specifications [12] which merit our consideration and

possible adoption. They recommend that the path

length be specified to ± 0.02 mm and the end windows

be flat over a defined beam area to four Newton fringes

per centimeter in mercury green light. It should be

feasible for greater accuracy, especially for the 10 mm

cuvets, to calibrate a limited number by appropriate

metrology techniques to ±0.01 mm.

III. Evaluation of Selected Liquid Filters

This section is a report of our efforts to develop

well characterized liquid absorbance standards. Three

general types of liquid filters are discussed. These are

(1) individual solutions of high purity materials, (2) empirical mixtures and (3) aqueous solutions of organic

dyes. Much of the emphasis has been placed on deter- mining the optimum conditions for preparation of these

filters and, once established, what the absorbing

species or ionic compositions of these systems are. By combining this information with the instrumental

considerations developed in the first part of this paper,

it is hoped that the resulting data represent a step

toward obtaining more meaningful absorptivity

measurements.

A. Instrumentation

Absorbance measurements at a fixed wavelength were performed manually on a high precision doublebeam spectrophotometer provided with a double

monochromator. The accuracy of the photometric

scale of this instrument was established with the

NBS high-accuracy spectrophotometer described by

Mavrodineanu [13]. The wavelength scale was checked

with a mercury lamp. Potassium halide solutions [9, 10] were used to assess stray light in the ultraviolet. Room temperature was maintained between 24 to 26 °C. The cuvet compartment and jacketed holders were thermostated by a circulating water bath. Copperconstantan thermocouples were used to measure

temperature differences between the bath and the

sample solution. An electronic feeler gauge [13] was used to measure

the path length of the 10 mm rectangular cuvets normally used. Solutions, assumed to obey Beer's

law, were used for calibrating the 1 mm cuvets. The accuracy of the microbalance was established

with NBS-calibrated weights. Class A, 1 -liter volu- metric flasks were checked gravimetically. All dilutions were subsequently made by volume. To minimize

errors from cell positioning, borosilicate, Pasteurtype pipets were used to transfer solutions to and from

the cuvets.

B. Individual Solutions of High Purity Compounds

1. Potassium Dichromate. Numerous attempts

have been made to use chromium (VI) solutions

as ultraviolet absorbance standards. In the early

studies [7, 14, 15, 16, 17] alkaline solutions of potassium

chromate were preferred. More recently, potassium

dichromate in slightly acidic media has been described

[18, 19, 20]. The absorption spectra of these two sys- tems are quite different with the latter giving the more

desirable arrangement of maxima and minima (fig. 4). The approximate composition of chromium (VI)

solutions may be derived from the following equilibria:

220 250 300 350 400

WAVELENGTH, nm

Figure 4. Absorption spectra of 10"4 M Cr (VI) in 0.05 N KOH

and 10-3N HCIO4.

H2 Cr04 = H+ + HCrOi (13)

HCrOi" = H++CrOf (14)

2HCr04- = Cr2 Of + H2 0. (15) Some values reported for the equilibrium constants

Ki, K2, and Kz at 25 °C are as follows:

/id = 0.16 [21]

#2 = 3.2 x 10-7 [22] and 3.0 X 10~7 [23]

#3 = 43.7 [21], 35.5 [24] and 33.0 [25].

Thus, at a pH greater than 10, chromium (VI) exists almost entirely as CrOf ions while, in weakly acidic

solutions, the predominant species is HCrOj which

partially dimerizes to C^Of. The amount of dimer

will increase with increasing chromium concentration. The formation of H2Cr04 or CrOf can be es- sentially eliminated by maintaining the acidity near pH 3.

99
Much of the uncertainty that has arisen from the use

of acidic potassium dichromate solutions as potential

absorbance standards can be attributed to the dimerization reaction (eq (15)). It is primarily this equilibrium which leads to the observed deviations from

Beer's law. Because of the range of values reported

for K3 , one of the first objectives of the present study was to redetermine this constant. The spectrophotometric method was chosen and is described in some

detail below.

a. Spectrophotometric Determination of the

Dimerization Constant for: 2HCr04" = Cr2 Of

+ H2 0. If HCrOj and Cr2Of are the only Cr(Vl)

species present with molar absorptivities ei and 62, respectively, the apparent molar absorptivity, e m, will be given by

em = (1 — a)ei + 1/2 • ae2 (16)

where a is the fraction of total chromium in the

dichromate form. From eq (15) the thermodynamic

equilibrium constant of the dimerization reaction is

K; [Cr2Of ] 72 [HCr04-] 2 ' y\

or, in terms of a

K3 =

a 72 2(l-a) 2Crr

' 7?

(17)

(18)

where 71 and 72 are the activity coefficients of HCrO-T

and Cr207=

, respectively, and Crr is the total chromium concentration. Because the ionic strength never exceeded 0.01 , the activity coefficients may be treated

by the Debye-Hiickel expression [25]

log 7,

: AZ2^2 T+Ti/2 (19)

where Z is the ionic charge, I is the ionic strength and

A has a value of 0.509 1/mol at 25 °C. Equations (16)

and (18) can be rewritten to give, respectively,

em = a(l/2 e2 — ei) + ei

and

log K3 = log

2AW2 2(l-a)2Crr l+I1 ' 2

(20)

(21)

Assuming a value of K3 , one can calculate from eq

(21) the a values at various total chromium concentrations. If the choice of £3 is correct, the measured

values of em should he on a straight fine of slope (1/2 e2 — ei) and intercept 61, as seen from eq (20). The

best value of K3 is then determined by the method of

least squares.

1. Experimental Procedure

The chromium (VI) solutions used in the subsequent

studies were prepared from NBS SRM-136c, potassium dichromate, which had been dried at 110 °C for two hours. The distilled water was shown to be free of reducing impurities by titration with a dilute potassium permanganate solution. Perchloric acid was used to maintain the pH at 3.0 ±0.1.

The optimum wavelengths for the determination of K3 were chosen as follows: Two solutions were pre- pared, one containing 391 mg K2 Cr2 07/l and the other,

40.0 mg K2 Cr207/l. The former was transferred to a

1.023 mm cuvet and the latter to a 10.00 mm cuvet. A

differential scan was then made on a recording spectrophotometer equipped with a 0-0.1 absorbance (full scale) slidewire, using the less concentrated solution as the reference. The resulting spectrum is shown in figure 5. Relatively large differences in absorbance are seen in the wavelength regions near 275 and 385 nm

even though the total number of chromium atoms in each light path is the same. Thus, it is in these two

regions that the largest deviations from Beer's law

should be observed. Three wavelengths were subsequently selected in each region for the determination

of #3-390, 385 and 380 and 280, 275 and 270 nm,

respectively.

300 WAVELENGTH, nm

400

Figure 5. Difference spectrum of 391 mg K2 Cr207/l in 1.023 mm

cuvet versus 40.0 mg K2 Cr>07/l in 10.00 mm cuvet; pH = 3.

2. Results

The apparent molar absorptivities calculated

from absorbance measurements on the four chromium

concentrations used for the determination of K3 are given in table 4. Using the method of least squares, these e m values were plotted against the a fractions

calculated from eq (21) for various assumed values of K3 . The molar absorptivities were weighted according

to the reproducibility of the absorbance measurements. A computer was used for all calculations.

100
A typical graph of the residual standard deviation 36. The minimum in this curve is the best estimate

of the experimental e m values from the best straight of K3 and, for the case shown, is 32.6. The K3 values

line fit is shown in figure 6 for K3 values of 28 through at the six wavelengths are summarized in table 5.

Table 4. Apparent molar absorptivities, em, of K^C^O? solutions at 25 °C; pH = 2.9 (HCIO4)

K2Cr207ConC. e m 1/mol-' cm- 1 mg/1

100.06 400.48

699.86

1000.27

390 nm 385 nm 380 nm 280 nm 275 nm 270 nm

589.2 748.7 865.1 1579.3 1718.6 1881.0

714.1 878.6 997.8 1758.2 1914.4 2069.8

806.2 973.8 1094.0 1890.0 2053.5 2208.0

875.9 1045.6 1166.5 1988.2 2161.5 2313.6

2.0

a —

—! 0.8 —

IE —

0.4 -

Figure 6. Least squares determination o{K3 .

Table 5. K3 values at 25 °C as determined at several

wavelengths

A, nm

390

385 380

280

275

270

K3

32.4

33.3 33.8

33.5 32.6

31.6

Avg. 32.9

Std. Dev. 0.8

Rel. Std. Dev. 2.5%

b. Absorption Spectra of HCrOj and O2O7.

The absorbances of three solutions containing nominally 20, 40, and 60 mg K2 Cr2 07/l (pH = 3.0±0.1) were measured in a 10.00 mm cuvet at 5 nm increments over the wavelength range of 220-400 nm. A

close approximation of eCHCrOj) was obtained by

extrapolating the apparent molar absorptivities to zero chromium concentration. This extrapolation is simplified since the variation of a is nearly linear over

this concentration range. Similar absorbance measure- ments were performed on three solutions containing

1000 mg K2 Cr2 07/l using a 1.023 mm cuvet and the

corresponding a value was calculated from eq (21), with K3 = 32.9. The first approximations of 1/2- €(Cr2 Of) values were obtained by substituting the

extrapolated e(HCrOj) results and the above a

value into eq (20). The e(HCr04-) and 1/2 • e(Cr2 Of)

values were then refined bv successive approximations.

The calculated absorption spectra of these two ions are shown in figure 7.

250 300 350 WAVELENGTH, nm 400

Figure 7. Absorption spectra of HCrOr and Cr2 Of.

c. Molar Absorptivities of HCrO-f and Cr207- at 350, 313, 257, and 235 nm. As shown previously

in figure 4, the absorption spectrum of weakly acidic

solutions of K2 Cr2 07 exhibit maxima at 350 and 257 nm

101
and minima at 313 and 235 nm. These maxima and

minima do not shift significantly over the concentration range of 20-100 mg K^C^C^/l. This is con- sidered a practical range for most applications since it yields absorbances of about 0.1-1.5 for a 10 mm cuvet. The molar absorptivities of HCrOj and C^O? at 350,

313, 257, and 235 nm were determined by the pro- cedure described above and are given in table 6. The

e(HCr07) values are estimated to be accurate to ± 0.5 percent. Because of the imprecision of the K3 determination, the uncertainty of the dichromate

molar absorptivity values may be as large as ± 5 percent.

Table 6. Molar absorptivities, €, of HCrO^f and Cr2Of

at 25 °C

€, 1 mol-1 cm-1

350 nm 313 nm 257 nm 235 nm

HCrO- 1576 711 2100 1804

1/2 Cr 0= 1788 823 3156 2688

Table 7 shows the agreement between the experimental and calculated apparent molar absorptivities

for five concentrations of potassium dichromate in dilute perchloric acid (pH = 2.92 ± 0.02). The experimental values are the averages obtained on five individual samples measured at each concentration.

The 95 percent confidence limits for any given set of measurements was 0.1 percent. The a values used are

also given. These are seen to vary nearly linearly over

the range of chromium concentrations employed.

d. Optimum pH and Choice of Acid. The

chromium (VI) solutions used in this study were pre- pared in dilute perchloric acid media having a pH of about 3. This acidity was selected because the existing

equilibria data indicated this pH limited the formation

of either t^CrCX or CrOl to less than 0.1 percent of the total Cr (VI) concentration. Perchloric acid was

chosen instead of sulfuric to prevent the formation of chromate-sulfate complexes. Tong and King [24] and

Davies and Prue [25] noted slight differences in the

absorbances of dilute perchloric and sulfuric acid

solutions after correcting for differences in ionic strength. They attributed these differences to mixed

complex formation. More recently, Haight et al. [26] have shown that the conversion of HCrOf to CrSOf is quantitative in 1 M HSOf solutions and that absorption

spectrum of this complex is significantly different from

the HCrO^ spectrum.

Because most of the previous work reported in

literature has been done in sulfuric acid, it was con- sidered of interest to intercompare these two systems.

The results are shown in each acid between pH 2 and 3. At 350 nm the results obtained in perchloric acid are

consistently 0.1-0.2 percent higher than in sulfuric;

at 235 nm, they are lower. The relatively large increase

in absorptivity at 235 nm at pH 1.90, together with the

above considerations, suggest the formation of additional chromium (VI) species in systems employing

dilute sulfuric acid solutions. (Seejable 8.) e. Effect of Temperature. The absorbances of a

solution containing 60 mg B^C^Cv/l in dilute perchloric

acid (pH = 3) were measured over the temperature

range of 17 to 37 °C. The percentage change in absorb- ance for wavelengths of 350, 313, 257, and 235 nm is shown in figure 8. The results are uncorrected for

Table 7. Experimental and calculated values (E, C) of the apparent molar absorptivity of r^C^Cv at 25 °C; PH = 2.9(HC104 ) e m , 1 mol _1 cm' 1 K2 Cr2 07 Cone.

mg/1 a 350 nm 313 nm 257 nm 235 nm

20.22 0.0090 E 1577.6 713.5 2113.6 1814.7 C 1577.9 711.9 2108.7 1812.1

Diff. -0.3 +1.6 +4.9 +2.6

40.09 0.0178 EC

Diff.

1579.9 1579.8 + 0.1713.3 713.0 + 0.32120.8

2118.8 + 2.0

1820.2 1819.7 + 0.5

60.12 0.0262 EC

Diff.

1581.4

1581.5 -0.1

713.8

713.8

0.0

2127.2 2126.7 + 0.51827.1 1827.2 -0.1

80.17 0.0344 EC

Diff.

1584.0

1583.3 + 0.7

715.0

714.8 + 0.2

2137.6

2135.3 + 2.3

1835.8

1834.4 + 1.4

99.92 0.0423 EC

Diff.

1585.5 1584.0 + 1.5715.6

715.0 + 0.6

2144.0

2143.7 + 0.3

1841.5 1841.4 + 0.1

102
Table 8. Apparent absorptivities, a, of sulfuric and

perchloric acid solutions of K2 Cr20? at 25°C; K2 Cr2 07 cone. = 0.050 g/1

a, 1 g

_1 cm-1

pH 350 nm 313 nm 257 nm 235 nm

H2S04 1.90 10.71o 4.837 14.444 12.437 1.98 10.71 2 4.839 14.440 12,418 2.20 10.722 4.839 14.429 12.402 3.00 10.73s 4.844 14.427 12.38,

HC104 1.99 10.727 4.848 14.448 12.396 3.08 10.740 4.845 14.434 12.383

Table 9. Apparent molar absorptivity of potassium

chromate in 0.05 M Na2 HP04 and 0.05 M

KOH at 25 °C

0.05 M Na2 HP04

em , 1 mol 1 cm-1 K2 Cr04 Cone, —

M X 10= pH 373 nm 274 nm

7 9.2 4827 3703 14 9.1 4820 3697 21 9.1 4813 3692

0.05 M KOH

7 4830 3705 14 4824 3698 21 4814 369,

0.6

j. 0.4 o 0.2 <

-0.8

1 1 1

1

-^^^ 313 nm

"^X350a257nm

1

1

1 1

1 235 nm

1 15 20 25 30 35 40

TEMPERATURE, °C

FIGURE 8. Change in absorbance with temperature for acidic solutions of K2 Cr2 07 ; pH = 3 (HC104 ).

thermal expansion of the solvent. The absorption

minimum at 313 nm exhibits the smallest temperature

dependence (— 0.02 percent per °C) while the maxima

at 350 and 257 nm and the minimum at 235 nm have

temperature coefficients of — 0.05 percent per °C over

the range 20 to 30 °C.

f. K2 Cr2 07 in 0.05 M Na2 HP04 and 0.05 M

KOH. To overcome some of the difficulties in handling strongly alkaline chromate solutions, Johnson

[27] has recommended dissolving K2 Cr2 07 in 0.05 M

Na2HP04 at pH 9. The apparent molar absorptivities

obtained in this medium and in 0.05 M KOH are given

in table 9. As seen from the data, both systems appear

to exhibit deviations from Beer's law of about three

parts per thousand over the concentration range

studied. Although this deviation is the same magnitude and direction predicted from uncompensated

internal multiple reflections discussed previously, we

have not been able to demonstrate experimentally that such reflections are responsible for the observed results. Studies in which the cuvet windows are coated

with various anti-reflection layers are continuing

however.

From a consideration of the equilibrium data, the

absorbances in the two systems should be nearly

identical. The ionic strength of the 0.05 M Na2 HP04 is 0.15 and, from Neuss and Rieman's work [22], the

ionization constant for the reaction HCrOi = H++ Cr0 4 is approximately 9X10-7 mol/liter. Thus, at pH 9.1,

only about 1 in 1000 chromium atoms is present as HCrOj. Since this species is less absorbing than

CrOf at 373 and 274 nm, the apparent molar absorptivities in 0.05 M Na2 HP04 should agree with those

obtained in 0.05 M KOH to within 0.1 percent.

The absorbance of alkaline chromate solutions de- creases with increasing temperature at both absorption

peaks. Between 17 and 37 °C, the temperature coefficient at 373 nm is — 0.09 percent per °C; at 274 nm,

it is — 0.06 percent per °C. 2. Potassium Nitrate. Aqueous solutions of potassium nitrate exhibit a nearly symmetrical absorption

peak with Xmax at 302 nm. This system has been

studied extensively by Vandenbelt [14] and by the

British Photoelectric Spectrometry Group in two

collaborative tests [28, 29]. Molar absorptivities re- ported vary from 7.06 to 7.20 liter mol-1 cm-1

. This

range of values undoubtedly results in part from the

fact that KN03 solutions deviate significantly from

Beer's law. Careful reexamination of this system has yielded

the following apparent molar absorptivities (table 10). These are seen to decrease rather markedly with

inqreasing potassium nitrate concentration. Several

factors conceivably contribute to this behavior. In

addition to possible multiple reflection effects already

discussed some ionic interactions are undoubtedly

possible in this system because of the relatively high

concentrations of solute required coupled with the

fact that the N — O bond in the nitrate ion has some

polar character.

The potassium nitrate used in the above study was

analyzed for commonly encountered impurities. After

drying at 110 °C, the water content was less than 0.02 percent. Flame emission analyses [31] indicated the

following levels of alkali and alkaline earth impurities

103

502-591 O - 13 - 8
(in parts-per-million): Li (< 0.2); Rb (13); Mg (0.2); Ca (1); Sr(4) and Ba (< 2). At 302 nm, the absorbance of KNO3 solutions de- creases with increasing temperature. Over the range

17 to 37 °C, the temperature coefficient is —0.14 per- cent per °C. Solutions stored for up to six months showed no measurable change (^ 0.1 percent) in absorbance.

Edisbury [29] has cautioned, however, that some solu- tions may develop bacterial whiskers on standing.

Sterilization may be effected by prior boiling of the

distilled water.

Table 10. Apparent molar absorptivity, em , of aqueous

solutions of potassium nitrate at 25 °C

Cm, 1 mol-1 cm-1

KN03 Conc.,M 302 nma

0.028 7.16o

.056 7.142 .084 7.127 .112 7.106 .140 7.09,

a Spectral bandpass = 1.0 nm

3. Potassium Hydrogen Phthalate. High purity

potassium hydrogen phthalate (KHP) is readily available. It is presently issued by the National Bureau of Standards as a primary acidimetric standard. Its use

in the preparation of buffer solutions of known pH is well established. It has also been used as a spectral

standard in the comparative evaluation of spectrophotometers [11, 32]. The absorbance of KHP solutions is dependent on

acidity, as shown in figure 9. In the present study,

dilute perchloric acid solutions were employed to minimize changes in ionic composition as a function

of pH. Acid concentrations much above 0.1 A' could

not be used because of the precipitation of potassium

perchlorate. Subsequently, all solutions were prepared

to contain 10 ml of perchloric acid per liter with a pH of 1.3. Ringbom [21] has given the first ionization constant

of phthalic acid as 1.6 X 10-3 at 25 °C at an ionic

strength of 0.1. At the pH used, the absorbing system

consisted of 97 percent phthalic acid and 3 percent

hydrogen phthalate ions. Table 11 summarizes the

absorptivity values obtained on these solutions. Absorbance decreases at the 275.5 nm maximum and

increases at the 262 nm minimum with increasing

temperature. Over the range 17 to 37 °C the temperature coefficients are —0.05 percent and +0.05 percent

per °C, respectively.

Perchloric acid solutions of KHP (NBS-SRM 84g) were examined for possible fluorescence, using a high

sensitivity spectrofluorometer. With 280 nm excitation, a faint fluorescence was detected with A.max at approximately 350 nm. The possible effects of this fluorescence on the absorbance data remain to be established.

300 350 WAVELENGTH, nm

Figure 9. Absorption spectra of KHP solutions at different

acidities.

Table 11. Apparent absorptivity, a, of potassium hydrogen phthalate in dilute perchloric acid

at 25 °C; 10 ml HC104/liter

a, 1 g" 1 cm -

1

NBS SRM 84g

Cone, g/1 275.5 nm 262 nm

0.034 6.332 4.49., .142 6.31 9 4.489

4. Cobalt and Nickel Sulfates and Perchlorates. Aquo cobalt (II) and nickel (II) ions have rela- tive broad absorption peaks with A. max at 512 and 394

nm, respectively (fig. 10). Solutions of either may serve

as useful absorbance standards. Most of the attention

to date, however, has been directed at cobalt sulfate which was first recommended by Gibson [1]. In this study, the apparent molar absorptivities of

Co(H2 0)^2 and Ni(H2 0)J

2 were examined in sulfate and

perchlorate media. All solutions were prepared from

the high purity metals by dissolution in H2 S04-HN03 or HCIO4-HNO3 acids. Nitrate was removed by

repeated fuming until the acidity was reduced so that on dilution to volume, a pH of 1 could be obtained. The

results obtained in the two acid media are given in table 12. The values in sulfate solution are significantly higher than in perchlorate media for both

cobalt and nickel, suggesting that some complexation

of these ions by SO 4 or HSO4 has occurred. A differ- 104
WAVELENGTH, nm WAVELENGTH, nm

Figure 10. Absorption spectra of Ni(H2 0)+2 and Co(H20)+ 2

.

Table 12. Apparent molar absorptivities, em of acidic

solutions of cobalt and nickel ions

at 25 °C; pH=1.0

em (512 nm)

1 mol-1 cm-1 Co Cone.

g/1 Sulfate Perchlorate

2.4 4.88, 4.803 12.0 4.869 4.79,

e m (394 nm)

1 mol-1 cm-1 Ni Cone.

g/1 Sulfate Perchlorate

2.3 5.I67 5.090 11.5 5.154 5.077

ential spectrum of 0.2000 molar cobalt solution in sulfate against the same cobalt concentration in perchlorate media is shown in figure 11. In sulfate

solution, the formation of a second cobalt species with Amax = 528 nm is indicated. Similar behavior was also observed for nickel. The absorbances of cobalt and nickel solutions at their maxima are temperature dependent and increase

with increasing temperature. Between 17 and 37 °C

the temperature coefficients are 0.18 and 0.14 percent

per °C at 512 and 394 nm, respectively. To explain

their nuclear magnetic resonance data on cobalt(II)

solutions, Swift and Connick [33] have suggested that the octahedral Co(H2 0)6+2 complex is in equilibrium

with the tetrahedral Co(H2 0)|2 complex and that for- mation of the latter is favored by increasing temperature. By analogy with halide complexes, the tetrahedral

Figure 11. Differential spectrum of cobalt sulfate versus cobalt

perchlorate; 10 mm cuvets; pH = 1.

configuration would be expected to be approximately one hundred times more absorbing than the octahedral

complex. A very small shift in this equilibrium could

result in a relatively large change in absorbance and

thereby account for the large temperature coefficient of this system. The same explanation may also be

valid for nickel solutions.

C. Liquid Filters of Improved Optical Neutrality

Two approaches have been used in an attempt to improve the optical neutrality and extend the wavelength range of liquid filters. One is based on the use

of empirical mixtures, while the other employs watersoluble, organic "black" dyes. In 1946, Thomson [34] described the preparation

of a grey inorganic solution consisting of chromic, cobal- tous and cupric sulfates and potassium dichromate.

This solution, however, does not transmit below 300 nm. To extend the wavelength range farther into the

ultraviolet, a second exploratory solution (henceforth

referred to as the NBS composite) was prepared in which the cupric and potassium dichromate components of the Thomson mixture were replaced by p- nitrophenol. The absorption spectra of these solutions are compared in figure 12 to a commonly used glass

filter (Chance ON-10). The NBS composite was pre- pared in dilute sulfuric acid and had a pH of 1. No

fluorescence was observed under these conditions.

Over the range 250-600 nm, the NBS composite

exhibits several broad maxima and minima whose

absorbances are less dependent on spectral bandpass

than the Chance ON-10 glass. Examples are given

in figure 13 at two wavelengths for each.

The possible use of either Thomson solution or the NBS composite as an absorbance standard is limited by two factors, both of which arise from the

presence of chromium (III) in these mixtures. First,

105
0.5 / v..

1 CHANCE ON -10 THOMSON SOLUTION NBS COMPOSITE

300 400 WAVELENGTH

,

500 600

Figure 12. Absorptt&n spectra of Thomas solution, an NBS

0.340

0.330

u

0.320 o

<

°? 0.310 o

in

CD< 0.300

0.290

0.280

composite and a Chance ON-10 glass.

1 1 1 1 1 1 1

10 (445 nm)

NBS (415 nm)

_^-^C)N-IO(560 nm)

y/NBS (460nm)

i i i 1 l 1 l 10 20 30 40 50 60

SPECTRAL BANDPASS, nm

70 80

Figure 13. Variation of absorbance with spectral bandpass for NBS composite and Chance ON-10 glass.

an initial "aging" of 6-8 weeks period is needed in order to achieve adequate spectral stability at room

temperature. Secondly, and even more critical for our purpose, is that the absorbances of the aged

solutions exhibit a nonreversible temperature de- pendence. They must therefore always be stored at the temperature at which they were aged.

Hall and Eyring [35] have studied the constitution

of chromic salts in aqueous solution and have sug- gested that during the aging process Cr(H2 0)J 3 ions

polymerize to yield species of the type

H

[ (H2 0)5 Cr—O—Cr(H2 0)5 1 ,

[(H2 0) 4Cr<

HO

O

H

r(H20) 4] +4

and

(H2 0)5—Ci

H

-O—Cr(H2 0)4- H-O—Cr(H2 0)5

in which the observed increase in acidity is explained

by the formation of —OH bridges. Recent studies [36] have shown that temperature, initial pH and the type

of anion present significantly affect the equilibrium

composition. Although refluxing can reduce the aging

period from several months to several hours, this is of no practical advantage since the high temperature

products, on cooling, are no longer stable. In addition to the investigation of empirical mixtures,

several water-soluble organic dyes have also been

studied. Some typical absorption spectra are shown

in figure 14. All of these dyes have high absorptivities

and the concentrations used were of the order of 50

milligrams per liter.

i.o

0.5

NEOLAN BLACK / \ CIBALAN BLACK / \ ALIZARIN LIGHT GREY \ LUXOL FAST BLACK L /

\ NIGROSINE /' ^

200 300 400 WAVELENGTH , nm

500 600

Figure 14. Absorption spectra of aqueous solutions of selected dyes.

Cibalan Black [37], Neolan Black [37] and Alizarin

Light Grey [38] were selected for further evaluation.

Aqueous solutions of these dyes were found insensitive

to pH over the range 2-9, not affected by heat and

relatively light fast. Solutions continuously exposed

to fluorescent light exhibited an absorbance change of about 1 percent over a period of two months. These

initial results appeared sufficiently promising to warrant purification and more extensive testing.

Members of the Organic Chemistry Section have

assisted in this effort. The purification of these dyes

has proven extremely difficult, however, and yields of only a few hundred milligrams of each have been

obtained. Much larger quantities are needed if these

dyes are to be used as Standard Reference Materials.

More efficient purification procedures are presently

being sought.

D. Standard Reference Material (SRM) 931

The first liquid absorbance standard to be issued by

the National Bureau of Standards is SRM 931. The

preparation and certification of this Standard Ref- erence Material are described below.

The filters were prepared by dissolving high purity

cobalt and nickel in a mixture of nitric and perchloric

acids. The weights of cobalt and nickel were chosen so

that the absorbances of their aquo complexes were

approximately equal at their absorption maxima.

The absorbance of nitrate was adjusted to a comparable level by evaporation and subsequent addition

106
Figure 15. Typical absorption spectrum of SRM 931.

of small amounts of nitric acid. The final pH of these

mixtures was about 1. An absorption spectrum of a typical filter is shown

in figure 15. The peaks at 302 and 512 nm are due to absorption by NO3 and Co(H2 0)6+2

, respectively.

The peak at 395 nm and the plateau at 650-700 nm

are due to Ni(H2 0)+ 2

. The filters are supplied in 10 ml ampoules which

have been prescored for easy opening. An SRM unit

consists of three sets of filters, each set containing

a "Blank" (0.1N perchloric acid) and three absorbance

levels of the filter. Each set is packaged in an individual

tray for added convenience.

The filters were calibrated by randomly selecting

ampoules from each lot. The certified absorbances

are as follows:

Table 13. Certification of NBS SRM 931

Net absorbance"

Wavelength and (Bandpass), nm

Filter 302(1.0) 395(1.7) 512(2.0) 678(6.5)

"A"-'Blank' 0.307± 0.003 0.304 ± 0.003 0.303±0.003 0.115 ±0.002

"B"-'Blank' 0.608± 0.005 0.605 ± 0.005 0.606 ±0.005 0.229±0.003

"C"- 'Blank' 0.906 ±0.007 0.907 ±0.007 0.911 ±0.007 0.345 ±0.003

a Net absorbances "A" — 'Blank,' "B" —'Blank,' and "C"— 'Blank' were determined using 10.00 mm cuvets at 25.0 °C.

The uncertainties are given at the 95 percent confi- dence level and include a possible systematic error of ±0.5 percent.

Absorbances are certified at 25.0 °C. Absorbances

at other temperatures in the range 17 to 37 °C may be

calculated using the formula:

At =A25[l + CA (t-25)]

where: At =Absorbance at temperature t (°C)

^25 = Absorbance certified at 25.0 °C

Ca = Fractional change in absorbance per °C

The values of Ca at the four wavelengths are given

below.

k, nm Ca

302 -0.0014

395 +0.0014

512 +0.0018

678 +0.0014

The absorbances of these filters will also depend on

the spectral bandpass used. To insure that the measured absorbances do not differ by more than ±0.1 per- cent from the true values, the effective spectral

bandpass should not exceed 1.5, 2.0, 3.3, and 8.5 nm at 302, 395, 512, and 678 nm, respectively.

IV. Conclusion

The availability of well characterized materials

from which the analyst prepares his own solutions is preferable to the issuance of ampouled liquids.

Although the former are not as convenient to use, they

offer potentially greater accuracy because of their

better stability.

The potassium dichromate and potassium hydrogen

phthalate used in this study are issued by NBS as primary oxidimetric and acidimetric standards (SRM

136c and 84g, respectively). The potassium nitrate is presently issued as a thermal analysis standard

(SRM 756). A product of comparable purity may be

107
obtained by repeated recrystallization of reagent-grade

material from distilled water. The cobalt and nickel

solutions are best prepared from the high purity metals

which are commercially available. To certify absorptivities for these materials, one must resolve the question as to how well these systems

obey Beer's law. For acidic potassium dichromate, the

increased dimerization of HCrO^ to Cr2 Of with

increasing chromium concentration primarily accounts

for the relatively large deviations observed. With

potassium nitrate, one can only speculate that ionic

interactions are enhanced at the higher concentrations! In neither instance are these deviations suffi- ciently large to limit the usefulness of these materials

as liquid absorbance standards. Such deviations do

require that the absorptivity data be used with proper

precautions.

Additional studies are needed to explain the 0.2-

0.3 percent deviations from Beer's law obtained for the various concentrations of alkaline chromate, po- tassium hydrogen phthalate, cobalt perchlorate and

sulfate, and nickel perchlorate and sulfate. Although

internal multiple reflection effects appear to be of the

proper magnitude to account for these differences,

initial attempts to measure such effects with magnesium fluoride-coated cuvets have not been successful. While extremely durable, magnesium fluoride reduces reflections by about 50 percent and more

efficient coatings will probably be needed to resolve

this problem. If internal reflections are responsible

for the observed deviations, the absorptivity data

given should not be corrected when using liquid

absorbance standards for checking the accuracy of the photometric scale since these reflections are included in every measurement. However, in the

determination of molar absorptivity, which is con- sidered a fundamental property of the material, ap- propriate corrections should be applied. Until this question can be resolved, it is recommended that these materials be used at concentrations which yield an absorbance of at least 0.5 when measured in a 10

mm cuvet. With this restriction, it is believed that the

uncertainty of the absorptivity data does not exceed

± 0.5 percent at the 95 percent confidence level.

The authors wish to express their appreciation to Radu Mavrodineanu for providing the calibrated

Schott filters and to David S. Bright for performing

the computer calculations on the HCrO-r/C^Of

equilibrium.

V. References

[1] Gibson, K. S., Nat. Bur. Stand. Circular 484 (1949).

[2] Harrison, G. R., M.I.T. Wavelength Tables (John Wiley & Sons,

Inc., New York, N.Y., 1969).

[3] Meehan, E. J., 'Treatise on Analytical Chemistry', Kolthoff,

I. M., and Elving, P. J., Editors, Part 1, Volume 5, Chapter 54

(John Wiley & Sons, Inc., New York, N.Y., 1964).

[4] Hardy, A. C, and Young, F. M., J. Opt. Soc. Am. 39, 365

(1949). [5] Eberhardt, W. H., J. Opt. Soc. Am. 40, 172 (1950).

[6] Broderson, S., J. Opt. Soc. Am. 44,22 (1954).

[7] Hogness, T. R., Zscheile, F. P., and Sidwell, A. E., J. Phys.

Chem. 41,379(1937).

[8] Perry, J. W., Photoelectric Spectrometry Group Bull. 3, 40

(1950). [9] Slavin, W., Anal. Chem. 35,561 (1963).

[10] Poulson, R. E., Appl. Opt. 3 , 99 (1964).

[11] Goldring, L. S., Hawes, R. C, Hare, G. H., Beckman, A. O., and Stickney, M. E., Anal. Chem. 25, 869 (1953).

[12] British Standard 3875, British Standards Institution (1965).

[13] Mavrodineanu, R., Nat. Bur. Stand. (U.S.), Tech. Note 584, 178 pages (1971), O. Menis and J. I. Shultz, Eds.

[14] Vandenbelt, J. M., Forsyth, J., and Garrett, A., Anal. Chem. 17,

235 (1945).

[15] Haupt, G. W., J. Opt. Soc. Am. 42, 441 (1952).

[16] Haupt, G. W., J. Res. Nat. Bur. Stand. 48, 414 (1952).

[17] Vandenbelt, J. M., and Spurlock, C. H., J. Opt. Soc. Am. 45,

967(1955).

[18] Gridgeman, N. T., Photoelectric Spectrometry Group Bull. 4, 67(1951).

[19] Ketelaar, J. A. A., Fahrenfort, J., Haas, C, Brinkman, G. A., Photoelectric Spectrometry Group Bull. 8, 176 (1955).

[201 Vandenbelt, J. M., J. Opt. Soc. Am. 50, 24 (1960).

[21] Ringbom, A., Complexation in Analytical Chemistry, (John Wiley & Sons, Inc., New York, N.Y., 1963), p. 294.

[22] Neuss, J. D., and Rieman, W., J. Am. Chem. Soc. 56, 2238

(1934).

[23] Howard, J. R., Nair, V. S. K., and Nancollas, G. H., Trans. Faraday Soc. 54, 1034 (1958).

[24] Tong, J., and King, E. L., J. Am. Chem. Soc. 75 , 6180 (1953).

[25] Davies, W. G., and Prue, J. E., Trans. Faraday Soc. 51, 1045

(1955).

[26] Haight, G. P., Richardson, D. C, and Coburn, N. H., J. Inorg. Chem. 3, 1777 (1964).

[27] Johnson, E. A., Photoelectric Spectrometry Group Bull. 17,

505(1967).

[28] Edisbury, J. R., Photoelectric Spectrometry Group Bull. 1, 10(1949).

[29] Edisbury, J. R., Photoelectric Spectrometry Group Bull. 16,

441(1965).

[30] Malitson, I., private communication.

[31] Rains, T. C, private communication.

[32] Ewing, G. W., and Parsons, T., Anal. Chem. 20, 423 (1948).

[33] Swift, T. J., and Connick, R. E., J. Chem. Phys. 37, 307 (1962).

[34] Thomson, L. C, Trans. Faraday Soc. 42, 663 (1946).

[35] Hall, H. T., and Eyring, H., J. Am. Chem. Soc. 72 , 782 (1950).

[36] Burke, R. W., and Deardorff, E. R., Nat. Bur. Stand. (U.S.), tech. Note 544. 153 pages (1970), O. Menis and J. I. Shultz, Eds.

[37] Ciba Chemical and Dye Company, Fair Lawn, New Jersey.

38] Industrial Chemicals Division, Allied Chemical Corporation,

Buffalo, New York.

(Paper 76A5-733)

108
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Accurate Measurement of Molar Absorptivities

Robert W. Burnett

Clinical Chemistry Laboratory, Hartford Hospital, Hartford, Connecticut 06115

(June 7, 1972)

The key to accurate measurement of molar absorptivities is a thorough understanding of the sources of error which appear throughout the measurement procedure. Sources of determinant error

will be listed with comments on estimating their magnitude and eliminating them where possible. Sources of random error will be discussed as well as the propagation of both random and determinant

errors. There is discussion of the need for accurate values of molar absorptivities using examples from

clinical chemistry. Finally, the proper use of accurate absorptivity values in the clinical chemistry

laboratory will be considered. Here, emphasis is on the need for a quality assurance system which

includes routine checks on such things as wavelength calibration and photometric accuracy of spectro- photometers, calibration of analytical balances, and quaUty of incoming reagents. Key words: Molar absorptivity in clinical chemistry; random errors in molar absorptivity; systematic

errors in molar absorptivity.

I. Introduction

Absorption spectrophotometry in the visible and

ultraviolet is undoubtedly one of the most valuable

tools available to the analytical chemist. In clinical chemistry, roughly 60 percent of all determinations

performed utilize this technique. It is natural, therefore, that in the quest for more meaningful data,

considerable attention is focused on accuracy in spectrophotometry, and specifically on accurate meas- urement of molar absorptivities. In this paper the important sources of error which affect molar absorptivity measurements are reviewed and the propagation of the individual errors is discussed. In addition, some of

the rationale underlying the increasing interest in accuracy in spectrophotometry is discussed, with

particular reference to clinical chemistry.

The key to accurate measurement of molar absorptivities is careful attention to the various error sources which are present in the measurement

process. The problem discussed here is that of es- tablishing a molar absorptivity uncertainty which is no greater than one part per thousand. With this in mind, we will be concerned with individual sources of error down to a level of one or two parts per ten thousand, recognizing that many errors contribute to the final uncertainty. This point is demonstrated more

formally in the brief discussion of error propagation.

Much of the material presented on errors is in the nature of a review; it is thought that this may be useful

since discussions of the various errors are somewhat

scattered in the literature, and the mathematical

complexity of some of the original discussions may

obscure their practical implications. Reference is

made to earlier papers for many of the mathematical

details. Molar absorptivity, e, may be defined by Beer's law as

e =M a,

be

where A is absorbance, M is the molecular weight of the solute, b is the effective path-length in centi- meters and c is the solution concentration in grams

per liter. The units of e are thus 1 • mol-1

• cm-1

. Two restrictions apply to eq (1); first, the equation is valid only for monochromatic radiation, and second,

the ratio of absorbance to concentration is constant

only for ideal solutions. The former restriction will be commented upon later and the latter is not usually a problem because measurements are ordinarily made at concentrations below 10~3 mol 1 _1 where deviation

from ideal behavior may be ignored. A related problem

sometimes encountered is commonly known as a chemical deviation from Beer's law, which occurs when the

concentration of the absorbing species does not increase linearly with solute concentration. Examples

of this behavior are well known and the effect will not be commented upon further, except to note that the

acid chromium (VI) system, which has been recently

studied by Burke, et al. [I] 1

, is a particularly important system of this type because of its common use

as an absorbance standard. It is emphasized that adherence to Beer's law over a range of concentrations
should never be assumed in the absence of sup- porting experimental data. In contrast to the chemical

sources of error, the items discussed below may be

classed as instrumental and procedural sources of error in the measurement process.

1 Figures in brackets indicate the literature references at the end of this paper.

II. Systematic Errors

A. Gravimetric and Volumetric Errors

Although these error sources may seem rather

obvious, they deserve mention both for completeness

and because certain components are probably often

ignored. Analytical balances must be calibrated using

weights certified by the National Bureau of Standards.

The solution temperature at which absorbance meas- urements are made must be known and the solvent

density at this temperature should be used in calculating the solution concentration. Accurate temperature measurement is especially important when the

solvent has a high coefficient of thermal expansion;

for example, chloroform shows a volume expansion of

1.3 parts per thousand per °C near room temperature.

In addition, it is interesting to note that the relatively

low heat capacity of many organic solvents means that they are much more sensitive to heating effects than

is water. Chloroform shows 27 times the volume ex- pansion of water for an equal heat input.

Where gravimetric technique is employed in measuring both solute and solvent, it is necessary to take

the apparent specific volume of the solute into account

in calculating the concentration, the relationship being

the cell path length must obviously be known with very

high accuracy. Electromechanical devices known as electronic gauges have been used for this purpose. In expert hands and with careful calibration and repetitive measurements, these instruments are capable of establishing the path length of a cell to ±0.0001 cm,

which for a standard one-cm cell is a relative accuracy

of one part per ten thousand. In addition, the measure- ment may be made at any point on the cell window so

that the parallelism of the windows is also readily

checked. The magnitude of the error which may be

introduced by accepting the nominal value for cell- path length is not commonly appreciated. At the

National Bureau of Standards, in a series of measure- ments of fourteen cells with a one-cm nominal pathlength, thirteen deviated from the nominal value by

greater than one part per thousand and five were in error by greater than 1 percent [4]. As will be seen,

this may easily be the most significant error source in a molar absorptivity measurement. An accurate measure- ment of cell path length will not suffice to eliminate all path length errors, however. Several additional factors which can influence the effective path length are discussed below.

C. Beam Alignment Errors A significant error due to nonparallel incident

radiation appears whenever the most extreme angle

which the light rays make with a fine normal to the

cell windows exceeds about 3°, as shown in figure la.

_ 1000 Wxp

~Ws +WxPv

(2)

©

where c is concentration in grams per liter, Wx is weight of solute in grams, Ws is weight of solvent in grams, p is solvent density in g/cra3 and v is solute apparent specific volume in cm3/g. This is especially

important for solutes of relatively high molecular

weight and low molar absorptivity, in which case the

correction due to v can approach one part per

thousand.

Finally, it must be remembered that in any weighing

process which involves displacement of air, a buoyancy correction must be applied. The magnitude of

this correction depends on the difference in density

between the weights used and the material being

weighed. For water, the correction is about one part

per thousand and it is only slightly smaller for most

solids. Buoyancy corrections for single-pan balances

have been discussed in detail by Burg and Veith [2] and

by Lewis and Woolf [3],

B. Path Length Errors To obtain highly accurate absorbance measurements,

(«0 (b)

FIGURE 1. (a) Increase in effective path length due to nonparallel incident radiation, (b) Increase in effective path length due to improper cell orientation.

This increases the effective path length and, therefore, the observed absorbance. At an absorbance of one, the error is five parts per thousand when the most

extreme rays deviate from the normal by 10°; if the

maximum deviation is 5° the error is under one part

per thousand. Exact corrections depend on the refractive index of the solution and must be obtained by

numerical integration [5]. Proper cell orientation is also critical since cell windows must be normal to the

incident radiation. Any other orientation will result in a longer light path through the cell, as shown in figure lb. This effect is significant at deviations greater than

1° and increases with B2

, the approximate relation being

7T2 0

2

64,800 n

2 110
where 8 is the fractional path length error, 0 is the

angle of incidence of the beam, in degrees, and n is the refractive index of the solvent. The error function

is shown graphically in figure 2 for n = 1.33.

2.0-

Cla

IO

z

LU 1.0 X

<

a.>- 0.5 <

LU

0

where T is the solution transmittance,/i is the fraction

of light reflected at the rear window (considering both

interfaces) and fz, the fraction reflected at the forward

window. Equation (5) may be rewritten in terms of the

relative absorbance error 8,

(6)

Figure 3 shows how 8 varies with A for the condition

/i =y2 = 0.04. This should be recognized as the minimum error which is possible since only the reflections

ANGLE OF INCIDENCE (Degrees)

(0.434 L (fnA

Figure 2. Relative path length error (parts per thousand) due to improper cell orientation, for n = 1.33. From eq (3).

D. Reflection Errors When fight passes from one medium with refractive

index ni to another with refractive index n2 , a fraction

of the fight is reflected at the interface. If the angle of incidence is zero (fight beam normal to the inter- face) the Fresnel relation for the fraction reflected, is

\ni + n2 / An air-glass interface may be roughly represented

by using nair =1.0 and raglass =1.5, in which case /= 0.04, i.e. 4 percent of the incident fight is reflected. Reflection losses can be partially compensated by subtracting the apparent absorbance of pure solvent from

the absorbance of the solution in an identical cell. Unfortunately, this still leaves an important error which is due to multiple reflections within the cell. The major component of this error is due to light which

enters the cell, is reflected back into the solution from

the rear window (the window nearest the detector),

is reflected again at the forward window, and reaches

the detector having traversed the solution three times.

This problem has been considered by Goldring,

et at. [6] who derived the following approximation for AA, the increase in apparent absorbance due to multiple reflections,

AA = 0.434(1 -T2

) (fz4) ( 5 )

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 ABSORBANCE

Figure 3. Relative absorbance error (parts per thousand) due to multiple reflections at cell windows. From eq (6) with /,=/* = 0.04.

at the air-glass interface are considered. In practice,

it may be found that reflections from other surfaces

close to the light path are significant, and f\ and/or /2 may be much greater than 0.04 [6] , although this should not be the case in well-designed instruments.

E. Effect of Finite Slit Width

Since nomenclature in this subject is not completely

standardized, the following definitions are given: slit function plot is the plot of photon flux emergent from

the exit slit versus wavelength; spectral bandwidth is the width at half-height of the slit function plot; natural

bandwidth is the width at half-height of the absorption

band; bandwidth ratio is the ratio of the spectral bandwidth to the natural bandwidth. Since Beer's law is valid only for monochromatic fight, the use of a band of

radiation, albeit narrow, introduces an error unless the

absorption spectrum is extremely flat in the region of

interest. The mathematical formulation of this problem

has been dealt with by several authors [7-9] and

usually involves the assumptions of a triangular slit function plot and a Gaussian shape for the absorption

band. The results obtained show that the observed

absorbance is lower than the true absorbance and that the error is a function of the bandwidth ratio, as shown

in figure 4. Strictly speaking, the error magnitude is 111
0 .01 .02 .03 04 .05 06 07 .08 .09 BANDWIDTH RATIO

FIGURE 4. Relative absorbance error (parts per thousand) due to

finite slit width (9, 10).

also a function of absorbance, but this dependence is negligible as long as the bandwidth ratio is less than

0.4 [9].

F. Stray Radiation

As used here, stray radiation means radiation ap- pearing at the detector with wavelengths outside the

envelope defined by the slit-width and monochromator

dispersion. This radiation may be either unabsorbed

or partially absorbed by the sample. The variation

with wavelength of source intensity, monochromator

transmission and detector sensitivity are all factors which affect the amount of stray radiation present.

For the case where the radiation is unabsorbed it can be shown [11] that the observed absorbance is less than the true absorbance by an amount, AA,

equal to AA = \og(l-r+lOA

r) (7) where r is the fraction of stray radiation present. Table

1 shows the relative absorbance error caused by the

presence of 0.1 percent and 0.01 percent unabsorbed

stray radiation. It must be emphasized, however, that

if a solution has intense absorption bands at other

than the wavelength for which the monochromator is set, the mathematical formulation is considerably more complex. The error may be somewhat less than

is shown in table 1, although there is also the case where the fraction of stray radiation absorbed is greater than the fraction absorbed at the wavelength

of interest. In this case, the observed absorbance is greater than the true absorbance. Measurement of stray fight has been discussed by Slavin [12] and

Poulson [13] among others and extensive correction

tables given by Opler [14]. Naturally, the fraction of

stray light appearing at the detector will be relatively

high in those regions where source output or detector

sensitivity is low, or when working at high absorbances.

Table 1. Absorbance error due to unabsorbed stray

radiation

A —X 1000 (parts per thousand)

r= 0.001 r= 0.0001

0.1 1.12 0.11

.2 1.27 .13

.3 1.44 .14

.4 1.64 .16

.5 1.88 .19

.6 2.15 .22

.7 2.48 .25

.8 2.87 .29

.9 3.34 .33 1.0 3.89 .39

G. Wavelength and Absorbance Accuracy

The spectrophotometer should be calibrated for both wavelength and absorbance as close as possible to the points where they will be used. Obviously the

measurements will be no better than the uncertainty

of the calibration system. The error in absorbance at an absorbance maximum, due to a systematic bias in wavelength setting, is a function of the width of the

absorption band of the compound of interest. The

relative absorbance error is shown in figure 5, and was

calculated assuming a Gaussian band shape and

0 .1 .2 3 .4 5 6 7 S .9 WAVELENGTH OFFSET (nm)

Figure 5. Relative absorbance error (parts per thousand) due to wavelength scale inaccuracy, for absorption bands of various

natural bandwidths (NBW).

neglecting the effect of finite slit-width. With regard to absorbance calibration, it may be noted that if it is feasible to use solutions as absorbance standards, the

systematic errors due to path-length, beam alignment

and multiple reflections may be partially compensated

in the calibration. On the other hand, if the accuracy

of the photometric readings is established by other, more sensitive means such as testing the summation

of photon flux (light addition principle), then these

112
errors must be accounted for separately.

The important sources of systematic error in spectrophotometry have been summarized in table 2. As used

in the table, a positive error is one for which the

apparent absorbance is greater than the true absorbance.

Table 2. Systematic error sources in spectrophotometry

AA/A Range

(parts per thousand)

obtained near A = 1.0 and does not vary significantly

between A = 0.5 and A — 1 .5. It should be kept in mind that the random error is actually a complex function of several variables, and

the consensus is that the procedure to be recom- mended is to experimentally determine AA/A as a

function of absorbance for the particular instrument

to be used. After appropriate steps have been taken to minimize the error associated with each measurement,

the residual random error component may be reduced

further by performing multiple measurements. This

will be commented on in the following section.

Solution temperature

Solute apparent specific volume Buoyancy correction

Cell path-length

Cell orientation Multiple reflections Finite slit-width Stray radiation Wavelength offset

-2 to +2

0 to-1 + 0.2 to + 1 -10 to +10

0 to + 1 + 0.5 to +3

0 to-1

0 to-1

0 to-1

III. Random Errors

In any analytical process, random errors occur whenever an instrument setting is made or a reading is taken. In the simplest type of measurement of a molar

absorptivity, significant random errors can be intro- duced at two points; first, the weighing of solute and

solvent, and second, the measurement of the solution

absorbance. The approach which should be taken is first to consider means of minimizing the errors and

then to estimate the magnitude of the residual errors. The random errors associated with weighing are minimized simply by using a balance of adequate sensitivity

in the range of interest. If the residual error is esti- mated to exceed one part in ten thousand, the actual

magnitude should be determined by experiment.

Random error associated with absorbance measure- ments is a rather difficult topic to treat theoretically,

and the simplifying assumptions found in many dis- cussions may be misleading. The objective is to deter- mine the point on the absorbance scale where the

relative absorbance error, AA/A, is a minimum. Of the

many components which can influence this, the one which often represents the limiting factor is the

detector. The most commonly employed detector in high precision spectrophotometers is the photomultiplier. Briefly, the principal advantage of this device is that for low pfiotocathodic currents the signal-to-noise

ratio varies with the square root of the current, while

photodiodes show a direct proportionality between

these two quantities. Thus, at the relatively low levels of light intensity commonly encountered in spectrophotometry, it can be shown that photomultipliers

operate at a higher signal-to-noise ratio than do photodiodes. Mathematical derivations for a single-beam

instrument have been recently presented by Ingle and

Crouch [15]. An analysis has also been given for a double-beam photomultiplier instrument [16] where it is shown that the minimum relative absorbance error is

IV. Propagation of Error

After all errors discussed above have been minimized and measured, it is necessary to ask how the

residual error components combine to give an overall uncertainty in the molar absorptivity. The simplest case is that where only single measurements are made,

in which case the errors in weighing and in reading

solution absorbance will be propagated as systematic

errors. If F is any function of three independent

variables, F=f(a,b,c), with associated bounds of systematic error, Aa, Ab, and Ac, the bound for the

error in F is given [17] by

AF =

dF

da

Aa +

dF

db

Ab +

dF

dc

Ac (8)

Applying this relation to eq (1) yields the expression

for the uncertainty in e,

A€ =(^) AA +

A

b

2 c Ab + C Ac (9)

Because of the particular form of eq (1), a simplification obtains from writing the result as a relative uncertainty

Ac _ A/4

| Ab| Ac

e A b c

(10)

This equation expresses the important result that the

relative error in e is just the sum of the relative errors

in absorbance, path length and concentration.

Finally, it is noted that the random error component

of Ae can be reduced by performing repetitive measurements. If multiple absorbance measurements are made on the same solution, AA may be decreased, but

A6 and Ac are unaffected and contribute to Ae as before. If many solutions are prepared and measured

independently, both AA and Ac may be reduced, al- though since the reduction of the random error is proportional to Vrc, an improvement by a factor of

two or three is all that may be practical. Moreover, it is clear that systematic errors are unaffected by

repetitive measurements and will always contribute a constant amount to the final uncertainty in molar

absorptivity.

113
V. Spectrophotometric Accuracy in

Clinical Chemistry

Although much attention has been focused on pre- cision in clinical analysis, there is increasing recognition of the importance of accuracy. In a thoughtful

discussion on the status of the clinical laboratory

sciences, Young, et al. state [18]:

"While day-to-day . . . reproducibility . . . is necessary for results to be meaningful

within a single institution, it is essential

that the results are also accurate if they are

to have long-term validity or are to be compared with results from other laboratories."

A prerequisite for accuracy in many clinical analyses

is an accurately known molar absorptivity. An important example is found in clinical enzymology where

measurements of enzyme activities in blood serum

account for 15 to 20 percent of all tests performed in the clinical chemistry laboratory. These activities are measured spectrophotometrically as the rate of utiliza- tion of substrate or rate of appearance of product of the

enzyme-catalyzed reaction, and reported values are given in units of micromoles per minute per unit volume of serum. In these tests, standardization in the usual sense is not feasible and accuracy of results

is directly dependent on the accuracy of absorbance

measurements and accuracy of the value assumed for the molar absorptivity of substrate or product.

The problem is complicated further by the fact that a molar absorptivity is not a solute property, but

may vary markedly with changes in solvent, pH or concentrations of other components in the solution. This is pertinent because of the many different conditions of temperature, pH and buffer composition which

are routinely employed in clinical enzymology. A

panel appointed by the International Federation of

Clinical Chemists is currently engaged in considering

referee methods for enzyme assays in clinical chemistry. It is significant that the goal of the panel is to recommend methods with a systematic bias no greater

than ± 1 percent. If this is to be achieved, it will probably be necessary to know the molar absorptivity

of the substrate or product to within one or two parts

per thousand.

A compound of particular interest in this connection

is nicotinamide adenine dinucleotide (NAD) which is a required coenzyme in many reactions of clinical importance. NADH, the reduced form of NAD, has a

characteristic absorbance maximum at 340 nm, and

rate reactions involving this couple are commonly

followed by measuring absorbance at 340 nm as a function of time. The accepted value of 6220

1 moH • cm-1 for the molar absorptivity of NADH

at 340 nm was determined in 1948 by Horecker and

Kornberg [19]. It is not widely appreciated that their seven experimental values ranged from 5930 to 6310.

These authors considered 6220 to be a minimum value

and estimated a possible error of 2 percent in their assigned value. Sources of error connected with the

spectrophotometer were not discussed. Thus, our best

estimate of this important constant may be more realis- tically stated as 6200 ± 200, and this uncertainty alone

may account for as much as a 2 to 3 percent systematic

bias in many of our enzyme assays. Another area where accurate molar absorptivities

could be used to advantage is that of monitoring quality

of incoming reagents, especially those used as standards. Bilirubin is an example of a compound which is widely used as a standard, but which has been notorious for variations in purity among the several commercial sources. The availability of purified bilirubin from the National Bureau of Standards (SRM 916) and

the determination of its molar absorptivity makes

possible the elimination of an important source of systematic error in this test and makes interlaboratory

comparison of results feasible. It is emphasized that monitoring incoming reagent quality is only one aspect

of an effective quality assurance program in clinical chemistry. Other items which should be routinely

checked include wavelength and absorbance ac- curacy of spectrophotometers, following the practical

suggestions of Rand [20], calibration of balances and

thermometers, and reagent water purity. Each of these

items deserves attention in order to make further progress toward the goal of increased accuracy in clinical chemistry.

VI. Summary

The determination of molar absorptivities with an

overall uncertainty not greater than one part per

thousand is possible only if consideration is given

to the several sources of error which have been dis- cussed. Systematic errors whose magnitude and

direction are both known may be corrected. This

leaves random errors and systematic errors for which

only an estimation of magnitude is available, which are used in estimating the overall uncertainty of the molar

absorptivity value from eq (10). The need for accurate molar absorptivities has been discussed, using ex- amples in clinical chemistry. These values, used in conjunction with a comprehensive quality assurance program, form the basis for increasing accuracy in many clinical chemistry tests, which will result in

clinical laboratory data which are more useful than that presently available.

VII. References

[1] Burke, R. W., Deardorff, E. R., and Bright, D. S., in Nat. Bur. Stand. (U.S.), Tech. Note 584, O. Menis and J. I. Shultz, Eds. (1971), p. 27.

[2] Burg, W. R., and Veith, D. A., J. Chem. Educ. 47, 192 (1970).

[3] Lewis, J. E., and Woolf, L. A., J. Chem. Educ. 48, 639 (1971).

[4] Matwey, J., Analytical Chemistry Division, National Bureau

of Standards, personal communication.

[5] Meehan, E. J., in Treatise on Analytical Chemistry, I. M. Kolthoff, and P. J. Elving, Eds., Part I, Vol. 5, (John Wiley &

Sons, New York, N.Y., 1964), p. 2776.

[6] Goldring, L. S., Hawes, R. C, Hare, G. H., Beckman, A. O., and Stickney, M. E., Anal. Chem. 25 , 869 (1953).

[7] Hardy, A. C, and Young, F. M., J. Opt. Soc. Am. 39,265 (1949).

[8] Eberhardt, W. H., J. Opt. Soc. Am. 40, 172 (1950). 114
[9] Broderson, S., J. Opt. Soc. Am. 44, 22 (1954).

[10] Cary Instruments, Model 16 Spectrophotometer Instruction Manual.

[11] Meehan, E. J., in Treatise on Analytical Chemistry, I. M.

Kolthoff and P. J. Elving, Eds., Part I, Vol. 5 (John Wiley &

Sons, New York, N.Y., 1964), p. 2776.

[12] Slavin, W., Anal. Chem. 35, 561 (1963).

[13] Poulson, R. E., Appl. Opt. 3, 99 (1964).

[14] Opler, A., J. Opt. Soc. Am. 40, 401 (1950).

[15] Ingle, J. D., and Crouch, S. R., Anal. Chem. 43, 1331 (1971).

[16] Optimum Spectrophotometer Parameters, Applied Physics

Corporation Application Report AR14 — 2 (1964).

[17] Ku, H. H., J. Res. Nat. Bur. Stand. (U.S.), 70C (Eng. and Instr.), No. 4, 263-273 (Oct.-Dec. 1966).

[181 Young, D. S., Scott, C. D., and Cole, E. B., Clin. Chem. 17,

818(1971).

[19] Horecker, B. L., Kornberg, A., J. Biol. Chem. 175, 385 (1948).

[20] Rand, R. N., Clin. Chem. 15 , 839 (1969).

(Paper 76A5-734)

I

115

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Problems Associated With the Need for Standardization in

Clinical Spectrophotometric and Fluorometric

Measurements

James R. Penton, Graham M. Widdowson, and George Z. Williams

Research Institute of Laboratory Medicine Institute of Medical Sciences, San Francisco, Calif. 941 1

5

(June 5, 1972)

There is a growing demand in clinical chemistry for analyses to be performed in a manner allowing

comparisons of results among laboratories and, from time to time, in the same laboratory. Reliable comparability requires adequate procedures of standardization for spectrophotometric and fluoro- metric instruments and methods. Problems with chemical and instrumental standardization are discussed. For assays where the substance to be measured is available in suitable form, primary chemical

standardization is justifiably popular. Relatively unsophisticated instrumentation can be used to compare measurements of unknown samples with such standards. Because primary standards meeting

all necessary criteria are not available for many assays of clinical significance, standardization must depend on precision and accuracy of the instrumentation used, and on accurately compiled values of chemical-optical properties for the materials of interest. The task of compilation is outside the capability of the routine laboratory and should be provided by a reliable central agency. If an individual laboratory is to use the agency's compiled values, that laboratory must have available precise, accurate and reasonably inexpensive instrumentation along with reliable absorbance, fluorescence, and wavelength calibration standards.

Key words: Clinical standards; standard reference materials; standardization, spectrofluorometric;

standardization, spectrophotometric.

I. Introduction

Spectrophotometric analyses in the routine clinical chemistry laboratory are performed within a unique

framework. This framework imposes conditions re- sponsible for many of the problems of inaccuracy

and lack of standardization in clinical chemistry.

The factors comprising this framework include:

a. Workload— many clinical chemistry laboratories are performing in excess of a quarter of a million analy- ses each year, and this number has been growing in the United States at a rate as high as 15 percent per annum.

b. Time — to enable prompt action by a physician,

results are nearly always required on the day the

specimen is collected; quite often results are needed

within an hour. A twenty-four hour service must

usually be maintained.

c. Range of analyses — as many as 40 different deter- minations are commonly available on request with a wide range of "special investigations" available by

arrangement.

d. Type of sample — analyses are most commonly

performed on serum or plasma, a viscous, proteinrich complex matrix available in restricted quantity.

e. Cost — clinical chemistry determinations contribute to the rising costs of health care; therefore the

laboratory director must be cost-conscious, though

not at the expense of precision and accuracy.

f. Personnel — the competent clinical chemistry

technician must master a growing number of tech- niques, and operate a wide range of instrumentation

that depends on many different principles.

As long ago as 1947, Belk and Sunderman [l] 1 suggested the need for standardization in the clinical chemistry laboratory. They distributed aqueous solu- tions of glucose, chloride, urea and calcium, serum

for measurements of total protein and albumin, and

citrated whole blood for measurements of hemoglobin

concentration, to a number of laboratories in Pennsylvania. They revealed wide discrepancies between

results from different laboratories analyzing identical

Figures in brackets indicate the literature references at the end of this paper. 117
specimens. For example, 27 of 41 laboratories obtained urea nitrogen results more than 5 mg/100 ml from the

actual concentration of 45 mg/100 ml. That poor

accuracy of results was a worldwide problem was

demonstrated by similar reports from Australia [2], Britain [3], Canada [4], and New Zealand [5]. A major change in the operation of most routine

clinical chemistry laboratories during the last 15 years has resulted from the introduction of mechanized

and automated analytical equipment, both of the dis- crete sample and continuous flow type. That the

introduction of such equipment can improve day-today precision of laboratory results has been demonstrated by Mitchell [6]. Gowenlock [7], reporting on

an interlaboratory trial in Britain in 1965, concluded

that apart from potassium determinations in serum,

laboratories using AutoAnalyzer 2 (Technicon In- struments Corporation, Tarrytown, N.Y.) methods

produced more consistent results. Many laboratories now have quality control schemes usually checking

the precision of results either by using pooled human

sera, commercial control sera or, in the United States,

by participating in the College of American Pathologists' Quality Assurance Service.

It might be assumed that the introduction of mechanized and automated equipment, together

with the use of quality control techniques, would by now have improved accuracy and thus interlaboratory

agreement. The results of the latest surveys are not always reported in a comparable way to those of Belk

and Sunderman [1], making such judgment difficult. For example, the College of American Pathologists'

Quality Evaluation Program for 1971 divided the

results for a given determination into groups according

to the type of analytical method used; no overall range, or range for results within a group, was given.

The results of the national quality control scheme in Britain, however, give the individual results obtained

by each laboratory. Figure 1 (taken from the British

scheme for a week in September 1969) shows the

wide distribution of results from different laboratories

when aliquots of identical serum specimens were

analyzed for uric acid. Even if there has been a slight improvement in the

accuracy of results since 1947, the performance of

clinical chemistry laboratories still falls far below

that which should be expected.

A precise analytical method is not necessarily accurate and herein probably lies a major cause of

the problem. Lack of accuracy in spectrophotometry methods of analysis may be caused by several

factors including lack of suitable standards, inadequate

colorimeters and spectrophotometers; and poor

specificity of the chemical reaction. The latter factor

is outside the scope of this discussion. Though there

is frequently an interdependence between the chemical

and instrumental aspects of analysis, we will consider

them separately.

URIC ACID mg/100 ml

Less than minimum t

NUMBER OF LABORATORIES

Greater than maximum

3.1 TO 3.19 3 T XXX

3 2 TO 3 29 3 T XXX

3.3 TO 3 39 0

3.4 TO 3.49 o

3.5 TO 3 59 7 I XXXXXXX

3.6 TO 3.69 6 [ XXXXXX

3.7 TO 3.79 3 [ XXX

3.8 TO 3.89 10 [ XXXXXXXXXX

39 TO 3.99 7 [ XXXXXXX

4.0 TO 4 09 11 [ XXXXXXXXXXX

4.1 TO 4 19 18 r

L Axxxxxxxxxxxxxxx A A A A A A A A A A A A A A AyxxA A

4 2 TO 4 29 17 r xxxx xxxxxxx xxxxx

x

L A A A A A A A A A A A A A A A A A

4.3 TO 4.39 13 T[ Axxxxxxxxxxxxx A A A A A A A A A A A A

4 4 TO 4.49 14 r xxxxxxxxxxxxxx A A A A AA A A A A A A A A

4 5 TO 4.59 12 ' XXXXXXXXXXXX

4 6 TO 4 69 r YYYVVV AAAAAA

4.7 TO 4 79 3 [ XXX

4 8 TO 4.89 8 [ XXXXXXXX

4.9 TO 4 99 3 [ XXX

5.0 TO 5 09 2 t XX

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the

best available for that purpose.

Figure 1. Histogram of uric acid results obtained when aliquots of a serum specimen were analyzed by 164 laboratories in the United Kingdom, during September, 1969. Courtesy of the Department of Health and Social Security, United Kingdom

Committee on Standards and Quality Control in Clinical Bio- chemistry.

II. Instrumental Considerations

To function appropriately within the described

framework of clinical chemistry requirements, a spectrophotometric analytical system must satisfy

certain criteria. Those criteria concerned with eco- nomics, i.e., initial cost, operating cost and analytical

rate, will, for the purpose of this discussion, be

considered satisfied; only those criteria directly

affecting standardization will be considered. Generally,

these criteria are:

a. Instrument specifications adequate for standardization of the analysis.

b. Instrument meets specifications.

c. Instrument can be easily operated to verified attainable performance.

d. Instrument performance routinely verified in quality control program.

e. Operators understand behavior of instrument

if measurement limits are exceeded.

Satisfaction of the criterion of adequate specification implies a thorough knowledge of the measurement

characteristics of the instrumental system and of the

interaction of those characteristics with those of the

chemical system. It is essential to know: the range of absorbance which the spectrophotometric device will be required to measure, the wavelength at which meas- urements must be made, spectral band widths of

absorption spectra to be investigated, temperature

U8
characteristics of both instrumental and chemical

systems, and calibration stability of the instrumental

system.

Once the necessary characteristics of the instrument

system have been defined, performance must be

verified. An adequate evaluation of instrument

performance requires reference materials of independently verified quality. These materials may be

used to check wavelength accuracy, spectrophotometric linearity and, if necessary, spectrophotometric

accuracy.

A. Wavelength

Several wavelength tests are available. In instru- ments where its use is convenient, Rand [8] recom- mended the mercury vapor lamp as a calibration source because of its many well defined emission

lines. Where use of a mercury vapor lamp is inconvenient because of mechanical considerations, the

sharp absorption bands of didymium and holmium

oxide glass filters [8, 9] can furnish useful calibration

points. These same filters, however, must not be used

for spectrophotometric linearity and accuracy tests. Their narrow absorption band widths, which suit them

for wavelength testing, make their transmission much

too sensitive to wavelength setting accuracy.

B. Spectrophotometric Linearity

Reule [10] pointed out that spectrophotometric

linearity is a necessary, but not a sufficient, condition

for spectrophotometric accuracy. He noted that scale

readings could be linear with light absorbance change

but differ from true absorbance by a nonunity factor. On this basis, he objected to the use of absorbing

materials to test spectrophotometer performance;

and he suggested a precise test method using carefully

controlled additions of light. Reule's method is not

suitable for general clinical laboratory use because of

its complexity. However, the information he presents

is fundamental to understanding of linearity and

accuracy tests. Only spectrophotometric linearity is required in many clinical chemistry assays. In these cases, Reule's objection to tests based on substances which

obey the Lambert-Beer law is negated and stable solutions of substances with broad absorption bands

near the wavelengths of interest may be employed.

Acidic solutions of cobalt ammonium sulfate and

alkaline solutions of potassium chromate [11] have

been suggested by NBS. Rand [8] favored acidic

potassium dichromate (available from NBS as Standard

Reference Material 136c). Buffered solutions of reduced nicotinamide adenine dinucleotide (NADH)

may be useful if their marginal stability is taken into account. Solutions carefully prepared from materials

of known ancestry can serve as useful spectrophotometric linearity benchmarks in both wide and narrow bandwidth spectrophotometers.

An instrumental characteristic which can markedly

reduce the linear range of a photometric device is stray light [12]. Results of an experiment in our

laboratory illustrate the effect of stray light and are shown in figure 2. These data were obtained on an AMINCO Rotochem (American Instrument Company,

Silver Spring, Md.) from determinations of the trans- mittance of solutions of NADH in 0.1 mol-1" 1 phosphate buffer (pH = 7.4). Measurements were taken at 340 nm, both with and without a minus-visible 360 nm

cutoff filter in the photo detector window. The fall-off of linearity seen at low transmittance without the filter results from stray heterochromatic light "sneaking

past" the monochromating element and into the photo

detector without being attenuated by the absorption

bands of the sample. The filter recommended by the

manufacturer in this example is typical of the "stray

light" and "order-sorter" filters found on prism and

grating monochromators. Mis- or disuse of such

filters can degrade the performance of even the best

spectrophotometer to. a level far below the specifications upon which its procurement was based.

with filter

.6 .8 1.0 1.2 .4 1.6 1.8 2.0 2.2 2.4

true absorbance

Figure 2. Absorbance of NADH solutions measured at 340 nm

with and without stray light filter.

C. Spectrophotometric Accuracy

When the goal of instrument testing is verification of spectrophotometric accuracy, several pitfalls appear

beyond those associated with linearity. Even though

linearity tests may be satisfied in characterizing the

spectrophotometric performance of wide bandwidth

devices, in most cases these instruments are not capable of spectrophotometric accuracy. The data in figure 3 were obtained in our laboratory from solutions

of buffered p-nitrophenol measured at 404 nm in an AMINCO Rotochem. The curves show that increasing

spectral bandwidth causes apparent absorbance

decreases from the "true" absorbance measured on

the Beckman DU (Beckman Instruments Inc. , Fullerton,

Calif.) at 0.6 nm bandwidth; linearity remains good.

If the instrument under test is of adequately narrow 119

502-591 0-73
1.2r

1.1 -

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 1.1 1.2 true absorbance

Figure 3. Absorbance of p-nitrophenol solutions measured at 404 nm with bandwidth of 40 nm, 20 nm and 10 nm; plotted against true absorbance.

bandwidth to warrant investigations involving spectrophotometric accuracy, then the materials used to assess this must be considered. In obtaining the

NADH results described in figure 2, we noticed that the measured absorbances were lower than predicted

from weight concentration and the accepted molar

absorptivity of NADH reported by Horecker and

Kornberg [13]. Solutions of NADH (Sigma > 98%

purity) from two lot numbers were prepared in phosphate buffer, and their absorbances measured at 340 nm on a Beckman DU spectrophotometer. Our

calculated molar absorptivities were approximately

two-thirds of the expected value. When the indirect

determination based on enzymatic conversion of

nicotinamide adenine dinucleotide (NAD) to NADH

described in [13] was duplicated, molar absorptivities

in good agreement with the reference value were

obtained. The source of this discrepancy has not yet

been resolved, but it is obvious that direct use of NADH as a spectrophotometric accuracy-test material

must be approached with caution.

Slavin [14] discussed the relative merits of liquid

and solid test materials for spectrophotometric

accuracy assessment and concluded that the requirements of convenience and stability favored use of

solid materials. He described applications of the

Chance-Pilkington (England) ON-10 neutral filter glass and of perforated metal screen filters. Copeland,

King and Willis [15] described the use of the NBS

carbon yellow filter glass as absorbance standard

for the 540 nm assay of cyanmethemoglobin but

lamented the strong wavelength dependence (0.0083

A/nm) of the filter at the desired wavelength.

Wavelength dependence is a criticism common to most reference materials proposed for use in spectrophotometric accuracy tests, especially when such

tests are conducted in the environment of the clinical chemistry laboratory. Each material requires considerable discretion in choice of wavelength range

for which it is used. If the material is used where its absorbance is strongly wavelength-dependent, then

seemingly trivial errors in wavelength calibration can lead to decidedly nontrivial errors in spectrophotometric accuracy assessments. Since the Beckman

DU used by Copeland, King, and Willis [15] has a wavelength accuracy of about ± 0.4 nm, this measure- ment on the NBS carbon yellow filter could have been

in error by 0.0035 A units from wavelength inaccuracy

alone.

D. Functional Considerations

Problems resulting from inadequate wavelength

accuracy, spectrophotometric linearity and spectrophotometric accuracy may be minimized by using

extreme care in specifying and verifying a spectrophotometric system, but we ask what are the implications of poor performance of these functions in the

nonideal world of the clinical chemistry laboratory. In enzyme assay by kinetic measurements, the rate of substrate conversion is calculated from molar

absorptivity and absorbance change with time. When

substrates and conversion products have relatively narrow spectral bandwidth absorption curves, accurate

calculation of the concentration from absorbance and

molar absorptivity will depend on accuracy of wavelength setting. To illustrate the effect of wavelength

dependence, we performed assays of serum alkaline

phosphatase using conversion of p-nitrophenyl

phosphate to p-nitrophenol. Measurements of kinetic

rates were made at the correct wavelength of 404 nm

and at "erroneous" wavelengths of 414 nm and

424 nm. The results in table 1 show that the apparent

activities are considerably lowered by errors in wavelength.

Table 1. Effects of wavelength on apparent activity of alkaline phosphatase

Activity* (LU./L) at 30 °C

404 nm 414 nm 424 nm

38.3 35.4 28.8

68.9 64.3 51.4

59.8 56.3 45.4

38.3 34.7 28.5 57.4 53.2 41.5

50.0 46.3 36.6

* Activities are the average of two assays. Even in the assays in which comparison measure- ments are made between standard and unknown

samples, spectrophotometric linearity is required,

except in the trivial case where the standard and the

unknown have exactly the same fight transmittance.

With instruments of nominally acceptable linearity,

there is still danger that unknown samples may be

encountered with transmittance outside the verified range of linearity, and routine treatment of such

samples will cause reduction in precision of the

measurement. This loss of precision stems from either

120
or both of two sources. The more severe effect results from measuring in a range of transmittance below

that for which the spectrophotometer is linear. Necessity for verification of linearity for each individual

instrument is obvious because some commonly used

clinical colorimeters have significant deviations

from linearity at absorbances as low as 0.5 (Association

of Clinical Biochemists' (A.C.B.) reports [16, 17]).

Another serious, but less apparent, source ot precision loss results from measuring at transmittances

far removed from 0.368 (Absorbance = 0.434). Near

this transmittance Beer's law calculations yield the

lowest relative concentration error (AC/C) for any given

uncertainty in transmission measurement [18, 19]. Willard, Merritt and Dean [20] show the relative concentration error, resulting from a transmittance

measurement uncertainty of 0.4 percent, to range from

8.2 percent at 0.95 T through 1.1 percent at 0.40 T to 8.7 percent at 0.01 T. The assay of enzymes by kinetic rate determination

often requires measurement of small changes in absorbance. Many spectrophotometers commonly

used for direct concentration assay do not have

absorbance resolution adequate to measure these

small differences precisely. Absorbance is proportional to concentration of the absorbing species in direct measurements. Therefore, for a given relative error in absorbance measurement, the relative error in concentration is the same; e.g., if an absorbance

measurement is 0.500 ± 0.002, then the relative error is ±(100 percent X 0.002/0.500) or ±0.4 percent, and the

relative error in concentration calculated from this absorbance measurement also will be ±0.4 percent.

However, if the same spectrophotometric error (±0.002) is present in an instrument used to measure

a kinetic rate, the relative error on the assay results may be much worse. When the enzyme aspartate

aminotransferase (SGOT) is determined by a common

kinetic rate method [21], the absorbance change observed for samples from normal subjects is of the order

of 0.01 per min at 50 °C. Typical absorbances lor two

readings taken 2 min apart are 1.000 and 0.980 and the

difference, 0.02. In the worst case, when the measure- ment errors for each of the two readings are additive,

the relative error could be ± (100 percent X 0.004/

0.020) = ±20 percent. The fundamental problem is that of measuring precisely small differences in large

numbers. Thus, small relative errors in each of the

two measurements can lead to large relative errors in the value derived from their difference.

This fundamental problem of precisely measuring

small differences is further aggravated by the widespread use of instruments with digital display or print- out of readings. While digital displays do much to reduce likelihood of operator reading error, they conceal the fact that precision of absorbance measurement

is more difficult to obtain at low transmittances. It is apparent on a meter scale, linear for percent transmission, that the range of 1.0 to 2.0 absorbance occupies

only 9 percent of the entire transmission scale; hence

a technician may be leery of making readings in this range. But if a digitial display shows 1.931 for one

absorbance reading and 1.968 for another, the operator

is likely to interpret those readings as exact when, in

fact, they are very probably quite inaccurate.

If an analog (continuously variable) signal is to be

displayed digitally, an analog-to-digital conversion

must occur. The accuracy with which the number

displayed represents the original analog signal depends

on the sophistication of conversion circuitry; that accuracy is not absolute and can be quite poor. As an example, we calculated the absorbance resolution of General Medical Sciences — Atomic Energy

Commission (GeMSAEC) spectrophotometric systems.

These systems employ analog-to-digital conversion

circuitry with resolutions of 1/2 10 ~0.1 percent (ten

bit) or 1/2 12 = 1 in 0.025 percent (12 bit). The best

absorbance resolution deliverable by these two converters at several absorbance levels is shown in table 2. The difference in resolution between the two converters is apparent.

Table 2. Absorbance resolution for 10 and 12 bit analog-to-digital converters

A/1 A 10 Bit 12 Bit

0.0 0.00054 0.00014

.3 .00108 .00027

.7 .00174 .00043

1.0 .00546 .00135

1.5 .01773 .00436

2.0 .05798 .01378

E. Routine Instrument Performance Verification

Although the necessity for initial verification of

instrument performance is undeniable, relatively little has been done to incorporate separate instrument

checks into the clinical laboratory quality control

program. For example, this omission may permit an

undetected malfunction to reduce the linear range of

the spectrophotometer. Samples of normal concentration could still give accurate readings, but abnormally

high concentration samples would produce readings

falling outside the range of the instrument's linear response. To be sure, use of control samples simulating

abnormal assay values would reveal this fault, but at the expense of complete re-run of all samples assayed

preceding discovery of the difficulty.

Several wavelength and absorbance verification devices are available in readily usable form. Examples

are the convenient Gilford (Gilford Instrument Laboratories, Oberlin, Ohio) calibration device for their

300-N Microsample Spectrophotometer, and the

Arthur H. Thomas (Arthur H. Thomas Company,

Philadelphia, Pa.) didymium and holmium oxide filter glasses supplied with an adapter to fit into any sample

carrier designed to hold standard 1 cm square cuvettes. However, it must be emphasized that it is appropriate

use, and not mere possession of verification devices

such as these, which standardize laboratory per- formance.

121
F. Operator Considerations

Even an instrument system which is capable of

meeting all necessary specifications will not reliably

perform accurate analyses if that system is not designed to be easily operated in the proper manner. However, it is difficult to visualize a more ergonomically efficient spectrophotometer than the Gilford

300—N. We have seen this instrument used to measure

absorbances greater than 3.0; used to measure

absorbance at two wavelengths without readjusting

the zero setting; calibrated with the zero-set control

and even operated from a cold start with no adjustment checks; all by experienced and presumably well- trained technicians who should know better. An

undetermined but undoubtedly large contribution to poor quality control arises from improper operation of laboratory equipment, due to poor instrument design

and poor operator understanding.

III. Chemical Standardization

Radin, in 1967, [22], reviewed the definition and

use of standards and the standard materials available

to the clinical chemist, and discussed the limitations of various so-called standards. His view that "there is actually very little in the way of official definitions of standards for the clinical chemist" was reinforced by a

joint report of the National Academy of Sciences, National Research Council Subcommittee on Reference

Materials [23]. The situation has improved since that time, as evidenced by the reports of Young and

Mears [24], Meinke [25], and Zief and Michelotti [26]. The improvement has been due to the introduction by

the National Bureau of Standards of Standard Refer- ence Materials (SRM) for use in clinical chemistry. To

July, 1971, these include cholesterol, urea, uric acid,

creatinine, calcium carbonate, bilirubin, D-glucose,

potassium chloride, tris (hydroxymethyl) aminomethane, tris (hydroxymethyl) aminomethane hydrochloride

and glass filters for spectrophotometry.

The importance of the SRM's must not be underestimated; however, they represent a solution to only

part of the problem. The clinical laboratory worker

must be aware of errors which may occur despite the

availability of SRM's; stability of standard solutions is particularly important. The variable stability of solu- tions of uric acid is well known, while Gowenlock [7] showed that in part, the lack of interlaboratory agree- ment for the determination of serum urea could be

attributed to deterioration of aqueous urea standards

containing no preservative. In a few cases, SRM's

when used in aqueous solution are not ideal standards

in clinical chemistry. Recently, we were investigating

the determination of uric acid by an adaptation of the

supposedly specific uricase neocuproine method [27],

both manually and on the Beckman DSA-560. Unable

to get 100 percent recovery of uric acid added to pooled

human serum, we discovered that the slope of the

standard uric acid curve (using SRM) can be varied

according to the amount of bovine albumin added.

When the concentration of bovine albumin is approximately 7.0 g/100 ml, 100 percent recovery of added

uric acid is attainable. A large number of routine clinical chemistry laboratories are using multichannel analyzers of both con- tinuous flow and discrete sample type. Aqueous stand- ard solutions containing a mixture of known amounts

of all the substances commonly being determined in the

routine clinical chemistry laboratory are not available. Multichannel machines are therefore being calibrated

with commercially supplied calibration sera for which

values for each determination have been assigned by

the manufacturers. That wide differences may occur between the values obtained on the same machine

with sera from different manufacturers, and even be- tween lots from the same manufacturer, has been re- ported by Childress [28] and Helman, Reingold and

Gleason [29]. These differences may occur for a variety of reasons, e.g., variation in weight or homogeneity of material. The Scientific and Technical Committee of the Association of Clinical Biochemists

(A.C.B.) [30] reported variation in dry content from

vials (all of the same lot number) of some manufacturer's control sera to be as high as 13.80 percent

C.V. Six of the 10 brands investigated had coefficients of variation of over 2.0 percent. The deviation of the

weight of the contents of these vials could be correlated

to the deviation in analytical results obtained when

these vials were reconstituted.

Data from our laboratory (table 3) shows the percentage deviation from the mean dry weight content of 10

vials of control sera compared with the percentage

deviation from the mean analytical value for several

determinations performed on the contents of each

vial. The 10 vials were selected at random from a box of 40 vials (all of the same lot number); great care was

taken to assure the correct amount of water was added

to each vial. Replicate aliquots from each vial were

analyzed in the same run. Excellent agreement was

obtained between replicate analyses from the same

vial for all the determinations. The variation in dry

weight content of the vials is unacceptable, however. If the contents of each vial is representative of a homogeneous batch, one would expect the deviations from

the mean assay values to be the same as the deviations

of the weight values; this is not the case. Most batches

of control sera from manufacturers are not subject to the large errors shown here. The large errors, however,

pose the question of how frequently smaller and less easily recognized errors occur. The results showing

variability of vial content were obtained on what was

described by the manufacturer as control serum, not

calibration serum. However, the same manufacturing

standards are required for both control and calibration purposes. Amador, Massod, and Franey [31] compared the

values they obtained for aspartate aminotransferase in three commercial control sera with the values assigned

by the manufacturer and found differences of 11 to 17 percent (possibly due to the manufacturer failing to provide sufficient details of the method they used to assign the values). In a more recent paper, Dobrow

and Amador [32] reported a similar study for lactate

122
Table 3. Percentage deviation from the mean dry weight content x of 10 vials of control sera compared with

the percentage deviation from the mean analytical value for several determinations performed on the contents of each vial

Percent deviation from x analytical value Percent deviation from x dry Total

Vial content protein Phosphate Uric acid Calcium Magnesium

1 -1.4 -3.2 -3.3 -0.9 -1.5 10.2

2 1.9 1.4 2.7 5.6 0.7 -5.5

3 2.1 1.4 2.7 5.6 0.7 -10.8

4 -2.1 -1.7 -3.3 -3.0 0.7 10.2 5 . -2.1 -1.7 -3.3 -5.2 -1.5 5.0 6 1.8 4.4 5.7 3.4 0.7 -5.5

7 -2.2 -1.7 -3.3 -5.2 -1.5 5.0 8 -2.2 -1.7 -3.3 -5.2 0.7 5.0 9 1.8 2.9 2.7 3.4 0.7 -5.5

10 2.3 -1.7 2.7 3.4 -1.5 -5.5

dehydrogenase, aspartate aminotransferase, alkaline

and acid phosphatase, and amylase. They concluded

that the activities stated by six of the eight manufacturers were doubtful or unacceptable in more than

50 percent of the sera tested. Bowers, Kelley and

McComb [33] have shown that in some assay pro- cedures, animal alkaline phosphatases (often used in commercial control sera) do not behave identically to human serum alkaline phosphatase.

These findings clearly indicate that currently availa- ble calibration sera should not be used directly as

calibration materials for any analytical procedure.

Cali [34] suggested recently that SRM's make verifi- cation of commercial calibration sera possible; this statement is correct only if referee methods of analysis are available for use in conjunction with the SRM's.

NBS will publish the first referee method for the

determination of calcium shortly. The availability of

referee methods and SRM's will do much to improve

standardization in large laboratories. However, in small laboratories, where the problem is greatest,

shortage of manpower and equipment does not permit

analysis of calibration sera by referee methods. We

suggest that NBS consider the production of reference

sera with values for the common routine determinations assigned through use of their own SRM's and

referee methods. These NBS reference sera would be

used by laboratories to check commercial sera used

for daily calibration of multichannel analyzers.

An alternative to the production of reference sera by

NBS may be a certification program for commercial

products. An example of the way in which an independent laboratory can be used for standard and

reagent certification has been demonstrated by the

cyanmethemoglobin certification program of the

College of American Pathologists, as described by

King and Willis [35]. In the meantime, the situation could be improved if the manufacturers used greater care in the preparation of calibration sera. When assigning values to calibration sera, they should indicate the exact methods

used for this purpose. Values for aspartate aminotransferase and alkaline phosphatase, for example,

are frequently said to have been assigned using the

Karmen [36] or Bessey, Lowry, and Brock [37] methods, respectively. Almost certainly, modifications of

these methods (with regard to substrate concentration

buffer type, etc.) have been used, resulting in quite

different activities from the original methods.

IV. Conclusions

1. Spectrophotometric analyses in the routine

clinical chemistry laboratory are performed within a unique framework.

2. The accuracy of results in clinical chemistry

laboratories falls far below the level that should be

expected.

3. Choice of instrumentation must be based on a thorough understanding of both the characteristics of

the instrument and the assays which must be per- formed.

4. Except in the case of inadequate absorbance

resolution, instrument availability does not generally limit clinical laboratory performance.

5. Materials for verification of instrument per- formance are often not available in a form suitable for routine use in the clinical laboratory.

6. Referee analytical methods are needed for use in conjunction with NBS standard reference materials.

7. The only materials currently available for calibration of multichannel analyzers are commercially

prepared sera. Because of ill-defined methods of assigning values and poorly understood properties of enzymes

from non-human sources, unverified use of these materials for calibration is highly undesirable.

8. The situation with regard to calibration sera could

be improved by:

a. Greater care in the manufacture of calibration sera and provision of information concerning the exact method used to assign values to the material.

b. Certification of commercial calibration sera by an independent laboratory using NBS standard reference

materials and referee methods.

c. Preparation by NBS of reference sera, to which

values are assigned using standard reference materials

123
and referee methods. Such reference sera could be

used by laboratories to assign values to new batches

of commercial sera.

9. The most sophisticated and versatile spectrophotometric system can yield standard results only

when operated within its limitations. Chemical and serum reference materials must be used with due re- gard for their limitations. It is axiomatic then that, to understand and fully control what he can do, the laboratory operator must also understand what he cannot do

with analytical apparatus and methods. He must be

able to recognize bad results as bad and be able to interpret these mishaps to correctly evaluate laboratory performance just as the physician should interpret

good laboratory results in diagnosis of the patient's

physiologic performance.

V. References

[1] Belk, W. P., and Sunderman, F. W., A Survey of the Accuracy of Chemical Analyses in Clinical Laboratories, American

Journal of Clinical Pathology 17, 853 (1947).

[2] Hendry, P. I. A., Proficiency Survey in Clinical Chemistry, 1961. Report of Adelaide Meeting, p. 8. College of Pathologists of Australia, 1961.

[3] Wootton, I. D. P., and King, E. J., Normal values for blood constituents: Inter-hospital differences, Lancet 1, 470 (1953).

[4] Tonks, D. B., A Study of the accuracy and precision of clinical chemistry determinations in 170 Canadian laboratories,

Clinical Chemistry 9, 217 (1963).

[5] Desmond, F. B., A clinical chemistry proficiency survey. New Zealand Medical Journal, 63 , 716 (1964).

[6] Mitchell, F. L., Quality Control in a Large Laboratory, Pro- ceedings of the Association of Clinical Biochemists, 4, 38

(1966).

[7] Gowenlock, A. H., Results of an interlaboratory trial in Britain, Annals of Clinical Biochemistry, 6, 126 (1969).

[8] Rand, Royden H., Practical spectrophotometric standards.

Clinical Chemistry 15 , 839 (1969).

[9] Edisbury, J. R., Practical Hints on Absorption Spectrometry

(Hilger and Watts, London, 1966).

[10] Reule, Alfred, Testing spectrophotometric linearity, Applied

Optics 7, 1023 (1968).

[11] Haupt, Geraldine W., An alkaline solution of potassium chro- mate as a transmittancy standard in the ultraviolet. Journal

of the Optical Society of America 42, 441 (1952).

[12] Poulson, Richard E., Test methods in spectrophotometry:

Stray-light determination, Applied Optics 3, 99 (1964).

[13] Horecker, B. L., and Kornberg, Arthur, The extinction coeffi- cients of the reduced band of pyridine nucleotides, Journal of Biological Chemistry 175, 385 (1948).

[14] Slavin, Walter, Photometric standard for ultraviolet-visible spectrophotometers, Journal of the Optical Society of America

52, 1399 (1962).

[15] Copeland, B. E., King, J., and Willis, C, The National Bureau

of Standards carbon yellow filter as a monitor for spectro- photometric performance, The Journal of Clinical Pathology

49, 459 (1968).

[16] Colorimeters with Flowthrough Cells — A Critical Assessment

of Four Instruments, Scientific Report No. 1. Association of Clinical Biochemists, England (1965).

[17] Colorimeters — A Critical Assessment of Five Commercial

Instruments, Scientific Report No. 2. Association of Clinical Biochemists, England (1966).

[18] Ayres, Gilbert H., Evaluation of accuracy in photometric

analysis. Analytical Chemistry 21, 652 (1949).

[19] Ringbom, Anders, Uber die Genauigkeit der Colorimetrischen Analysenmethoden I. Zeitschrift fur Analytische Chemie

115, 332 (1939).

[20] Willard, H. H., Merritt, L. L., and Dean, J. A., Instrumental Methods of Analysis (Van Nostrand, New Jersey, 1965).

[21] Amador, E., and Wacker, W. E. C, Serum glutamic-oxalacetic transaminase activity. A new modification and an analytical assessment of current assay techniques, Clinical Chemistry

8 , 343 (1962).

[22] Radin, N., What is a standard, Clinical Chemistry 13, 55

(1967).

[23] Report by the Sub-Committee on Reference Materials, Committee on Analytical Chemistry Division of Chemistry and Chemical Technology, National Academy of Sciences —

National Research Council: Standard reference materials

as viewed by the laboratory supervisor — A status report, Analytical Chemistry 40, 24 A (March 1968).

[24] Young, D. S., and Mears, T. W., Measurement and standard

reference materials in clinical chemistry, Clinical Chemistry

14, 929 (1968).

[25] Meinke, W. W., Standard reference materials for clinical measurements. Analytical Chemistry 43, 28 A (May 1971).

[26] Zief, M., and Michelotti, F. W., Clinical chemistry: A challenge

for high purity standards and reagents, Clinical Chemistry

17, 833 (1971).

[27] Morgenstern, S., Flor, R. V., Kaufman, J. H., and Klein, B., The automated determination of serum uric acid, Clinical Chemistry 12 , 748 (1966).

[28] Childress, C. C, An Improved Technique for the Standardization of the SMA 12 AutoAnalyzer, Advances in Automated

Analysis, 1970, p. 167, Technicon International Congress.

[29] Helman, E. Z., Reingold, I. M., Gleason, I. O., Plea for standardization of commercial calibration materials for automated

instruments, Clinical Chemistry 17, 1144 (1971).

[30] A note from Scientific and Technical Committee, Association

of Clinical Biochemists, News Sheet, United Kingdom,

p. 11 (Aug. 1971).

[31] Amador, E., Massod, M. F., Franey, R. J., Reliability of glutamicoxalacetic transaminase methods, American Journal of

Clinical Pathology 47, 419 (1967).

[32] Dobrow, D. A., arid Amador, E., The accuracy of commercial enzyme reference sera, American Journal of Clinical Pathology 53. 60 (1970).

[33] Bowers, G. N., Kelley, M. L., and McComb, R. B., Precision estimates in clinical chemistry. Variability of analytic

results in a survey reference sample related to the use of a nonhuman serum alkaline phosphatase, Clinical Chemistry

14, 595 (1967).

[34] Cali, P. J., SRM's and verification of commercial standards,

Clinical Chemistry 18, 172 (1972).

[35] King, J. W., and Willis, C. E., Cyanmethemoglobin Certifica- tion Program of the College of American Pathologists, American Journal of Clinical Pathology 54 , 496 (1970).

[36] Karmen, A., A note on the spectrophotometric assay of glutamicoxalacetic transaminase in human blood serum, Journal of

Clinical Investigation 34, 131 (1955).

[37] Bessey, O. A., Lowry, O. H., and Brock, M. J., A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum, Journal of Biological Chemistry

164, 321 (1946).

(Paper 76A5-735)

124
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). The Role of Spectrophotometric Standards in the Clinical

Chemistry Laboratory

Royden N. Rand

Department of Pathology, University of Pennsylvania, and William Pepper Laboratory, Hospital of the University

of Pennsylvania, Philadelphia, Pa. 19104

(June 7, 1972)

It is obvious that erroneous data reported to a physician may adversely affect patient welfare. Currently, acceptable limits of accuracy and precision are poorly defined. It should be recognized,

however, that the spectrophotometric measurement step in an appropriate analytical procedure is critical and inapparent error may occur. Spectrophotometric measurements, both manual and auto- mated, are extensively used in the clinical chemistry laboratory. At least 1,000,000 such measurements

per day on rather diverse equipment are made in this country; yet, few standards exist. Results of

intra-lab surveys suggests that performance could be improved. The various ways in which spectro- photometry is used will be illustrated and a discussion of possible errors resulting from nonstandardized instrumentation will follow. There is pressing need for well defined and easily usable standards

for wavelength, photometric accuracy, photometric linearity, stray light, as well as NBS specifications

for optical cuvettes.

Key words: Clinical spectrophotometry, accuracy, precision; optical cuvettes; spectrophotometric

standards, clinical.

I. Introduction

It is not widely appreciated that spectrophotometric methods represent the principal measurement

techniques used in the clinical chemistry laboratory.

A recent text lists 147 analytical techniques; of these,

84 require the use of spectrophotometry [l]. 1 The

twenty-five most widely used techniques at the

William Pepper Laboratory are spectrophotometric.

The numbers of spectrophotometric analyses per- formed in any active hospital are impressive. At the

Pepper Laboratory, for example, of 450,000 analyses

per year about 300,000 require the measurement of the absorbance of light. We, therefore, make 800-

1,000 spectrophotometric measurements per day.

Other information allows us to estimate that more

than 1,000,000 spectrophotometric tests are performed

daily in the clinical laboratories in this country [2]. When it is realized that the growth-rate of clinical chemistry is approximately 15 percent per year, then a perspective of present requirements for ac- curacy and precision is immediately recognized.

Given the information that spectrophotometric

measurements are so widely used, it seems strange

that suitable standards are not readily available and

in widespread use. It seems appropriate, therefore,

for this communication to discuss:

1 Figures in brackets indicate the literature references at the end of this paper.

1. The accuracy and precision requirements for

clinical chemical measurements.

2. The use of spectrophotometry in the laboratory.

3. The present "state of the art" of spectrophotometric measurements.

4. The various types of spectrophotometric errors and how such errors may relate to the accuracy

and precision requirements discussed in section III. 5. The type of standards which are required.

II. Nature of Clinical Laboratory Data

For the fields other than medicine, it is relatively

easy to describe how the quantitative data of the analytical laboratory are used. Several examples can be

cited:

1. The analysis of steel yields information that helps control quality, composition, and costs.

2. The analysis of gold is required to protect the

purchaser's investment.

3. The analysis of the products of organic synthesis

may verify composition and help optimize

yield of desired products.

4. Quantitative analysis of biochemical systems

helps elucidate reaction mechanisms.

125
In general, therefore, quantitative analysis can be used

to obtain data that are used theoretically or empirically,

in rather well defined ways. By comparison, it is difficult to show clearly and un- ambiguously how quantitative clinical laboratory infor- mation is used in the practice of medicine. For example,

while a change in the butterfat content of milk of 0.2 percent can be recognized as economically significant, a comparable change in the total protein content of serum, although easily measured, cannot be so easily

interpreted. In general, small changes in the levels of medically important substances are difficult to inter- pret. Nevertheless, the clinical chemistry laboratory

does quantitative analysis. It may be instructive, therefore, to describe how these data may be used.

A. Generalized Use In Diagnosis

Although recent studies of logic of diagnosis avoid

dealing with the precise role of laboratory tests, it is generally recognized that they play a key role in medical decisionmaking [3]. When a physician sees a patient presenting certain

signs and symptoms, he forms a hypothesis about the

possible diseases compatible with them. He may then

order one or more laboratory tests, the result of which

will help confirm or negate the hypothesis. Given ther likelihood of a disease, the expected increase or de- crease in specific serum constituents helps confirm

the presence of the disease. This use of laboratory data

is essentially qualitative. That which is sought, is evidence of change in blood level clearly outside the

normal range. Such use of laboratory information implies only moderate requirements of precision and

accuracy. From the viewpoint of the analyst, however, the per- spective changes considerably. The concentration

ranges of biologically important substances varies

from micrograms to grams per deciliter. Further, the

maximum concentration ranges encountered in disease

may vary from 1.5 times the mean normal value to as much as 50 or greater. Because of this, the only possi- ble means of assuring consistent information within

and between laboratories, between analyst and be- tween days, is to perform analyses with well defined

and characterized methods, making measurements

with the use of high purity standards and instruments

known to be calibrated correctly.

B. Specific Diagnosis

To a limited extent, quantitative analysis provides

specific diagnostic information. This is obviously true when the analytical procedure provides information

concerning the amount of an abnormal substance —

such as a paraprotein — present in a body fluid. There

are, though, only a limited number of instances where

this is the case, and indeed, few of these analyses use spectrophotometric procedures. Some substances

normally in blood appear to vary within narrow limits. Examples of this are calcium and other electrolytes as well as total protein and albumin within an individual

[4, 5]. Further research in this area using measure- ments of high accuracy and precision may show how

minimal abnormalities in levels of such substances are

related to disease.

C. Therapy Related Decisions

An extremely important aspect of quantitative blood

analysis is in relation to therapeutic decisions. The de- termination of such substances as sodium, potassium,

hemoglobin, bilirubin, and certain drugs are commonly

encountered examples. The accurate quantification of these substances seems to be required for consistent

and reliable medical decision-making. For example, in fetal Rh incompatibility, pediatricians feel that a bili- rubin level of 20 mg/dl suggests a high risk of signifi- cant brain damage and, thus, will intervene with ex- change transfusion. Similarly, routine control of electrolyte levels following surgery requires impressive

quantitative support from the laboratory. Another ex- ample is the quantification of the blood levels of thera- peutic drugs to insure optimal response without toxicity.

Accuracy and precision seem most clearly related to patient welfare for this application of quantitative analysis, where therapeutic decisions may depend on a blood level of some substance.

III. Accuracy and Precision Requirements

The discussion, to this point, suggests that accuracy

and precision requirements of clinical laboratory de- terminations have not been rigorously defined. Several

recent articles, however, have discussed the problem.

The most comprehensive are by Barnett, entitled "Medical Significance of Laboratory Results," and Campbell

and Owen, entitled "Clinical Laboratory Error in Per- spective" [6, 7]. In both, the authors attempt to derive

acceptable limits of variability for commonly used tests on the basis of the consensus of highly qualified physicians,2 table 1 summarizes some of their findings.

The range of values for one standard deviation seems

to be rather broad. For example, in Campbell and Owen,

glucose was thought to require a reproducibility of 0.8-

3.6 mg/dl; Barnett concludes that 5.0 mg/dl is acceptable. This variability may be due to the fact that the

requirements for reproducibility were obtained by a consensus technique. Since there seems to be no cur- rent theoretical or experimental approach to the problem, consensus is the only means of arriving at these

specifications.

Neither paper makes an explicit distinction between

accuracy and precision. The medical decision levels represent the value at which a decision may be made

and the variability represents the allowable precision

limits. Although these are stated to be one standard

deviation, it should be at least questioned as to whether

these would not better represent two standard devia2 Using operations research techniques in his doctoral dissertation, Cavanaugh has

studied the probable effects of laboratory error for three commonly used tests [8]. He, too, found it necessary to use a consensus of informed medical expertise to elucidate the impact

of laboratory error. He concludes that a "cost per laboratory error" can be computed and

that this has meaning in terms "loss of life and limb." The concept is intriguing and Cavanaugh's challenging approach should be further explored. 126
Table 1. Acceptable Analytical Reproducibility

From Barnett [6] and Campbell and Owen [7]

Test

Decision

1CVCI |UJ mg/dl

Acceptable

reproduci"

bility 2 [6] mg/dl

Acceptable

reproducibility2 [7] mg/dl

Glucose 120 5.0 0.8 -3.6 Urea 27 2.0 0.9 -2.4 Calcium 11 0.25 0.03-0.33

Chloride 3 90 2.0 0.6 -2.7 Phosphate 4.5 0.25 0.14-0.25 Sodium 3 130 2.0 1.0 -2.6

1 Level at which a medical decision may be made.

2 1 S.D. in appropriate units.

3 Units for chloride and sodium are milliequivalents per liter, mEq/1.

tion limits. In any case, the listed precision limits are

certainly achievable as one standard deviation limit and probably for two standard deviation limits, by careful work.

For our purposes, it is not necessary to decide

whether one figure or another is acceptable and

correct. We may use them as guidelines to maximum

acceptable error. In fact, we shall use Barnett's

figures later in this paper to help elucidate some

aspects of analytical error and, perhaps, better understand some of the requirements of spectrophotometric

practice.

IV. Spectrophotometry in the Clinical

Laboratory

A. Instrumentation

1. Wide Band Instruments — Wide band instruments

are of many types and varieties with spectral bandwidths greater than 10 nm. These may be simple

filter colorimeters or reasonably sophisticated spectrophotometers. Older instruments are null point type,

whereas newer models are direct reading. Some are very simple; some are relatively complicated with

highly stabilized electronics and digital absorbance

readouts.

2. Narrow Band— The more expensive grating and

prism ultraviolet visible type, narrow spectral bandwidth instruments are less commonly encountered.

These may be single or double beam.

3. Devices Used in Automation — The most commonly

encountered are double beam, interference filter, transmission type equipment.3

B. Type of Analyses Performed With Each Kind

1. Wide Band—The wide band instruments are primarily used for manually performed procedures in the visible area of the spectrum. In general, reagents

are added to react with the substance being quantified

3 Strictly speaking these are not true double beam systems. One beam, of course, passes

through the colored sample; the other passes through a filter of the same transmittance and is sensed by a separate photocell. Thus only variations in the output of the source can

be compensated.

either directly in the fluid being analyzed or in a protein-free filtrate. A color is produced, sometimes

after the application of heat, and its light absorbing

characteristics are compared to those obtained

with standards similarly treated.

2. Narrow Band — The most important applications

of the narrow band instruments are: (1) the deter- mination of enzymes, (2) toxicologically significant

substances, (3) the direct absorptiometry of compounds

such as bilirubin or uric acid, and (4) identification of

unknown substances. In general, for these uses, absorptivities are used to compute the unknown concentrations.

3. Photometric Instruments Used in Automation —

These instruments are used in a manner similar to the wide band instruments. They generally operate in' the visible area of the spectrum and colors of standard

compounds are compared to colors obtained with

biological specimens.

V. "State of the Art" Spectrophotometric

Measurement in the Clinical Laboratory

A. Wide Band Instruments Few studies have been published on the signifi- cance of wide band instruments. A major study,

however, was "Colorimeters — A Critical Assessment

of Five Commercial Instruments" by Broughton and

colleagues [9]. The participants studied two models

of five manufacturer's instruments. The most signifi- cant performance factors were thought to be: (1) reproducibility (2) sensitivity (3) linearity. The overall design of the instrument, the accompanying sample

cells and accessories were also critically discussed.

Stable solutions yielding absorbances of 0.05—

0.55 were used to evaluate reproducibility. Typically, ten readings were made without resetting zero, then

ten additional readings completed the series. Linearity was studied over a wide range of concentrations for

five different chemical procedures, using five separate

wavelengths. Sensitivity was defined as the ratio of

the slope of the calibration curve to that obtained in a narrow band instrument.

A summary of their findings is found in table 2. Detailed examination of the paper makes clear that of

the five wide band instruments examined, only one

could be relied upon to yield calibration curves linear

to an absorbance of 1, as well as sustaining a sensitivity

in the range of 0.7 to 1. Although it is obviously

unfair to extrapolate conclusions from this study to include all wide band instruments, it may be that many instruments of this type will exhibit some

limitations on performance. It seems evident that the

presence of such problems may limit the reliability of

any analyses performed with such equipment. Standards for linearity and sensitivity evaluation are sorely

needed.

Further evidence supporting this point may be found

in a survey for wide band instruments performed in New York State [10]. Relevant data are found in 127
Table 2. Performance of Selected Wide Band

Colorimeters

Reproducibility — when expressed as coefficient of variation, it varied from 1-3 percent at an absorbance of 0.1; above an absorb- ance of 0.4 it was 1 percent or less. Linearity— varied from a continuous curve to linearity through an absorbance of one. Many instruments are linear only to absorbance

of 0.5-0.6. Instruments of same make and type frequently showed

significant differences in performance.

Sensitivity— widely variable, from a low of 21 percent to a high of 120 percent. Instruments of same make and type frequently showed significant differences.

table 3. The three types of instruments listed are ones commonly found in clinical laboratories in this country. Three points are important:

1. The range of results reported for any solution,

for any type of instrument is distressingly wide.

For example, for instrument 1 and solution B,

95 percent of the instruments fall between 0.413

and 0.513.

2. There are significant deviations from linearity for instruments 2 and 3.

3. The relative sensitivity for instruments 2 and 3

are unimpressive.

The reasons for these poor results are not clear. Again, the need for appropriate standards and their Summary of data found in Broughton, et al. [9]. frequent use seems very clear.

Table 3. Performance of Wide Band Spectrophotometers 1

Absorbance

Instrument 2 Bandpass nmNumber of instruments Solution A 3 0.0500M

Solution B 3 0.100M

Deviation in linearity

percent

Relative

sensitivity

percent No. 1 20 75 0.228 ±0.012 0.463 ±0.025 + 1.5 95 No. 2 10 mm 30 34 .191 ±0.017 .382 ±0.039 0 81

12 mm 35 58 .197±0.011 .386 ±0.022 -1.8 82

19 mm 35 67 .193 ±0.013 .368 ±0.027 -4.9 79 No. 3 10 mm 20 33 .202 ±0.022 .395 ±0.044 -2.5 84

12 mm 20 74 .214±0.011 .416 ±0.018 -1.8 82

19 mm 20 51 .206 ±0.008 .400 ±0.019 -3.2 85

1 Vanderlinde, R., Instrumentation Survey— Visible Spectrophotometry. Report of Laboratories for Clinical Chemistry, New York

State Dept. of Health, June 7, 1971.

2 Millimeter designation refers to the size of the test tube used to hold sample.

3 Solutions A, B = 0.0500 M. 0.100 M cobaltous ammonium sulfate in 1 percent (v/v) H2 S04 read at 510 nm; all values corrected to 10 mm

light path.

B. Narrow Band Instruments

It might be supposed that the performance of narrow band instruments would be more impressive

than the wide band types. Unfortunately, this is a

generalization not substantiated by published data. Several surveys bear this out:

1. Even if one looks only at the data for potassium

nitrate, data from the world survey conducted by

the Photoelectric Spectrophotometry Group [11], indicate a spread of results for both absorbance

and peak wavelengths that is unacceptably broad.

2. In mid 1971, the College of American Pathologists (CAP) surveyed narrow band spectrophotometers in a number of clinical laboratories

in the United States [12]. Solutions in sealed

ampules included potassium dichromate (25, 50,

100 mg/1 in 0.01 N H2 S04 ); Thomson Solution

i, \ full strength); alkaline potassium chromate

(40 mg/1 in 0.05 N KOH). Data for some of these

solutions are summarized in figure 1. The means

and two standard deviations, as well as coefficients of variations are graphically shown. At

257 nm, the intra-instrument variability is aston-

-2S.D. MEAN ABSORBANCE +2 S.D. 257 nm

350 nm

450 nm

550 nm

650 nm

0.419 0.673 0.927 50 mg/l Acid Dichromate

CV= 183%

I

+2S.D.

0452 0.524 0.596

50_mQ/l Acid Dichromote

oLs3 0I759 0'

o!627o!e95oL

0.835

CV» 6.9 %

CV« 5.0 %

CV=4.9 %

CV« 6.0 %

0.594 0.676 0:758 THOMPSONS SOLUTION- Full Strength

Figure 1. Performance of narrow band spectrophotometers in the United States. Data from the report of the Subcommittee on Instrumentation,

College of American Pathologists (1971). 128
ishingly high; at the other wavelengths, the coeffi- Table 4. Absorptivity of Potassium Dichromate in cient of variation varied from 5-7 percent. 0.01 H2 SO^

By contrast, a number of laboratories in New York

State were surveyed at about the same time by the (350 nm)

New York State Department of Health [10]. Some 25mg/l High 12.24 of these data are shown in figure 2. By compari- Mean 10.48 son, the variability of instruments in the New Low 8.72 York State survey was remarkably low. Pepper Lab 10.36

MEAN ABSORBANCE

257 nm (492 mg/l) Q ^

REFERENCE "2 S.D. 0703-1-0.723 +2 SB CV= 07 %

LABS 1 0709

FIELD LABS 0.674—

I 0.744 CV = 2.5 %

350 nm (49.4 mg/l) Q528

REFERENCE 0.520-r-0.536 CV= 0.8 %

LABS 1 0524

FIELD LABS Q494 1 0554 CV= 2.9 %

Acid Dichromata Figure 2. Performance of narrow band spectrophotometers in New York State.

Data from Vanderlinde. R., Report of a Survey, N.Y. State Depart- ment of Health (1971).

If the CAP results are representative of the range of spectrophotometric performance in this country, then we need to be concerned with the effect of such

variable performance upon accuracy and precision.

This point is amplified in table 4. The data represent

the absorptivity of acid dichromate computed from

the CAP survey for three concentrations. Shown are the high, low and mean values as well as data obtained

in our laboratory. It is obvious that any analyses

requiring an absorptivity for conversion of absorbance

to concentration would be seriously in error, if a

literature value were used.

It is also worth noting that the currently accepted

value of 10.69 for the absorptivity of acid dichromate

(in 0.1 H2 S04) was not met even at the mean [13]. Our values were also somewhat low. By contrast, we

obtained 10.65 and 10.63 for the 50 mg/l and 100

mg/l solutions in the New York State survey.

50 mg/l High 11.92 Mean 10.48 Low 8.04 Pepper Lab 10.58

100 mg/l High 11.83 Mean 10.09 Low 8.35 Pepper Lab 10.38

1 College of American Pathologists, Instrumentation Survey,

May 1971 [12].

It is difficult to feel complacent about the reliability

of measurements on narrow band instruments in the

clinical labs in this country. When variability of ± 10 percent to ± 39 percent can occur, then surely

the accuracy of analyses performed on such equipment

must be severely questioned.

C. Automatic Instrumentation

To this author's knowledge, no studies of the

photometers used in automatic instrumentation have

been published. However, intra-laboratory surveys of analytical variability have been conducted [14]. Results have been analyzed by separation of the data

from those laboratories using automatic methods

from those using manual techniques. Typical data are shown in table 5. The reasons for the scatter in both

instances include poor standardization and photometric variability. It would seem in order, therefore,

to suggest that the photometric performance of auto- matic instrumentation should be studied in order to

Substance 2

Phosphorus

Number

A. 131

B. 135

C. 134

Table 5. Results of an Intra-Laboratory Trial in Britain 1

Manual methods Mean

concentration

3.037

7.758

4.458

S.D.

0.406

1.231 0.469

Range 3

1.2 to 9.3

3.1 to 10.4

3.1 to 5.5

Number

3535

35

Autoanalyzer methods Mean

concentration

2.860

7.906 4.666

S.D.

0.273 0.576

0.243

Range 3

2.3 to 3.7 6.6 to 9.0 4.0 to 5.3

Urea A. 59 121.80

B. 61 67.03

C. 60 79.73

15.33 15 to 164 113

7.94 53 to 86 112

9.60 52 to 123 112

126.23 6.62 108 to 140

65.32 4.89 45 to 92

81.96 4.81 63 to 100

1 From Gowenlock, A. H., Ann. Clin. Biochem. 6, 126 (1969).

2 Samples A + B were dried sera; Sample C, aqueous solution.

3 Range includes reported results; mean and S.D. are best estimates. 129
assess the magnitude of the photometric component

of analytic error. At the least, linearity specifications

and standards seem to be required.

VI. Common Sources of Spectrophotometric

Error and Their Possible Effect on the

Accuracy and Precision of Clinical Chemical

Analyses

A. Wide Band Instruments

1. Wavelength — Wavelength error is of considerable

significance if colored substances with sharp absorption bands are analyzed. This is true, of course, because the rate of change of absorbance with wavelength will probably be high enough to cause a signifi- cant absorbance error. Usually, however, colored

materials with appreciable broad absorption bands

are analyzed in wide band instruments. Hence, clinical chemical analysis of this sort is little affected by

wavelength error over moderate intervals, particularly

if standards are run concurrently. On the other hand,

it is well known that the optimum wavelength for any

procedure is at the peak of absorbance. It follows that evidence of wavelength accuracy is implicit for

reliable definition of this optimum wavelength.

2. Photometric Accuracy— It is difficult to give

photometric accuracy unambiguous meaning in wide

band instruments [13]. Thus, accurate absorbance

measurements are not really possible in this type of instrument.

Some idea of the magnitude of the errors involved can be gained by study of table 6. This table summarizes an experiment in our laboratory in which a

dilute solution of reduced nicotinamide adenine

dinucleotide (NADH) was made in phosphate buffer

at pH 7.4. Concentration 1 was twice concentration 2. These solutions were placed in 19 mm internal diameter test tubes and read against a phosphate blank in wide band grating instruments of 10 and 20 nm

bandpasses. The same test tubes were used in both

instruments and the readings were completed within a few minutes.

Table 6. Absorbance of NADH in Two Wide Band

Instruments at 340 nm. 1 > 2 > 3 -

Instruments Bandpass Concentration 1 Concentration 2 AA

1 10 nm 0.304 0.156 0.148

2 20 nm 0.233 0.126 0.107

1 NAHD dissolved in 0.1 M phosphate buffer at pH 7.4.

2 Concentration 2 was one-half concentration 1.

3 10 mm round test tubes used for photometric readings.

Several conclusions are immediately obvious from

these data: (1) Instrument 1 shows a more nearly

linear relation of absorbance to concentration. (2)

Instrument 2 gives absorbances significantly below

instrument 1. (3) If NADH coupled enzymes were

being assayed and the NADH concentration changed

from Ci to C2 , instrument 2 would have given an

apparent enzyme activity 20 percent below instru- ment 1. Such differences, traceable to instrumentation, are most distressing. This is further evidence

that the accuracy of spectrophotometric measure- ments in wide band instruments is questionable and

may lead to significant error.

3. Photometric linearity— The wide band instruments

used in most laboratories are assumed to be linear through an absorbance of 1.0. Earlier in this paper were cited data indicating variable performance of wide band instruments with respect to linearity [8]. It seems clear that the percentage error due to un- suspected nonlinearity is a function of the actual

deviation and the absorbance at which nonhnearity

becomes evident. Absorbance errors of more than 10

percent could be due to nonlinearity.

4. Photometric Sensitivity — Again, the British are to be credited for recognizing that linearity and photometric sensitivity are both important instrument

parameters [9]. If an instrument is linear through an absorbance of one, then the slope of the line relating

absorbance to concentration is a function of a number

of variables. These include: (1) sample size, (2) absorptivity of colored complex, (3) optical path, (4) dilution factor, (5) photometric response. Since the

slope and sensitivity may be altered by more than one

factor, it is difficult to weight the photometric component and assign its contribution to analytical error. Indeed, there seems to be no general agreement as to what constitutes optimum sensitivity.

Some indication of the scope of the photometric

problem may be obtained by reference to data found

in table 7. This table lists for a few determinations the

Table 7. Required Sensitivity of Absorbance

Measurements

Analyte Medical

decision

level mg/dl.

Absorbance

sensitivity required

(Autoanalyzer)

Absorbance

sensitivity required

(Manual)

Glucose 120 ±5 0.013 0.005 BUN 27 ±2 0.010 0.080

Uric Acid 6 ±0.5 0.067 0.022 Calcium 11 ±0.25 0.035 0.080 Phosphate 4.5 ± .025 0.016 0.080

From Barnett [6].

acceptable uncertainties pointed out by Barnett and

shown in table 1 [6]. If we use actual absorbanceconcentration relationships found for each of these,

then for each of Barnett's uncertainties, we may compute a minimum change in absorbance which must be

sensed and sensed repeatably. These are shown in 130
column 3 for Autoanalyzer 4 procedures in our laboratory and in column 4 for manual procedures as described in a text [141. Inspection of these data

show the following; (1) The minimum sensitivity

required is 0.005 absorbance and the maximum is 0.080. (2) Of the 10 values listed, five require a sensitivity of 0.022 or less. It seems likely that few current wide band instruments can consistently measure

differences of 0.005; by the same token differences of

0.080 should easily be measured. It should also be

realized that absorbance differences of 0.022 require,

in general, measurement of transmittance„values that

differ by 1 percent or less. Thus, even to meet Barnett's broad tolerances requires spectral measurements of

high sensitivity and precision.

B. Narrow Band Instrumentation

Narrow band spectrophotometers are used for the

determination of a number of substances, some of which are listed in table 8. In addition to these, the

Table 8. Determinations Performed With the Use of

Narrow Band Spectrophotometers

Bilirubin NADH— coupled enzymes

Alkaline phosphatase (kinetic — usingsodium dinitrophenol phosphate)

Barbiturates Diphenylhydantoin Doriden Tolbutamide Hemoglobin derivatives Uric acid at 292 nm

partial characterization of the purity of standards by

the determination of molar absorptivity is growing in importance. This use is discussed in this Journal by

Burnett. (See figs. 1 and 2). Our purpose in this section is to assess the signifi- cance of commonly encountered spectral errors and

their effect on the analytical reliability of the deter- minations involved. It would be beyond the scope of

this article to exhaustively catalog each of the various

determinations. Rather, we may take two different procedures and discuss some aspects of each.

First, let us consider the spectrophotometric deter- mination of bilirubin [1]. Typically, a 1 to 50 dilution

of serum may be made and the absorbance determined

at two wavelengths, (455 and 575 nm). The 455 nm

wavelength represents the bilirubin peak; the 575 nm

peak may be used to correct the bilirubin absorbance

for the contribution of hemoglobin. In table 9 some

computations are based on two assumptions: (1) what we are dealing with is a hypothetical serum in which

no interference from hemoglobin or other absorbing

substances exist, (2) that the absorptivity of bilirubin

in serum at 455 nm is 60.1.

If we refer to Barnett's paper we may note that the

usual medical decision level for serum bilirubin in the

newborn infant with hemolytic disease is 20 mg/dl

and that the uncertainty of the bilirubin measurement

at this level should be ±1.5 mg/dl. One can, then,

compute the expected absorbances that might be

obtained at upper and lower limits; and this is shown

in table 9, column 4.

It is to be noted that on the high side, the expected

absorbance is II percent above the nominal and on the

low side is 8 percent of the nominal. The "allowable"

spread is in the range of 15-20 percent of the mean.

In the previously cited CAP survey, the range of values

reported for Thomson's solution at 450 nm, was 10

percent of the mean. Since one-half of the "allowable"

variation could be accounted for by spectrophoto

metric error, only 5—10 percent remains for all other sources of error. It seems evident that reduction of the measurement error could significantly reduce

analytical error. If this were so, the allowable limits might be set even lower than stated by Barnett.

Similarly, in table 10 some hypothetical computations with respect to NADH are made. That which is assumed is: (1) the "true" molar absorptivity of NADH

is 6.2 X 103

, (2) a 10 percent high or low absorbance error

Table 9. Absorbance of Bilirubin Solutions and Medical Decisions 1

Substance Decision level Upper and lower Absorbance Absorbance Percent of

limits 2 at limits difference decision level mg/dl

Bilirubin 21.5 0.457 0.046 11 20 mg/dl. .411

18.5 .379 .033 8

1 Assumes a direct spectrophotometric procedure reading at 455 nm, an absorptivity of 60.1 and a dilution of 1/50 and no hemoglobin

interference.

2 See Barnett [6].

4 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the best

available for that purpose.

can occur in any instrument, (3) the "true" absorptivity is used to compute enzyme activity in inter- national units.

If such absorbance error occurs, then in column 3

131
Table 10. Spectrophotometric Error and Effective Molar Absorptivity of NADH and Its Effect on Computed

NADH-Coupled Enzyme Levels

NADH level Absorbance Molar absorptivity

Normal enzyme concentration

7.8 mg/dl. 0.700

10 percent low 0.630

10 percent high 0.770 Elevated enzyme concentration

10 percent low

10 percent high

6.2 X103 7 X103

5.7 X 103

6.2 x 103

7 X 103

5.7 X103

Apparent enzyme

activity (U)

(Expected)

32.4

32.4

32.4 (Expected) 404

404

404

Enzyme activity

• (corrected for apparent absorptivity)

(U)

28.6 35.0

358

437

The high and low absorbance values are assumed as possible errors. See figure 1 for justification.

are listed the apparent molar absorptivities corresponding to the erroneous readings. In column 4 are listed

activities for a hypothetical NADH coupled enzyme in the high normal range and a typical elevated enzyme

activity. If we ignore the error occurring in the absorb- ance measurements of the enzyme activity itself, then in the last column are listed the "true" values assignable

to these two enzyme activities based on NADH

absorptivity corresponding to the high and low errors. The large differences are obvious.

Now this has the appearance of a quantitative

argument; it is not intended to be so. It is, rather, a model to show the effect of spectrophotometric error on a clinically important determination. That such

error can occur is attested to by the data in figure 1, from the CAP survey. Two coefficients of variation

at 350 nm were observed to be 13.8 percent.

The common sources of spectral error in narrow band instruments are well understood. Wavelength

error, nonlinearity, photometric inaccuracy, and stray

fight are major contributors. Standards for each of

these are needed urgently. 4

C. Automatic Instrumentation

Let us consider the photometers in use in automatic

analyzers. The basic photometric requirements seem

clear enough: (1) Given a chemistry which is linear,

it is expected that the photometer output would exhibit

basic conformance to the Beer-Bouguer Law over the

range of transmittance of 0.1 to 1.0. (2) The sensitivity

should be such that the desired or optimum absorbance

concentration relationship is achieved and sustained

from determination to determination.

In our hands, the various automatic analyzers seem

to yield adequately linear calibration lines through an absorbance of one.5 Linearity should not be considered

* It is recognized that there are other sources of error which also contribute. Among

these are variability in optical path dimensions, scatter of sample, fluoresence of sample,

reflections, etc. Some of these were reviewed in a classic paper which indicates compelling

obstacles to the determination of absolute absorbance [16] Our argument here is that elimination of major sources of error would allow analytical accuracy at a level rarely approached. The question of the measurement of absolute absorbances and the elimination of all sources of error is a most difficult matter. The major effort now, however, should be

to reduce within and between instrument variability and error to less than 1 percent.

5 This statement does not imply that there is evidence that all Autoanalyzer determinations are inherently linear. Some are; some are not.

apart from sensitivity since considerable flexibility exists with respect to possible ratios of sample to reagent. We may define the appropriate sensitivity as

that which will yield a change in absorbance per

defined detection limits (see table 2) clearly within

the capability of the photometric system. This can be

done by selection of the appropriate sample size; however, one may be hampered because the concentration of analyte which spans the 0-1 absorbance range

may be so small as to require a large number of

dilutions and re-running of samples. Some compromise

is usually accepted.

An experiment was performed to evaluate the ability of an Autoanalyzer to discriminate small changes in concentration. Phosphate solutions in the range 3.23 to 4.73 mg/dl were prepared by dilutions of weighed

reagent grade phosphate salt. These were analyzed on

an Autoanalyzer (modified Sumner technique [17]) , on

three separate days. On each day, the standards used

in the routine lab were utilized to prepare calibration curves. These were linear over the concentration

range 1 mg/dl to 12 mg/dl (absorbance range 0.065 to 0.710). The equation of the standard line was computed by linear regression; this equation was then

used to compute the values obtained for the weighed

materials. Some of the data are summarized in table 11. Over the three experimental days, the determined

values group very closely with the maximum range not exceeding 0.22 mg/dl. Although all estimates are lower

than the weighed-in values, the worst case underestimates by only 0.18 mg/dl. (See day 2-3.12 mg/dl.)

Thus it would appear that at least for aqueous material, we can easily estimate phosphorus within 0.25 mg/dl.

This falls within the specifications cited by Barnett [6]. 6

VII. Current Needs for Spectrophotometric

Standards

The National Bureau of Standards has a current program related to spectrophotometric standards.

9 It is possibly significant to mention that a skilled operator used the instrument. Further- more, there is no evidence that the same sensitivity would apply to proteinaceous materials To obtain data of this quality, standards must be run each time the analyzer is operated, at the very least. 132
Table 11. Ability of an Autoanalyzer to Distinguish

Small Changes in Concentration Using

Aqueous Phosphate Standards

Weighed

concentration Found High-low range

mgldl. mgldl. mgldl. Run 1 Run 2 Run 3

3.23 3.27 3.15 3.25

3.27 3.05 3.16 0.22 3.15 3.25 Mean of 3 determinations = 3. 19

3.46 3.50 3.42 3.42 3.43 3.51 3.42 0.16

3.42 3.34 Mean of 3 determinations = 3.43

3.69 3.69 3.60 3.59 0.19

3.69 3.60 3.50 Mean of 3 determinations = 3.61

4.32 4.26 4.34 4.19 0.15 4.26 4.34 Mean of 3 determinations = 4.29

4.73 4.73 4.70 4.61 0.12

4.73 4.61 4.61 Mean of 3 determinations — 4.66

Current research has been summarized in this Journal

and in another publication [18]. A major contribution of this program is the current

availability of calibrated Schott NG Glass. These

glasses, mounted in a convenient holder, are most

useful for checking photometric accuracy in the visible range. Other standards are also needed, if we are to hope

for spectrophotometric measurements of uniformly

high accuracy and precision. A summary of these

follows:

A. Wavelength Standards

Suitable wavelength standards of universal applicability are strongly indicated. These should be usable in both narrow and wide band instruments.

B. Photometric Accuracy and Linearity

Although the current NG glasses are excellent, it appears that a more neutral glass, optically more

homogeneous, would allow calibration to ±0.1 per- cent relative transmittance. The capability of use in the ultraviolet would be a most important specification.

Additionally, these glasses should be usable in wide

band instruments.

Chemical standards are also needed for both narrow and wide band instruments for primary use in linearity

and sensitivity evaluation. These would also be useful

when necessary to prove that accurate spectrophotometric measurements can be made with liquids. This

in contrast, of course, to proof that the photometric

accuracy of an instrument, itself, is acceptable. Chemical standards should also be available for checking the

linearity and sensitivity of the photometers used in automatic analyzers.

Stray light, of major significance in ultraviolet measurements, needs a standard method for its measurement.

Spectrophotometric grade cuvettes need to be specified with respect to optical path length and wedge. A

standard method of measurement needs to be established. Dr. Barnett's paper, I believe, indicates the

importance of cuvette error in spectrophotometric

systems.

Last, but not least, it should be emphasized that independent means for defining and measuring photometric accuracy need continued research. The pres- ence of the high accuracy instrument at the Bureau

should afford a useful tool for study of the problems

related to this fundamental question.

VII. Summary

This paper has discussed the role of spectrophotometric standards in the clinical laboratory. Its underlying thesis is that errors in the color measuring step

of photometric analysis have largely been ignored. Errors occurring in this step can and do contribute

significantly to analytical error. It can be shown that errors traceable to the color measuring step can be of a magnitude such that medical decisions are made more

difficult or may cause harm to the patient.

The assistance of Mrs. A. Ritz in obtaining some of the data in this paper is recognized with pleasure. The

criticism of D. Arvan, who read the entire manuscript, was most helpful.

IX. References

[1] Tietz, N. W., Ed., Fundamentals of Clinical Chemistry (W. B. Saunders Co., Philadelphia, Pa., 1970).

[2] Brownfield, R. L., personal communication.

[3] Feinstein, A. R., Ann. of Int. Med. 61, 564 (1964).

[4] Gordan, G. S., Loken, H. F., Blum, A., and Teal, G. S., Metabolism 11, 94 (1962).

[5] Cotlove, E., Harris, E. K. and Williams, G. Z., Clin. Chem. 16,

1028(1970).

[6] Barnett, R. N., Amer. J. Clin. Path. 50, 671(1968).

[7] Campbell, D. G. and Owen, J. A., Clin. Biochem. 1, 3 (1967).

[8] Cavanaugh. E. L.. Doctoral Dissertation, Univ. of Calif., Berkeley, Calif. (1968).

[9] Broughton, P. M. G.. Riley, C, Cook. I. G. H., Sanders, P. G., Braunsberg, H., Colorimeters — A Critical Assessment of Five Instruments, Assoc. Clin. Biochemists (1966).

[10] Vanderlinde, R., Report of A Survey, N.Y. State Dept. of Health

(1971).

[11] Anon., Photoelec. Spectrometry GrouD Bull. 16. 441 (1965).

[12] Anon., Report of the Subcommittee on Instrumentation,

Coll. ofAmer. Path. (1971).

[13] Rand, R., Clin. Chem. 15 , 839 (1969).

[14] Gowenlock, A. H., Ann. Clin. Biochem. 6, 126 (1969).

[15] Anon., Clinical Methods Manual, Bausch & Lomb, Inc., Rochester, N.Y. (1965). 133
^ j cu h T T Fds Nat Bur. Stand. (U.S.) Tech.

Gobbing L. S., Ha.e,. R. C B- G. H.

A- O. M "fcfc^^fcJML - SS4. » <»»• SfSTiSi i*.

~ m"' ,Paper 76A5"736)

134
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973).

Spectrophotometric Standards

W. H. Venable, Jr.

Institute for Basic Standards, National Bureau of Standards, Washington, D.C. 20234

(May 31, 1972)

To be useful, spectrophotometric measurements must be believable and practical. The basic standard tor any believable spectrophotometric measurements is the ability to accurately compare

fluxes of radiation within the framework of a well-defined geometry. The emphasis in the program

proposed for the Institute for Basic Standards is to develop such ability over the broadest range of spectrophotometric measurements. Establishing such a basis will enable the National Bureau of Standards to render real assistance to those who deal with the problem of making practical measurements Key words: Photometric accuracy; standards, spectrophotometric.

Spectrophotometric standardization is probably

best described as elusive. This is not to say that it is an animated being which goes skulking from one

hiding place to another, nor is it something which

cannot be obtained. Rather, its elusiveness lies in the extreme care required to define what is to be

measured and to make sure that something else is not being measured instead. Very seldom is there a

one-to-one relationship between the spectrophotometer output and the desired quantity. To standardize

a useful spectrophotometric measurement is to deter- mine what actual relationship exists. This assertion can best be illustrated in terms of very high accuracy measurements. The careful worker

in spectrophotometry, as it is presently applied, can

in general feel confident of an accuracy of plus or minus 0.005 in transmittance in the visible range and,

under very favorable circumstances, attain a confidence to 0.002. Very high accuracy measurement for the purposes of this discussion would correspond to confidence to within less than 0.001 in transmittance

in the visible, with corresponding degradations in the more difficult wavelength regions and for more

difficult geometries. At this level, the effects to be

discussed are very important and can determine

success or failure in achieving the desired accuracy.

It should also be borne in mind that where the ac- curacy really counts is in determining the desired

property of the material being investigated, which is seldom completely or even partially an optical property. If the coupling between the property of interest

and the measured spectrophotometric quantity is weak, as is the case in many health and environmental

measurements, it may be necessary to achieve a very

high spectrophotometric accuracy in order to measure

the desired property even crudely. Consider as an

example some of the factors which enter into the

problem of determining the concentration of a solute in a solution, such as: a. Linearity — Although it is important to know the

linearity of an instrument, knowing it is not sufficient

to assure accuracy in measuring transmittance, since

many other factors contribute to producing the

spectrophotometer output. It should also be noted

that gages, such as filters, which have been calibrated on one instrument cannot provide a highly accurate means of determining the linearity of another instru- ment, since gages have a number of properties which

are measured in different ways by different instruments.

b. Image Shift — Since inserting a medium in the beam changes the optical path length, the focus,

and hence the way the receiver is illuminated, will be changed. Also, if the sample is tipped, as is sometimes done to reduce the effect of interreflections,

the fight reaching the receiver will be shifted laterally. If the receiver is at all sensitive to such varia- tions in illumination, the spectrophotometer will measure this effect as well as transmittance.

c. Reflections — In the usual arrangement of a

solution in a cuvette, there will be four surfaces at which reflections occur, and multiple reflections

between these surfaces contribute to the spectrophotometer output in most cases. If there are any

transmitting glasses such as lenses or windows in the spectrophotometer, there can be additional effects due to reflections from these. d. Index of Refraction — The index of refraction of a solution fluctuates strongly in the wavelength

range occupied by an absorption line. This change

in refractive index will in turn cause image shifts and

changes in reflections within that range of wavelengths.

502-591 O - 73 - 10 135
This again will appear as a part of the spectrophotometer output.

e. Identification of properties — Even if a spectrophotometer were made to measure trans- mittance perfectly, transmittance per se is often not

the property of interest. Transmittance is a complex

quantity which depends upon the measurement

geometry and several properties of the sample, and

thus is not simply related to a single property such as the concentration of a solute in a solution. The foregoing discussion has dealt with some of the

interactions between spectrophotometer and test sample. There are, of course, other sources of diffi- culty such as scattering of light by the sample, changes

in the sample with temperature and stray light from

the monochromator. All of these also contribute to the spectrophotometer output.

The point to be made here is that if high spectrophotometric accuracy is to be achieved, these effects must be accounted for. The electronics, optics, detectors and readouts of the spectrophotometers available

have been greatly improved, so that these instruments can be made much more stable and more sensitive

than they were five or ten years ago. The result has

been more than an order of magnitude improvement

in precision, but there has been no corresponding

improvement in accuracy. Improving accuracy can

only come about through concerted efforts by instru- ment makers, standards laboratories, and those who

need to perform very accurate spectrophotometrie

measurements.

Instrument makers should at the very least make

the optical layout of each instrument known in complete detail so that the owner or prospective owner

can see what stands in the way of his achieving the

accuracy he needs. It would also be a great contribution if each new instrument intended for high accuracy

work were designed for accuracy in a particular type

of measurement, without undue concern that it give

the same "numbers" for a given filter that last year's

instrument gave. The standards laboratory has a many-faceted role

in standardization for high accuracy. Research should

be conducted to find how the magnitude of each of the

various contributions to error can be determined, to find in which case corrections can be made, and in such cases give practical procedures for making the

corrections. When special gages are needed to aid in a particular step of calibration, the standards laboratories should see that these are available and that all the pertinent properties of these gages are made

known. For example, for very high accuracy work, the

absorbance, index of refraction, thickness and scatter- ing properties of a filter should be supplied by the

standards laboratory instead of just a transmittance

value. For such work it makes no sense for a standards

laboratory to pass out gages with a "best value" of some single property attached. The standards laboratory must maintain a versatile, thoroughly evaluated,

and carefully documented measurement system in order to perform its own work and evaluate the work

of others. Also, the standards laboratory must constantly be aware of new developments in spectrophotometry and maintain a close liaison with all groups involved in the measurements in order to assist everyone, including the standards laboratory itself, to achieve the needed measurements.

The ultimate responsibility for standardization of very high accuracy measurements lies in the person or persons performing the measurements. The user must

take the steps necessary to determine what stands in the way of his obtaining the particular data he desires

with his particular instrument and make appropriate

corrections. The operator of the spectrophotometer

may have the knowledge and experience to do this on

his own, or he may have to rely heavily on instructions

provided by others, but he is the one who must standardize his measurements.

What has been described is spectrophotometrie

standardization for very high accuracy— standardization for the future, since very high accuracy spectrophotometry is not being used at the present. However,

factors such as those fisted in the early part of this discussion contribute to any measurement, and they

should be considered when making measurements at any level of accuracy. The cost in effort to obtain

very high accuracy measurements will be great, so

it is extremely important to attempt them only where

the anticipated benefits will be correspondingly

great — they certainly should not be attempted when

not really needed. Very high accuracy measurements

can be obtained with diligence on the part of those

making the measurements, and support for such

measurements can be provided by instrument makers

and standards laboratories, but a change in attitude

and approach by all may be required before they are achieved.

To be useful, spectrophotometrie measurements

must be believable and practical. The basic standard

for any believable spectrophotometrie measurements

is the ability to accurately compare fluxes of radiation

within the framework of a well-defined geometry.

The ability to relate the results of such measurements

to a property which is to be determined can only be

achieved through careful interpretation of the spectrophotometrie output based on considerations such as those described above. The emphasis in the program

being proposed for the Institute for Basic Standards

is to develop a strong measurement capability over the

broadest possible range of spectrophotometrie measurements. Establishing such a capability will enable

the National Bureau of Standards to render real as- sistance to those who deal with the problem of making

practical measurements for a given application.

(Paper 76A5-737)

136
PART II. LUMINESCENCE MEASUREMENTS

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Absolute Spectrofluorometry

W. H. Melhuish

The Institute of Nuclear Sciences, Department of Scientific and Industrial Research

Private Bag, Lower Hutt, New Zealand

(July 11, 1972)

The last 10 years has seen the increasing publication of the emission spectra of organic, inorganic and metal-chelate compounds, but there is no agreed method of presentation of such spectra. In the few cases where corrected emission spectra have been published, there is often no mention of the units used for the intensity coordinate or the method used for correcting spectra. A method of reporting absolute fluorescence spectra originally put forward in 1962 will be reexamined and improved. The

two best known methods for calibrating spectrometers for absolute spectrofluorometry: (a) standard

tungsten lamp, (b) quantum counter method, will be critically examined, and the limitations and possi- ble improvements in accuracy will be proposed. The criteria for an emission standard will be examined

and the use of emission standards for calibrating spectrofluorometers discussed. It is suggested that the distribution of emission standards to laboratories measuring corrected fluorescence spectra and

the analysis and publication of the results should be done in the near future.

Key words: Actinometers; calibration of spectrofluorometers; detectors, absolute; fluorescence spectra, corrected; quantum counters; spectrofluorometers, design; spectrofluorometry, absolute; standard

lamps; standards, fluorescence; thermopiles.

I. Introduction

Although fluorescence from organic molecules or molecular complexes has been used for many years as

a method of trace analysis especially in biochemical

and medical work, it was not until the introduction of

commercial spectrofluorometers in the 1950's that fluorescence spectra began to appear in the literature

in any great numbers. The selectivity and accuracy of

analysis were greatly improved by measuring fluores- cence spectra and also it became possible to recognize

organic molecules in very dilute solution from their

fluorescence spectra. The use of fluorescence spectra

in many photophysical investigations such as determination of quantum efficiencies of fluorescence, studies on the configuration of ground and excited states, elec- tronic energy transfer and so on, has become increasingly important in the last 10 or 15 years [1, 2]. 1 The emission spectrum recorded by a spectrofluorometer, unlike an absorption spectrum, is greatly

distorted by the response function of the instrument

(sensitivity as a function of wavelength), and by

artefacts such as scattered light in the analyzing monochromator. The response function of the spectrofluorometer is determined by the transmission of the

monochromator as a function of wavelength, the spectral sensitivity of the detector (usually a photomultiplier tube) and the variation in transmission and

1 Figures in brackets indicate the literature references at the end of this paper.

focusing properties of lenses (if used) with wavelength.

The response function can only be reliably measured

by using a source of known spectral energy distribution. Absolute spectrofluorometry, therefore, is mainly

concerned with the problem of obtaining sources with

accurately known spectral distributions.

Despite increasing publication of fluorescence spectra, especially in the last 10 years, it is disappointing to find that most spectra have not been corrected in any

way and are thus of limited use to other workers. In

the few cases where corrected spectra have been published, it seems customary to give the intensity "in

arbitrary units" and provide no information on the

dimensions of the units or the method used for correction. Those who publish fluorescence spectra should

take note of a joint statement made by a number of

investigators in 1962 [3] which stresses the impor- tance of reporting the method of correction, subtracting

"background fluorescence" and using appropriate

units (e.g. quanta s

-1 per unit wave number interval).

This list has been extended by Demas and Crosby [4]

to include those factors which are important when

measuring quantum efficiencies of fluorescence from

emission spectra. In view of the fact that absolute calibration of spec- trofluorometers is an exacting task requiring equipment not available in many laboratories, it would seem

important to encourage the use of fluorescence emission standards. These can be used in three ways: (a) for presentation alongside the spectrum being investiL37
gated so that the reader can correct the spectrum from

the most recently known figures for the standard, (b) for calibrating spectrofluorometers, (c) for use in a

ratio spectrofluorometer to give the ratio of the un- known to standard at each wavelength. Some standards have already been proposed [5, 6, 7], but new ones need to be found, especially in the ultraviolet and near

infrared regions. In this account of absolute spectrofluorometry, no

attempt has been made to completely review the litera- ture. The reader will find further references in the recent review article on photoluminescence quantum

yields by Demas and Crosby [4]. Rather the emphasis

will be on the experimental methods in absolute spec- trofluorometry with a critical examination of methods

of calibration, suggestions for improving accuracy and

recommendations on nomenclature.

II. Definitions

A. Units

The recommended radiometric and photometric

units as defined by the Systeme International d'Unites

(SI), are the joule (J), the watt (W) and the lumen (lm). The symbols and defining equations for quantities

used in emission studies are:

radiant energy

radiant flux, <p irradiance, E

spectral irradiance, E(k)

= Q0) = dQldt (W) = ebp/dA (W m-2

) = dE/dk (W m-2 nm- The spectral irradiance, E(k), is commonly used to record the output of black bodies and tungsten lamps.

Unfortunately, there are no SI units for photon quantities and in this review we will use the nomenclature

recently recommended by Muray, Nicodemus, and

Wunderman [8]. The authors propose placing a "q"

as a subscript on radiometric quantity to denote a photon quantity. Thus the photon units for some of the

emission quantities would be:

photon energy

photon flux, ipq photon irradiance, Eq photon spectral irradi- ance, Eq (k)

or Eq (v)

Qa(hv)

dQqldt (hv s" 1 ) difqldA (hv s

_1 m-2

) = dEqldk (hv s

_1 m-2 nm_1

) = dEqldv (hv s1 m-2 (cm"1 )" 1 )

In the past, the quantity Eq

(i>) has been variously

labelled /, q, B, Q( v) , etc., but the new symbol would

appear to be less confusing and we recommend its adoption.

B. Presentation of Emission Spectra

The integrated corrected emission spectrum is pro- portional to the fluorescence efficiency. The most useful quantity for photochemists is the quantum fluoresor I Eq (v)dv. Thus the most useful spectra for publication are Eq (k) or Eq (v) spectra in which the maximum of the curve is given as Eq {v) — 1.00. Many

workers report fluorescence spectra on a wavelength

scale, but a wave number scale is to be preferred especially when vibrational structure is present. It is important to remember that the shapes and positions of the peaks of fluorescence spectra depend on the units used. This is illustrated in figure 1. Here E(k) and

Eq(v) for quinine in 1 N H2SO4 are plotted on the same

graph. The relation between the various units for emission spectra can be shown from the work of Ejder [9] to be:

k

3E(k) = k2Eq (k) = kE(v)=Eq(v).

Thus emission spectra can be readily transformed by

multiplying by the appropriate power of A.; e.g. an Eq (v) spectrum can be converted to an Eq (k) spectrum

by multiplying by A. -2

.

10

0-8

°« 0-6 EW

0-4

y / \\e(a)

\v

02

00 1 1 1 1 1 1 1 1 1 J 20 25

104

x \j

cence efficiency which is proportional to Eq (k)dk

Figure 1. Eq

("v) and E(\ ) spectra of quinine (10"4 M) in /V H2 S04 .

C. Glossary

The following abbreviations will be used in the

following text, diagrams and tables:

FS = front surface

SV = side view

<Pf — quantum efficiency of fluorescence

0D = optical density or absorbance

PM = photomultiplier

M= monochromator

QC = quantum counter TP — thermopile

5(i>) = spectrofluorometer response function re- quired to give Eq (v) emission spectra

k = wavelength (nm)

P=l/\ = wave number (cm-1

). 138
III. Equipment

A. Excitation Sources

Undoubtedly the most popular exciting source for fluorescence is the high pressure xenon lamp in combination with a wide aperture grating monochromator. In order to get sufficient light at wavelengths shorter than

300 nm, it is common to use lamps of 450 watts or

larger and these have the advantage of being somewhat

more stable than the lower wattage lamps. If a very

intense exciting source is needed, a medium or high

pressure mercury lamp can be used with appropriate

niters. When measuring the fluorescence spectra of concentrated solutions, powder or single crystals

where front surface illumination is necessary (Sec. V),

stray light from the exciting monochromator can be a problem, especially if the fluorescence emission from

the sample is weak. Stray light can either be light ap- pearing in the second or higher orders, or instrumental

scatter of many wavelengths. This second type of

instrumental scatter is easily demonstrated by placing

the eye at the exit slit of the excitation monochromator

when its wavelength is set in the UV region. Interfer- ence from second order stray light can be greatly re- duced by using suitable nonfluorescent cutoff filters or bandpass filters [10]. Unfortunately few commercial

spectrofluorometers have filter holders and it is necessary to have them fitted. Double grating monochromators will reduce instrumental scattered light to a large

extent, but the throughput is also reduced. Good practi- cal discussions on the use of filters and monochromators is given by Kortiim [11] and Parker [2].

The amount of stray fight from a Gillieson type monochromator (as modified by Schroder [12]) when a mercury lamp is placed at the entrance slit is shown in figure 2, dashed line. The wavelength setting was 365

-6 10 ^ 1 ' 1—

1-5 20 2-5 3-0

10 4 xV cm"1

Figure 2. Stray light in excitation and analyzing monochromators. # - mercury lines. Dashed line: stray light from excitation monochromator set as 365 nm with a mercury lamp at entrance slit, and a 2c filter at exit

slit.Solid line: spurious peaks due to stray light in analyzing monochromator set at 365 nm, with a mercury lamp at the entrance slit.

nm, the bandwidth was 20 nm and the 390 lines/mm

grating was blazed at 350 nm. The light output was

scanned with a Jarrell-Ash 2 1/4 meter grating monochromator. The units are <pQ for the mercury lines and

Eq (v) for the continuum. A Wratten 2 C filter (\ cutoff

at 390 nm) was placed at the entrance slit of the 1/4

meter monochromator to eliminate errors due to scat- tering and multiple diffraction of the intense line in the

analyzing monochromator (see Sec. Ill, B). The stray

mercury lines may be further reduced by choosing a

suitable bandpass filter for the exciting line.

B. Analyzing Monochromators

The most readily available and least expensive monochromator is the single grating instrument based on

the Ebert-Fastie or Czerny-Turner mountings (fig. 3,

b and c). The Gillieson mounting (fig. 3a) is sometimes used

in monochromators intended for excitation purposes.

Most commercial instruments use single grating monochromators and provided one is aware of their limitations, they are adequate for measuring the rather

broad fluorescence spectra given by organic molecules

in solution. The advantages of grating monochromators

over prism ones are: (a) linear wavelength dispersion,

(b) high aperture and (c) easy interchange of gratings

for different spectral regions. However, the grating

monochromator generally has higher scattered light

and the overlapping of orders can be a problem.

The most important source of stray light in a grating

monochromator, particularly one of large aperture, is

1 /

(a)

G

1

(b)

G

(c)

Figure 3. Types of grating monochromators: (a) Gillieson, (b) Ebert-Fastie, (c) Czerny-Turner. (G = plane grating).

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no

instances does such identification imply endorsement by the National Bureau of Standards,

nor does it imply that the particular product or equipment is necessarily the best available

for that purpose. 139
multiple diffraction. One possible pathway for double

diffraction is shown by the dashed lines in Figure 3c. This well known problem has not been quantitatively

investigated until recently [12, 13]. Multiple-diffracted

light can be reduced with a mask, but this reduces the

transmission of the monochromator. An experimental

determination of stray light in the Jarrell-Ash 1/4 meter

grating monochromator with a grating blazed at 600 nm

is shown in figure 2, solid line. The exciting wavelength was 365 nm and the baffle inside the monochromator was removed for these measurements. Many of the

spurious peaks shown are due to multiple diffraction as may be proved by removing the exciting fight with

a Wratten 2 C filter; the peaks disappear, but the stray

mercury lines from the excitation monochromator

are not affected. This spurious light is particularly

troublesome when scattered exciting fight cannot be

avoided as when measuring multicrystalline material

from the FS or measuring solutions with a very low

fluorescent intensity. In these cases it is advisable to remove the exciting light with a sharp cutoff filter and make corrections for any fluorescence absorbed

by the filter. The minimizing and correction of stray

light in spectrofluorometry will be further discussed

in Section V, A.

C. Detectors and Amplifiers

The detector used in spectrofluorometry is the photomultiplier. End-window photomultipliers are to be

preferred, since there is no grid in front of the photosurface to obstruct the narrow fight beam from the exit

slit of the analyzing monochromator. A quartz window

photomultipfier with an S-20 photo surface has been

found to have good sensitivity over the range 220 to 800 nm. A modern tube such as the EMI type 9698

QB has a very low dark current (~10~ 10 A) and does

not need cooling.

For fluorescence spectral measurements in the

range 800 to 1100 nm, a cooled photomultipfier with

and S— 1 sensitivity curve is commonly used. It is possible that an uncooled P—I— N silicon diode may

prove to be a more useful detector in this wavelength

region [14], but unfortunately the surface areas of

silicon photodiodes presently available are too small to be useful in spectrofluorometry.

For amplification of photocurrents of S5 10-12 A,

solid state electrometers of the type described by Weinberger [15] are quite adequate and inexpensive.

Selectable time constants ranging from 0.1-2 s. should

be included in the amplifier to improve the signal-tonoise ratio. Photocurrents less than 10~ 12 A are best

measured by photon counting. Not only is it possible to use simple amplifiers and discriminators [16, 17] but

'

greater accuracy with shorter sampling times is achieved [18]. It should be noted that the photocathode should be at ground potential when the photomultipfier is used for photon counting to minimize

noise due to current leakage from the photocathode.

D. Design of Spectrof luorometers

Parker [2] has discussed the design of spectrofluo- rometers and several instruments have been described

in the literature [19-22]. Few instruments, however,

are completely satisfactory if they are required to perform a number of different functions, and we

believe the following criteria should be examined

before building a spectrofluorometer:

(a) There should be space around the sample so

that heating and cooling baths, flow cells, liquid

helium Dewars, etc., may be fitted. (b) The instrument should be double beam in order

to eliminate fight source fluctuation and have the

ability to measure excitation spectra.

(c) The sample should be capable of being observed

from the front face and from the side.

(d) Excitation fight source should be easily exchangeable.

(e) There should be space for fitting filters at the

exit beam of the excitation monochromator and

at the entrance slit of the analyzing monochromator.

(f) Space should be

discs, e.g., for spectra.

(g) The scanning speed should

adjustable over a wide range.

(h) Amplifiers should have adjustable time

stants to improve the signal-to-noise ratio.

available for fitting chopping

measuring phosphorescence

be accurately

A design used in this laboratory which meets all of these requirements is shown in figure 4. Three conM1

(a)

O 10cm.

1

|M2

*

(b)

(c)

FIGURE 4. Spectrofluorometer design. Ml, M2 = monochromators,

Q = quartz beam splitter, f = filters, M = spherical aluminized

mirror, P = photocell, PM = photomultiplier, QC = quantum

counter, S = scatterer, L = quartz lens. 140
figurations are shown: (a) for measuring fluorescence

spectra using FS excitation, (b) for measuring fluo- rescence spectra with SV and (c) configuration for calibrating the analyzing monochromator and photomultiplier, using the quantum counter method (Sec.

IV, C). Light shields have not been shown on the dia- gram. Excitation spectra can be measured by replacing

P in figure 4a with a quantum counter. The two signals

from the monitoring photocell (or quantum counter

photomultiplier) and the analyzing photomultiplier are amplified by electrometers and led to a ratio recorder

of the type described by Haugen and Marcus [19]. Components are held rigidly on a series of optical

benches contained in a hinged box between the two

monochromators.

The spectrum recorded by spectrofluorometers is usually an uncorrected one plotted on a wavelength

scale. The correction of the spectrum and replotting on a wave number scale is very laborious and instru- ments for the direct correction of spectra, as they are recorded, have been described [22, 23]. However these

corrected curves must still be replotted if a wave number scale is required. The use of small, inexpensive

on-line computers is becoming increasingly common

and in the future it is likely that these will be used to correct both ordinates and plot the spectrum directly.

The only additional equipment needed for the spectrofluorometer is a digital voltmeter with binary coded

decimal (BCD) output and several stepping motors.

Interfacing to a small computer such as the PDP-8

presents no problems.

IV. Calibration Methods

A. Introduction

The emission spectrum recorded by a simple spectrofluorometer, i.e., one without built-in automatic

correction devices, is distorted not only by the scat- tering artefacts described in Section III, B, but also by the wavelength dependence of the detector and

monochromator transmission. Only two calibration

procedures for determining this response function

have so far been developed. The first method depends

on the assumption that a tungsten lamp operated at a

color temperature, Tc , has a spectral distribution of a black body at Tc K multiplied by the emissivity

(e( A.)) of tungsten:

E(\) =

Zirhc \- 5 e(\)

[exp (0.01438/ \TC )]-1 (1)

where A is in meters. This emission standard is then used to calibrate the

system as described in Section IV, B. The second method makes use of an intense source

of light and a monochromator. A detector of known

response measures the watts (or quanta s _1

) emerging

from the exit slit at various wavelengths. This cali- brated source is then used to determine the response

function of the spectrofluorometer. These two methods

will be critically examined in Sections B and C,

following.

It is important to note that the spectrofluorometer

is not being calibrated under precisely the same conditions that pertain to the measuring of fluorescence

spectra. Errors may arise if: (a) different areas of the

grating or prism are illuminated; (b) different areas

of the photomultiplier photocathode are illuminated;

or (c) if the calibration curve depends on the plane of

polarization of the light.

B. Tungsten Lamp Calibration

Vavilov [33], one of the first to measure a corrected

fluorescense spectrum, made visual spectrometric

comparisons between the output of a tungsten lamp

of known color temperature and the radiant flux from

a fluorescent solution. The greatest source of error in the use of tungsten lamps appears to be the uncertainty in the spectral emissivity (e(X)) of tungsten. To

overcome this error, Stair et al. [24] at the National

Bureau of Standards, Washington, D.C., individually

calibrate tungsten strip lamps against a high temperature black body, and it is probable that these lamps

(type U90) are the most accurate spectral radiant sources presently available. The uncertainty in out- put ranges from a maximum of 8 percent at short

wavelengths to 3 percent at long wavelengths [24]. A description of the use of a standard tungsten lamp

is given by Christiansen and Ames [25]. Certain

precautions must be taken in the use of standard

tungsten lamps. The lamp housing dimensions, the

position and the current used must be precisely as specified and the radiance must be taken normal to the tungsten strip from a small area in the middle.

The output of tungsten lamps is very low in the near UV region and care must be taken to correct for stray

light by the filter subtraction method [25]. We have

found in practice that it is not possible to use the NBS

standard lamp below about 340 nm when calibrating a

single grating monochromator and photomultiplier,

owing to excessive stray light.

A good standard lamp, such as the NBS type U90

is expensive, difficult to use and has a limited lifetime. The usual practice is to compare the standard lamp

against an inexpensive lamp which is used as a secondary standard. Such lamps are commercially available. Care must be taken with secondary standards that errors are not introduced by reflections from the glass

envelope. Lippert et al. [5] made a secondary standard

lamp which was filled with argon and backed by a horn-shaped absorber of the sort used in Raman

spectroscopy.

C. Calibrated Emission Monochromator

The idea of taking a xenon lamp and monochromator

and measuring the radiant flux from the exit slit in order to calibrate a monochromator and photomultiplier appears to have first been advanced by White

et al. [26]. They used fluorescein in NaOH to detect

the number of quanta s _1 emerging from the mono141
chromator over the wavelength range 250 to 300 nm.

Melhuish [27] and Parker [28] describe the method in more detail. Since the accuracy of the method depends

primarily on the detector used to measure the radiant

flux, we will discuss detectors in more detail below

(Sec. IV, D). A fixed fraction of the flux from the exit beam of the exciting monochromator is reflected [27] or scattered [28] into the analyzing monochromator and

the photomultiplier current measured at each wavelength. Eastman [29] scatters the light into the analyzing monochromator with colloidal silica (Ludox) in order to have a geometry close to what will be used

when measuring fluorescence spectra. In calculating

the correction factor, S(v), it is important to allow for the wavelength dependence of the scatterer and the

transmission characteristics of the quantum counter window and any beam splitters used in the optical

system. For the apparatus used in this laboratory

(fig. 4c),

S(v) = <p q (v)M(v)W1 (P)

r2 ( v) jR(i>)di>

where <p q (v) — relative number quanta s -1 observed by

the detector

M(~v) = reflectivity of scatterer Wi(v) — transmittance of the quartz beam

splitter W<l ( v) = transmittance of the quantum counter window

R(v) — recorder deflection

Equation (2) is strictly valid only if S{i>) is constant over the bandwidth of slit used in the analyzing monochromator, typically 4 to 6 nm. A preliminary calibration

is made using equation (2) to determine S(v). The

correct calibration factor is then calculated from

if>q {v)M{v)Wl {v)

v) \l [S(

(3) v0 )IS{i>)]R(v)dv

where S(i>o) is the correction factor at the center of

the scanned band. Scorr. (i>) will usually differ from

S(i>) by not more than 2 or 3 percent, except in the

region of 700 to 800 nm where the photomultiplier

sensitivity changes very rapidly with wavelength.

This procedure allows one to choose any slit width for the excitation and analyzing monochromator and is the

most reliable method to use. Very little error is introduced if peak heights instead of I R{v)dv are

measured provided, (a) the bandwidth of the light

leaving the excitation monochromator is 4 or 5 times

less than the bandwidth of the analyzing monochromator and, (b) S(i>) does not change rapidly over the

bandwidth defined by the slit of the analyzing monochromator. Calibration by measuring peak height was

used by Melhuish [27] and Parker [28], a procedure

which was justified because S(v) did not change

rapidly with wavelength over the wavelength range

investigated.

It has been pointed out by B0rresen and Parker [30] that any silica focusing lenses in the excitation monochromator system can cause calibration errors because

a constant area of the scatterer receives varying

amounts of the exciting flux depending on the wavelength, while the quantum counter measures the total photon flux in the beam.

The accuracy of this method of calibration rests on

the accuracy of the response of the detector measuring

<fq(v) (Sec. IV, D) and on the accuracy to which the

spectral reflectivity factor M

(

v ) is known. Aluminum

films were used in the calibration technique described

previously [27] because if these are prepared by rapid

evaporation at low pressure, the reflectivity is constant over a wide wavelength range [31]. The reflectivity of

magnesium oxide seems to be less well known. Values

published by Benford, Schwartz and Lloyd [32] were

obtained using an integrating sphere. Multiple scatter- ing in the sphere could result in a lower reflectivity at some wavelengths, than for a single scattering. We

have recently found that the spectral reflectivity of

fresh MgO (1 mm thick) to be the same as a freshly

deposited aluminum film to within ±1.5 percent from

340 to 800 nm (fig. 5). There was a slight decrease

(~3 percent) at 300 nm, the shortest wavelength

investigated. Good values of M( v ) for single scattering

from MgO are clearly needed, especially since the

MgO scatterer is used in the determination of absolute

quantum efficiencies of fluorescence [43]

.

100

M (a) 0-98

0-96

o o o

1- o O oooo°o 0 oo-i

300 500 700

A nm

Figure 5. Reflectivity (M(k)) of freshly deposited MgO versus a

fresh rapidly deposited aluminum surface.

D. Detectors The calibration of the emission monochromator by

measuring the energy of quanta s _1 from the exit beam

requires a detector with an accurately known spectral response. Three detectors have been used for this and

each of these will be critically examined.

(1) Thermopiles: These detectors are coated with

a layer which absorbs the radiation, causing the heating

of a number of thermopiles. The thermoelectric current must be amplified with a high gain, low drift current

amplifier. Thermopiles are generally compensated for changes in ambient temperature, but warm objects,

such as the human body or a hot lamp housing, can

cause changes in output. The absorbing coating on

142
thermopiles (lamp black or blackened gold) is often

assumed to absorb all UV, visible and infrared light,

but this is not true. The reflectivity of lamp black is known to increase in the UV region and so for a properly

calibrated thermopile, the reflectivity of the actual

coating on the junctions ought to be measured. The

response curves of three thermopiles measured by

Christiansen and Ames [25] differ considerably from

each other (40-80 percent at 250 nm) so that it cannot

be assumed that all thermopiles have a constant

sensitivity over a wide wavelength range. The chief

disadvantage of the thermopile is its low sensitivity.

The measurement of the output of the emission monochromator becomes very difficult as a result of the narrow bandwidth required (1 to 3 nm) for calibration

of the analyzing monochromator and photomultiplier.

The wide wavelength response of a thermopile might

actually be a disadvantage since stray infrared energy

in the beam would also be detected. White et al. [26]

and Rosen and Edelman [21] have used thermopiles

for calibration purposes on the assumption of the flat energy response.

(2) Quantum Counters: Early investigations by

Vavilov [33] showed that the energy efficiency of many

dyes in solution was approximately proportional to the

exciting wavelength (i.e. <pf independent of X) except

near the absorption maximum. Further work by

Anderson and Bird [34] , Harrison and Leighton [35]

and Bowen [36] showed that for many dyes, <£/ was

independent of wavelength over a wide range and that such solutions could, therefore, be used to determine

quanta s -1 in the UV and visible region by measuring

the fluorescent flux from a fairly concentrated solution

of the dye. Rhodamine B (4 g/1 in ethylene glycol) was

first tested and used as a wide range quantum counter by Melhuish [37] . An important innovation of this new

quantum counter was to move the photomultiplier so

that no directly transmitted light could be detected.

It was now possible to reliably measure the quanta s _1

in a broad fluorescence band even if the tail of the band

extended beyond 600 nm. The preferred geometry for a quantum counter is that of Parker [38] which not only avoids errors from directly transmitted light, but

uses the full thickness of the quantum counter as a

filter (fig. 4c). By comparing the rhodamine B quantum

counter with a thermopile [27, 39] it has been shown

that the response of the quantum counter is constant

to within about ± 5 percent from 220 to 600 nm. Deviations which are observed may in fact be due to j the

thermopile and not the quantum counter [27]. Another

method of testing a quantum counter is to use one for measuring excitation spectra. For an ideal quantum

counter, the excitation spectrum should agree with the

absorption spectrum, provided that <ff is independent

of A. and the OD of the fluorescent solution being

measured does not exceed 0.05. A series of substances

may be selected to cover a wide wavelength range

and by limiting the excitation range to only one

electronic band, the probability that iff changes with

wavelength is reduced. Figure 6a shows the percent

deviation of the excitation spectrum from the absorp-

(a)

t5%-\ . • • . v •• ••• • .

-5%- • •

-

i i i i_

300 400 500 600

X nm

(b)

4

1

l~PM

I---V---

i i ->

Figure 6. (a) Deviation of excitation spectra from absorption spectra (±3%).

(b) Apparatus for measuring excitation spectra.

tion spectrum for solutions of 2 aminopyridine,

quinine, 3 aminophthalimide, proflavine, fluorescein,

rhodamine B and methylene blue using a rhodamine B

quantum counter in the apparatus shown in figure 6b. Solutions were freshly prepared since deterioration

occurred when the solutions were exposed to fight.

The <pf of quinine in 1 N H2 S04 , for example, was

25 percent too low at 310 nm if left for three days in the light. The dashed line in figure 6a shows the

comparison of a thermopile with the rhodamine B

quantum counter [27].

Methylene blue has been used as a quantum

counter by Seely [40]. A 1.5 g/1 solution of methylene

blue in ethylene glycol can be used from 520 to 700

nm, but it is about 50 times less sensitive than the

rhodamine B quantum counter when both are used

with an S-20 photomultiplier.

The advantage of the quantum counter over the

thermopile is its high sensitivity. Moreover, if several

different fluorescent compounds are used as quantum

counters, all of which give the same response, we can

be reasonably assured that we are actually measuring

the quanta s

_1 in the beam. The apparatus needed

for calibrating an analyzing monochromator and photomultiplier using a quantum counter is illustrated in figure 4c. The excitation monochromator has a bandwidth of 1 to 2 nm and the analyzing monochromator

a bandwidth of 4 to 8 nm. A filter cutting off at 350 nm

is inserted at / (fig. 4c) when calibrating between 400

and 700 nm. The outputs from the amplifier connected

to the quantum counter and the photomultiplier

amplifier are fed to a ratio recorder. It is important to refer back to a particular wavelength from time to time, to correct for drift in the system.

143
(3) Actinometers: At least one author [26] has

calibrated the output of an emission monochromator

using the uranyl oxalate actinometer. The quantum

yield of the more sensitive ferrioxalate actinometer

has been measured as a function of wavelength by

Parker and Hatchard [42] and could probably be

used for calibration, although long exposure times

would still be required. The wavelength response of the actinometer is not constant and its yield at different wavelengths must be measured against a detector

such as the thermopile. Thus, the accuracy of the

yield versus wavelength of the actinometer can be no

better than the thermopile, which, as we have seen

above, may not be very well known especially in the

UV region.

E. Polarization Effects The transmission of a grating monochromator de- pends on the direction of polarization of light incident on the entrance slit. Thus the correction factor,

S(v), which has been measured for completely unpolarized light will not apply to plane polarized light.

Figure 7 shows the correction curves for a Jarrell-Ash

V4 meter monochromator (blaze = 600 nm) and a 9698- QB (S-20) photomultiplier. If the fluorescence

spectrum being measured is not completely de- polarized the correction factor S(v) obtained for unpolarized light cannot be used for correcting the

spectrum. The true spectrum might be obtained using

a polarizer at the entrance slit of the analyzing monochromator and recording || and _L spectra, correcting

each by the appropriate S(v) curve and adding. It is also important to recognise that the S(v) depends on

the plane of polarization when investigating polar- ization of emission spectra using a grating

monochromator.

J I L 300 500 700

A nm

FIGURE 7. Correction curves [S(t>)] for a grating monochromator

and photomultiplier = unpolarized light, — polarization || to grating rulings, = polarization ± to grating

rulings.

The light from the exit slit of a grating monochromator is also polarized to some extent, the degree of polarization varying with the wavelength setting. Hence,

in using this light to calibrate an analyzing monochromator as described in Section IV, C, it is important

to depolarize the light by scattering off a magnesium

oxide scatterer. The use of an aluminum reflector can sometimes give errors of 10 percent or more and,

therefore, should not be used.

V. Methods of Measuring Spectra

A. Dilute Solutions

When the absorption spectrum overlaps the fluo- rescence spectrum, a true fluorescence spectrum, free from reabsorption effects, can only be obtained in dilute solution. It is important that the cell be blackened with matt black paint on three sides, if observation is from the front surface, or two sides, if viewed

from the side. The use of side viewing reduces stray

exciting light which is particularly important if the

exciting wavelength is within the emission band. Stray

light is due to the following causes:

(a) fluorescence of the solvent and cell material

(b) Raman and Rayleigh scattering by the solvent

molecules

(c) scattering of light at interfaces or from dust in the solution.

Stray exciting light can often be reduced by filtering

the solution to remove solid particles and this should

be done as normal practice. Sometimes it is possible

to choose a light filter which prevents stray exciting

light from reaching the inlet slit of the analyzing

monochromator, but does not absorb any of the

fluorescence. Stray background light should always

be checked after the measurement of the fluorescence

spectrum by filling the cell with clean solvent and

rescanning. Any stray light is then subtracted from the

observed fluorescence spectrum.

Most spectrofluorometers measure fluorescence

emitted at right angles to the exciting beam (SV mode).

The decrease of fluorescence in these instruments as the concentration of the fluorescence is increased,

sometimes attributed to concentration quenching, is actually a geometric effect caused by a shift in the

region of absorption in the cell towards the front surface where it cannot be seen. Thus, at high con- centrations of solute, the fluorescence must be

observed from the same side as the exciting light enters (FS mode). FS viewing may also be used for dilute solutions; in fact, more fluorescence can be

collected by the analyzing monochromator than in the

SV mode. However, stray exciting light scattered

from the front face of the cell will be much larger

than in the SV mode. We have found that a 5 X 10"6 M

(3 parts per million) solution of quinine in H2SO4

excited at 365 nm to be the lowest concentration that can be used in the FS mode before stray light becomes

excessive.

144
Before correcting the fluorescence spectrum it is important to see if the refractive index of the solvent

varies over the emission band scanned since the

amount of light entering the slit of the analyzing monochromator from the fluorescent solution is proportional

to re 2

. For a fluorescent solute in benzene, for example,

the photon irradiance increases by 3 percent from

434 to 588 nm as a result of dispersion in liquid

benzene.

B. Concentrated Solutions and Crystals

When the absorption coefficient of the sample for the exciting light is so high that absorption is virtually

complete in the first millimeter or less of the sample,

fluorescence must be observed from the front surface. The scattering of exciting light into the analyzing monochromator now becomes a problem, especially if the

sample is multicrystalline. Furthermore, it is not usually possible to measure a blank for crystalline

samples. Although the use of a double monochromator

for analyzing the fluorescence helps, the only real solution is to use a sharp cutoff filter to remove the

exciting light. If the fluorescence emission falls within

the region where the filter absorbs, the transmission

of the filter must be measured and the observed

fluorescence spectrum corrected for absorption by

the filter. A more serious error arises from reabsorption of fluorescence if the absorption and fluorescence spectra

overlap. This error can be corrected by making use

of the equation for reabsorption of fluorescence for the special case of front surface viewing [43]. For

highly absorbing solutions where shifts in the region

of absorption can be neglected, this equation becomes

dB(k')=Af(k')dk'ax (l + <pK'+<p2 K"+. . .)

f exp [— (ct\ + aK x)z]dz

Jo

(4)

A

/(V)

dB(k')

where a prime refers to fluorescence, unprimed to the

exciting light, = constant = true (corrected) fluorescence , spectrum

= observed (corrected) fluorescence

spectrum a = 2.303 eC where € = molar extinction

coefficient and

C= molar concentration

ac = sec [sin -1 (sin 8/n)] where 6= angle

between the exciting beam (J. to the

cell face) and the observing beam

and re = refractive index of the solvent

/= thickness of the cell 2= distance from the front face K' , K", . . . = reabsorption-emission factor [43]

absolute quantum efficiency of

fluorescence.

«p=,

Equation (4) can be simplified further if absorption

of the exciting light is sufficiently high that

exp— («x + aK -x) < 1

and it is assumed that all the K factors are equal.

Then

dB(k') = [AI(l-<pK)][aKl(aK + aK x)]f(^') dk'

.

(5)

Since the first term in square brackets is independent

of A., the observed spectrum dB(k') may be corrected

by multiplying by (a\ + aK 'x)/aK at each wavelength.

Perylene in ethanol has a large overlap between

absorption and fluorescence spectra and this com- pound has been used to test eq (5). The fluorescence

spectra of 5 X 10~4 M and 10~6 M solutions of perylene

in a 10 mm cell is shown in figure 8. The solution was excited and observed from the front surface.

The spectrum was corrected using the rhodamine

B quantum counter method (Sec. IV, C and D) using an MgO scatterer. The cell was blackened on 3 sides

and corrections were made for stray light with solvent

in the cell. The bandwidth of the analyzing monochromator was 1.5 nm. The spectrum of the 5 X 10~4 M solution, when corrected by eq (5) (Open circles,

fig. 8) agrees well with the 10~6 M spectrum.

2 0 2-1 2-2 10 4 xv cm"1 2-3

Figure 8. Fluorescence spectra of perylene in ethanol, k e = 365 nm, FS. = 10-6 M, = 5 X lO"4 M, = absorption

spectrum.

By using a very thin cell and choosing an excitation

wavelength where a>, is as large as possible, the re- absorption effect can be minimised. Thus, 5 X 10~4 M

perylene in a 0.2 mm-cell when excited at 404 nm gives a fluorescence spectrum almost identical (±3%) with

the 10"6 M spectrum in the 1-cm cell.

C. Narrow Bands

The measurement of a fluorescence spectrum with narrow bands require narrow slits and a slow scanning

speed. As a rough guide to the settings needed to re- cord a spectrum without significant error, we may use 145
the equation of Petrash [44] which states that the

error in a recorded band of Gaussian or dispersion

shape is approximately proportional to

(y-tdXIdt) 4'9 (6)

where dk/dt= scan rate and y= half-line width. Thus,

if the time constant of the spectrofluorometer is ad- justed so that the error in the recording is no greater

than 1 percent, the scanning rate would have to be

reduced nearly 100 times to record a band of onetenth the width with the same accuracy. An incorrect

setting of scanning rate or time constant is easily ob- served by stopping the scan on the steeply rising part of the band and observing any subsequent movement of the pen to a higher reading.

D. Reporting Corrected Fluorescence Spectra

The most useful fluorescence spectrum for publication is one measured in very dilute solution where

errors due to reabsorption of fluorescence are negligible. However, there are several other factors which

could influence the shape of the spectrum and, therefore, the author should give a complete description of

the measuring conditions. These are listed below (see Ref. 3). Conditions (a) to (d) are the most important,

but the complete list should be given if the spectrum is suggested as a standard.

(a) If a standard tungsten lamp is used for cali- bration, describe the origin and the accuracy of

its spectral distribution. Were corrections made

for scatter in the monochromator? (See Sec. IV, B.)

(b) If a standard fluorescent substance is used, give

the author and list the factors (d) to (m) below.

(c) If a quantum counter, thermopile or actinometer is used for calibration, give full details of the detector. Describe the system used with particular reference to the spectral reflectance of

the scatterer used.

(d) Clearly state the units used for the fluorescence

flux; Eq {v) = quanta s

_1 per unit wave number

interval is preferred.

(e) Were corrections made for stray background

light using the pure solvent? (See Sec. V, A.)

(f) State the geometry of the apparatus, whether

front surface or side viewing and the dimensions of the cell.

(g) Give the bandwidth of the analyzing monochromator.

(h) The source, purity and concentration of solute should be stated.

(i) The solvent used and method of purification

should be noted.

(j) The temperature should be given,

(k) Was the cell blackened on any of its faces?

(1) State the excitation wavelength and its spectral

purity,

(m) Give (pj , if known.

Some commercial spectrofluorometers record

absolute fluorescence spectra. It is important to know

what units are used for the fluorescence flux and

convert to quanta s

_1 per unit wave number interval,

if necessary. These instruments use thermopiles or correction cams in order to automatically correct the emission spectrum and their accuracy is, therefore,

uncertain. In reporting fluorescence spectra obtained

with these instruments, it would seem desirable to include the spectrum of one or more standards.

E. Future Developments

If the time comes when there are a sufficient number

of precisely known standards covering a wide wavelength range, a double beam spectrofluorometer,

which takes the ratio of the fluorescence fluxes of the unknown and standard at such wavelength,

would overcome the necessity of calibrating the analyzing monochromator and detector. If the observed ratio of unknown to the standard at the frequency is R(v),

then

Eg {v) x = Eq (p) s XR('v ) where subscript x = unknown, and s = standard.

Lippert et al. [5] describe a double beam spectrofluorometer of this design using a 13 Hz chopping

frequency. The advantage of a double beam system

is that fluctuation in the exciting light or in the gain

of the photomultiplier does not influence the recorded

spectrum.

VI. Standards

A. Introduction

An absolute emission standard is ideally one which

satisfies the following criteria:

(a) Broad wavelength range with no fine structure

(b) Small overlap between absorption and fluores- cence spectra

(c) Easily obtained and purified

(d) Stable

(e) High (pf

(f) Spectrum independent of exciting wavelength

(g) Clear, free from scattering centers

(h) Completely depolarized emission.

A standard with a broad emission band with no peaks is important so that the spectrum does not de- pend on the resolution of the analyzing monochromator.

A high iff and a reasonably high OD at the exciting

wavelength are both important to reduce errors due

to stray fight (Sec. V, A). A number of absolute standards have been proposed, notably by Lippert et al. [5], but the quinine spectrum is the only one measured by

a number of authors. The new standard 2-amino

pyridine proposed by Rusakowicz and Testa [45] covers approximately the same wavelength range as 2-naphthol and is to be preferred since it does not have the temperature effects shown by the latter. 146
The effect of reabsorption of fluorescence on the

emission spectrum has been discussed in Section V,

B. Although standards should be measured in very

dilute solution where reabsorption has no effect,

sometimes spectra are determined at high concentrations. It is important, therefore, to have some idea of what region of the standard spectrum might be affected

by reabsorption. The upper limit of the error due to reabsorption is given by eq (5). The term A = aJ

(a\ + a\x) has been included in tables 1 to 5 to show the wave number region where reabsorption

errors can be expected.

B. Individual Standards

1. Quinine in sulfuric acid (fig. 9). Table 1 lists the determination since 1941 of the absolute fluores- cence spectrum of quinine. Many of these figures have

been taken from rather small diagrams and are therefore not accurate to better than ± 3 percent. All spectra

have been converted to Eq {v) spectra using the appropriate conversion factors. We have found that for a 10"4 M solution of quinine

viewed from the FS, the spectrum is unaffected by: (a) varying the sulfuric acid concentration from 0.1 to 2 N,

00 1 ' 1 1 1 ' 1 1 1 1—

2-0 2-5

10"4 x \5 cm"1

Figure 9. Quinine, 10-" M in N H2 S04 (25 °C, ke = 365 nm). # = Lippert [5].

(b) varying the exciting wavelength from 260 to 390 nm,

(c) measuring three different brands of quinine. Thus

the differences between the published quinine spectra

are almost certainly due to calibration errors. The

largest deviations are found for those spectra deter- mined from a tungsten lamp calibration.

Table 1. Absolute fluorescence spectrum of quinine in H2SO4 (X<> = 365 nm)

10"3 X v cm" 1 A" Eq

(i>)

25 0.95 0.07 0.105 1 0. 105 0.135 0.105 0.15 0.10 0.16 0.14 0.15 0.152 24 .98 .36 .43 .375 .37 .375 .46 .37 .47 .46 .49 .480 23 1.00 .78 .80 .740 .79 .74 .80 .78 .80 .78 .83 .810 22 .99 .99 .980 .99 .98 1.00 .99 .99 .99 .99 1.00 21 .92 .91 .950 .92 97 _ 0.92 .97 .92 .97 .93 0.915 20 .56 .67 .715 .68 .75 .70 .74 .70 .77 .70 .710 19 .35 .40 .460 .40 .47 .42 .41 .42 .51 .42 .405 18 .20 .215 .240 .20 .26 .21 .21 .24 .26 .21 .200

17 .104 .140 .09 .16 .10 .11 .11 .08 .095

Ref. 47 6 5 48 7 27 21 46 29 22 41 Solvent 2N 2N 0.1/V 0.1/V 1/V IN IN 0.5/V 0.1N 1/V Mode FS FS FS sv FS SV SV SV SV SV FS

Conc(M) 10-5 10- 5 10-3 2 x 10-6 5 x 10-3 2 x 10-« 10- 5 2 x 10-5 10-4

Calibration 1" L L L L L L + Q T <? Q Q Q

a A = aJ{ak + ayx)

b L = tungsten lamp calibration, Q = quantum counter calibration T= thermopile calibration

2. 2-Naphthol (fig. 10). Lippert et al. [5] have

published the absolute fluorescence spectrum of

2X10-4 M 2-naphthol in a 0.02 N, pH 4.62 acetate

buffer at various temperatures. In using this standard,

the temperature and the composition must be known,

and the concentration and pH of the buffer must be

exactly as specified by Lippert. B0rresen [46] has

published the absolute emission spectrum of a 10~4 M

147
solution of 2-naphthol using an instrument calibrated

with the rhodamine B quantum counter. The results are given in table 2. We have also checked the spectrum using the quantum counter method of calibration and found good

agreement with B0rresen's results, but rather poor

agreement with Lippert.

3. 3-Amino phthalimide (fig. 11). A comparison of Lippert's spectrum with the absolute spectrum

recently measured in this laboratory is given in table 3.

Table 2. Absolute fluorescence spectrum of 2-naphthol in 0.02 /V acetate

10 3 Xu cm -1 A a A

(pH 4.62 -4.68) (\e = 313 nm)

_

31) 0.51 0.220 0.380 0.220 0.36 0.370 0.220 0.345 29 .93 .860 .900 .860 .90 .900 .860 .930

28.5 .99 .980 .995 .980 .97 .980 .980 1.00 28 1.00 .980 .955 .980 .97 .970 .985 .950 27 .745 .680 .740 .71 .745 .750 .715 26 .625 .590 .620 .62 .628 .630 .620 25 .780 .800 .770 .72 .750 .790 .825 24 .915 .930 .900 .80 .842 .930 .895 23 .810 .785 .795 .65 .630 .825 .670 22 .550 .515 .535 .41 .390 .565 .405 21 .300 .245 .295 .22 .224 .305 .235 20 .140 .092 .135 .095 .125 .145 .135

Ref. 5 49 5 46 41 5 41 Temp. (°C) 24 24 23 22.7 22.7 25 25 Conc.(M) 2X10"4 10- 5 2X10-" io-4 5X10"5 2X10-4 5X10-5 Mode FS SV FS SV FS FS FS

Calibration b L Q+L L 0 Q L Q

b L = tungsten lamp calibration. Q = quantum counter calibration

2-0 2-5 104 x\i cm"' 30

E,(5)

"ICT* x \J cmFigure 10. 2-Naphthol, 5 X 10-5 M in 0.02 N acetate, pH 4.62 Figure 11. 3-Aminophthalimide, 2 x 10"4 M in N H2 S04 (25 °C,

(25 °C, ke = 313 nm). • = Lippert [5]. Xe = 365 nm). • = Lippert [5].

148
Table 3.v Absolute fluorescence spectrum of 3-amino phthalimide

in H2SO4 at 25 °C XP = 365 nm

10-3 x a cm-1 A* Eq ( v)

23 0.97 0.01 22.5 .99 .03 0.04 22 1.00 .110 .125 21.5 .280 .308 21 .530 .560 20.5 .775 " .790 20 .945 .955

19.5 1.000 .990 19 0.950 .875

18.5 .825 .725 18 .650 .565

17.5 .500 .402 17 .385 .290

16.5 .295 .207 16 .220 .151 15.5 .160 —

Ref. 5 41 Cone. (M) 5x 10- 4 2 x 10-4 Mode FS FS

Solvent 0.1 N 1 N

Calibration'' L Q

3 A = aJ(otK + otx x)

h L = tungsten lamp calibration, Q = quantum counter calibration

4. m-Dimethylamino nitrobenzene (DNB) (fig.

12). Lippert et al. [5] measured the absolute emission

spectrum of DNB in 30 percent benzene and 70 per- cent hexane. Table 4 compares this spectrum with the one measured in this laboratory. The spectrofluorometer was calibrated with rhodamine B and methylene

blue quantum counters. A disadvantage of this standard is its low <ff and this, coupled with the fact that the

instrumental sensitivity is decreasing in the region of

TABLE 4. Absolute fluorescence spectrum of m-dimethylamino

nitrobenzene at 25 °C in 30 percent benzene. 70 percent hexane

(

\

f = 365 nm)

10 :'Xv cm-i A

B. E„( i>)

22 0.93 0.03 0.10 21 .98 .235 .41 20 1.00 .610 .80 19 .935 1.00 18 .975 0.86 17 .825 .68 16 .615 .48 15 .405 .31 14 .235 .17 13 .110

Ref. 5 41 Cone. (M) 10-" 5 X 10~4 Mode FS FS'

Calibration b L Q

i> A = aKl(ax + aK 'x)

b L = tungsten lamp calibration, Q = quantum counter calibration

1-5 2-0 104

x \) cm"1

Figure 12. m-Dimethylamino-nitrobenzene, 5 X 10~4 M in 30

percent benzene, 70 percent hexane (25 °C, Ae =365 nm). # =

Lippert [5],

the emission spectrum, results in a poor signal-tonoise ratio. 5. 2-Aminopyridine (fig. 13). The corrected fluo- rescence spectrum of 2-aminopyridine determined by

Rusakowicz and Testa [45] using a tungsten lamp for calibration is given in table 5. The spectrum measured

Table 5. Absolute fluorescence spectrum of 2-aminopyridine in H,S04 at 25 °C

10-3 X v cm-1 EQ (u)

31 0.64 0.03 0.05

30.5 .82 .145 30 .94 .17 .325 29.5 .97 .540 29 1.00 .575 .686

28.5 .840 28 .93 .960

27.5 1.000 27 .99 0.980

26.5 .920 26 .805 .790 25.5 .660 25 .52 .530

24.5 .405 24 .26 .310 23.5 .222 23 .138 .148 22.5 .102 22 .068 .075

Ref. 45 41 Solvent 0.1 N 1 N

Cone. 10-5 10-5 Mode sv FS

Calibration b L Q

Af nm 285 313

a A = aJ(aK + aK x)

b L = tungsten lamp calibration, Q = quantum counter calibration

502-591 O - 73 - 11 149
in this laboratory using the quantum counter method of

calibration is in good agreement with their spectrum.

00

20 2-5 3-0 cm

Figure 13. 2-Aminopyridine, 10"5 M in N H2 S04 (25 °C, A. f = 313

nm). 0 = Rusakowicz and Testa [45].

C. New Standards

The greatest lack, at present, is for standards in the

ultraviolet and near infrared regions of the spectrum.

Reisfeld et al. [50] recommend thallium activated

potassium chloride as an emission standard in the

UV region. For the red and near infrared regions,

rhodamine B and methylene blue might be used since

these substances have a high tp/. Preliminary measure- ments in this laboratory (fig. 14) indicate that these

two would cover the range 18,000 to 12,000 cm-1.

E,(5)

20

10^ x \) cm"

Figure 14. Rhodamine B, 5 X 10~6 M and methylene blue, 5 X 10~5 M in ethanol (25 °C, k e = 436 nm).

VII. References

fl] Birks, J. B., Photophysics' of Aromatic Molecules (John

Wiley and Sons, Inc., New York, N.Y., 1970)

.

[2] Parker, C. A., Photoluminescence of Solutions (Elsevier, New

York, N.Y., 1968).

[3] Chapman, J. H., Fiirster, Th., Kortiim, G., Lippert, E., Melhuish, W. H., Nebbia, G., Parker, C. A., Phot. Soc. Group

Bull. No. 14, 378 (1962), Appl. Spectroscopy 17, 171 (1963);

Z. Anal. Chem. 197 , 431 (1963).

[4] Demas, J. N., and Crosby, G. A., J. Phys. Chem. 75, 991 (1971).

[5] Lippert, E., Nagele, W., Seibold-Blankenstein, I., Staiger, W.,

Voss, W., Z. Anal. Chem. 170, 1 (1959).

[6] Kortiim, G., and Hess, W., Z. Phys. Chem. NF 19, 9 (1959).

[7] Melhuish, W. H., J. Phys. Chem. 64, 762 (1960).

[8] Muray, J. J., Nicodemus, F. E., Wunderman, I., Appl. Opt. 10,

1465 (1971).

[9] Ejder, E., J. Opt. Soc. Amer., 59, 223 (1969).

[10] Bennett, H. F., and McBride, W. R., Appl. Opt. 3, 919 (1964).

[11] Kortiim, G., Kolorimetrie, Photometrie u. Spektrometrie 4th

ed. (1962) (Springer-Verlag, Berlin, 1955).

[12] Mitteldorf, J. J., and Landon, D. O., Appl. Opt. 7, 1431 (1968).

[13] Pribram, J. K., and Penchina, C. M., Appl. Opt. 7,2005 (1968).

[14] Fisher, R., Appl. Opt. 7, 1079 (1968).

[15] Weinberger, R. G., Elect. Aug. 30, 58 (1971).

[16] Akins, D. L., Schwartz, S. E., Bradley-Moore, C, Rev. Sci. Instrum. 39, 715 (1968).

[17] Lampton, M., and Primbsch, J. H., Rev. Scient. Instr. 42, 731,

(1971).

[18] Jones, R., Oliver, C. J., and Pike, E. R., Appl. Opt. 10, 1673

(1971).

[19] Haugen, C. R., and Marcus, R. J., Appl. Opt. 3, 1049 (1964).

[20] Turner, G. K., Science 146, 183 (1964).

[21] Rosen, P., and Edelman, G. M., Rev. Sci. Instrum. 36 , 809,

(1968).

[22] Cundall, R. B., and Evans, G. B., J. Sci. Instr. (J. Phys. E.) 1,305(1968). .. [23] Lipsett, F. R., J. Opt. Soc. Amer. 49, 673 (1959).

[24] Stair, R., Johnston, R. G., Hallbach, E. W., J. Res. Nat. Bur. Stand. (U.S.), 64A, 291 (1960).

[25] Christiansen, R. L., and Ames, I., J. Opt. Soc. Amer. 51, 224

(1961).

[26] White, C. E., Ho, M., Weimer, E.. Anal. Chem. 32 , 438 (1960).

[27] Melhuish, W. H., J. Opt. Soc. Amer. 52, 1256 (1962).

[28] Parker, C. A., Anal. Chem. 34 , 502 (1962).

[29] Eastman, J. W., Photochem. and Photobiol. 6, 55 (1967).

[30] B0rresen, H. C, and Parker, C. A., Anal. Chem. 38, 1073 (1968).

[31] Hass, G., and Waylonis, J. E., J. Opt. Soc. Amer. 51, 719 (1961).

[32] Benford, F., Schwartz, S., Lloyd, G. P., J. Opt. Soc. Amer. 38,

964(1948).

[33] Vavilov, S. I., Phil. Mag. 43,307 (1922); Z. Phys. 42,311 (1927).

[34] Anderson, W. T., Bird, L. F., Phys. Rev. 32 , 293 (1928).

[35] Harrison, G. R., Laighton, P. A., Phys. Rev. 38 , 899 (1931).

[36] Bowen, E. J., Proc. Roy. Soc. 154A, 749 (1936).

[37] Melhuish, W. H., N.Z. J. Sci. and Tech. 37B, 142 (1955).

[38] Parker, C. A., Nature 182, 1002 (1958).

[39] Yguerabide, J., Rev. Scient. Instr. 39, 1048 (1968).

[40] Seely, G. R., J. Phys. Chem. 73, 125 (1969).

[41] Melhiush, W. H., unpublished. Apparatus used shown in figure

4a.

[42] Hatchard, G. G., Parker, C. A., Proc. Roy. Soc. 235A, 518

(1956).

[43] Melhuish, W. H., J. Phys. Chem. 65, 229 (1961).

[44] Petrash, C. G., Opt. and Spect. 6, 516 (1959).

[45] Rusakowicz, R.. and Testa, A. C, J. Phys. Chem. 72, 2680

(1968).

[46] Borresen, H. C, Act. Chem. Scand. 19, 2089 (1965).

[47] Kortiim, G., and Finckh, B., Spect. Act. 2, 137 (1941).

[48] Parker, C. A., and Rees, W. T., Analyst 85, 587 (1960).

[49] Unpublished (1962) using the apparatus in Ref. 27.

[50] Reisfeld, R., Honigbaum, A., and Velapoldi, R. A., J. Opt. Soc. Amer. 61, 1422 (1971).

(Paper 76A6-741)

150
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Absolute Quantum Efficiencies

G. A. Crosby*

Department of Chemistry, Washington State University, Pullman, Washington 99163

J. N. Demas

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901

and

J. B. Callis

Department of Chemistry, University of Washington, Seattle, Washington 98105

(July 11, 1972)

Recent developments in several areas of chemistry, laser technology, photodetector instrumentation, and calorimetry are surveyed, and their probable impact on the measurement of quantum yields

is assessed. Chemical developments include: (a) synthesis and design of new luminescent molecules

that could possibly serve as standards, (b) application of improved separation techniques to provide

samples of extreme purity, and (c) advances in photochemistry that portend the development of wide- range chemical actinometers. The potential use of lasers in quantum-yield measurements and their advantages over conventional sources for application in both optical and calorimetric techniques are pointed out. New methods of quantum-yield measurements, based on the novel characteristics of laser pump sources, are suggested, including the feasibility of measuring yields under time-resolved conditions and of employing internal standards. The possible lifting of wavelength restrictions on both

laser sources and detector devices and the implications of these developments for extending the

spectral range of quantum-yield measurements are discussed. The current status of calorimetry for determining yields is surveyed, and the impact of recent technology on the feasibility of developing

calorimetric methods competitive with optical methods is assessed.

Key words: Calorimetry in quantum yields; laser, use in quantum yields; photodetectors in quantum

yields; quantum efficiencies.

I. Introduction

The explosive developments of science and technology in the last quarter century have been mirrored

in the steady rise of optical methods in chemistry,

physics, and biology. Today the use of fluorescence and

phosphorescence in fundamental and applied investi- gations is routine. Initially focussed on frequency and

relative intensity measurements, the effort during the

last few years has begun to encompass determinations

of efficiencies of large classes of luminescent materials.

Such a measurement, the quantum-yield determination, is one step removed in difficulty from recording

energy, measuring relative luminescence intensity, or monitoring rate of decay (at least for times beyond

a microsecond) of excited species. The result is that developments in quantum-yield technology have lagged

'Prepared during the tenure of AFOSR (NChOAR, USAF, Grant No. AFOSR- 72-2207.

behind those in the other areas. Recently, however,

the technological advances relevant to quantum-yield

measurements and the increased interest in obtaining

efficiencies signal that a comprehensive analysis

should be carried out on the present needs for ac- curate quantum efficiencies and an assessment be

made of the current developments in the methods of determining them.

The present article is not an attempt to review the

field of quantum-yield measurements. Although considerable space is devoted to optical and calorimetric

methods for determining yields, no attempt has been

made to review these techniques adequately either. (For a comprehensive review of optical methods, see

ref. [I]. 1

) Rather the thrust of the present article is to point out a few of the obvious needs for accurate yield

information, to note some of the current progress in

1 Figures in brackets indicate the literature references at the end of this paper.

151
instrumentation technology and chemistry that may

lead to better yield determinations, and to suggest a few avenues of investigation that appear ready for development. Our remarks are restricted mainly to solutions and rigid glasses. For readers interested in yields from solid materials, we direct them to the re- view article by Lipsett and to the original literature [2-10]. Gas phase determinations are also not dis- cussed, although recent publications show that the

field is expanding [11—13].

II. The Importance of the Quantum Efficiency

The luminescence quantum efficiency of a compound

(<P) is defined as the fraction of molecules that emit a photon after direct excitation by the source. The terms

absolute yield, quantum yield, yield, and efficiency are all used interchangeably throughout this article. Recent research developments in many areas of

physical, chemical, and biological research have

reached a stage where the lack of absolute quantum

efficiencies is seriously hampering further progress. We enumerate here a few areas where such information could be decisive. In theoretical chemistry,

studies of radiative and especially radiationless

processes have been proliferating [14-18]. The role of various terms within the Hamiltonian (vibronic,

spin-orbit) [19-22] and the effect of substituents on

both radiative and radiationless rate constants of

isolated molecules are under scrutiny [23-25]. The

interactions of excited species with the host molecules

in condensed phases are not well understood, but some

information is beginning to accumulate [26, 27]. Reliable efficiencies are needed on many classes of molecules to test the predictions implicit in the

formalisms and to unearth new problems.

An especially important fundamental problem con- cerns the limits of applicability of the Strickler-Berg

equation [28] for obtaining radiative lifetimes of excited

states from the inverse absorption processes [29]. Serious discrepancies exist between the measured

natural radiative lives (Tom ), as obtained from the

equation To — Tflfyf where T/ is the fluorescence

decay time and is the fluorescence quantum yield,

and those calculated by the Strickler-Berg relation. These discrepancies exist for simple molecules (e.g., SO2) [30] and for larger systems such as diphenylpolyenes [31]. The lifetimes can be anomalously long

[32, 33 ] or anomalously short [34]. Large intermolecular

[33] or intramolecular configurational changes [34] have

been invoked to rationalize the results. More quantum

efficiencies are required to quantify the experimental

situation. A second problem in the application of the Strickler- Berg equation involves the selection of degeneracy

factors for upper (gu) and lower (gi) states involved

in the transitions when application is made to formally

forbidden transitions (S —> T). For hydrocarbons at the

usual refrigerant temperatures (2= 50 K) a value of gu — 3 is reasonable, since the level splitting is much

smaller than kT and thermal equilibrium is established

among the nearly isoenergetic levels. For systems

where spin-orbit coupling is large, such as encountered

in d~d [35] and charge-transfer [36, 37] excited states, the splittings may be comparable to or even greater

than kT. Then measured quantum yields and lifetimes are expected to be temperature dependent, and the

Strickler-Berg relation can only really be applied and

tested after careful studies of temperature-dependent

decay times [38] and quantum yields [39] have been

carried out. Reliable quantum efficiencies for many

model systems are needed.

Another area where reliable quantum efficiencies are lacking, and one where the role of experimental

yields can be decisive, is in determining the effects of deuteration on the radiationless rate constants for degrading excitation energy. Theoretical classifications of radiationless processes according to "strong" and

"weak" coupling limits have been made. In the first case, no important deuterium effects on radiationless

rates are predicted; in the latter, large changes due to deuteration are expected [40]. Few systems have been

studied experimentally to distinguish these coupling

limits. It is manifest that major decisions about the

relative importance of various mechanisms for fundamental radiationless processes could be made from

quantum-yield comparisons of deuterated and non- deuterated molecules [41-43].

The present status of organic photochemical re- search also warrants the effort of measuring accurate luminescence yields. Particularly important is a knowledge of efficiencies for organic molecules that can be

used as energy donors or acceptors in photosensitization experiments. Reliable yields on series of closely

related molecules whose singlet or triplet states span

important energy regions would aid substantially in the

design of experiments to test mechanisms of energy

transfer and to control photochemical products. The

need for yields of organic systems is compounded by

the fact that mixed solvents and unconventional pure

solvents are sometimes necessary. Thus, the known

efficiencies of compounds must be extrapolated into systems where conditions differ radically from those

prevailing during the original determinations.

Not only total quantum yields, as defined initially,

but yields from particular levels in molecules exhibiting

multiple level luminescence are needed. Such quantities are of paramount importance for assessing relative quenching rates of excited singlet and triplet levels, for obtaining efficiencies of intersystem crossing, and

for relating measured quantities to theoretically defined

parameters. The processes associated with a typical

organic system are illustrated in figure 1. In terms of

the rate constants displayed on the figure, we can

define: 4>f = ks\ (kf + k

Qf + &iS ) , $p = kP l(kp + kQp), and

4>is— kj (kiS + kf + kq

f

). It is obvious that O, the total measured quantum yield as defined earlier, equals

(f)f+(f>p provided the only processes occurring after excitation are those depicted in the figure. It is also clear that the sum of 4>f and $iS may or may not be

unity depending upon the existence of singlet quench152
FIGURE 1. Principal pathways of degradation of an excited organic molecule. *, radiative decay; \a.<m> , radiationless decay. Ay=

radiative rate constant for depopulating Si; kq = radiationless rate constant for depopulating Si; ki, = radiationless rate constant for converting from Si to 7Y; kp = radiative rate constant for depopulating Tu kq^ = radiationless rate constant for depopulating 7\.

kfl (kf + k

qj.+ his) = <f>f (quantum yield of fluorescence from Si). kPl (kp + kq^) =<f)P (quantum yield of phosphorescence from Ti).

kisl(ki$ + kf + kQj) =<j)is (quantum yield of intersystem crossing).

ing (kq

f

). A particularly important quantity is (f)i S

. It measures the efficiency of populating the triplet state, thus producing a reactive species responsible for many

photochemical reactions and frequently serving as an

efficient energy donor.

Several methods have been proposed to determine

<f)iS [44-51]. Different photophysical properties anc processes have been utilized and ingenious techniques

have been devised to extract <£jS from the data. None

theless, the methods rely eventually on the measure ment of a photoluminescence quantum yield. A funda

mental question raised by these measurements ol intersystem crossing yields is whether the sum of

(/>/+(/>iS is indeed unity. The equality has been shown

to hold for a number of aromatics [52-57] and for porphin and its derivatives [58]. Exceptions abound,

however, [59] and the experimental situation is still unclear [60]. Accurate and extensive quantum-yield

measurements are necessary in order to establish or

refute the stated equality as a fundamental (or rarely

violated) law of luminescence. For pyrene, k/

Q is re- ported to be negligible at 77 K but competitive with

kf at room temperature [61]. The role of quantum-yield measurements in pro- viding information on biologically important systems is expanding rapidly. Reports of quantum yields of naturally occurring bases [62] as functions of pH are pro- viding insight into the possible fundamental physiological functions of these materials. Useful information on

conformations and the nature of the excited states of

biopolymers [63-65] has been provided from fluo- rescence measurements on oligonucleotides and polynucleotides containing fluorescent purine analogs.

Some of the reported quantum yields are extremely low

dictating new demands for accurate quantitative

measurements at low light levels. The real power and usefulness of fluorescent probes

in biological systems are becoming appreciated. A

typical example is 2-p-toluidinyl-6-naphthalenesulfonate (TNS), one of a series of compounds that are

practically nonfluorescent in water but strongly fluo- rescent in organic solvents or when bound to protein

molecules. This substance can be used as an extrinsic

fluorescent chromophore for the study of protein

conformations [66, 67], in the study of nitrogen antibody

interactions [68] , in following protein conformational

changes produced by metal ion binding [69], and in elucidating enzyme activation processes [70]. In spite

of its obvious importance it is significant that the

mechanism by which the fluorescence of this molecule

is changed by the medium is still not well understood.

Spectroscopic studies, including detailed and accurate quantum-yield determinations, are of fundamental

importance for molecules of this type and should be

actively sought.

The need for quantum yields of inorganic molecules

is also manifest and growing. Especially those materials

classed as inorganic coordination compounds, usually

containing some organic moieties, deserve considerable

attention. This class of molecules, long utilized empirically in fluorescence metal analysis, has only relatively

recently been recognized as providing candidates for electro-optical devices [71], for fundamental studies of

radiative and radiationless transitions [35, 36], and for energy donors in inorganic photochemical studies

[72]. The real possibility of engineering these molecules to have predetermined frequency ranges, high yields, and appropriate chemical properties

[73] opens up a whole range of new experiments that were not possible just a few years ago. Needed, however, is detailed information on their spectroscopic

properties, especially their absolute efficiencies, and

the temperature dependences thereof. The near infra- red region, virtually inaccessible at present to quantum-efficiency measurements can possibly be made

accessible by the use of complexes possessing lowlying emitting levels, particularly those molecules

displaying charge-transfer luminescence. Yet, few

yields of these compounds have been measured, and

none, to our knowledge, of compounds emitting beyond

800 nm. We digress here to present some information on a few representative transition-metal complexes whose

unique luminescence properties are not yet well recognized. Complexes of ruthenium(II), osmium(II), and

iridium(III) with 7r-conjugated ligands display visible and near-UV absorption spectra dominated by strong

charge-transfer absorption bands of high intensity

(2000 s£ e =£ 20,000, where e is the molar absorptivity)

(figs. 2, 3, 4). In the case of osmium(II) complexes, the

absorption bands extend well into the red region of the

spectrum. Upon irradiation these molecules emit light

with good efficiencies at low temperature (> 10%),

and some are still reasonably efficient at room temperature in fluid solution. In figures 2 and 3 it is seen that the quantum yields are independent of wavelength,

a property of fundamental and applied importance.

Other spectroscopic properties of interest of these

molecules are evident in figure 4. One sees that each

emission spectrum consists of a unique electronic band.

This is a feature characteristic of all these systems at 153
uj w |.05 >>:

I— 1.00 < z

uj H 0.95

a. z

< 0.90 o

75

ro

iO

x 50

25

300

-i 1

o

400 500 nm

i 1—i—i

<~ 400 500 600 800 nm

(b)

V

(c)

40

roO

10 X

30 25 20 kK

FIGURE 2. Relative quantum yield (a) and absorption spectrum (b, c) of tris(2,2'-bipyridine)ruthenium(II) chloride in methanol at room

temperature: (ajl 0.2 g/5 ml in a 1-cm cell, (b, c) 6.7 X 10-5 M in a 1-cm cell. The first and last yield points are less accurate than the

others. [Ref: J. N. Demas and G. A. Crosby, J. Amer. Chem. Soc. 93 , 2841 (1971).]

to

I o 8

x

to2 8

X

u, 4

.(a)

i

i ii

i

i

i i.i [IrClgfdi^bipy)^

A

!

1 M

- A

\ 1 w » V ! \

\ • v V i ^

I i Vj i i ^

.(b)

-

[IrCI 2

(di;2fphen);) ]+

\ h\

i

\ 1

\

1 V V i \ - \ 1 \

28 24 20 16 1'

Figure 4. Absorption (-

12 kK

-) and luminescence (- - -) spectra of phenyl-substituted iridium(III) complexes in ethanol-methanol glass

(4 : 1, v/v) at 77 K. (a) cts-dichlorobis(4,4'-diphenyl-2,2'-bipyridine)-

iridium(III) chloride, (b) cts-dichlorobis(4,7-diphenyl-l,10-phe"nanthroline)iridium(III) chloride. [Ref: R. J. Watts and G. A. Crosby,

J. Amer. Chem. Soc. 94,2606(1972).]

400 500

3 1.051-

... (a) CKSOQ

2(S)Qo3

0.95 < Z< 1.00 O 0.95

20

? 15 o

x 10

5

600 nm 1—i

1—

(b)

-i 1

1—

r

0 0%ee0

0 9 77 K

15 kK

0.90

0.85

< 0.80

0.75

» [IrCI 2(di^bipy)2

] +

• [IrClgldl^phenlg]'1

"

20 40 60 80 100 120 °K

Figure 5. Temperature dependence of quantum yields. X, cis- dichlorobis(4,4'-diphenyl-2,2'-bipyridine)iridium(HI) chloride. O,

cis-dichlorobis(4,7-diphenyl- 1 ,10-phenanthroline)iridium(III) chloride. Both molecules were dissolved in poly(methylmethacrylate) and irradiated with ~ 365 nm radiation.

FIGURE 3. Relative quantum yields (a, b) and absorption spectra (c) or tris(l,10-phenanthroline)osmium(II) iodide in ethanol-methanol

(4:1, v/v): (a) 4.5 X 10-7 M in a 1.76-cm cell at room temperature;

(b) 4.5 X 10-7 M in a 1.76-cm cell at 77 K (glass); (c) - - -, 9.0 X 10"6 M

in a 10-cm cell at room temperature; , 7.12 X10~5 M and

1.42 X 10-5 M in 1.76-cm cells at 77 K (glass). [Ref: J. N. Demas

and G. A. Crosby, J. Amer. Chem. Soc. 93, 2841 (1971).]

temperatures above 77 K [35-37]. Furthermore, the

measured decay times are on the order of microseconds, thus falling between those limits normally

encountered for established fluorescences and phosphoresences. Finally, the capability of tuning the fre- quency by ligand change is apparent.

Additional spectroscopic features of importance are evident in figure 5. Not only are the yields of some of the molecules extremely high but, also, for some of them, the efficiencies are virtually temperature independent over large ranges. Because of the favorable

chemical properties of these substances and their unique luminescence behavior, considerable fundamental information concerning the excited state properties of transition-metal complexes can be gleaned

154
from studies of their photoluminescences. Particularly important are accurate determinations of quantum

yields as a function of temperature [38, 39]. Their

possible value as fluorescence standards and as components in chemical actinometers is discussed

below.

III. Standards

The absolute determination of quantum yields is a measurement fraught with difficulties. Understandably,

workers have resorted to the much more convenient

and rapid technique of measuring yields relatively,

i.e., by comparison with known standards. Perforce,

this procedure shifts a major burden of the accuracy of the final figure to the standard. Thus, the real need for more and better standards. Compounding the problem

is the necessity to have standards for various solvents,

for various temperature ranges, for wide ranges of wavelengths, and for different states of matter (solid,

plastic, glasses, solutions). This is a stiff order. When

one considers the importance of standards, the small

number of well-studied ones, and the disconcerting

disagreement of reliable workers on the actual reported

efficiencies, the magnitude of the problem of obtaining

good standards is revealed. [Relative yield measure- ments are so much easier to perform than absolute

ones, however, that the demand for absolute standards

will continue to increase.] An account of commonly used

standards and an assessment of the reliability of their

yield values are available [1]. Additional information on common standards and some interesting suggestions

for new ones are contained in the article by Testa [74]. Many workers choose standards possessing favorable

chemical properties for relative measurements even

though the accuracy of some of the "standard" yields

is not well substantiated.

The incidence on the market of commercial instru- ments that can measure yields routinely also increases

the need for standards. In fact, institution of a program

to remeasure old standards and search for new ones

has considerable merit, in our opinion. If a bank of

reliable standards could be built and marketed, which

would provide checks on both research results and

instrumentation throughout the country, a mass of

misinformation would be clarified, and future additions

of shoddy yield data to the literature would be measurably reduced. Such a program seems appropriate for the National Bureau of Standards.

At this juncture we wish to point out again the un- usual optical properties of inorganic complexes,

especially those of ruthenium, osmium, and iridium

described above. It is our contention that they deserve

serious study as candidates for standards, as well as

for numerous other applications in spectroscopy and

photochemistry. Because their properties can be modified by ligand substitution, ligand modification, and

change of counter ion, it is possible to design them to have specified frequencies, high yields, and desirable

solubilities. Thus, molecules can be engineered to dissolve in most common solvents. Their potential as

quantum-yield standards should be studied, especially if internal standards (see below) were employed.

Finally, we wish to call attention to recent developments in purification procedures that bear on the accu- racy of quantum-yield measurements. Chemical purity

of any fluorescent compound is always desired; for quantum-yield standards it is a stringent requirement.

This aspect of quantum-yield work has been repeatedly emphasized [1, 29, 75], not only for quantum-yield

measurements but for emission spectroscopy in general. Intense light sources commonly employed to measure low yields exacerbate the impurity problems.

The use of lasers dictates adherence to criteria for chemical purity far stricter than those normally applied

[76]. Increases in detector sensitivity and the employment of high aperature equipment to monitor weak

emitters place still greater demand for high standards

of chemical purity. The availability of image intensifier tubes adds a new dimension to the impurity problem

[77].The virtues of thin-layer chromatography, column

chromatography, and zone refining as supplements to the classical chemical techniques for obtaining high

purity are relatively well known. Not commonly appreciated, however, are the remarkable chemical separations obtainable with the use of dextran molecular

sieves in column chromatography. Although usually

employed for biochemical separations, the effectiveness

of molecular sieves for purifying inorganic materials

is not common knowledge [78) Indeed, the usual con- ception of the primary mechanism responsible for such

separations (dominant role of molecular size) leads one

to predict that molecules of similar size and shape

should not be separable with such procedures. Yet,

separations of isomeric, six-coordinated complexes of

transition metals have been accomplished [73]. In

fact, the discriminatory ability of the Sephadex gels to separate the components of reaction mixtures is so acute that surface adsorption activity must be at work

in addition to steric effects [79]. Because of the many desirable features of gel filtra- tion chromatography as a purification technique we

strongly recommend its use both for purifying fluores- cence standards and for preparing samples in general.

For transition-metal complexes where reaction mixtures can produce a host of chemically and spectroscopically similar molecules, efficient separations

are mandatory. In many cases gel filtration provides

a superb answer to the problem.

IV. Quantum Efficiencies by Optical Methods

The first reliable method for measuring quantum

yields by an optical technique was introduced by

Vavilov in 1924 [80]. The method is a substitution

technique employing a solid scatterer as a standard.

The experiment requires two basic measurements.

First, the detector monitors the sample luminescence

that is generated by total absorption of the excitation

light focussed to a point in the cell Then the detector

155
records the light diffusely scattered by a magnesium

oxide surface substituted for the original cuvette. From these data and some ancillary measurements, the

absolute quantum yield can be computed. A detailed

description of the technique including the derivation of equations is given in reference 1. Basic improvements

on the method were the introduction of a quantum

counter by Bowen in 1937 [81] and the incorporation of the refractive index correction by Fbrster in 1950 [82]. Refinements by McClure et al. [83] in the early 50's and the introduction of the rhodamine B quantum

counter in 1955 by Melhuish [84] resulted in a method

of measurement that has produced some of the most

accurate quantum yields available. In 1957 Weber and Teale [49] made a step forward

by the introduction of solution scatterers for standards.

This technique introduces its own special problems

and limitations but is easier to carry out than the basic

Vavilov method [1]. Numerous workers have devised

improvements and modifications of both the Vavilov

and the Weber-Teale methods for determining quantum yields, yet the measurements are still relatively

tedious to perform and have many associated problems.

The difficulties inherent in absolute procedures and

the demand for yield measurements on hosts of compounds have led workers to devise simple relative methods for performing the measurement. In these

methods the yield of an unknown is compared with the

"known" quantum yield of a standard material.

Relative methods fall into two categories, optically

dense and optically dilute. In the former, concentrated

solutions are employed to absorb all the incident flux; in the latter, dilute solutions insure negligible diminution of the excitation beam across that part of the

sample viewed by the detector. Optically dense

quantum-yield measurements relative to a standard can be made extremely rapidly and are subject to moderate errors provided the substances satisfy

somewhat rigid requirements. The accuracy can be

low due to effects associated primarily with high

concentrations. Optically dilute methods generally

avoid problems created by solute-solute interactions

and, due to the availability of high quality spectrofluorometers and their components, have become the

preferred techniques for obtaining yields.

An analysis of optically dilute methods, the useful

equations and their limits of validity, and the problems

associated with this kind of measurement are contained

in reference 1. We refer the interested reader to this review and the references therein. We wish to point out here, however, two problems associated specifically with yields obtained by optically dilute proce- dures that modern laser technology should admirably

solve, (a) inaccurate (high) yields caused by strong

light from a broad band source leaking through the

filter system and (b) inaccurate yields produced by

uncertainties in the effective absorbances of samples

over the finite bandpass of the filters or monochromator excitation system (see Lasers, below).

For routine determinations of quantum yields by

optical methods, the use of optically thin solutions

versus optically thin standards is probably the best

method available. The reported reproducibility is quite good (1-2%), and the agreement between laboratories is excellent (<5%) for some molecules. Considerable improvement in accuracy is to be expected from

substituting lasers for conventional sources and from

incorporating modern electronics and detectors into the systems. Although frequently reported with high

precision [35-37], the accuracy of quantum yields

obtained from substances dissolved in rigid glasses

may not be high in spite of the good reproducibility.

The relative importance of polarization errors is especially vexing to assess for rigid systems, and more

work on this aspect of the optically dilute methods is badly needed [85]. Finally, we reiterate that both the optically dense

and optically dilute techniques are still relative ones. Whatever systematic errors are built into the standards

will be reflected in the results. Accurately measured

standards are urgently needed.

A. Recent Developments in Instrumentation

We turn now to highlight some recent instrumentation advances that bear on optical methods of determining quantum yields. Lasers are by far the most important development, but the significance of available

high sensitivity Raman instrumentation and recent improvements in photodetectors deserve attention.

1. Lasers

Lasers are now available in a wealth of types, powers,

and configurations. The salient features of the most

common varieties are reviewed in table 1. This list is by

no means complete. In many cases, commercially

available items are listed, and data have been drawn

frequently from the manufacturer's literature. It should be further noted that specialized design fre- quently can improve many of the listed characteristics. In addition, because of the rapid advances in laser technology, this table will surely be obsolete by the

time it is printed. The interested reader should consult

the latest issues of the laser trade journals (e.g., Laser

Focus and Laser Sphere) and other technical journals

for up-to-date information. An especially valuable source is reports on advanced seminars in laser technology [86]. Most significant for luminescence and quantumyield work is the high continuous wave (cw) powers

available in the near UV and visible from argon lasers. The continuous tunability of the cw pumped dye lasers and their remarkably narrow line widths are also particularly noteworthy. Pulsed nitrogen lasers with their extremely short duration pulses and UV frequencies

are ideally suited for most lifetime measurements and

even for nanosecond photolysis. By driving a dye laser with a nitrogen laser and selecting the wavelength with an intracavity grating, one can preserve the extremely

short durations and relatively high powers (one to two

orders of magnitude in power are sometimes lost), but

the added feature of continuous tunability from the

156
Table 1. Characteristics of selected lasers

Continuous Wave Lasers TYPE

Discrete Line Lasers

Ar a

Kr a

Tunable Lasers

cw pumped dyes with intra- cavity \ selection a

TYPE

Discrete Line Lasers

Nd3+-YAG

Ruby

Tunable Lasers

Flashlamp pumped dyesc

N2-laser pumped dyes b

Nd3+ -YAG pumped dyes e

Parametric oscillator e

\, nm

351.1+363.8; 457.9; 472.7; 476.5;

488.0; 496.5; 501.7; 514.5; 528.7

350.7 + 356.4; 476.2 ; 482.5; 520.8;

530.9; 568.2; 647.1; 676.4; 752.5; 799.3

520-690

POWERS

20 mW to 2 W

20-500 mW

10-160 mW

\, nm

337.1

266 (frequency quadrupled), 13 lines from 473 to 679 (frequency doubled),

13 lines from 946 to 1358

694 (Q-switched)

347 (frequency doubled)

340 -> 650

360 -> 650

540-670 and 270-340

650-3000

Pulsed Lasers

POWERS

1-10

1-100 kW peak

0.5 W average

1-5 kW peak

10-100 mW average

100 MW peak

1 J per pulse

10 MW peak

0.25 J/pulse 600 kW peak

25 mW average

2-150 ^ij/pulse 1-10 kW peak

1 -50 m W average

2C 700 W peak

0.2-20 mW average

50-700 W peak

1-10 mW average

MONOCHROMATICITY

< o.ooi A

< o.ooi A

0.1 A normal to 0.001 A

MONOCHROMATICITY DURATION,

nsec

~1 A

< i A

o.i A

o.i A

< 1-10 A

< 1 -20 A

<i A

=s i A to o.ooi A

5-10

100-400

20

20

100-500 d

2-10

70-150

d Data for lasers from Spectra-Physics and Coherent Radiation. d Mode locking can yield picosecond pulses (< 0.1 nsec), although

b Data for AVCO Everett Research Lab. lasers. spectra bandwidth will usually be increased. See references [90, 91].

c Data for Synergetics Research, Inc. lasers and from references e Data for Chromatix laser. [88,89,90,91].

157
near UV through the visible is obtained. These characteristics are ideal for carrying out time-resolved

spectroscopy, a powerful fundamental and analytical

tool. The large pulse energies available from the flash- lamp-pumped dye lasers are also attractive for some

studies, and systems can be built for minimal cost [87-89].

From table 1 the benefits accrued from replacing

conventional light sources with a laser source are manifest. The powers available far exceed those

normally obtained from even wide bandpass con- ventional sources. This power gain, however, can be

obtained at no loss in spectral purity, and for most

molecular systems one can treat the sample ab- sorbance as constant over the excitation line. Thus,

one of the significant sources of error mentioned

above in quantum-yield and excitation-spectra de- terminations is eliminated. The higher powers available

from the lasers also permit the use of more dilute

solutions; this reduces the trivial errors of self ab- sorption and secondary emission.

In addition to the high powers and narrow fine widths, lasers have other advantages. Typically, the

laser beam is of very small size. A cw argon laser, for example, has a beam diameter of 1 to 2 mm. It is highly

coherent and can be focussed to an essentially diffrac- tion limited spot. This high degree of focusability is commonly exploited in Raman work where the laser

is directed down the long axis of a microcapillary.

The use of a microcapillary cell would also yield

significant advantages in luminescence spectroscopy.

There would be negligible reabsorption, and the source composed of the emitting volume would be a near

line. Thus the emissions could be focussed extremely

efficiently onto the entrance slit of an emission monochromator, even for very narrow emission bandpasses. Such a system could not be used with even

moderately photosensitive materials, however, unless

a flow system were employed. Microcapillary cells have a further disadvantage for comparative work

due to the higher precision required for reproducible

alignment.

There seems little doubt that the wavelength range,

the pulse duration, and the available powers of most

of the types of lasers will be greatly improved. With

progress in mode-locking [90-92] it seems reasonable

to anticipate that the dye lasers will yield repetitive,

stable, and continuously tunable subnanosecond pulses

extending from the UV to near-IR regions. Such a tunable, short duration source would represent truly a delta function excitation flash for luminescence life- time measurements and time-resolved spectroscopy,

with little sacrifice in power or spectral purity. For dye

lasers especially, considerable improvements in peak

and average power are anticipated as the pulsed nitro- gen lasers become more efficient. Wavelength range

limitations of tunable dye lasers will almost certainly

become relaxed as the dyes are frequency doubled into the UV, and new dyes will surely extend the available

range well into the infrared. Already the entire range

between 7000 and 11,700 A has been covered by pumping photographic film sensitizer dyes with Q-switched

ruby lasers [93]. A number of parametric oscillators has been constructed that exhibit continuous tunability from 7000 to 20,000 A, and efficiencies of 45 per- cent have been reported [94]. Referring to the optical quantum-yield methods

sketched above, we cannot think of a technique that would not benefit substantially from the replacement of the conventional light source with the laser. We have

no doubt that where low cost is not a prime factor, but

accuracy is of paramount importance, lasers will eventually become the excitation source of choice. This statement applies to calorimetric methods also.

2. Raman Instrumentation

The developments of laser Raman spectroscopy have

yielded a whole new line of optical components that are admirably suited for luminescence and quantumyield measurements as well. One of the principal areas of progress is monochromator technology. Quite

fast, extremely-low stray-light, double monochromators

have been produced. Stray fight in these devices is so low that one can scan to within a few wave numbers of the laser line pump. Such superb stray light rejection

makes them a natural choice for luminescence and

quantum-yield measurements, especially when laser excitation is employed. It is noteworthy that much of the impetus for the development of very high power

krypton and argon lasers also stemmed from the needs

of the Raman spectroscopist.

Photomultipliers have also undergone enormous

improvements. By reducing the effective photocathode

size and cooling the tubes to reduce the dark current,

the dark backgrounds have been reduced to a few

counts per second (e.g., tubes of the IT&T2 type). All this has been accomplished with an extended red

response S-20 photocathode, which may exhibit a quantum efficiency of several percent beyond 800 nm. Thus the modern unmodified Raman instrument

using an argon laser source represents a nearly ideal

excitation-detection system for luminescence, at least when the molecules absorb in the region of the laser

lines. The ready availability of these Raman spectrometers within many laboratories also adds to their attractiveness for such work. Almost any further ad- vances in Raman technology are certain to be adaptable

with benefit for making luminescence measurements

and for determining quantum efficiencies.

3. Photodetectors

In addition to the restricted area tubes used in Raman spectroscopy, there have been other signifi- cant advances in detector technology. RCA, for example, has introduced a line of gallium arsenide and

indium-doped gallium arsenide extended red-response

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no

instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the best available

for that purpose.

.158
photomultipliers. The gallium arsenide tubes typically

have quantum efficiencies of 1.4 percent at 860 nm. With indium doping, the red response is further ex- tended, and RCA will supply tubes having efficiencies of 1 percent at 950 nm, 0.2 percent at 1000 nm, and 0.1 percent at 1060 nm. In addition to having remarkably

deep infrared penetration, these tubes have quite

low dark currents even at room temperature; in most

cases they can be regarded essentially as a quantum

jump better than the old infrared tubes with S-l

response. Further improvements seem likely since

gallium arsenide photosensitive surfaces have been

prepared having maximum quantum efficiencies of

80 percent [95]. Semiconductor diodes are also improving. Ger- manium diodes of the type used in nuclear instrumentation make superb optical detectors [96]. They can

have large areas (> 1 cm X > 1 cm) and sensitivities comparable to photomultipliers. Their noise equivalent

power (NEP) is ~ 10"15 W/Hz1/2 from the visible to beyond 1600 nm. Their stability and near quantum flat response of ~ 0.6 has led to the suggestion that they be

used as replacements for thermopile radiation

detectors [96]. When operated in the avalanche mode, these photodiodes have many of the characteristics of photomultipliers. They still have the high quantum efficiencies of

the solid state photodiodes but also exhibit a limited

gain multiplication. Typically, their sensitivities stretch

well into the near infrared. Texas Instruments, for example, has introduced a series of silicon and ger- manium photodiodes packaged with amplifiers

(TIXL 74-76). For the silicon system, the NEP at 900

nm is less than 5X 10~ 13 W/Hz1/2

. For the germanium

ones, the NEP is ~ 2 X 10~12 W/Hz1 ' 2 at 1060 nm or ~6X 10- 12 W/Hz1 ' 2 at 1540 nm. With properly biased

photodiodes, single photon counting is even possible.

With germanium photodiodes, NEP's of ~ 10~ 13 W/Hz1/2 at 1500 nm have been realized. Germanium

devices are usable to beyond 1700 nm [97]. It seems

reasonable that further improvement in these avalanche

devices will yield even higher sensitivity in the near- IR region. It is even possible that they will become competitive with photomultipliers in the visible-UV

regions. In a spectrophotometry application weak

transient absorption during flash photolysis has been

detected in the 0.6—1.1 /i region with the aid of an

inexpensive silicon photodiode [98]. If the major source of noise in an optical experiment is due to the photon statistics, then the signalto-noise ratio depends upon the square root of the

quantum efficiency of the detector. Thus for high light level experiments the high quantum efficiency of photodiodes is a distinct advantage for noise discrimination,

especially in the near infrared.

B. New Experimental Techniques

1. Internal Standardization

Internal standardization is frequently used in analytical chemistry. To date, however, we are aware

of no use of an internal standard for quantum-yield

measurements. This failure has been a result of the

experimental difficulties involved rather than because

of any undesirable fundamental features of the tech- nique itself. In fact, internal standardization is probably one of the more elegant and useful techniques

for accurate relative quantum-yield determinations.

The method appears especially feasible in view of

modern instrumental developments.

In the internal standardization procedure both the

unknown and standard would be contained in the same solution. The emission spectra of the two com- pounds would then be run in a single scan of the emission instrument. From the relative emission intensities

of the standard and the unknown and their absorb- ances one would then calculate the efficiency of the

unknown relative to the standard by normal pro- cedures. The advantages of such a procedure are numerous. Since the standard and unknown are in the same solvent and cell, refractive index corrections are

all but nonexistent, and radiation trapping is essentially identical between both samples. Errors intro- duced by cell mismatch are eliminated. There are, of course, serious restrictions on the method. The

standard and the unknown must emit in well-resolved

spectral regions (by means of time-resolved spectro- scopy, even this restriction may be lifted). They must

also absorb at the same wavelength.

Several experimental difficulties must be considered

closely if satisfactory results are to be obtained

using an internal standard. Because the standard

and unknown are present in the same solution, great care must be taken to prevent any bimolecular quenching or energy-transfer processes, since their occurrence would modify the yields of the species present [99]. As long as one is using optically dilute solutions, there

is no problem with long-range energy transfer of the

Forster-type. This would not, however, always be

true if optically dense solutions were employed.

Diffusion-controlled processes must also be considered. One can readily show that even if a sample

has a component with a lifetime as long as 1 (jls that

is quenched by the other one at the diffusion controlled

limit (10 10 M~l s" 1 ) then the concentration of the

second species must exceed 10~~6 M to cause 1 percent

quenching of the first. Since optically dilute meas- urements are usually carried out at optical densities

less than 0.01, then any second component with

e 3= 104 at the exciting wavelength would introduce a

negligible error. Of course, if the first compound has

a lifetime of less than 1 (jls or the quenching proceeded

with a rate less than the diffusion-controlled limit,

then this already weak restriction would be relaxed

still further. For example, if the standard's lifetime were 10~8 s (a typical fluorescence lifetime), then the

maximum permissible concentration of the second

species would be 10~4 M; this would permit molecules with e 3= 102 to be measured. The same argu- ments apply to the second component. Most materials

and standards would have coefficients satisfying this condition in some region. Thus, because of bimolecular

159
quenching it appears that the internal standardization

technique is largely limited to optically dilute measurements. Consequently, all of the restrictions and limi- tations on any optically dilute method are present [1]. The remaining serious difficulty or source of error en- visioned in a procedure employing an internal standard

is that inherent in accurately calibrating any spectrometer over a wide wavelength range. Also, some

compounds have extremely wide emission bandwidths;

then difficulty will be encountered in obtaining

satisfactory standards. Transition-metal complexes,

as discussed above, may possibly be designed for ap- propriate standards. Sensitivity, even when working

with low yield substances under optically dilute conditions, does not appear to present a severe

limitation for the method.

In summary, we believe that the use of an internal

standard in relative quantum-yield determinations

deserves serious consideration for investigation and

development. If well-defined standards become readily

available, the method could eventually become one

of the preferred ways of running high-accuracy yield

determinations.

V. Quantum Efficiencies by Actinometric

Methods

The principle of the actinometric method for measuring quantum yields is conceptually simple. One

merely surrounds the sample completely with an

actinometer solution, measures the apparent intensity

of the emitted radiation, and then, using the same type

of actinometer solution, monitors the intensity of the

excitation beam. If the irradiated solution is totally

absorbing, the ratio of the two intensities is the absolute quantum yield. Whereas actinometers are capable

of measuring absolute intensities, this technique is, in fact, a relative method since the absolute yield of the actinometer need not be known. To our knowledge,

this method has not been used for measuring optically

excited luminescence efficiencies. It has, however,

been utilized in a suitably modified form for measuring

chemiluminescence yields [100]. The principal reason for the nonadoption of an

actinometric method is experimental. Most actinometers have quantum yields that are strongly wavelength dependent, and their absorption characteristics

make it difficult to arrange to have all of the emitted

light absorbed over the very wide wavelength ranges

encountered in molecular luminescences. Because of these factors it is difficult to obtain an accurate measurement of the intensities of both the excitation

beam and the broad molecular emission with the same

actinometer. With the advent of new sources and acti- nometers, however, actinometric methods may achieve

a useful place in absolute quantum-yield measurements.

before launching into speculative discussion we

point out that the potassium salt of trans- [Cr(NH3)2(NCS).,] (Reinecke) is an excellent widerange actinometer covering the 300 to 600-nm region

[101]. Its yield has been measured over an extended

wavelength range, and it is reasonably sensitive. Unfortunately, it is not without its shortcomings. To

absorb most of the emitted light in the 300 to 600-nm

range, rather concentrated and/or thick solutions are required (about 1.5 cm at a concentration of 5X 10~2 M). The compound also exhibits a slow but bother- some thermal decomposition. Finally, there is a slight,

although perhaps not real, variation of the quantum

yield with wavelength, a variation that is, however,

small enough that reliable corrections could be made.

No one has confirmed the original yields, and the

initial data were taken at somewhat greater wavelength

intervals than would be desirable for precise corrections. Notwithstanding these objections, Reinecke's

salt is even now usable as a wide-range actinometer

for yield determinations.

Recent photochemical investigations portend the

development of additional wide-range actinometers.

Demas and Adamson [102] have found that tris(2,2'- bipyridine)ruthenium(II) sensitizes, by a diffusion- controlled process, the redox decomposition of tris- (oxalato)cobaltaie(lll). Since the donor has a broad, in- tense absorption, it is quite easy to obtain, with thin

solutions, total absorption of incident fight from the

UV to beyond 500 nm. The decomposition of the [Co-

(C^O^]3- may be readily monitored spectrophotometrically (unfortunately not with quite the high sensitivity of the ferrioxalate). Furthermore, since the efficiency of population of the donor level is believed to be unity

and to occur virtually instantaneously following excitation, the system has a decomposition yield that is in- herently wavelength independent and that has a broader

range than most other actinometers. In its present

form, their system is not, however^ a useful actinometer. With modifications it, or a related system, may

become practical. Lasers make the actinometric method for measuring

yields appear even more attractive. The excellent

focusability of lasers permits an extremely small entrance aperture to be used and thus minimizes

the error due to loss of radiation through the excitation port. Also, because the laser beam is inherently

well collimated, one could use relatively dilute solu- tions in very long cells and irradiate down the cylin- drical axis. In this way trivial reabsorption effects would be minimized. A device built along these lines might look much like a condenser with the outer

cooling jacket filled with the actinometer solution. Of course, any measurement based on an optically

dilute method would be subject to the normal corrections, and the optically dense method would be prone

to its usual inherent errors. In spite of these objections, common to all optical quantum-yield measurements, it seems that the actinometric method has

distinct possibilities even now with potassium

Reinecke, and it will probably become more attractive with time, especially as the developments in modern inorganic photochemistry gain momentum and

uncover new photochemical reactions and sensitizers.

160
VI. Quantum Efficiencies by Calorimetric

Methods

Although measurement of quantum efficiencies optically involves generally familiar instrumentation,

this statement cannot be made about calorimetric

procedures. Recently, however, developments in technology point toward a gaining importance of

calorimetry in the field of quantum yields, and we

wish to discuss some of them. Because of their rela- tive unfamiliarity, we present here a more detailed

account of the various methods employed in calorimetry than was included above for the optical methods.

Calorimetric methods of measuring quantum yields are based on the assumption that energy absorbed from

a beam of light incident upon a solution of fluorescent

molecules is either lost through reemitted photons or degraded to heat by radiationless processes. The objective of calorimetric techniques is to measure the

energy yield of these radiationless processes, Yh,

which is defined as the ratio of heat energy produced

to incident photon energy absorbed. Yh is obtained by

measuring the ratio of the heating produced by irradia- tion of the fluorescent sample to the heating produced

by irradiation of a nonfluorescent sample that absorbs

the same fraction of the excitation energy.

In the absence of photochemistry, the fluorescence

[103] energy yield is the complement of Yh and the

quantum yield of fluorescence is related to it by the

formula

<P=(valvf)(l-Yh ) (1)

where va and V/ are the average frequencies (cm-1

) of absorbed and fluorescence photons, respectively,

defined by the formulas

Va= UIa(\)A(k)dkl\]IUla (\)A (k)dk] (2)

Vf= [f If(k)dk/k]l[flf(k)dk] (3)

If(k) and Ia (k) are the fluorescence and excitation

intensities at wavelength k, A(k) is the percent

absorbance of the sample with a pathlength equal to the cell dimension, and the integrals are over the

fluorescence and excitation bands, respectively.

Since V/ and va are easily measured with high precision by standard photometric techniques, they will not be discussed further here [1, 29]. Several important factors favor calorimetric methods

for the determination of absolute quantum yields: (a) The method serves as a valuable independent check of

the assumptions used in photometric techniques for measuring <P/; in particular, corrections for experimental geometry, index of refraction changes, and

polarization effects are largely eliminated, (b) Using

relatively crude apparatus, some workers have obtained values of Yh with a precision comparable to those obtained by other methods [29]. (c) The calori- metric method works well with samples that emit in

the near infrared where the sensitivity of optical tech- niques is relatively low. Recent developments in photodetectors are alleviating this problem, however

(see above).

With the usual procedures and components, the

principal disadvantages and limitations of the method

have been the following: (a) The insensitivities of

the calorimeters used thus far have forced workers to use highly absorbing samples; consequently, large

corrections for reabsorption of the fluorescence [29,

104] are required, (b) The method has been limited to substances possessing high quantum yields, (c) Measurements have been confined to liquid samples at room temperature, (d) Methods devised thus far have

been tedious and time consuming to perform.

Recent advances in calorimetry, especially in the

technology of light sources and transducers, and improvements in methods of data acquisition portend

wider acceptance of the technique for obtaining

quantum efficiencies. The generality, sensitivity, and

ease of performance of calorimetric methods will undoubtedly improve. Our purpose here is to review some of the advances in methods and components

and to indicate possibilities for still greater improvements.

A. Calorimeters

The calorimetric apparatus consists of an excita- tion source, a means for selecting a bandwidth of the

incident radiation, and a calorimeter to measure the

heat energy produced during irradiation. The science

of calorimetry is a mature field of endeavor, and the

reader desirous of a general background in the subject

is referred to the several books and recent reviews now available [105-108]. Let us begin oui discussion

of calorimetric apparatus by considering the constraints on the calorimeter imposed by the requirements for measuring quantum yields. First, provision

must be made for admitting the exciting light. Second,

the calorimeter must be designed so that all fluores- cence escapes the cell. Third, since the sample will not be uniformly irradiated, the average or total heating

must be detected. Fourth, the calorimeter must be very

sensitive. In general, the sensitivity requirements will depend on the energy of the incident radiation, the

absorbance of the sample, and the value of the quantum

yield to be measured. A straightforward analysis of the

propagation of errors in the calorimetric method shows

that in order to measure a value of <P/ = 0.5 with a pre- cision of 1 percent, the heating of the nonfluorescent

sample must be measured with a precision of 0.1 percent.

It is shown later that most calorimeters detect the

heating power produced by steady state irradiation.

If the incident radiation power is 100 mW, and we are using samples that absorb 10 percent of the beam or

less to avoid reabsorption effects, then our calorimeter

must be capable of measuring a heating power of 10

/iW with a signal-to-noise ratio of unity.

It is now obvious that the method for detecting heat

flow in the calorimeter must be very sensitive. Most

161
calorimeters use temperature transducers to measure

heat flow. One figure of merit, useful for evaluating

temperature transducers, is their noise equivalent

temperature (NET) given by the following expression

NET= [N/S(Afyi2

] deg/Hz1 ' 2 (4)

where N is the noise voltage developed by the trans- ducer, S is the voltage sensitivity of the transducer,

and Af is the noise bandwidth. The NET is defined as the temperature change required to generate a signalto-noise ratio of unity when the detector noise is re- ferred to a one cycle bandwidth.

One common method for measuring temperature

changes in calorimetry is by means of a thermopile.

Generally, the limiting noise in this device is due to the

Johnson noise of its resistance. For a 100-junction

copper-constantan thermopile of 100 fl resistance,

the NET is calculated to be 0.2 /udeg/Hz 1/2

. The tiny

voltages associated with these devices can be con- veniently measured by means of a commercially

available nV amplifier such as the Keithley Model

148. The construction of thermopiles is an art and is discussed by Hill [109], Howarth [110], and Evans [111]. Resistance thermometers have also been used for calorimetric work. Larsen [112] has shown that the

sensitivity of platinum resistance thermometers op- erated by an ac bridge approaches the theoretical

limit imposed by the Johnson noise of the device. He

gives a NET of 20.5 /u.deg/Hz1/2 for a commercially

available 500 Cl platinum resistance thermometer.

These tranducers are very stable with time, are smaller

in size than thermopiles, and do not require reference

junctions.

The use of thermistors in calorimetry has been dis- cussed by Meites et al. [113]. Previously, these temperature transducers have been criticized for calorimetric

work because of long term instabilities [112]. Meites

et al. found, however, that operation of selected and

aged 100 K thermistors at low power levels i(18/u,W)

allowed reliable detection of temperature changes of 10 (jl deg by means of a dc bridge and recording

potentiometer. The NET as predicted by consideration

of the Johnson noise is even lower, 0.7 /ideg/Hz1 ' 2

. Attainment of this resolution will probably require use

of an ac bridge and phase sensitive detection. Thermistors also offer advantages due to their small sizes and fast response times. In addition to temperature transducers, one can also employ volume transducers to measure heat flow. The

volume change, AV, produced at constant pressure in response to a heat change, A^, is given by the formula

AV=aAQICpp (5)

where a is the thermal expansion coefficient of the

sample, p is its density, and Cp

is its heat capacity per

gram. A simple dilatometer for measuring volume

changes in calorimetric experiments has been used by

Seybold, Gouterman, and Callis [29]. In their apparatus, the sample is contained in a cell open to the

atmosphere only through a capillary tube. The volume

of the solution is visually monitored as it expands up

the capillary. Volume changes as small as 7X10-5 cm3 can be measured; for a 10-cm3 ethanol sample this corresponds to a temperature rise of 7X10-3 °C. Further increases in sensitivity in capillary dilatometers can be obtained by using a travelling microscope [114] or a photocell to measure changes in the

height of the liquid in the capillary.

A greatly improved technique for measuring

volume changes has been developed by Callis, Gouter- man, and Danielson [44]. These workers use a capacitor microphone to detect volume changes. They

claim a noise equivalent volume (NEV) change of

4X10-10 cm3 referred to a 1-Hz bandwidth, which

corresponds to a NET of 0.04 /ideg/Hz 1/2 for heating

10 cm3 of ethanol. In addition to its sensitivity, this technique has other advantages: (a) The volume

change is independent of the size of sample irradiated,

whereas the magnitude of the temperature change

depends on the size of the sample, (b) Equation (5)

is valid even though a uniform temperature is not established if, at all points, AT is small enough so

that the equation holds locally. Thus, the sample need

not be stirred, (c) The response time of this apparatus is limited only by the transit time for sound in the cell. Disadvantages of the volumetric technique are: (a) The electronics, as originally devised by Callis et

al. are ac coupled. Use of a capacitance bridge can overcome this disadvantage, however, (b) The ap- paratus is sensitive to vibration and acoustical noise

and therefore must be isolated from both, (c) For water, an important solvent, ajpCp

is ~ 10 times lower than

for organic solvents.

All of the calorimeters we shall discuss in this paper

are surrounded by an isothermal shield, and thus

all can be characterized by the same energy flow equation

dH/dt = FYhP(t)-KH(t). (6)

H(t) is the heat energy retained in the sample, F is the fraction of incident power absorbed by the solute,

P(t) is the incident radiation power, and K is the first- order heat loss constant under the assumption that

all heat losses are newtonian. We have neglected

heating due to absorption by the solvent and reabsorption of fluorescence photons. These effects are dis- cussed elsewhere [1]. Two cases of eq (6) may be distinguished: (a) When

the heat loss term is negligible, the calorimeter is an

adiabatic one. (b) When the heat loss term is large,

the calorimeter is a conduction calorimeter. When

the heating input is exactly balanced by an equal

cooling, then the calorimeter is known as a compensation calorimeter. Let us first consider the use of the conduction type

calorimeter as employed by Alentsev [104] for measuring Yh. The calorimeter consists of a thin cell sus- pended in a chamber whose walls are maintained at a constant temperature. Temperature changes are measured by means of a thermopile. The measuring

162
junctions are arranged symmetrically around the lateral wall of the cell, and the reference junctions are buried in the isothermal wall. The major source of

heat loss is through the thermopile itself. A schematic

of the apparatus is shown in figure 6. Since temperature changes are measured, we can use the relation- ship H(t) = Cp [T(t) — To] to convert eq (6) to a form

involving the instantaneous temperature.

constant

/-temperature ¥ wall thermopile

Figure 6. Conduction calorimeter.

dT(t)ldt= [FYh P(t)/Cp ] —K[T{t) - To] (7)

where To is the temperature of the wall. For steady

state irradiation (P constant), the solution to eq (7) is

T(t) -To = FYhP(KCp ) - 1 [1 - exp ( - Kt) ] . (8)

After a time t > 1/K the maximum temperature rise,

ATm , is reached.

ATm = FYh P(KCp ) (9)

The ratio of ATm for the fluorescent sample to ATm

for a nonfluorescent sample that absorbs the same

fraction of incident light gives Yh, provided K and

Cp are the same. For a given energy input, ATm can

be maximized by using the lowest value of K and

Cp. As used for determination of Yh, the conduction

calorimeter may be thought of as a power measuring

device, and thus a factor of merit is its noise equivalent

power (NEP), which is defined as the power input

required to generate a signal-to-noise ratio of unity

when the detector noise is referred to a 1-Hz bandwidth. For the conduction calorimeter

NEP = NET(K)CP (10)

where the NET is that of the temperature transducer

used. For a NET of 1 /udeg, a heat capacity of 10

cal/°C and a K 1 of 10 min, one obtains a NEP of

0.07 /x.W. In practice, the best conduction calorimeters

[115] exhibit a NEP of 0.2 /xW; but even this value is far better than that obtained by Alentsev. The limit of

sensitivity of this apparatus is apparently determined

by the temperature stability of the environment of the calorimeter [115]. Design and construction of

conduction type microcalorimeters is discussed ex- tensively by Evans [111] and by Calvet and Prat

[115, 116]. In the most advanced designs [111, 115, 116],

the experiment is conducted differentially, i.e., the

temperature difference between the irradiated sample

in one cell and an identical unirradiated sample in a twin cell is measured. This technique greatly increases

the long term stability of the apparatus and is to be

recommended for any type of dc calorimetry.

As employed for conventional calorimetric measurements, conduction calorimeters are not stirred. This

is possible if a large number of thermocouples is distributed evenly over the surface of the calorimeter

so that the average surface temperature is measured.

For fluorescence measurements, however, it is ideal

to have all of the fluorescence escape the cell. Thus,

the thermopiles should occupy only a tiny fraction

of the cell surface. The cell must be stirred. Unfortunately, stirring produces its own heating, which

gives rise to additional noise in the system [111].

Perhaps the major disadvantage of using conduction

calorimeters is that long times are required for measurement. For a K~l equal to 10 min, one must wait

at least 1 h for the sample to come into equilibrium,

and additional time will be taken waiting for the

temperature to stabilize. Some recent work by Chastel and Tachoire

[117] suggests that the commercially available and

highly perfected Tian-Calvet conduction microcalorimeter may be used in a virtually unmodified form in conjunction with a pulsed light source. The calorimeter is shown schematically in figure 7. The cell is made of glass or quartz, which is surrounded by a

metallic "can" whose inside is coated with a totally

absorbing black substance. The thermocouples are

light path

constant temperature

wall

metallic container

(blackened) glass cell sample

thermopile

blank

cell experimental

cell

Figure 7. Tian-Calvet conduction microcalorimeter modified for quantum-yield determination.

AT

^fluorescence energy ^radiation less energy

Figure 8. Thermogram from Tian-Calvet microcalorimeter.

163
affixed to the metallic cell. Irradiation of the sample

produces a double flux of light and heat. The light is converted to heat through absorption by the metallic

cylinder. A recording of the temperature as registered

by the thermocouples versus time in the form of a thermogram would be expected to yield the results shown in figure 8. The first peak is due to light ab- sorbed at the cylinder; the second is due to heat

diffusing from the sample produced by radiationless processes. If the response times of the apparatus to heat and light are determined, then it is easy to separate the two responses and obtain the energy

yields. Chastel and Tachoire have obtained excellent

results on energy yields of flash sources for photography in this manner. A second type of calorimeter, which can be used for measurements of Yh, is a heat flow compensation

calorimeter. The design of such calorimeters has been

discussed by Sturtevant [118]. In these devices, heat- ing produced by irradiation is balanced by an equal

amount of cooling power supplied by a thermoelectric

module. The null heating point is sensed by a temperature transducer. Thus, the cooling power supplied is a direct measure of the energy input power. An ap- paratus is shown schematically in figure 9. This device

isothermal wall

calorimeter -

pOmplif ier

temperature sensor

thermoelectric heat pump

FIGURE 9. Heat flow compensation calorimeter.

is also essentially a power measuring apparatus, and

its factor of merit is given by the NEP, which is related

to the NET of the temperature sensor, as follows: NEP = NET{Cp

)t- (11)

where r is the time constant of the temperature detection. To minimize the NEP, one desires the lowest

values of heat capacity and NET and the longest time

constant. For a NET= 1 fideg, Cp = 10 cal/°C , and r=10

the NEP is 4.2 fxW. This type of calorimeter may also be used in a differential mode where the difference

between an irradiated and unirradiated cell is measured.

Adiabatic calorimeters are a third possibility for Yh

measurements. They have been employed in this manner by Seybold, Gouterman, and Callis [29] and

by Gudmundson et al. [119]. In these devices one at- tempts to eliminate heat transfer between the calorimeter and its surroundings. Because of the tiny temperature changes involved, this ideal is best approached

by conducting experiments in a constant temperature

jacket calorimeter for short time periods so that heat

losses are negligible. As with conduction calorimeters,

adiabatic calorimeters can be used with steady or pulsed sources. If the sample is irradiated by a steady

light source, the temperature rise can be measured as

a function of time. In this case, the calorimeter may

be thought of as a power measuring device, and the

minimum detectable power level will be related to the

NET of the temperature sensor by the formula

NEP = NET(Cp )Ar (12)

where Af is the maximum time after the start of ir- radiation at which a measurement can be made. For

the isothermal jacket calorimeter this condition will be K&t < 1 where K is the rate of newtonian heat

loss. For a NET of 1 fxdeg, a Cp of 10 cal/°C, and a A* of 100 s, a NEP of 0.42 /iW is obtained. If a pulsed

light source is used, the net temperature rise is measured. In this case, the calorimeter determines

the integral of the incident power, and thus a figure of merit is the noise equivalent energy change, NEE,

which is just the NET of the temperature sensor

times the heat capacity of sample and calorimeter.

Thus, for a NET of 1 fideg and a 10 cal/°C heat capacity, we obtain a NEE of 10 /xcaL This method is attractive because pulsed light sources of high energy are

readily available (e.g., pulsed lasers), and heating due

to absorption of fluorescence by the calorimeter can

be separated by its time dependence.

Thus far, all of the types of calorimeters we have

discussed are, in principle, capable of measuring very

low power levels and will allow elimination of reabsorption corrections. They all have a common single

disadvantage, however. The measurements are per- formed near zero frequency and are tedious and time

consuming. Further, the temperature of the calorimeter and surroundings must be controlled to the NET

of the temperature sensing device if the lowest possible

NEP of the calorimeter is to be realized. Currently, a new technique for calorimetric measure- ments of Yh, which can overcome some of the dis- advantages of conventional calorimetry, is under

development. Essentially the apparatus is the flash calorimeter of Callis, Gouterman, and Danielson

[44] shown in figure 10. It consists of a glass cuvette, one wall of which is the compliant diaphragm of a capacitor microphone. The valve attached to one

side of the cell serves as a capillary leak so that the

liquid can slowly enter or leave the cell. This prevents

slow pressure changes that would occur in a completely

closed cell subject to temperature drifts and the dc

component of repetitive excitation. Because of the fast response time of this apparatus,

the exciting light is modulated, and the resultant heating is detected at the frequency of the modulation

by phase sensitive techniques. The advantages of ac methods of doing experiments is well known

[120]. Briefly, all experiments are contaminated with

noise, some of which has a 1//" frequency dependence.

This latter noise is dominant in calorimetric measure164
STATIONARY ELECTRODE

Figure 10. Flash calorimeter.

ments, which are performed at low frequencies where

thermal drifts are important. By modulation one can

shift the frequency domain of the experiment to a point

where noise due to thermal drifts is no longer important and thermostating can be eliminated. By means of phase sensitive detection, very narrow bandwidths

can also be obtained, further discriminating against

noise. Due to these advantages, it is worthwhile to consider

in more detail this new method for determining Yh

. Assume that the light intensity has the time dependent

form

P(t)=P0 {l + cos <ot). (13)

Po is the steady state power in cal/s and o> is the modulation frequency. The heating in the cell in response

to the excitation is given by eq (6). We may combine

eqs (5) and (6) to obtain a differential equation for the

time dependence of the volume change

dV(t)/dt= [aFYhl PCp]P(t) -K'V(t) (14)

where K' is the first-order rate constant for removal of the excess volume change and is given by

K' = aKICp + K". (15)

In this expression, K" is the rate constant for the capillary leak. Integration of eq (14) gives for the steady

state solution

V{t) = YhPo(cos<1)t)lpCp(t> (16)

where we have assumed that <t>> K'

.

The amplitude of the signal will, therefore, be

inversely dependent upon the frequency of modulation.

In practice, the experiment should be done at the

lowest frequency commensurate with unimportant

llf noise. This apparatus, like the other calorimeters

discussed, is also a power detector, and its NEP will

be given by

NEP = NEV(Cp )p(ola. (17)

For a NEV of 10" 10 cm3

, pCp ja = 4 X 102 cal/cm3

, and &>=1 s _1

, we obtain a NEP of 0.2 ^W. In addition to its great sensitivity, the advantages of this technique over others we have thus far discussed are the elimination of elaborate thermostating and the

execution of measurements rapidly.

An alternative method for using the flash calorimeter

is to employ a pulsed exciting source. If a fairly high

repetition rate is achieved, the volume change can be

stored in a computer and averaged over many repetitions. If the noise is random, then the improvement in signal-to-noise ratio will increase as the square root of the number of experiments averaged.

B. Light Sources

The ideal light source for calorimetric measurements

must be of high intensity, stable with time, and of

high spectral purity. Thus far, only conventional

noncoherent light sources have been used. Tungsten

lamps are very stable with time but give useful

output only in the visible region of the spectrum;

further, their high infrared output is difficult to remove. High pressure mercury and mercury-xenon arc lamps

appear to be the most useful of the noncoherent light sources. They generate high-intensity pressure- broadened lines in the visible and ultraviolet, produce

a tolerable infrared output, and can be easily stabilized. Monochromatization of light from noncoherent

sources has been achieved through use of solution

filters, but complete removal of infrared light is not

possible. In contrast, interference filters can be

fabricated to specification and will remove most

unwanted infrared radiation.

Potentially the most useful light sources for calo- rimetric yield measurements are lasers, just as they

are for optical quantum-yield measurements. As

discussed above, there are now commercially available

several different types of cw gas lasers that lase at a

variety of frequencies in the spectral region 320 to 650 nm. The 2-W argon ion laser is a good example

(see table 1). Other gas phase lasers commercially

available and of possible use in calorimetry are Kr, He-Ne, He-Cd, Se, and Xe. The amplitude stability

of these lasers can be made as high as 0.1 percent.

Generally, there is some noncoherent plasma radiation

in the beam, but this may be removed by interference

filters and the use of long pathlengths. The principal

drawback to these devices is that their outputs are limited to specific lines, and it is always desirable to excite a molecule at the maximum of its absorption.

The availability of the laser-pumped dye lasers should

remedy this problem. The narrow line widths, stable outputs, and tuning capabilities over wide spectral

regions make them prime candidates for sources in calorimetric quantum-yield determinations.

502-591 0-73-12 165
If a pulsed light source can be used, then the flash- lamp-pumped dye laser is an ideal choice. As discussed

previously, these sources can be tuned continuously in the region 340-600 nm using several different dyes,

and their extension farther into the infrared is already

at hand. Good short term amplitude stability and high

energies per pulse are commercially available in coaxially pumped systems.

C. Conclusions

Calorimetric methods for measuring quantum yields

have been little used in the past because of the diffi- culty of making the measurements and, to some extent,

because of the average photochemist's unfamiliarity

with the techniques. Recent advances in the technology

of calorimetry, the progress in transducer design, and

the availability of laser excitation sources promise

considerable improvements in the rapidity and accuracy

of calorimetric methods. These improvements should

lead to wider appreciation of this powerful technique

for attacking the fundamental problem of measuring

radiative and radiationless efficiencies of excited

molecular systems.

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(Paper 76A6-742)

167

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Phosphori metry *

J. D. Winefordner

Department of Chemistry, University of Florida, Gainesville, Florida 32601

(July 26, 1972)

Phosphorimetry in the past has received limited use because the precision of reproducibility was

inadequate, there were solvent limitations, and preparation of test specimens was difficult and time consuming. Detection limits have now been lowered by more than two orders of magnitude by using a

rotating capillary sample cell, a more stable excitation-source power supply, and aqueous solvents. These steps have also increased precision by more than an order of magnitude. Considerable reduction

in time and effort of sampling and measurement has been effected compared to phosphorimetric measurements made with standard procedures and commercial equipment. Twenty microliters of aqueous solution is all that is required to fill a quartz capillary cell by capillary action. Capillary cells

filled with aqueous solutions do not crack when cooled to 77 K or when returned to room temperature.

Rotation of the sample cell minimizes effects due to cell orientation and thus improves precision. Reduction of background phosphorescence results in improved accuracy of analysis. A study was made

of the influence of methanol-water mixtures and of sodium-halide aqueous solutions on the magnitude

of phosphorescence signals from several substances and of the effect on signal-to-noise ratios. The

optimum solvent system for many phosphorimetric measurements is discussed. Analytical results are given for several organic substances measured by phosphorimetry at 77 K. These results are compared

with those from previous studies by older methods.

Key words: Aqueous solvents in phosphorimetry; phosphorimetry; rotating capillary cell; solvents for phosphorimetry.

I. Introduction

Phosphoresence is a type of photoluminescence in which radiation is emitted by a molecule following

radiational excitation of the molecule by means of

radiation of higher energy than the emitted radiation. Phosphorescence involves two singlet-triplet transi- tions and as a result has a longer lifetime than fluores- cence which involves two singlet-singlet transitions. Because the first triplet level of a molecule with an

even number of electrons has lower energy than the

first excited singlet level of the same molecule,

phosphorescence also involves lower energy transitions

than fluorescence, and so the phosphorescence spec- trum of a given molecule occurs at longer wavelengths

than the fluorescence spectrum of the same molecule.

The mechanism of the production of phosphorescence

is well known [1-3] 1 and so will not be discussed.

This manuscript is concerned with the analytical uses

of phosphorescence for quantitative analyses of organic

molecules—called phosphorimetry. Zander [4], Winefordner, McCarthy, and St. John [5], Winefordner,

St. John, and McCarthy [6], McCarthy [7], and Winefordner, Schulman, and O'Haver [8] have reviewed and

*Research was carried out as part of a study on the phosphorimetric analysis of drugs

in blood and urine, supported by a U.S. Public Health Service Grant (GM- 11373-09).

1 Figures in brackets indicate the literature references at the end of this paper.

described the instrumentation, the methodology, and

the uses of phosphorimetry.

Phosphorimetry has been used during the past

decade for a limited number of quantitative analyses,

e.g., the analysis of impurities in polycyclic aromatic

hydrocarbons [9-11], in coal tar fractions [12], in air [13-15], and in petroleum fractions [16—18], the

analysis of inhibitors in petroleum products [19], the

analysis of pesticides, fungicides, oils, etc., in foods

[20-25], and the analysis of amino acids and pharmaceuticals in biological fluids [27-35]. It is evident that up to now, phosphorimetry has been utilized only

sparingly as an analytical tool for quantitative analysis

and has been confined primarily to those cases where

other analytical methods, such as fluorimetry and

absorption spectrophotometry, either cannot be used

due to lack of sensitivity or presence of interferences

or give ambiguous results, and so additional analytical

information is desirable. In fact, fluorimetry and ab- sorption spectrophotometry have often been used for quantitative analyses which could quite well have been

performed with greater precision, accuracy, and speed

by means of phosphorimetry. The lack of use of phosphorimetry (with commercial spectrofluorimeters with

phosphoroscope attachments) for quantitative analysis

has been a result of the following factors: (i) the

greater complexity and time to carry out an analysis

due to the use of small sample cells which are difficult

169
to clean, fill, align, and empty, the need for solvents

which form clear, rigid glasses at liquid nitrogen

temperatures, and the use of liquid nitrogen for sample cell cooling; (ii) the poor precision and accuracy

of measurements due to the difficulty of alignment

of the sample cell in the Dewar flask containing the

coolant, due to the poor source stability—particularly

long term drift, and due to the ease of contamination

of glassware, cells, and solvents; and (iii) the marginal

sensitivity (limits of detection) for many molecules

due to light losses resulting from the excessive number

of optical surfaces, the small sample cells, and the use of the phosphoroscope (mechanical chopper)

which cuts out more than 75 percent of the measured

signal as compared to continuous excitation-measure- ment. In this manuscript, the instrumental methods

utilized in the author's laboratory to eliminate or minimize the above limitations will be described and

typical analytical data indicating the improvement

in results will be given.

II. Typical Commercial Phosphorimeter

operated xenon arc lamp driven from a well-regulated

dc power supply so as to minimize long-term drift. A

portion of the source radiation is introduced into the

excitation monochromator to produce a small spectral

band of excitation radiation which is focused upon the

sample cell, usually a quartz tube (containing sample)

which is placed in a quartz Dewar flask which contains

a coolant (usually liquid nitrogen) for freezing the sample (see fig. 2). A fraction of the phosphorescence

emission is observed by the emission monochromator,

and the wavelength region of interest is thus presented

to the transducer, usually a multiplier phototube with an S—4 or S-5 response. In the averaging dc measure- ment systems, frequently employed in commercial

spectrophotofluorimeters, the current (usually 10~ 6 to 10~9 A) is amplified and either read out directly from

a meter face or on a time base or %—y recorder (the

x—y recorder is most frequently employed when utiliz- ing one of the monochromators in the scanning mode

for recording of spectra, and the time-base recorder is often used for quantitative measurements at one set of excitation and emission wavelengths).

In order to compare the improvements made in phosphorimetric instrumentation, it is necessary to describe the typical commercial phosphorimeter. A

block diagram of a typical commercial spectrophotofluorimeter with rotating can phosphoroscope which

has been used by the author (e.g., AMINCO SPF2 with

phosphoroscope assembly) is shown in figure 1. The

source of excitation energy is generally a continuouslySAMPLE

'COMPARTMENT SAMPLE

TUBE -DEWAR FLASK EXCITATION MONOCHROMATOR A

EMISSION MONOCHROMATOR

PHOTOTUBE

ELECTROMETER

LIQUID NITROGEN

SAMPLE IN RIGID SOLVENT

UNSILVERED QUARTZ

Figure 2. Schematic diagram of typical commercial sampling

system for phosphorimetry.

RECORDER

FIGURE 1. Block diagram of typical commercial analytical spectro- photofluorimeter with phosphoroscope attachment.

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no in- stances does such identification imply endorsement by the National Bureau of Standards,

nor does it imply that the particular product or equipment is necessarily the best available

for that purpose.

A means for terminating exciting light and allowing

measurement of a portion of the phosphorescence

radiation without interference from the intense,

scattered, incident radiation and the faster decaying

fluorescence radiation must be included in the phosphorimeter design. The instrument diagrammed in figure 1 utilizes a typical rotating-can phosphoroscope,

shown in greater detail in figure 3. In operation, the

sample is excited for a short time through a window in the can; the can rotates to terminate excitation, after

170
EXCITING

RADIATION

Figure 3. Schematic diagram of a typical rotating can phosphoroscope.

which another window is presented to the detection

axis. The phosphorescence signal is observed through

this window until the window moves off-axis, then the

process is repeated. The instrumental response time

constant employed in the dc measurement systems

used with such phosphorimeters is usually made suffi- ciently large so as to provide a steady phosphorescence

signal at all but the slowest phosphoroscope speeds.

Other means of modulation, such as the Becquerel

disc phosphoroscope and pulsed source-gated detector

systems, are not used in commercially available phosphorimeters. The pulsed source-gated detector system

is by far the most versatile and potentially the most

useful analytical system for phosphorimetry. All improvements discussed in this manuscript will be

with respect to the system described above and shown

in figure 1.

III. Instrumental Improvements in

Phosphori metry

A. Solvent Selection and Sampling Technique

Improvements

Phosphorescence is generally not observed in liquid

solutions at room temperature because radiationless

deactivation due to collisions which occur within the

lifetime of the excited molecule result in low phosphor- escence quantum yields [36] producing phosphor- escence signals buried within the phosphorescence

background noise [37]. Therefore, all analytical phosphorimetric studies have been carried out in rigid

media [4-8]. The criteria previously used to select a

solvent for phosphorimetry have been: solubility of the

analyte; low phosphorescence background (easy to

purify); and formation of a clear, rigid glass at 77 K

(boiling point of liquid nitrogen which is a safe, inexpensive, optically transparent coolant).

Unfortunately few solvents, particularly very polar ones, form transparent glasses at 77 K [38-40]. Ethanol

is an excellent polar solvent for phosphorimetry.

Small volume percents of water, mineral acids, and

strong bases can also be combined with ethanol to produce clear rigid media at 77 K. The most popular

mixed solvent has been EPA (a 5:5:2 volume ratio of

diethyl ether, isopentane, and ethanol). Unfortunately,

these solvents either do not have good solubility

characteristics or result in irreversible changes of

many organic species, particularly those of biological

importance. Solvents, which are crystalline or severelycracked at 77 K, have been used for some qualitative

studies [41-44] but have generally been ignored or discounted as solvents for quantitative measurements.

Scattering [45] and surface reflections [46] have been

cited as reasons for not using snowed media [4-8] in analytical studies. The cracks or crystals create

optical inhomogeneities in the medium which result

in nonreproducible phosphorescence signals. Freed

and Vise [29], as well as Parker and Hatchard [47], have suggested the use of an internal standard (spectrally removed from the molecule of interest) to be

used as a reference to compensate for variations in sample positioning and cracking of the medium.

The use of an internal standard offers a reasonable

solution to the problem of sample positioning and

occasional cracking; however, the choice of an

internal standard satisfying the above criteria and

not undergoing chemical or physical interaction with

the compound being studied is extremely difficult and

time consuming. In addition, the use of an internal

standard is not applicable to the case of snowed or cracked media because the phosphorescence signal

may be influenced by the extent of scattering, which

can be wavelength dependent as long as the effective particle size is small [48, 49]. An alternative to the internal standard technique was suggested first by Hollifield and Winefordner

[50] and later improved upon by Zweidinger and

Winefordner [51] and still later by Lukasiewicz,

Rozynes, Sanders, and Winefordner [52], and by

Lukasiewicz, Mousa, and Winefordner [53, 54] all of whom used a rotating sample cell assembly to average

all orientations of the sample cell, thus resulting in greater precision and accuracy of phosphorometric

measurements in clear, rigid glasses [50], in rigid

organic snows [51], and in rigid, aqueous snows

[52-54]. In addition, there are no difficulties en- countered with sample positioning and alignment.

Lukasiewicz, et al. [52-54] have added another

great improvement to allow the measurement of aqueous or predominately aqueous solutions at 77 K by means of phosphorimetry. These workers used an open quartz capillary cell as the sample cell. Sample solutions (~20 yu.1) fill the cell by capillary

action, and the sample cell is then rotated as described

above. As a result of the open capillary cell, these cells with predominately, or pure, aqueous solutions do not crack when rapidly cooled to liquid N2 temperatures

or when warmed back to room temperature by use of a heat gun. Of course, the major advantage of using

aqueous or aqueous-organic solutions in phos171
phorimetry are: most biochemical species are more

soluble in aqueous solutions than in the solvents

needed to produce clear, rigid glasses at liquid nitrogen

temperatures; the solution conditions, e.g., pH, ionic

strength, etc., can be varied for optimal analytical

results; water is easier to obtain in higher phosphorimetric purity than most organic solvents; and

contamination problems are less when water can be

used to clean glassware, cells, and equipment.

The rotating sample cell assembly currently being

used is a modification of the one described by Hollifield and Winefordner [50]; the present system,

described in detail by Zweidinger and Winefordner [51] and modified by Lukasiewicz, et al. [52-54], is shown

in figure 4. The rotating assembly consists primarily of

a Varian A60—A high resolution NMR spinner assembly

(Varian Associates, Palo Alto, Calif.) mounted on an AMINCO phosphoroscope sample compartment in

rru

E

o

1

7

E=3

FIGURE 4. Schematic diagram of rotating sample cell assembly [51]. A. Quartz sample cell B. Varian (909614-04) spinner assembly for NMR

C. Aminco light cover mount

D. Aminco quartz Dewar flask

Reprinted from Anal. Chem. 42 , 643 (1970). Copyright (1970) by

the American Chemical Society. Reprinted by permission of the copyright owner.

place of the usual lid. The pressure cap of the spinner

assembly is covered with black tape to ensure a completely light-tight sample compartment. The rotating

sample cell is driven by a flow of nitrogen gas or air taken from a compressed gas cylinder. The sample cell must have a 6 mm o.d. and be about 25 cm long. The

sample rotation is maintained at some constant speed

between 450 and 1400 rpm; the actual speed is unimportant as long as it is constant during a series of measurements and can be assured by means of a normal two-stage regulator and a rotameter flow meter

to monitor the gas flow rate. The normal quartz sample

cells used by Zweidinger and Winefordner [51] were

5 mm o.d. and 3 mm i.d., whereas the quartz capillary

cells used by Lukasiewicz, et al. [52-54] were 5.0 mm

o.d. and 0.90 mm i.d. The cells were made of synthetic,

high-purity, optical-grade quartz (Quartz Scientific Co., Eastlake, Ohio for the capillary tubing and

Amersil, Inc., Hillside, New Jersey for the normal

tubing).

The inherent photoluminescence of the quartz

resulted in an appreciable "phosphorescence" background, especially with the capillary cells. However,

the long-lived luminescence of the quartz cells could be

minimized by mounting an excitation polarizer oriented

perpendicular to the sample cell length between the

sample cell housing and excitation monochromator

entrance slit. Luminescence excitation spectra of the

long-lived component of the quartz capillary sample

cells are shown in figures 5 and 6. It can be seen from

the results in figure 6 that the lowest background was

obtained when both polarizers were used. For quantitative work, however, use of both polarizers is undesirable because each polarizer has a low transmittance

and the loss of sensitivity is compounded. The background resulting when only one polarizer was used in the excitation beam was slightly higher than that obtained with two polarizers but still approximately 100

times lower than the background obtained without the use of the excitation polarizer.

The improvement in precision of measurement when

the normal sample cell was rotated as opposed to not rotated is given in table 1. It should be stressed that the

rotating sample cell holder, even when used in the

stationary aligned mode (no rotation), can provide and

increase in precision of as much as tenfold over the same cell in random orientation. When the normal

sample cell is rotated, a further improvement in precision of about twofold is obtained (see table 1). It is interesting to note that the precision for snowed and

cracked matrices is not significantly different from

that for an ethanolic, clear matrix. The stationary,

randomly oriented sample cell measurements were

considerably more precise than those obtained by

Hollifield and Winefordner [50] because of the close tolerances of the present rotating sample cell system.

One great advantage of using the rotating sample

cell is the simplicity of positioning the cell. After the

normal sample cell is cleaned and filled (requires

about five minutes), the cell with the Teflon (du Pont)

turbine is simply dropped into place and spun by flow

172
(D

200 300 400 500 600

Wavelength (nm)

Figure 5. Excitation and emission spectra of long-lived component of quartz capillary sample tube (no polarizer used) spectra 1, 2, 3, 4, 5, and 6 corresponding to excita- tion at wavelengths 210, 240, 230, 250, 220, and 280 nm respectively.

Reprinted from Anal. Chem. 44, 238 (1972). Copyright (1972) by the American

Chemical Society. Reprinted by permission of the copyright owner.

Table 1. Precision of phosphorescence measurements a for clear glasses and snows using rotating sample cell assembly [51]

Relative percent standard deviation

Number of Nature of Stationary 3 '" Stationary

,c Rotat- determinations matrix random aligned inga,f orientation

4 Clear" 8.7 1.3 0.8 6 Clear" 6.0 0.5 0.8 5 Cracked" 13.7 3.4 1.4 11 Clear" 3.1 0.9 0.3 10 Clear" 2.9 2.8 1.0 5 Snowc 3.6 1.6 0.9 10 Snow0 2.8 0.8 0.6 10 Snow0 2.7 2.4 0.7

a Phosphorescence measurements were made on 1.6 X 10~5 M

sulfanilamide solutions which give a signal five orders of magnitude

above phototube dark current. The normal rotating sample cell was

used. "Ethanol solvent.

0 Isooctane : ethanol, 4 : 1 , vjv mixture as solvent.

d This column is indicative of the reproducibility obtainable with

the commercial sampling system.

e This column gives results for the rotating normal sample cell assembly but with the normal cell stationary in the assembly.

f This column gives results for the rotating normal sample cell assembly but with the normal cell rotating in the assembly.

of compressed air. As a result of spinning the cell, the

inner filter effect is also minimized.

A series of 10 replicate determinations of 1 /xg/ml

2-thiouracil in 10 percent v\v methanol-water were measured to determine the precision obtainable with

the rotating quartz capillary cell. The relative standard

deviation obtained was 1.5 percent. This is an improvement of nearly an order of magnitude over the

stationary quartz capillary tube system used by

Lukasiewicz, Rozynes, Sanders, and Winefordner [52], and compares favorably with the standard deviations

of results obtained with normal rotating sample cells (see table 1). The major noise contribution in the

system with the rotating capillary cell arises from

wobble of the cell caused by the loose fit of the turbine

sleeve in the spinner assembly. If the capillary cell were held rigidly by the turbine sleeve so that no wobble occurs on rotation the precision would probably

be comparable to the best data reported in table 1 for the larger, normal quartz, sample cell.

B. Improvements in Precision and Accuracy of Phosphorimetry

The relative percent standard deviations obtainable

with the typical commercial system shown sche173
200 300 400 500 600

Wavelength (nm)

Figure 6. Excitation and emission spectra of long-lived component of quartz capillary sample tube, using polarized radiation; orientation of polarizers with respect to vertical axis of cell as follows [52]:

Excitation Emission

1. ± II 2. II II 3. 1 1

4. II 1

5. II no polarizer

6. 1 no polarizer

7. no polarizer 1

8. no polarizer

Spectra 1 thru 6 were obtained with a gain of 5-fold; full-scale approximately equal to twice level of dark current of multiplier phototube. Spectra 7 and 8 were obtained

with a gain of unity.

Reprinted from Anal. Chem. 44, 239 (1972). Copyright (1972) by the American

Chemical Society. Reprinted by permission of the copyright owner.

matically in figure 1 are given in table 1. By using the

rotating sample cell system as well as a more stable xenon lamp power supply, the precision of phosphorimetric measurements can be increased by as much as tenfold (see data in table 1). In addition, the

contamination of glassware, solvents, and sample

cells with phosphorescent impurities seriously limits precision of measurements, particularly at low concentrations of analyte. Zweidinger, Sanders, and

Winefordner [55] have described techniques for

cleanup of sample cells and glassware. These authors

recommend cleaning the sample cells and glassware

used in solution preparation in an ultrasonic cleaner

for about 30 seconds or in concentrated nitric acid if there is serious contamination; when contamination

is extremely serious the item can be heated in an oven or placed directly in a flame. Of course, solvents

used in phosphorimetry should be purified by distilla- tion, zone freezing, or some other separation method.

It should be stressed that distillation of demineralized

174
water results in a solvent of extremely low phosphorescence background compared to ethanol, EPA,

or any other commonly used organic solvent. By use of an elliptical source condensing system

and a more stable xenon lamp power supply, it was

possible to reduce drift and noise by about tenfold as compared to those obtained with the standard power

supply in the commercial phosphorimeter. The

elliptical source condensing system is commercially

available (in our case, from American Instrument Co.,

Inc., Silver Spring, Maryland). The xenon lamp power

supply system was assembled using a highly regulated

power supply (Harrison Lab Model 6?68 A, HewlettPackard, Palo Alto, Calif.) operating in the constant

current mode at 7.5 A (150 W lamp) with the addition

of the starter circuit shown in figure 7. A comparison of phosphorescence signals and signalto-noise ratios obtained with the rotating normal

sample cell and the Zweidinger, Sanders, and Winefordner [55] sample cell cleanup procedure with the

earlier cleanup procedure of Tin and Winefordner

[56] is given in table 2.

rb Spark

Table 2. Comparision of signals and signal-to-noise ratios with

rotating normal sample cell and with or without new cleanup

procedure [55]

Figure 7. Starter circuit for xenon arc lamp (51). C„ C2 , C3 —0.5 fi¥ (300 VDC)

C4 —1300 mF (300 VDC)

C5 —0.01 fiF (30,000 VDC)

C6 —100 /u.F (300 VDC)

R, —1 fi R2 —1 k n

R3 —1.8 k a

51 —Momentary contact switch 52 —15 Ampere switch —to be closed only after lamp

ignites L] —300 mH (10 A, hand wound on ferrite core) L2 —30 mH (15 A, hand wound on ferrite core) D —Diode (15 A, 300 PIV)

Ti —Autotransformer T2 —10 : 1 Ratio transformer

Reprinted from Anal. Chem. 42, page 643 (1970). Copyright (1970) by the American Chemical Society. Reprinted by permission of the copyright owner.

C. Decrease in Limits of Detection in Phosphorimetry

Limits of detection in phosphorimetry are reduced

if either the signal level can be increased and/or

Signal (nA)a - b

Signal-to- background

fluctuation b,c

Signal-toConventional clean- up procedure

New cleanup

procedure

1.5(0.05) c - f

0.3 '

2.5

25

100[15] e

45

a Signal is due to impurities in ethanol (solvent) background.

b All signals, signal-to-background fluctuations, and signal-to- noise ratios are approximate. Excitation is at 262 nm and emission is measured at 380 nm.

c Background fluctuation is percent standard deviation of five background readings.

d Time constant for all measurements was 0.3 s. e Values in parentheses were obtained without the Aminco ellipti- cal condensing system (American Instrument Co., Inc., Silver Spring,

Maryland).

1 Dark current is approximately 0.02 nA.

the noise level can be reduced. By use of the more

stable xenon lamp power supply, the elliptical source condensing system, the rotating sample cell assembly,

and the improved cleanup procedure as well as the use of aqueous solutions for some molecules, the

noise level is reduced and thus detection limits are

also reduced. By use of more intense continuously

operated or pulsed with gated detector, light sources phosphorescence signals can also be increased and

detection limits are decreased even more. The improvement in detection limits, resulting

primarily from noise reduction due to the processes

described in the above paragraph, is given in table 3

for the measurement of several organic molecules

using normal sample cells in the rotating sample cell assembly. Similarily, detection limits obtained for several organic molecules measured using the quartz

capillary cells in the rotating sample cell assembly are given in table 4. The range of linearity and limits of

detection reported in table 4 compare favorably with

those previously obtained in clear, rigid glasses in the

normal sample cell (see table 3), even though the

sample volume is an order of magnitude smaller with

the capillary cell. Because of the small volume of sample solution retained in the capillary tube, the total number of grams of analyte necessary for a determination is in the picogram region, and thus phosphorimetry can be considered both a trace and micro method of

analysis. This should be of particular importance in biological and clinical applications where sample size

is often limited. The average slopes of the analytical curves (logarithm of phosphorescence signal versus

logarithm of analyte concentration) for the molecules

in tables 3 and 4 are 0.94 which approaches the ideal

slope of unity for phosphorescence signals in snowed

matrices [51]. Both positive and negative deviations

175
Table 3. Phosphorescence characteristics of several organic molecules studied [51]

Compound Solvent Concentration

range (M)

of near

linearity

103 103 Toluene Ethanol b Toluene Propylene glycol: c water/

(9: l,vlv) Toluene Isooctane 0 103 Sulfanilamide Isooctane: 0 Ethanol/ 105 (4:1, v/v) Oxythiamine HC1 Isooctane: 0 EthanolA 102

(4:1, v/v) 3-Acetylpyridine Isooctane 0 105

Slope of

linear portion

0.94

.76

.89 .96

1.01

1.03

Relative percent standard

deviation a

1.5

d 2.5

e 0.9 f (6.1)

e .8 a (1.1)

1.9

1.9

Linear

correlation

coefficient

0.9999

.9999

.9998

.9998

1.0000

1.0000

Limit of detection

(/xgl ml B

)

Limit of detection

(/xg/ml h

)

0.03

.03

.02 .0004 1 .012

.34

.012 '3.6

a For samples giving signals 5 times the limit of detection.

h Formed clear glasses.

c Formed snowed samples.

d Harrison power supply not used.

e Linear portion of the analytical curve.

f Nonlinear portion of the analytical curve.

e Limits of detection in this study [51].

h Best previous limits of detection using standard commercial

phosphorimetric equipment.

1 From Reference 57.

Table 4. Phosphorescence characteristics of molecules studied [541

Compound

Slope of linear portion

Limit of detection (/ng/ml)

Number of grams

at limit of detection

Concentration range of near

linearity

p-Nitrophenol 0 0.88 Sulfamethazine 3 1.01 2-Thiouracil a 0.81

7 X 10"3

6 X 10 3

7 X 10-"

a Solvent 10 percent (v/v) methanol water, all solutions formed snowed samples.

b Volume of sample retained in capillary tube calculated as 17.8 fiL

0 Solvent 10 percent (vjv) methanol water with 1 percent by volume saturated NaOH added.

1.24 X 10- 10

1.07 X 10-'°

1.24 X 10-"

104

104 104

from a slope of unity have been observed; however,

much more data is necessary before a conclusion can be drawn as to whether an individual molecule perturbs

the crystalline matrix and alters the slope or whether

the deviations can be attributed to instrumental parameters influencing the precision and accuracy of signal

measurements. Present theory assumes the scattering

coefficient of the sample is not a function of solute molecule or concentration [51, 53].

D. Pulsed-Source Gated-Detector Phosphorimetric

System

Fisher and Winefordner [58] have described a pulsed- source gated-detector system for phosphorimetry; such

a system was shown to allow an increase in selectivity

of analysis due to the use of time resolution [59], which will not be discussed here, and an increase in sensitivity of analysis due to the increased signal-tonoise ratio. The latter increase is a result of an in- creased signal and a decreased noise. The basis of

pulsed-source systems and the system of Fisher and

Winefordner [58] will be briefly described in the

following paragraphs.

A pulsed-source gated-detector phosphorimetric

system consists of much of the same equipment found

in the commercial continuously operated source mechanically modulated phosphorimeters; the exception being that no mechanical phosphoroscope is required in the pulsed system. Also, the source of

excitation energy is a short-arc high-pressure xenon

flashtube rather than a continuously operated xenon

arc lamp (usually 150 W); pulsed sources are operated

from rather simple electronic circuits and power

supplies and have operating characteristics suitable

for the excitation of (fast) phosphors, e.g., a blackbody temperature of around 7000 K, a pulse rate as high as several thousand hertz, and most important,

a pulse duration on the order of several microseconds

(a duty cycle of about 10~ 3

). With too short a pulse, not enough molecules will be excited per excitationemission cycle to permit the observation of analytically useful phosphorescence signals. With too long

a pulse, the early portions of the emission decay

curves of fast phosphors will be buried in source

scatter noise. Also, with long pulses, the phosphorescence background signal, often a result of slow

phosphor species in the sample mixture, may become

increasingly significant.

176
Readout devices for pulsed-source systems are more complex than dc systems employed in commercial phosphorimeters; three principal readout

methods have been used. The method proposed by

O'Haver and Winefordner [60], involved phototube

pulsing (gating) as well as source pulsing and is schematically illustrated in figure 8; it is seen that the first event in the excitation-observation-of-emission

cycle for a pulsed-source system is the intense burst

of energy from the source with a flash duration half- width, t f . During this time, the phosphorescence

intensity climbs to a value Io and begins to decay

exponentially. At a delay time, td, preferably after the source flash has decayed significantly, the multiplier phototube is turned on (a high-voltage negative

pulse is applied to the dynode chain), and the lumi- nescence signal is monitored. The phototube is then

turned off by termination of the negative "on" pulse:

if an integrating dc measurement system is used to monitor its output, the integrated luminescence

intensity / is measured during the on-time t p of the

phototube. If desired, td may be varied in such a

^Jf/JL— t d .

/ \ \V / \

/ \

/ \ / \

\ \ \

Figure 8. Schematic diagram of events occurring during one cycle

of sample excitation and observation in a pulsed source phosphorimeter system [60].

Key to Symbols and Lines in Diagram

tf — half-intensity width of the source flash

td = delay time after the end of the excitation pulse (assuming

excitation pulse to be a rectangular) to the beginning of observation of the phosphorescence signal ("on" time of the detector or the readout system)

t// = "on" time of detector or readout system

Dashed line represents buildup and decay of phosphorescence

Solid line tailing peak represents flash intensity temporal

distribution Solid line rectangular peak represents "on" time of detector

or readout system Cross-Hatched area represents measured integrated lumines- cence signal per source pulse

Reprinted from Anal. Chem. 38, page 1259 (1966). Copyright (1966) by the American Chemical Society. Reprinted by permission of the copyright owner.

manner that the gate tp

is scanned along the decay

curve, providing the readout device with a display

of the phosphorescence decay. This method was more

difficult to achieve instrumentally than the other two

methods discussed in the following paragraph.

Two other readout methods for observation of fast phosphor emissions were used by Fisher and

Winefordner [58]. In the first method, the phototube was operated continuously in the conventional manner,

but an electronic gate examined the integrated

phosphorescence intensity after a time delay of td over the time period tp ; this gate may also be scanned

to display the decay curve. This method is termed the

box-car integrator method. In the second method, the

phototube was also continuously operated, but the

signal for the entire decay curve was presented to a

fast multichannel readout device. This method is called the signal averager method. The latter method

is the simplest one of the three to use for pulsed

source studies although the problems of measurement

of fast electronic signals accompanied by noise are more important with this method.

The integrated luminescence intensity / (crosshatched area in fig. 8) achieved with a pulsed-source

gated-detector system is given [6] by

hjtf[exp( -tjT)] [1-expUp/r)]

[l-exp(-l//r)]

assuming a dc type readout is used to measure /

within the phototube "on" time tp (terms are defined

in fig. 8). If an ac system is used, / will not be directly

proportional to /, the source pulse repetition fre- quency. Figure 9 gives the variation of relative reI0

5 r

10"

1 io- 7

CO

bJ

CO

lil g

^ -9

CO lu o

X

_L

10" 10 10 10 DECAY TIME, s

-2

Figure 9. Plot of phosphorescence signal ratio (ratio of integrated

intensity / to the steady state integrated intensity Ipo ) versus sample phosphorescence decay time t[60] for case of pulsed source

gated detector (see text for values off/, t p , and td). Reprinted from Anal. Chem. 38, 1259 (1966). Copyright (1966) by

the American Chemical Society. Reprinted by permission of the copyright owner.

177
sponse (ratio of integrated luminescence intensity /

to the steady state integrated intensity h) with phosphor decay time r for parameters typical of an analytically useful pulsed-source gated-detector system

(tf = 10~6 s, tp = 10-5 s, t d = 10-5 s). It is evident by the

existence of the maxima in figure 9, that the pulsed- source system is capable of providing selective

response to short-lived phosphors over long-lived

phosphors. This is of considerable importance, be- cause the background phosphorescence signal, which

often defines the limits of detection and precision for

fast phosphors in alcoholic/hydrocarbon/ether solutions, is usually due to a long-lifetime phosphor impurity. For example, the pulsed system (represented

in fig. 9) for a typical fast (t= 1 ms) sample phosphor

in the presence of a slow (t= 1 s) background phosphor

interferent should have a 200-fold greater rejection of

background than the commercial mechanical phosphoroscope phosphorimeter.

Therefore, the principle advantages of pulsed-source

phosphorimetry over conventional continuous operating sources with mechanical modulation are: the possibility

of achieving higher peak source intensities during the

measurement period and thus lower detection limits and the possibility of achieving greater selectivity for a given short-r phosphor over a long-lived inter- ferent. Lesser advantages also exist. For example,

since a flashtube pulse may terminate in several microseconds, whereas a typical phosphoroscopic mechanical disk chopper has a termination time (transit

time) tt of 3 X 10 -5 and a typical phosphoroscopic

mechanical can chopper has a tt of 1 X 10~4 s, shorter- lived phosphors (t's as short as 10 /as) can be measured.

In addition, since the temporal parameters tf, ta, and

tp may be more readily varied with a pulsed-source

gated-detector system, larger signals can be measured

and greater selectivity achieved. Gating the detector

so that it is on for only a predetermined time results

in an improvement of the signal-to-noise ratio and a reduction in detection limits, an increase in precision,

and an improvement in the accuracy of measurement.

Finally, because it is easy to determine the linearity of a log / versus ta plot, a rapid check on the purity of the phosphor under study can be obtained as well as an estimate of the phosphor decay time, which may

be useful for structural (qualitative) studies. A block diagram of the pulsed-source gated-detector

phosphorimeter used by Fisher and Winefordner [58]

is given in figure 10; a schematic diagram of the flash- tube trigger circuit is shown in figure 11. To compare

the standard commercial (AMINCO in our case)

spectrophotofluorimeter with rotating can phosphoroscope with the pulsed-source gated-detector system,

it was decided to measure the limit of detection of a

fast phosphor (4-bromoacetophenone, t = 6.8 ms) in purified ethanol which was known to contain a phosphorescence background due to a slow phosphor

(unknown material, t = 0.8 s) having phosphorescence

excitation and emission spectra which overlapped

considerably the corresponding spectra of the fast phosphor. The limit of detection of 4-bromoacetopheA B C

1

Figure 10. Block Diagram of Pulsed Source Phosphorimeter [58]. Key to Symbols

A. Pulse generator (Model 100, Datapulse Inc., Culver City,

Calif.) B. 300 V power supply (Model C-281, Lambda Electronics,.

Melville, N.Y.)

C. 1400 V power supply (Model 403 M, John Fluke Mfg.,

Co., Seattle, Washington)

D. Trigger circuit (see fig. 11) E. Xenon flashtube compartment (Laboratory Constructed)

F. Sample cell compartment (identical to Aminco phosphoroscope accessory minus rotating phosphoroscope

can) G. Emission monochromator (Model EU-700, Heath Co., Benton Harbor, Michigan)

H. Phototube power supply (Model EU- 701-30, Heath

Co., Benton Harbor, Michigan)

I. RCA 1P28 photomultiplier tube

J. Load resistors (500 fl to 3M CI) and fast switching diodes

K. Oscilloscope (Model 545,Tektronix Inc., Orlando, Florida)

L. Preamplifier (Model 465A, Hewlett-Packard, Orlando,

Florida) M. Signal averager (Biomac Model 1000, Data Laboratories,

Ltd., London, England), or Boxcar Integrator (Model 160, Princeton Applied Research, Princeton, New Jersey) N. x-y Recorder

Reprinted from Anal. Chem., in press. Copyright (1972) by the American Chemical Society. Reprinted by permission of the copyright owner. none was 10 -6 M when measured using the standard

commercial spectrophotofluorimeter with rotating can

phosphoroscope. However, with the pulsed-source

gated-detector system of Fisher and Winefordner [58],

the detection limit was found to be 10 -8 M, even though

178
Figure 11. Schematic diagram of flashtube trigger circuit [58]. Value of Components

C, 0.001 /xF R, ioo kfi L ~ 100 C2 0.1 /i.F R2 33 kfi S SPST switch C3 0.022 /xF R3 56 fi T EG & G TR 132 Trigger Transformer C4 0.002 nF R4I.I kO V, 300 V dc power

supply

C, 1.0 /xF (600 V) R5 6.8 kfl v2 15 V dc power

supply

C 0.1 (jlF (5000 V) R6 190 kO v3 1400 V pulse power

supply

Reprinted from Anal. Chem. 44, 953 (1972). Copyright (1972) by the American Chemical Society. Reprinted by permission of the copyright owner.

the average power of the continuously operated xenon

source in the commercial system (150 W) was 75 times

as great as the average power of the pulsed xenon

flashlamp (2 W). The main reason for the 100-fold reduction in the limit of detection with the pulsed

system is the reduction in noise, i.e., essentially no

phosphorescence signal due to the long-lived background component was observed with the pulsed

system, and so the noise level was only about twice

the dark current shot noise level. One other factor which contributes to phosphorescence background should be mentioned. The

diethyl ether used in the Fisher and Winefordner [58]

studies was Baker and Adamson anhydrous reagent

grade. Listed as impurities in this solvent are "0.001

percent carbonyl (as > C = 0)." As expected, this also produced a fast decaying phosphorescence background to which the pulsed instrumentation was

responsive. Fortunately, the ether background was

very short-lived, and this interferent decayed before

introducing error into the phosphorescence signal of

the analyte with the pulsed-source system.

IV. Noninstrumental Improvements in

Phosphorimetry

Lukasiewicz, Rozynes, Sanders, and Winefordner

[52] showed that phosphorescence signals remain

nearly constant for a variety of analytes in snowed

matrices of mixtures of ethanol/water (methanol/

water) at 77 K over the range of 10 to 50 percent v/v; as the" volume percent of ethanol (or methanol) in- creased from 0 percent to about 10 percent, the phosphorescence signals of 2-thiouracil, p-nitrophenol, or sulfamethazine increased by about 3 orders of magnitude and simultaneously the physical appearance of

the frozen matrix changed from a translucent severely

cracked medium to an opaque completely snowed

matrix. The phosphorescence signal levels were

nearly the same in either methanol/water or ethanol/

water (10-50) percent v/v of alcohol/water), but the

phosphorescence background level was considerably

lower in the former than the latter solvent mixture

(spectrograde methanol versus 95 percent ethanol).

The increased phosphorescence signals of analytes

in 10 to 50 percent v/v of alcohol/water mixtures at 77 K as compared to those in pure aqueous solutions was correlated with the physical nature (snow versus cracked glass) rather than with chemical properties

of the analyte and/or the solvent as has been further

verified by Lukasiewicz, Mousa, and Winefordner

[53] (see below).

Lukasiewicz, Mousa, and Winefordner [53], in a more recent study, have made careful phosphorescence

measurements of the influence of solvent (methanol/

water and alkali-halide salt solutions) composition

upon the phosphorescence of 3-indole acetic acid,

hippuric acid, and sulfacetamide at 77 K. Typical

results for the methanol/water mixtures at 77 K are shown in figure 12. The three analytes all show the same

solvent dependency, i.e., a region where there is an

abrupt increase in phosphorescence signal between 0

and 3 percent v\v methanol/water, followed by a plateau

region between about 5-30 percent v/v, and a region

where the phosphorescence signal decreases slightly for solutions with more than 30 percent methanol.

Little or no spectral shift was observed for the phosphorescence excitation and emission spectra as a function of solvent composition. Perhaps the most

significant observation was that each region corre- sponded to a visible change in the nature of the matrix.

Pure water solutions were severely cracked, but nearly

transparent. Addition of small amounts of methanol to water produced an opaque matrix which corresponded

to the region of greatest slope in the solvent dependency curves. Between approximately 5 and 30 percent

v/v methanol/water, the frozen matrix became

completely snowed, and the signal remained nearly

constant. Increasing the percentage of methanol

even more resulted in a signal decrease which corre- sponded to the reappearance of a cracked, translucent,

179
V)

10'" I 1 1 1 1 1 1 0 10 20 30 40 50 60 V/V % METHANOL / WATER

FIGURE 12. Phosphorescence Signal as Function of Solvent Composition (methanol-water) [53]. O 2.0 X 10-W 3-indole acetic acid A 4.7X10 ~ eM hippuric acid 1.7X10-6M sulfacetamide Reprinted from Anal. Chem. 44, 965 (1972). Copyright (1972) by the American Chemical Society. Reprinted by permission of the copyright owner.

rigid matrix; the precision of measurement of phosphorescence signals for methanol/water solutions with

40 percent v\v or more methanol is poor because of the

nonrandom cracking of the frozen solvent. The shapes of the phosphorescence signal versus

solvent composition curves when sodium chloride is added to water rather than methanol (or ethanol) are

identical to those for the alcoholic solutions (fig. 13), and the previous conclusions regarding the physical nature of the matrix seem to be justified. It should be

stressed, however, that the maximum phosphorescence

signal for the NaCl solutions is about 3 times greater

than the methanol/water solutions (the salt solutions

at 77 K result in a rigid, snowed matrix with more

finely divided particles than the snowed matrix for the

methanolic solutions).

The use of the external heavy-atom effect to increase

the transition probability from the first excited-singlet

to the first excited-triplet state and therefore, to in- crease the sensitivity of phosphorescence, has also been reported [62, 63]. However, in the past, results obtained using the external heavy-atom effect have

been of limited analytical value because of the fol- lowing restrictions. First of all, the heavy-atom perturbant had to be appreciably soluble in the solvents used

WT % NaCl

FIGURE 13. Phosphorescence Signal as Function of Solvent Composition (sodium chloride-water) [53]. O 3.7 X 10"6M 3-indole acetic acid A 1.8 X 10-6M hippuric acid 3.9X10"6M sulfacetamide Reprinted from Anal. Chem. 44, 965 (1972). Copyright (1972) by the American Chemical Society. Reprinted by permission of the copyright owner.

for phosphorimetry and also had to form clear, rigid

glasses with the solvent. Second, the perturbant should

not absorb in the same spectral regions as the com- pounds being studied. Third, the perturbant had to be

available in extremely pure form, since impurities

could cause a large background signal. Ethyl iodide

has been most commonly used as an external perturbant; however, results obtained were limited because

ethyl iodide did not fulfill all the requirements stated

above, especially the last two. In aqueous solutions,

however, the use of simple perturbants, such as alkali halide salts is possible. Solubility and purity of these

salts make them desirable for phosphorimetric studies

of aqueous solutions. The effect of adding the alkali halide salts, Nal,

NaBr, and NaCl to pure water solutions of 3-indole

acetic acid, is shown in figure 14. The combined

physical matrix and heavy-atom effect result in the

greatest phosphorescence signals with added Nal.

All three salts (see background spectra in fig. 14) result in significant phosphorescence background

NaBr giving the largest. Also, both NaBr and Nal

solutions result in the same general shaped composition curve as for NaCl and alcohol with water, except that the Nal-water curve in figure 14 is somewhat sharper

than the others. An increase in phosophorescence

180
if)-" I I I I I

I 1 I I I 1 I 1 lu 0 2 4 6 8 10 12 14 16 18 20 22 24 WT % ALKALI HALIDE SALT

Figure 14. Phosphorescence Signal of 3.7 X 10- 6 M, 3-indole Acetic Acid in Alkali Halide Salt Solution: # aqueous Nal solution as solvent A aqueous NaBr solution as solvent aqueous NaCl solution as solvent Phosphorescence background of alkali halide salt crystals: O Nal crystals A NaBr crystals NaCl crystals Excitation Wavelength 285 nm, emission wavelength 435 nm [53]. Reprinted from Anal. Chem. 44, 965 (1972). Copyright (1972) by the American Chemical Society. Reprinted by permission of the copyright owner.

signal occurred for concentrations of Nal up to 1 M.

Therefore, the use of alkali-halide/ water solutions as solvents for phosphorimetric analysis of polar organic

molecules is advantageous for a number of reasons:

alkali-halide salts may be obtained in ultrapure form,

and thus phosphorescence background may be reduced

to extremely low levels; salt solutions have the same

matrix effect as alcohol/water solutions, while producing a much lower background signal; and, in the case

of sodium iodide, an additional factor, namely the

heavy-atom effect, enhances the phosphorescence

signal. In some cases, however, use of aqueous sodium

iodide solvents may be less desirable than other alkali halide salts. Phosphorescence decay time measure- ments for the compounds studied in this study by

Lukasiewicz, Mousa, and Winefordner [53] indicated

that decay times in aqueous sodium iodide solutions were considerably shorter than in methanol/water

j solutions. The changes in decay times for the compounds studied in aqueous sodium iodide and alcohol/

water solutions are summarized in table 5. These

observations are consistent with the heavy-atom per- turbation theory [61]. Phosphorescence signals, however, did not increase for all three compounds listed

in figure 13. A sixfold increase in phosphorescence

signal was observed for 3-indole acetic acid, and a

threefold increase for hippuric acid in 1 M sodium

iodide. Sulfacetamide, on the other hand, showed a threefold decrease in signal even though the phosphorescence decay time was lower.

Table 5. Phosphorescence decay times in aqueous sodium iodide and aqueous alcohol solutions

Lifetime(s)

Solvent Solvent 1 M

Compound 10 percent v/v aqueous sodium

methanol-water iodide

3-Indole Acetic Acid 7.0 0.90 Sulfacetamide 1.7 0.83 Hippuric Acid 3.0 0.97

It is therefore quite evident that proper selection of

the solvent for phosphorimetry can lead to an appreciable increase in phosphorescence signals of some

species and in some cases an increase in selectivity

of analysis. The advantages of using alkali-halide salt solutions over alcohol/water solutions as phosphorimetric solvents include: the availability and low cost

of high purity salts; increased signal levels; and the

high solubility of many biochemically related species.

V. References

[1] Hercules, D. M., Fluorescence and Phosphorescence Analysis,

D. M. Hercules, Ed. (Wiley, Interscience, New York, 1966).

[2] Parker, C. A., Photoluminscence of Solutions (Elsevier, New

York, 1968).

[3] Becker, R. S., Theory and Interpretation of Fluorescence and Phosphorescence (Wiley, Interscience, New York, 1969).

[4] Zander, M., The Application of Phosphorescence to the Analysis

of Organic Compounds (Academic Press, New York, 1968).

[5] Winefordner, J. D., McCarthy, W. J., and St. John, P. A., Methods of Biochemical Analysis, D. Glick, Ed., Vol. 15

(Interscience, New York, 1967).

[6] Winefordner, J. D., St. John, P. A., and McCarthy, W. J., Chapter on Phosphorimetry, in Fluorescence Assay in Biology and Medicine, Vol. II, S. Udenfriend, Ed. (Academic Press, New York, 1969).

[7] McCarthy, W. J., Phosphorimetry, Chapter in Spectrochemical

Methods of Analysis, J. D. Winefordner, Ed. (Wiley, New

York, 1970).

[8] Winefordner, J. D., Schulman, S. G., and O'Haver, T. C,

Luminescence Analysis in Analytical Chemistry (Wiley,

New York, 1972).

[9] Zander, M., Angew. Chem. Intern. Ed. Engl. 4, 930 (1965).

[10] Zander, M., Z. Anal. Chem. 226, 251 (1967).

[11] Clar, E., and Zander, M., Chem. Ber. 89, 749 (1956).

[12] Zander, M. , Erdoel Kohle 19, 279 (1966).

[13] Sawicki, E., Chemist-Analyst 53, 88 (1964).

[14] Sawicki, E., and Johnson, H., Microchem. J. 8, 85 (1964).

502-591 O - 73 - 13 181
[15]

[16]

[IT

[18]

[19]

[20]

pi;

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35;

[36]

[37]

[38]

[39]

[40]

Sawicki, E., Stanley, T. W., Pfaff, J. D., and Elbert, W. C,

Anal. Chim. Acta 31, 359(1964).

Sidorov, N. K., and Rodomakina, G. M., Uch. Zap. Saratovsk

Gos. Univ. 69,161,(1960).

Drushel, H. V., and Sommers, A. L., Anal. Chem. 38, 101

(1966). Drushel, H. V., and Sommers, A. L., Anal. Chem. 38, 19(1966). Drushel, H. V., and Sommers, A. L., Anal. Chem. 36, 836

(1964). McCarthy, W. J., and Winefordner, J. D., J. Assoc. Offic. Agr. Chemists. 48,915(1965).

Winefordner, J. D., and Moye, H. A., Anal. Chim. Acta 32, 278

(1965). and Winefordner, J. D., J. Agr. Food Chem. 13,

Winefordner, J. D., J. Agr. Food Chem. 13, 533

Moye, H. A. 516 (1965). Moye, H. A.,

(1965). Moye, H. A., J. Assoc. Off. Anal. Chem. 51, 1260 (1968). Latz, H. W., and Hurtabise, R. J., J. Agr. Food Chem. 17, 352

(1969). Latz, H. W., Madsen, B. C, Anal. Chem. 41, 1180 (1969). Freed, S., and Salmre, W., Science 128, 1341 (1958). Freed, S., Turnbull, J. H., and Salmre, W., Nature. 181, 1831

(1958). Freed, S., and Vise, M. H., Anal. Biochem. 5, 338 (1963).

Hollifield, H. C, and Winefordner, J. D., Anal. Chim. Acta 36,

362(1966).

Hollifield, H. C, and Winefordner, J. D., Talanta 14, 103 (1967). Winefordner, J. D., and Tin, M., Anal. Chim. Acta 32, 64 (1964).

Hollifield, H. C, and Winefordner, J. D., Talanta 12,860 (1965). Hood, L. V. S., and Winefordner, J. D., Anal. Biochem. 27, 523

and Winefordner, J. D., Anal. Chim. Acta 42,

(1969). Hood, L. V. S

199 (1968). McCarthy, W. J., Parsons, M. L., and Winefordner, J. D.,

J. Chem. Educ. 44, 136 (1967).

St. John, P. A., McCarthy, W. J., and Winefordner, J. D., Anal. Chem. 38, 1828 (1966). Winefordner, J. D., and St. John, P. A., Anal. Chem. 35, 2211

(1963). Scott, D. R., and Allison, J. B., J. Phys. Chem. 66, 561 (1962). Smith, F. J., Smith, J. K., and McGlynn, S. P., Rev. Sci. Instr. 33, 1367 (1962).

[41] Kanda, Y., and Shimada, R., Spectrochim. Acta. 17, 279 (1961).

[42] Sponer, H., Kanda, Y., and Blackwell, L., ibid. 16, 1135 (1960).

[43] Kanda, Y., Shimada, R., and Saka, Y., ibid. 17, 1 (1961).

[44] Kanda, Y., Shimada, R., Honada, K., and Kajigaeshi, S., ibid. 17, 1268 (1961).

[45] Von Foerster, G., Z. Naturforsch. 18a, 6201 (1963).

[46] Kubelka, D., and Munk, F., Z. Techn. Physik 12, 593 (1931).

[47] Parker, C. A., and Hatchard, G., Trans. Faraday Soc. 57, 1894

(1961).

[48] Klasen, H. A., Philips Res. Rep. 2, 68 (1947).

[49] Kortiim, G., Braun, W., and Herzog, G., Angew. Chem. Intern. Ed. 2, 333 (1963).

[50] Hollifield, H. C, and Winefordner, J. D., Anal. Chem. 40, 1759

(1968).

[51] Zweidinger, R. W., and Winefordner, J. D., Anal. Chem. 42,

639 (1970).

[52] Lukasiewicz, R. J., Rozynes, P. A., Sanders, L. B., Winefordner,

J. D., Anal. Chem. 44, 237 (1972).

[53] Lukasiewicz, R. J., Mousa, J. J., and Winefordner, J. D., Anal. Chem. 44, 963 (1972).

[54] Lukasiewicz, R. J., Mousa, J. J., and Winefordner, J. D., Anal. Chem. 44, 1339 (1972).

[55] Zweidinger, R. W., Sanders, L. B., and Winefordner, J. D., Anal. Chim. Acta 47, 558 (1969).

[56] Tin, M., and Winefordner, J. D., Anal. Chim. Acta 31, 239

(1964).

[57] Sanders, L. B., Cetorelli, J. J., and Winefordner, J. D., Talanta 16 , 407 (1969).

[58] Fisher, R. P., and Winefordner, J. D. Anal. Chem. 44, 948

(1972).

[59] Winefordner, J. D., Accnts, Chem. Res. 2 , 361 (1969).

[60] O'Haver, T. C, and Winefordner, J. D., Anal. Chem. 38, 1258

(1966).

[61] McGlynn, S. P., Azumi, T., Kinoshita, M., Molecular Spectros- copy of the Triplet State (Prentice-Hall, Englewood Cliffs, New Jersey, 1969).

[62] Zander, M., Z. Anal. Chem. (Fresenius), 226, 251 (1967).

[63] Hood, L. V. S., and Winefordner, J. D., Anal. Chem. 38, 1922

(1966).

(Paper 76A6-743)

182
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Measurements of Absolute Values in Biochemical

Fluorescence Spectroscopy

Raymond F. Chen

National Heart Institute, National Institutes of Health, Bethesda, Maryland 20014

(July 19, 1972)

Fluorescence spectroscopy is an important tool of the biochemist studying the structure and

interactions of proteins and nucleic acids. The four basic quantities to measure accurately are:

1. spectra (corrected excitation and emission), 2. quantum yields (Q), 3. fluorescence decay characteristics, and 4. polarization. Commercially available instruments, with little modification, can be

used to obtain these measurements, but the biochemist in this field is very dependent on the accuracy

of measurements of substances he uses as standards. Confusion arises from disagreement between

reported values for standards which may be used to calibrate a detector system to obtain quantum

yields, or to set up lifetime and polarization photometers. For instance, the protein chemist is fond of using tryptophan and quinine as quantum yield standards, but Q for tryptophan has been variously

reported as 0.13 and 0.20, and Q values for quinine bisulfate range from 0.4 to 0.7. The biochemist

should also be aware of the problems inherent in the use of commerically available instruments in absolute measurements, as well as the special complications arising in complex biochemical systems where the fluorescence is heterogeneous.

Key words: Absolute fluorometry; absolute fluorometry in biochemistry; standard reference materials in fluorometry.

I. Introduction

A. Applications of Fluorescence Spectroscopy in Biochemistry

Within the last dozen years, biochemists have

generally become aware of the value of fluorescence

spectroscopy as a tool for the study of macromolecules

such as proteins and nucleic acids. The marked in- crease in the use of fluorescence during recent years has also been due to improvements in instrumentation

and techniques. Before discussing the types of meas- urements which are needed, let us consider some of the

applications of fluorescence spectroscopy in physical

biochemistry:

1. Obtaining Information on the Size and Shape of

Macromolecules.

In 1952, Weber [1, 2] 1 pointed out that the polarization of fluorescence of dyes attached to proteins could

be used to give information on rotational relaxation

times. These times were an estimate of the Brownian

motion occurring during the lifetime of the excited

state; hence, information was obtained concerning the

size and shape of the proteins. The equation derived

1 Figures in brackets indicate the literature references at the end of this paper.

by Weber [1] to correlate the degree of polarization, P,

with the fluorescence decay time r, and the relaxation

time ph, was based on similar equations originally

formulated by Perrin [3] and Levshin [4] for simple

organic fluorophors:

H=

(?rt)(1+

;S v

where Po is the limiting polarization. Because the quantity rjph is proportional to the ratio T/tj (T, absolute

temperature; 17 viscosity), a "Perrin plot" of l/P — 1/3 versus T\y\ obtained from polarization data with

protein-dye conjugates at different temperatures and/or

viscosities, contains all the information needed to calculate the rotational relaxation time of the molecule,

provided the lifetime can be measured. Polarization

data are also useful for following conformational

changes such as unfolding or dissociation of a protein.

Such events can frequently be monitored by no other

known techniques [5]. Note that utilization of eq (1) requires both polarization and lifetime determinations.

2. Study of Binding Equilibria

The interaction of proteins with small ligands is a key function of proteins comprising certain physiological systems. This is especially true in the case of

183
enzymes, proteins with catalytic properties. Some of these ligands, such as coenzymes like reduced nico- tinamide adenine dinucleotide (NADH) are naturally

fluorescent, and the binding of the ligands is accompanied by a large (typically, 2- to 5-fold) enhancement

of the quantum yield, alteration of the emission

spectrum, and an increase in the polarization of fluores- cence. Other ligands such as flavin coenzymes are quenched on binding to proteins. Also, Velick [6] showed that the ultraviolet fluorescence of proteins is often quenched on binding of certain ligands. Any or

all of these changes in fluorescence parameters are useful in obtaining the stoichiometry and association constants of the binding.

3. Elucidation of the Nature of Binding Sites

The dielectric properties of different areas on

proteins differ markedly; such differences can be

signalled by the fluorescence behavior of fluorescent

ligands bound to these areas. Dyes which have been

useful in this regard have been dubbed "fluorescent

probes". The Stokes shift and quantum yield of

bound fluorescent probes have been used to estimate

the microscopic dielectric constant of binding sites in proteins. An example of how the nature of the binding

site affects fluorescence is given in figure 1. The

l-dimethylaminonaphthalene-5-sulfonyl (DNS) group

has emission properties which make it useful for a

variety of fluorescence studies, including polarization.

When DNS is used as a probe, a high quantum yield

and a small Stokes shift is indicative of a nonpolar

environment. Absorbed onto the active site of the

enzyme, carbonic anhydrase, DNS in the form of the

sulfonamide has a smaller Stokes shift than it does

when adsorbed to BSA (bovine serum albumin) or attached covalently to the enzyme (fig. 1).

CA(DNSA)

350 450 550 650 WAVELENGTH (m^)

FIGURE 1. Corrected emission spectra of proteins containing the l-dimethylamino-naphthalene-5-sulfonyl (DNS) group. CA(DNSA)

and BSA(DNSA) are adsorbates of carbonic anhydrase and bovine serum albumin containing l-dimethylamino-naphthalene-5-sulfonamide, and DNS-CA is a conjugate prepared by reacting carbonic anhydrase with DNS chloride. The curves are normalized to the same height. From Chen and Kernohan [7].

4. Study of the Environment Near Tryptophan and

Tyrosine Residues in Proteins

Since it was discovered in the late 1950's that proteins have an ultraviolet fluorescence due to aro- matic amino acids, much has been learned about the

types of molecules or groups on a protein which can

influence such fluorescence. The spectrum of tryptophanyl fluorescence is an indicator of the polarity of the environment near the emitting group. Changes in intrinsic fluorescence can be used to follow conformational changes or binding. Intrinsic fluorescence is a

useful optical parameter for kinetic studies, including

rapid reactions followed by stopped-flow or perturbation methods.

5. Determination of Intermolecular Distances by

Energy Transfer

The radiationless transfer of energy from an excited

chromophore to an acceptor molecule via a dipoledipole resonance mechanism has been shown to occur

in many intances. The theory of such energy transfer was studied by Fdrster [8] who developed a method for estimating the distance between donor and acceptor

moieties based on their spectroscopic properties and

the degree of energy transfer. Fluorometry is the best

method for studying energy transfer, because this proc- ess is manifested by a quenching of the fluorescence of the donor and/or a sensitized fluorescence of the aceptor. A common example is that of a dye attached to a

protein: the distance between the dye and the tryptophan residues may be estimated by the energy

transfer from tryptophan to dye.

6. Study of the Orientation of Molecules Attached to Asymmetrical Macromolecules

By the use of polarized excitation, it is possible to obtain information on the orientation of ligands on a larger molecule. Using polarized photoselection,

Lerman [9] determined that certain mutagenic dyes were attached with their planes perpendicular to the

axis of deoxyribonucleic acid (DNA) molecules; this was the basis for his theory that mutagens intracalate

between DNA base pairs. The orientation of dyes at- tached to proteins can similarly be studied by polarization measurements [10, 11]. The lack of a unique

orientation of a dye fixed on a protein can also be detected. Such would be the case if dyes were attached in a random manner, or if a dye were attached to a flexible segment of the macromolecule.

7. Study of Changes in Conformation of Macromolecules

This is perhaps the most important use of fluores- cence spectroscopy in biochemistry at present, and is accomplished by following changes in intrinsic fluorescence parameters as well as the fluorescence of attached groups. Such changes sometimes cannot be

followed by other optical methods, and fluorescence

184
becomes the method of choice, especially for kinetic

studies.

B. The Parallel Development of Fluorescence Spectroscopy and Instrumentation

From the preceding section, one can see that bio- chemical fluorescence spectroscopy requires the

measurement of a variety of parameters: excitation

and emission spectra, quantum yields, lifetimes, and

polarization. The needs of biochemists often exceed

those of individual physicists or physical chemists,

although in biological fields investigators frequently

are considerably less knowledgeable concerning in- strumentation than physical scientists and are not

qualified to construct their own instruments. However,

commercially available instrumentation has improved

and often has incorporated the best features of laboratory-built devices described in the literature. It is now possible to measure all of the above fluorescence

parameters with available instrumentation. The

development of such instrumentation has played an important role in the evolution of fluorescence

techniques.

Following the description by Bowman et aL [12] in 1955 of the first spectrofluorometer, several instru- ments based on their design were produced commercially. Such instruments contain, basically, a xenon

arc lamp, a sample compartment, two monochromators, and a photodetector system. The principal innovations by Bowman et al. were the introduction of the xenon arc as a source of energy continuum for fluores- cence excitation, and the incorporation of two monochromators for excitation and emission. In this country, at least half a dozen spectrofluorometers of

this type are marketed. There are probably thousands

of these spectrofluorometers in use. They may serve as a basic tool for fluorescence spectroscopy and are of

modest cost. More sophisticated instruments are now available

for determining spectra which have been designed to compensate for variations of exciting energy and

photodetector response. These instruments generally

cost 3 or 4 times as much as an uncompensated

spectrofluorometer.

Polarization measurements have been made on

light scattering jjhotojneters^ filter fluorometers, and

spectrofluorimeters [12]. Although specific instruments

for fluorescence polarimetry do not seem to be generally used, the modification of other available instru- mentation for such purposes poses no great problem.

Even in the field of lifetime measurements there are commercial instruments available for the purpose.

The TRW Instruments 75A (El Segundo, California) 2 fluorescence decay apparatus and the ORTEC

Model 9200 fluorescence spectrometer are the first instruments for determining lifetimes, which were

previously accessible only to those with considerable

engineering skill.

II. Measurement of Fluorescence Parameters

A. Definition of the Term "Absolute"

The term "absolute" as used here refers to a property of a substance rather than its measurement. For example, the absolute quantum yield of a substance is the ratio of the photons emitted to the

number of photons absorbed, in contrast to the

relative quantum yield, the number of photons emitted

by one substance as compared with another under

specified conditions. Confusion may arise, because

the phrase "absolute quantum yield" has been used

by some workers to denote the quantum yield which

has been measured by an "absolute" method; i.e., a method not requiring the use of a reference standard

of known quantum yield. Absolute measurements are

frequently required in certain biological fields like bio- luminescence, but will not be treated here.

1 . Excitation and Emission Spectra.

a. Definition.—The true, or absolute, fluorescence

emission spectrum of a substance is a plot showing

the relative number of photons emitted at different

wavelengths; i.e., dq/dX versus X, where q represents

the total number of photons emitted and X is the wavelength. Alternatively, the same information can be

plotted on a frequency scale, dq/dv versus v in cm-1

. For simple organic compounds the emission spectrum

is generally independent of the excitation wavelength, but for complex systems there is no single

absolute emission spectrum since the emission may

vary continuously with exciting light energy. The excitation spectrum is a plot of the relative number of photons emitted at a given wavelength

as a function of the exciting wavelength. The true, or corrected, excitation spectrum is compensated

for any variations of excitation energy with wavelength.

The corrected excitation spectrum is expected to coincide with the absorption spectrum in simple

compounds, but this relationship may not hold in large biochemical systems.

b. Need for Corrected Spectra.—Absolute

fluorescence spectra would be useful for comparing

results from different laboratories and have long been

advocated [14] as being the most suitable form of

data presentation. Expensive spectrofluorometers are

available for automatic recording of absolute fluores- cence spectra. However, many uncorrected spectra

are published even today, and some case can be made

for continued use of uncorrected data. Thus, when the

main point of a study is simply to show changes in fluorescence characteristics, there is little reason

to take trouble to correct the spectra. The thousands

of spectrofluorometers in use are equipped with similar

2 In order to adequately describe materials and experimental procedures, it was oc- casionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau' of Standards, nor does it imply that the particular product or equipment is necessarily the best available for that purpose.

185
gratings and photomultiplier tubes, so that uncorrected

spectra from one laboratory can, in fact, be used for comparison with results from another laboratory.

Absolute spectra are more informative, are required

for quantum yield determinations, and are necessary

for calculations of natural lifetimes or energy transfer

distances. Nonetheless, one can confidently predict

continued use of uncorrected spectra in the literature. c. Calibration of Lamp Output and Detector

Response. — Since most biochemists utilize un- compensated spectrofluorometers, it should be pointed

out that procedures for calibrating these instruments

have been published, e.g. [5, 15, 16]. The main steps

involve (a) Calibration and alignment of the monochromators with a line source such as a mercury pen

lamp, (b) Calibration of the exciting source with a fluorescent screen solution, such as Rhodamine B or some other dye, and (c) Calibration of the detector

response by comparing the recorded signal from a standard lamp with its known output.

An example of a detector response calibration curve

is given in figure 2. The response of an RCA 1P28

5.0

4.0

SIX)

3.0

2.0

1.0

300 400 500 600 WAVELENGTH (m/i) FIGURE 2. Detector calibration curve for Aminco-Bowman Spectro- fluorometer fitted with 1P28 phototube and a grating blazed at 300 nm. Filled circles: data obtained with a National Bureau of Standards tungsten filament lamp. Open circles: data obtained

with calibrated xenon arc lamp. S(\) is a function of the reciprocal

of the sensitivity. From Chen [16]

Photomultiplier in an Aminco-Bowman spectrofluorometer fitted with a grating blazed for maximum

response at 300 nm seems to be quite linear in the

region from 290 nm to 400 nm. The detector system

thus delivers "absolute" emission spectra for sub- stances like proteins which emit in this range. By

choosing suitable gratings and phototubes, it is sometimes possible to obtain linear response in regions of

visible fluorescence as well, thus avoiding the need for point-by-point correction of spectra.

The accuracy of detector calibration depends on such factors as the photometric accuracy of the

system and the accuracy of the standard lamp cali- bration. Lee and Seliger [17] have considered at length

all the possible errors involved in absolute calibrations

of detector systems. Their interest was principally the

determination of chemiluminescent quantum yields,

and it was necessary to know the relation between the

number of photons and the detector response. In

contrast, the determination of true emission spectra

require that the relative detector response at different wavelengths be specified. Hence, the uncertainty in Lee and Seliger's absolute calibrations, ±4.5 percent,

is an upper limit in the more usual calibrations. Calibration of spectrofluorometers by the use of

"standard spectra" of well-known substances has been

advocated [18]. In theory this would be a very con- venient method. Comparison of the apparent excitation spectrum of a simple compound could be made

with its absorption spectrum to yield the correction

factors for lamp output. The method eliminates the

need for a front-surface illumination attachment [15], which is needed for calibrations using the fluorescent screen method. Comparing absorption and emission

spectra has the disadvantage that the same dispersion

generally cannot be used in the absorption and

fluorescence spectrometers. Other disadvantages

make the comparison-of-spectra method less desirable

than the fluorescent screen method. The latter re- sults in a continuous plot of photon output versus wavelength compared with the discontinuous plot ob- tained by spectral comparison. Also, several absorption

spectra are required to achieve a desired optical

density at regions of maxima and minima, and several

fluorescence excitation spectra at different gain

settings will be needed as well. The use of "standard spectra" to calibrate the de- tector response is becoming common practice as more

corrected spectra are published. Unfortunately, the

reliability of any given published spectrum is un- known, and it would perhaps be helpful if some

spectra were certified by a committee of experts. In

the past, there have been many discrepancies in published emission spectra of quinine [19], although more

recent spectra seem to agree closely [20-23] and could

be used for detector calibration. One complication

which we pointed out several years ago is that the

emission spectrum of quinine shows a red shift when

excited in the long wavelength edge of the absorption

band [21]. This effect is shown in figure 3. The emission

shift has been postulated by Fletcher [22] to be related

186
I 1 I I I I I 1 300 350 400 400 450 500 550 WAVELENGTH (mji)

FIGURE 3. Fluorescence and polarization spectra of quinine in glycerol containing

0.1 N H2SO4 at 8 "C A. Excitation spectrum. B. Fluorescence polarization spectrum

obtained with varied exciting wavelength and emission monochromator set at 450 nm. C. Corrected emission spectra obtained with excitation at 340 nm (curve 1) and 390 nm (curve 2). D. Variation of polarization in the emission band with constant

excitation at 340 nm. From Chen [21].

to photoselection of quinine molecules with slightly different conformations. Other compounds also show

this effect [21, 22, 24], which may involve what Fletcher

calls rotatable auxochromes such as the methoxy

group at the 6-position of the quinoline nucleus in quinine. At low temperatures, emission spectral shifts also occur due to the presence of different solvation

states [25]. However, the spectrum of quinine under

usual conditions of excitation should be suitable for a standard.

2. Quantum Yield Measurements

a. The Comparative Method of Determination.—In biochemistry, the quantum yield is most

often obtained by comparison with a quantum yield

standard. A spectrofluorometer is required, and the

yield is calculated from the relation

Qst Fst Ax (f>x n\ (2)

where Q is the quantum yield, F is the area under the

corrected emission spectrum, A is the optical ab- sorbance at the exciting wavelength, is the relative photon output of the light source at that wavelength,

and n is the refractive index. The subscripts x and

st refer to the unknown and standard,

b. Factors Affecting the Accuracy.

1. Accuracy of the emission detector response

calibration. Any errors present may have little effect on the quantum yield calculations if the spectra of the

standard and unknown are similar. One should not use fluorescein as a standard to determine quantum

yields of ultraviolet fluorescence, for example.

2. "Bookkeeping" errors. Anyone who has at- tempted to perform quantum yield determinations

must surely be impressed by the large number of

factors which have to be inserted into the final calculations. Aside from the terms in eq (2), one must also remember that the photometer sensitivity may have

been set at different values for standard and unknown,

and the sensitivity scale may need recalibration.

The absorbance, A, is usually read for concentrated

solutions which then are diluted by some factor which

renders self absorption unlikely in the fluorescence

measurement; the dilution factor must be inserted.

One can suspect that some of the large errors which

have appeared in the literature are due to deficiencies

in bookkeeping.

3. Determination of the absorbance. Although it would seem a simple matter to determine the ab- sorbance of the test substances, errors arise because

the wavelength calibrations of the spectrofluorometer

and the spectrophotometer may be slightly incorrect.

Excitation then may occur at a wavelength different

from that at which the absorbance was measured.

The bandwidth of the monochromators also are usually

different, and the amount of error from this discrepancy

depends on the spectral characteristics of the compounds. We have found that our average deviations for quantum yield determinations have been smaller when

the unknown and standard are each excited at the

peak of their long wavelength absorption bands. With

187
this method, one never need worry about slight raono- chromator alignment errors which would produce

large errors in regions of sharply changing absorption.

4. Source output calibration. If sample and unknown

are excited at the same wavelength, the term (f)stl(f)x of eq (2) reduces to one. However, since different wavelengths are usually used, some error may intrude.

The lamp calibration curve for the xenon arc [16] is fairly smooth from 230-400 nm. In this region, if the

exciting wavelengths for x and st are not too wide, one would not expect much error to be introduced if there is slight inaccuracy in the knowledge of the exciting wavelengths. Between 400 nm and 520 nm,

however, there are some strong peaks in the xenon arc

output. In this region, perhaps it would be best to use the same wavelength to excite both standard and

unknown. The calibration curve itself might be

erroneous due to factors such as impurities in the

fluorescent screen substance. This may be checked

by using several different screens, or by determining

if the calibration curve allows for corrected excitation

spectra which match the absorption spectra.

5. Polarization effects. In protein and nucleic acid

fluorescence studies, the emission is frequently

polarized due to the large size of the molecules. The

resulting emission anisotropy can introduce an error

into the quantum yield calculations even when unpolarized excitation is used, since even here, in instru- ments utilizing right-angle viewing geometry, the

fluorescence is polarized. A number of papers have

considered this problem [26-29]. Taking the worst

possible case, Paoletti and Le Pecq [29] have esti- mated that a 15 percent error could occur in relative quantum yield measurements. However, an error this large could only occur with a sample showing maximum polarization in an instrument having large polarization artifacts, and where the fluorescence of standard

and unknown were compared at individual emission

wavelengths. Since the actual conditions for quantum

yield assays are normally quite different, the errors are usually neglected, and indeed have never emerged

as a great problem.

6. Accuracy of the value of the fluorescence

standard.

a. Characteristics of quinine fluorescence. Recent

reviews [30, 31] have pointed out the surprising dis- crepancies in reported quantum yields which exist in the literature and the controversy over the values of

several well-known quantum yield standards. The most

common standard is quinine in aqueous sulfuric acid

solutions. An absolute yield of 0.55 for quinine in 1.0

/V H2 S04 was reported by Melhuish [32, 33] as well as by Windsor and Dawson [30]. These workers employed

absolute methods for the determination. A value of 0.58 for quinine in 0.1 N H2 S04 was reported by

Eastman [34] using a technique which was probably

less accurate. The report by Drobnik and Yeargers

[35] that quinine probably had a yield of less than 0.4 must be dismissed, since Fletcher [36] was unable to duplicate their experiment and found a discrepancy

of a factor of 6 in their reported Q for 2-aminopurine.

Windsor and Dawson [30] pointed out that there were

already literature reports that quinine yield was

dependent on the concentration of sulfuric acid. Many

investigators, including ourselves, were guilty of using

0.546 for the yield of quinine in 0.1 TV H2SO4 rather

than 1.0 N acid. We find that the quantum yield ac- tually is 6 percent lower in the more dilute acid, so a

yield of 0.51 should be used. The reason for the de- pendence of yield on acid concentration is not clear,

but it must be an excited state phenomenon since we

have been unable to demonstrate any significant

change in absorption spectrum for quinine dissolved

in 10 N H2 S04 compared with 0.1 N H2 S04 . Figure 4 shows the titration of quinine which has a midpoint at about pH 4.3 by absorption measurements.

The optical density-pH titration curve shows the

normal flattening at both ends, but the titration by

fluorescence shows a slight continuing rise in the acid

region, although the midpoint of the titration curve is the same. Figure 5 shows the variation of quinine

fluorescence yield with sulfuric acid concentration.

Several features may provide a clue as to the processes

involved here. The fluorescence increases almost

linearly from 0.1 N to 10 N H2 S04 , although there is no change in absorption. However, we have also noted

that S04 =is a quencher of quinine fluorescence,

although Stern-Volmer kinetics were not obtained.

Thus the rise in quinine yield at high acid concentrations would be even larger except for the presence

.9

o

COo

08

.7

LU^ .6

S .3 o

CO

rO O .2

******

4 5

FIGURE 4. Titration of quinine monitored by fluorescence at 450 nm

(---) or by optical density at 360 nm ( — ).

188
oin|

100 O

QUININE

-

.01 .02 .04 0.1 0.2 0.4 1.0 2.0 4.0 10 H

2 S04

(N)

Figure 5. Effect of H2 S04 concentration on the fluorescence of quinine excited at 350 nm and monitored at 450 nm.

of sulfate ion. The slight increase in quinine yield

at low acid concentration is reproducible and may be

due to the lower concentration of sulfate ion. Quinine is a good quantum yield standard for bio- chemical studies for several reasons. It is used in aqueous solutions. There is little overlap of absorption

and emission spectra. The quantum yield is high. The

material is obtained in pure form readily and is stable

in acid solutions for months. There is little structure

in the absorption spectrum and none in the emission

spectrum, so high resolution equipment is not needed.

The temperature dependence of the yield is relatively small. Figure 6 shows the temperature dependence of

the yields of some compounds which might be useful as standards.

It is interesting that Rusakowicz and Testa [37] obtained a quantum yield of 0.46 for quinine in 0.1 N

H2SO4 when comparing it with 9,10-diphenylanthra- cene, assumed to have a yield of 1.0 in cyclohexane.

This value approaches Windsor and Dawson's value

of 0.50 [30] and a value of 0.51 which we now favor. The value of 0.46 is fairly good since a very large

refractive index correction (17%) was needed for comparing cyclohexane and water solutions. Nevertheless,

there is no compelling reason to favor use of 0.46

rather than 0.51 as the yield for quinine in 0.1 /V H2SO4.

The commonly accepted value for the quantum yield

of quinine has also been challenged recently by Scott

et al. [23], who claim that the yield in 0.1 /V sulfuric

acid is 0.70 ±0.02. This value was determined from a comparison of measured lifetime with calculated

natural lifetime, assuming the relation (? = T/Tnat. This

approach must be questioned, since the calculated

natural lifetimes based on equations such as those of Forster [38] or Strickler and Berg [39] are quite ap- proximate. Both Ware and Baldwin [40] and Birks and

Dyson [41] noted the discrepancy between the measured and calculated lifetimes of quinine and have

proposed explanations for this. The value of 0.70 must

be too high, because many studies have used 0.55 as

the value for quinine and have obtained values near

unity for other compounds by comparison with quinine.

Substitution of 0.70 for 0.55 would result in many

quantum yields of greater than unity.

b. Controversy over the quantum yield of tryptophan. The most common quantum yield standard

used in biochemistry for the ultraviolet region is tryptophan, because it has an emission which matches

closely the tryptophanyl fluorescence of proteins.

Here, too, there has been controversy over the quantum

yield. Shore and Pardee [42] estimated Q to be 0.09,

but used a rather crude method involving a spectrophotometer modified for fluorometry with filters. Teale

and Weber [43] subsequently reported a value of

0.20, which has since been widely used. We reported

that the value should be 0.13 [44] based on comparison

with quinine in 0.1 N H2 S04 assumed (incorrectly)

to have a yield of 0.55. Our value for tryptophan Q

should be revised downward to 0.12 in view of the lower

yield of quinine, 0.51. Also using quinine as a standard,

B0rresen [45] and Bridges and Williams [46] obtained

tryptophan yields of 0.11 and 0.15. Eisinger [47] determined the tryptophan quantum yield to be 0.14

based on comparison with p-terphenyl, whose yield was 0.87. If Eisinger's data are correct, there can be no doubt that the value of 0.20 is much too high, for then the quantum yield of p-terphenyl would greatly

exceed unity. We have convinced ourselves that 0.20 is, in fact, too high by similar comparisons

with 9,10-diphenylanthracene and 2-aminopyridine

(2AMP) which have very high quantum yields and have

been recommended as standards [37, 48]. This simple

experiment is recommended to anyone still in doubt

concerning the quantum yield of tryptophan. While

absolute measurements of quantum yield are difficult, relative quantum yields can easily be carried out with

a precision of 5 percent. Therefore, if a standard

with a supposed quantum yield of 1.0 is used, it is easy

to determine an upper limit for the yield of any given

unknown. Q determined in this way may be the real quantum yield, or, if the standard's Q turns out to be

less than 1.0, some smaller value.

c. Other useful standards. Some quantum yields

which have been measured in the author's laboratory

are listed in table 1. Several values are at variance

with those reported recently from Testa's laboratory

[37, 48, 49]: 1. The yield of 2AMP is found to be 0.73

rather than 0.60. 2. The yield of 2AMP is found to be 6, rather than 13, times that of tryptophan. 3. A

value of 0.55 for quinine in 1 N H2 S04 is consistent

with our relative measurements, and is slightly

higher than would be expected from the value of 0.46

reported for quinine in 0.1 N H2SO4.

Tryptophan with a quantum yield of 0.12 seems to be the best standard for protein quantum yields. The

idea of using a protein as a quantum yield standard

has occurred to us. However, when testing different

commercial preparations of serum albumin, the most

widely available protein in pure form, we have found

wide variations in yield. Fresh tryptophan solutions are more suitable; solutions are not stable for more than one day.

There are, of course, many other compounds which

might be suitable for standards, but the problem is to find compounds which have been examined in many

189
Table 1. Quantum yields of some possible standards

Compound Q

2-Aminopyridine, 0.1 N H2 S04 *0.74 Do 73 Do 74 Trytophan, water *.12 Do 12 l-dimethylaminonaphthalene-5-sulfonate, 0.1 .34 M NaHC03 4-Methylumbelliferone, 0.01 M NaOH 64

4-Methylumbelliferone, 0.01 M HC1 65

5- Hydroxyindole (serotonin), pH 7.4 .23 Tyrosine, water .13 Phenylalanine, water .022 Phenol, water .13 l-dimethylaminonaphthalene-5-sulfonamide, .051 pH 7.4

Standard, Qst

9,10-diphenylanthracene, 1.0 tryptophan, 0.12 quinine, 1 N H2 SC>4 , 0.55 9,10-diphenylanthracene, 1.0 quinine, 1 N H2 S04 , 0.55 Do.

Do.

Do.

Do. Da

Do.

Do. Do.

All values were obtained by the author with the Aminco-Bowman spectrofluorometer and

a Cary Model US spectrophotometer, except for those marked (*). The latter were deter- mined with a Turner Model 210 spectrofluorometer. The author is indebted to Dr. H. Edelhoch

for the use of this instrument.

different laboratories which agree on the quantum

yield values. Fluorescein in aqueous base has fre- quently been used with a yield of 0.85 [cf. 50], and this

is an excellent standard if the unknown has similar

spectra. Biochemists will most often require aqueous

standard solutions, since their samples are usually

in water. The correction factor for refractive index

differences between a given solvent and water may be

quite large (+ 17% for cyclohexane solutions, for ex- ample). The correction factor is only approximate for the actual geometry existing in a given spectrofluorometer, and should be avoided wherever possible.

d. On the "certification" of quantum yield standards. In spite of our own confidence in the quantum

yield data in table 1, we recognize that acceptance

of compounds as standards comes only after agree- ment is reached between a number of reliable laboratories. Several studies have supported the values listed for tryptophan, l-dimethylaminonaphthalene-5-sulfonate, quinine, 9,10-diphenylanthracene, and other

compounds. Although a caveat has been issued [31] against the use of tryptophan as a standard, because of the controversy over its yield, a careful review of the

data show that tryptophan Q is as well known as that of quinine. The use of quantum yield values for compounds over which controversy has not yet developed,,

because they have not been adequately investigated, seems irrational. The dearth of accepted quantum

yield values is well brought out by the review of Demas

and Crosby [31]. Perhaps one solution would be to carry out a cooperative study between several laboratories on a number of standards. This might speed the

"certification" of standards.

3. Polarization Measurements

a. Definitions.—The degree of fluorescence

polarization, P, is defined by

Iv + Ih

'

If a fluorometer utilizes right-angle geometry, Iy and

Ih refer to the intensities measured with the analyzer

oriented normal and parallel to the direction of

excitation. Fluorescence polarization is observed when

the excitation is either polarized or unpolarized,

provided that observation of the fluorescence is normal

to the direction of excitation. If polarization observed

with excitation polarized vertically (i.e., parallel to the

orientation of the analyzer when measuring Iy) is pp , the polarization observed with unpolarized, or

natural, excitation, pn , is given by

2Pn Pp= TTp-n

-

Most studies are carried out with polarized excitation

because the absolute values of the polarization are higher (— 1/3 + 1/2) than with natural fight

(— 1/7 ^ p„=£ + 1/3). Perhaps the main reason for

utilizing natural light is to obtain greater excitation

intensities. b. Measurement of polarization. —If a lightscattering photometer is adapted for polarization of fluorescence measurements, one is generally limited to excitation with certain mercury lines, and both

excitation and emission are selected by filters. The use

of a spectrofluorometer results in greater flexibility.

There seem to be no commercially available instru- ments designed primarily for fluorescence polarimetry, but polarizing attachments are offered by

makers of fight-scattering photometers and spectrofluorometers.

190
c. Artifacts in polarization measurements.

1. Defective collimation. The definition of P given

above is based on the assumption that the exciting

and emitted rays are perfectly collimated. In long

path-length instruments, such a condition is nearly

achieved. However, we have found that some simple

filter fluorometers are quite unsuited for polarization

measurements because the lamp is large and placed

close to the sample, and the photodetector is also adjacent to the sample chamber. Large lenses which

collect the fluorescence emitted at large angles to the

axis connecting the sample and the photodetector

may also invalidate the polarization data.

2. Polarizer defects. These can be several types.

Azumi and McGlynn [51] found that improperly

fabricated Glan prisms rotated the beam of light as the prisms were turned from one orientation to the

other. Errors in mounting can cause the ordinary

(unpolarized) ray to pass to the sample, or the polarizers

may be set at some angle to the desired orientation.

Some film polarizers may have incomplete polarization, especially at short wavelengths. Most polaroid

film polarizers do not seem to be light-fast and will rapidly bleach when exposed to a high intensity source. However, some light-fast film polarizers of

large aperture are available [13]. A convenient ar- rangement for spectrofluorometers is to use such filters in the exciting beam, and polaroid analyzers before

the photodetector, as shown in figure 7. This arrange- ment has been used by many workers with good results.

hi/

Figure 6. Recommended arrangement of polarizers in the AmincoBowman spectrofluorometer. The polarizers (Pol) and analyzers

(An) are in sliding filter holders. From Chen and Bowman [13].

3. Spurious polarization introduced by the instru- ment. Light reflected from surfaces in a grating monochromator instrument becomes partly polarized,

and such polarization must be corrected. In the exciting beam, this partial polarization is best rendered

completely polarized; i.e., pp rather than Pn is measured, especially in spectrofluorometers. The polarization introduced by the emission monochromator is wavelength dependent and related to variations in efficiency of the grating ("grating anomalies"). This

art'fact may be corrected by first determining the

relative response of the detector system at any wavelength to horizontally and vertically polarized light.

The grating correction factor, G, is obtained by ob- serving a source of unpolarized light with the analyzer

in the vertical and horizontal positions. A fluorescent

sample can act as such a source if excited with

horizontally polarized light. Polarization is then determined from

Iw — GIVH Iw + GIvh

where G =IhvIIhh, and the subscripts refer to the

orientations of the polarizer and analyzer.

G should depend only on the wavelength setting of the emission monochromator. For this reason, a calibration curve may be obtained with one or two highly

fluorescent solutions and may be applied to measure- ments on other substances. The monochromator

bandwidth should affect the G factor, but in our

tests, the available bandwidths made very little difference in G [52]. Figure 8 shows the G factor

A

5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40

TEMPERATURE (°C) Figure 7. Variation of the quantum yield, Qx , of some compounds

with temperature. Abbreviations: 4 MeU, 4-methylumbelliferone; FL, fluorescein; RB, Rhodamine B.

calibration obtained with a spectrofluorometer.

Although G could be measured for each sample, some

solutions have very low fluorescence yield, and there

may be large blank corrections required in determining

Ihv and Ihh. A calibration curve obtained under better

conditions is preferable. One of the interesting

features of figure 8 is that G varies sharply with wavelength. Some authors [51] have suggested that G

declines monotonically with wavelength, but this is clearly not the case.

191
4. Contributions from blanks. Like other measure- ments involving fluorescence intensity, polarization

measurements should be rigorously corrected for blank

contributions. If the G factor is known from prior

calibration, polarization measurements still require

four measured values: h v and Ivh for sample and blank

The magnitude of the blank relative to the sample

signal can be large, as shown in figure 9 for the example

involving bilirubin. Q for bilirubin bound to albumin is less than 0.01, and there is a background fluorescence

due to impurities on albumin. There are relatively

few instances where the blank can be neglected

completely. Such might be the case of high quantum

yield dyes dissolved in glycerol. A number of "auto- matic" fluorescence polarimeters have been described

[53-55] in which Ivh and Ivv are measured simultaneously and ratioed electronically to give a function

proportional to P. However, the common failing of these instruments is an inability to correct for the

blank.

G

0.8 -

0.7 1 1 1 1 1 1 1 1 300 350 400 450 500 550 600 650 WAVE LENGTH (nm)

Figure 8. Dependence of the grating factor, C, with emission

setting; i.e., calibration curve forG. The samples used were 10~ 4 M

2AMP in 0.1 N H2 S04 excited at 285 nm, and If)- 3 M DNSA (1- dimethylaminonaphthalene-5-sulfonamide) in 50 percent ethanol, excited at 330 nm. C was calculated from IhvIIhh- From Chen [52].

5. Scattering. If scatter signal reaches the detector,

the polarization will be anomalously high. Scatter can

be corrected with a suitable blank, but such blanks

may not be available. For example, in obtaining the

polarization of the intrinsic emission from a protein,

the blank should have the same light-scattering properties without being fluorescent. In turbid solutions, scatter may indirectly lower the observed fluorescence

by causing the excitation to appear to reach the

sample from many directions due to multiple reflections. For the most accurate measurements, the solu- tions should be clarified by centrifugation or filtration.

6. Depolarization by radiative energy transfer. In concentrated solutions of substances with considerable

overlap of their absorption and emission spectra, de- polarization can result from secondary fluorescence

which has been excited by self-absorption of radiation. The effect can be detected by the increase in r upon

dilution, and can be minimized by the use of small

path length cells. Dyes with high absorption coeffi- cients, like fluorescein and Rhodamine B, have been

reported to exhibit concentration depolarization in solutions as dilute as 4 X 10~6 M [56]. d. Standards .for Polarization.—Standards for fluorescence polarimetry would be helpful to evaluate

the performance of a given instrument, but such well- defined standards do not appear to exist. There are considerable variations in reported polarization values

for given solutions. The variations are as great as in the area of reported quantum yield values. In the

absence of such standards, it is necessary to be

certain that the apparatus registers zero polarization

for nonviscous solutions of compounds with long

lifetimes. Anthracene or other aromatic hydrocarbons

in ethanol or cyclohexane may serve this purpose. At

the other end of the scale, a sample having nearly

the theoretical maximum polarization of 0.5 is desir- able. An instrument could read zero for the polarization of a nonviscous solution, but might well give

erroneously low polarizations for viscous solutions. Alkaline fluorescein, or Rhodamine B, in glycerol were reported to have polarizations between 0.4 and

0.5 and are therefore often used to test the perform- ance of fluorescence polarimeters. The polarizations

reported by various workers are given in table 2. Note

Table 2. Polarizations of dilute dye solutions in glycerol

Exciting

Sample wavelength P Reference

(nm)

Fluorescein 366 0.28 Chen and Bowman

[13] Do 366 .424 Price at al. [57] Do 430-520 .477 ±0.01 Chen and Bowman

[13] Do 436 .440 Weber [56] Do 436 .446 Price et al. [57] Do 436 .4922 Szalay et al. [58]

Rhodamine B 366 —0.17 Chen and Bowman

• [13] Do 366 -.0655 Weber [56] Do 366 -.091 Price et aL [57] Do 366 -.17 Kaye[59] Do 436 -.020 Chen and Bowman

[13] Do 436 -.0244 Weber [56] Do 436 0.0 Price et al. [57] Do 436 -0.03 Kaye [59] Do 546 0.462 Chen and Bowman

[13] Do 546 .444 Weber [56] Do 546 .441 Price et al. [57] Do 546 .462 Singleterry and Weinberger [27] Do 546 .44 Kaye [59]

that there is considerable variation in these values. The best agreement is for Rhodamine B excited at

192
6 A

300 350 400 450 500 WAVE LENGTH (nm)

FniURE 9. Example showing significance of blanks in polarization measurements.

In A are shown the absorption (light line) and polarization spectrum (heavy line and points) of bilirubin bound to rabbit serum albumin. The polarization was calculated, using C = 0.960, from the data of Part B, which gives the apparent excitation spectra of the sample (I) and blank (B). Subscripts refer to orientation of the polari- zer and analyzer. The emission was monitored at 520 nm. From Chen [52].

546 nm: the P values are 0.45 ±0.01. Poor agreement

is found for the values of polarization of Rhodamine

B excited at 366 nm.

Useful polarization standards should have polarization which is independent of the emission wavelength. We have found this to be the case for Rhodamine

B, fluorescein, and other compounds. However, we

found [21] that quinine polarization decreases with

wavelength in the emission band (fig. 3). In many biochemical systems, the emission arises

from several fluorophors such as dyes or tryptophans

in different parts of a single protein. The polarization

therefore depends on both the exciting and emission

wavelengths. Much of the early work on the polarization of dye conjugates was obtained with fluor- ometers where the emission was isolated by a cut-off

filter. Such data cannot be duplicated on a spectrofluorometer which detects only a segment of the

emission band.

4. Fluorescence Lifetimes.

The fluorescence decay time, or lifetime, t, is defined by I = Ioe~ tlr where Io is the fluorescence in- tensity at time t=0 following a delta function exciting

pulse, and / is the intensity at subsequent time t. The

lifetime is thus the time required for decay of the

fluorescence to a value of Iole. The decay times of organic molecules in solution

which are of interest to biochemists lie in the range of

0.1 to 100 ns. The accurate measurement of such short

times requires complex electronic equipment, but

there have been many recent advances in this field. There were no commercially produced instruments

for the measurement of t until 1966. However, numerous laboratory-built instruments had been designed

and described. The features of such instruments have

been reviewed by Birks and Munro [60].

193
Two basic types of instrument have been employed,

(a) Phase and modulation fluorometers. Here the

sample is excited by light which is sinusoidally

modulated at megacycle frequencies. The fluorescence

thus also fluctuates at these frequencies, but lags

behind the excitation by a certain time which is detected electrically as a phase shift with reference

to the exciting light. From the phase shift, one can

calculate the lifetime value if fluorescence is assumed

to decay as a single exponential. The fluorescence

decay is also manifested as a decrease in the degree

of modulation, and the lifetime can be obtained from

this parameter quite apart from the electrical phase

shift. Miiller et al. [61] reported that a phase fluorometer could be constructed wholly from commercially

available components. Spencer and Weber [62] have

also constructed a modern phase and modulation

fluorometer. Both of these instruments are said to have a very high time resolution, of the order of

0.02 ns. Therefore, the accuracy of lifetime measure- ments can be said to exceed the accuracy of absolute

determinations of quantum yield. No phase fluorometers have ever been marketed, (b) Nanosecond pulse

instruments. By repetitively exciting a sample with a

short pulse of light, it is possible to obtain directly,

with the aid of a fast oscilloscope, the curve of the

fluorescence decay. Ideally, the light flash should be

infinitely short; the observed fluorescence should

then decay exponentially. Practically, lamp pulses

have a finite duration with widths at half maximum

intensity of 1 to 10 ns, and the pulse shape may be

irregular. The fluorescence decay is also a complex

function from which the lifetime must be extracted

by any of several mathematical procedures. Advances

in lamp design and the use of mode-locked lasers with picosecond pulses may in the future avoid the

need for such manipulations, since the excitation

will be much shorter than the fluorescence.

The two instruments which are currently avail- able from commercial sources for lifetime deter- minations are both based on the nanosecond pulse

method. In 1966, TRW Instruments introduced a decay

time apparatus consisting of a nanosecond fluorometer

with a "decay time computer" and requiring a dual- gun oscilloscope. One gun of the oscilloscope was used

to display the signal from the sample chamber, while

the other channel was connected to the computer,

which in turn consisted of a variable waveform signal

generator and an analog circuit. The computer was

used to compare the shapes of the waveforms derived

from the lamp pulse and from a fluorescent sample.

The difference between the waveforms was analyzed

by the computer to give the corresponding decay time

directly. The instrument has been described in some

detail [63, 64], is still available, and can be fitted with different kinds of lamps for excitation at different wavelengths. Although originally designed for long

lifetimes such as those associated with solid laser materials, the instrument was shown to be capable of

measuring lifetimes less than 2 ns [65].

Recently, ORTEC, Inc. (Oak Ridge, Tenn.) has

produced a nanosecond pulse instrument based on the

so-called single photon counting principle. The

important feature of the system is the method used to detect and record the signal from the sample chamber.

The low intensity, very fast, nanosecond flash lamp

is made weaker still by attenuators so that for each

repetitive pulse, no more than one photon which leaves

the fluorescent sample causes a response in the photodetector. By timing the interval between the flash and the arrival of the photon at the detector, a histo- gram of counts versus time is built up and stored in a multichannel analyzer. After sufficient counts have

been accumulated, the curve can be displayed on an

oscilloscope or digitized for computer analysis. The

advantage of the system is the extreme sensitivity,

the good time resolution, the use of a lamp, which

because of its low output can be made very fast, and

then recording the actual undistorted decay. The system

theoretically can be used to determine multiple life- times present in the fluorescence.

Lifetime measurements by any of the instruments

mentioned require only a single standard, namely, a

reflecting or scattering substance serving as a standard

with t = 0. No comparison with substances of known

lifetimes is needed, so each lifetime measurement is, in fact, an absolute determination. There are now

several compilations of lifetimes which can be used by

investigators to test the accuracy of their instruments

[60,66].

Some lifetimes of fluorescence standards are fisted

in table 3. Many developers of lifetime instruments

have examined fluorescein in aqueous base, and a

lifetime of 4.5 ±0.3 ns agrees with most of the values

listed. Curiously, the value of 3.83 ns was obtained

by Miiller et al. [61] with an instrument estimated

to be accurate to 0.02 ns. Several values for quinine

lifetime have been reported and are listed in table 3. In 1.0 N H2SO4 the lifetime of quinine obtained by

averaging the tabulated data is 19.9 ns with an average

deviation of 0.5 ns or 2.5 percent. The agreement for

this compound is as impressive as any listed by Birks

and Munro [60], so quinine is probably the best

lifetime standard now available. However, some caution is required in the use of

quinine, because we have now found that not only

the quantum yield, but the lifetime too, is dependent

on the concentration of sulfuric acid (table 3) present.

III. Summary and Conclusions

In biochemistry, many of the same principles for measuring absolute fluorescence parameters are used

which are employed in any other area of physical

chemistry. Perhaps the only differences are the general use of aqueous solutions, the greater need for commercially available instrumentation, and the diverse

types of measurement needed. The range and quality

of available instruments have improved. There are

still many problems regarding standards to be used for quantum yields, polarization, and spectra. Quinine,

194
Table 3. Lifetimes of some fluorescence standards

Sample

Quinine, 0.1 W H2 S04 Quinine, 1.0 W H2 S04 Do

Do

Do

Quinine, 10.0 WH2 S04 Quinine, 1.0/VH,SO4 Quinine, 0.1 WH2 S04 Quinine, 0.2 M 7Yjs-N03

-, pH 8. .

.

Fluorescein, dilute NaOH or KOH.

Do

Do

Do

Do

Do

Do

Do

Do

Do

Do

Do

Do

9,10-Diphenylanthracene,

cyclohexane, not de-aerated.

9,10-Diphenylanthracene,

cyclohexane, de-aerated.

Do

2-Aminopyridine,0.1 N H2 S04 ...

1-Dimethylaminonaphthalene5-sulfonate, 0.1 M NaHC03 . Do

Lifetime Reference

(ns)

19.5 Weber [67] 19.2 Berlman [66] lO A w are ana oaia win [t-uj 20.1 Birks and Dyson [41] 20.5 Metcalf, cited in [41] 21.8 Present work

20.4 Do.

19.0 Present work, also [65] 17.2 Do.

4.62 Ware and Baldwin [40]

4.6 Szymanowski [68] 4.6 Metcalf [69] 4.8 Bennett [70] 4.8 Brody[73]

4.8 Kirchhoff[74]

4.8 Maercks [75] 4.5 Bailey and Rollefson

[71]

A ^ 4.0 cnen et ai. |Ooj 4.2 Weber [67] 4.0 Brewer et al. [72] 4.0 Strickler and Berg [39] 3.83 Miiller et al. [1]

6.7 Present work

9.35 Berlman [66]

10.0 Amata et al. [76] 12.7 Present work

13.8 Chen et al. [65]

13.6 Forster and Rokos [77]

the universal standard, has been shown to undergo

changes in emission spectrum, quantum yield, polarization, and lifetime depending on factors such as the

wavelength of excitation and the sulfuric acid concentration. To determine what the "true" values are for a given compound, further reporting of such values is to be encouraged. It is suggested that collaborative

efforts might speed up the "certification" of values for fluorescence standards.

IV. References

[1] Weber, G., Biochem. J. 51,145(1952).

[2] Weber, G., Biochem. J. 51,155(1952).

[3] Perrin.F.J. Phys. 7,390(1926).

[41 Levshin, V. L., Z. Physik 26, 274 (1924).

[5] Chen, R. F., Edelhoch, H., and Steiner, R. F., in Physical Principles and Techniques of Protein Chemistry, Part A, S. J. Leach, Ed., pp. 171-244 (Academic Press, N.Y., 1969).

[6] Velick, S. F., J. Biol. Chem. 233, 1455 (1958).

[7] Chen, R. F., and Kernohan, J. C, J. Biol. Chem. 242 , 5813

(1967). [8] Forster, T., Ann. Phys. (Leipzig) 2, 55 (1948).

[9] Lerman, L. S., Proc. Nat. Acad. Sci. 49,94 (1963).

[10] Weber, G., and Anderson, S., Biochemistry 8,361 (1969).

[11] Witholdt, B, and Brand, L., Biochemistry 9, 1948 (1970).

[12] Bowman, R. L., Caulfield, P. A., and Udenfriend, S-, Science 122,32 (1955)

[13] Chen.R. F., and Bowman, R. L., Science 147,729 (1965).

[14] Chapman, J. H., Forster, T., Kortum, G., Parker, C. A., Lippert,

E., Melhuish, W. H., and Nebbia, G., Appl. Spectroscopy 17,

171 (1963).

[15] Melhuish, W. H., J. Opt. Soc. Am. 52, 1256 (1962).

[16] Chen, R.F., Anal, Biochem. 20,339(1967).

[17] Lee, J., and Seliger, H. H., Photochem. Photobiol. 4, 1015

(1965). [18] Argauer, R. F., and White, C. E.. Anal. Chem. 36,368 (1964).

[19] B0rresen,H. C, Acta Chem. Scand. 19,2089(1965).

[20] Gill, J. E., Photochem. Photobiol. 9, 313 (1969).

[21] Chen,R.F., Anal. Biochem. 19,374(1967).

[22] Fletcher, A. N., J. Phys. Chem. 72,2742 (1968).

[23] Scott, T. G., Spencer, R. D., Leonard, N. J., and Weber, G.,

J. Am. Chem. Soc. 92 , 687 (1970).

[24] Hughes, E., Jr., Wharton, J. H., and Nauman, R. V., J. Phys.

Chem. 75,3097(1971).

[25] Galley, W. C, and Purkey, R. M., Proc. Nat. Acad. Sci. 67,

1116 (1970).

[26] Weber, G., and Teale, F. W. J., Trans. Faraday Soc. 53, 646

(1957).

[27] Singleterry, C. R., and Weinberger, L. A., J. Am. Chem. Soc. 73,4574(1951).

[28] Almgren,M., Photochem. Photobiol. 8,231 (1968).

[29] Paoletti, J., and LePecq, J.-B., Anal. Biochem. 31, 33 (1969).

[30] Windsor, W. R., and Dawson, M. W., J. Phys. Chem. 72,3251

(1968). [31] Demas, J. N., and Crosby, G. A., J. Phys. Chem. 75, 991 (1971).

[32] Melhuish, W. H., J. Phys. Chem. 65,229(1961).

[33] Melhuish, W. H., N. Zeal. J. Sci. Technol. 37B, 142 (1955).

[34] Eastman, J. W., Photochem. Photobiol. 6. 55 (1967).

[35] Drobnik, J., and Yeargers, E., J. Mol. Spectr. 19, 454 (1966).

[36] Fletcher, A. N., J. Mol. Spectr. 23,221 (1967).

[37] Rusakowicz, R., and Testa, A. C, J. Phys. Chem. 72, 793

(1968).

[38] Forster, T., Fluoreszenz Organischer Verbindungen, p. 158 (Vandenhoeck u. Rupprecht, Gottingen, 1951).

[39] Strickler, S. J., and Berg. R. A., J. Chem. Phys. 37 , 814 (1962).

[40] Ware, W. R., and Baldwin, B. A., J. Chem. Phys. 40, 1703

(1964).

[41] Birks, J. B., and Dyson, D. J., Proc. Roy. Soc. A274, 135

(1963).

[42] Shore, V. G., and Pardee, A. B., Arch. Biochem. Biophys. 62,

355 (1956).

[43] Teale, F. W. J., and Weber, G., Biochem. J. 65,476(1957).

[44] Chen, R. F., Anal. Lett. 1 , 35 (1967).

[45] B0rresen, H. C, Acta Chem. Scand. 2 1 , 920 (1967).

[46] Bridges, J. W., and Williams, R. T., Biochem. J. 107 , 225

(1968).

[47] Eisinger, J. .Photochem. Photobiol. 9,247(1969).

[48] Rusakowicz, R., and Testa, A. C, J. Phys. Chem. 72, 2680

(1968).

[49] Weisstuch, A., and Testa, A. C, J. Phys. Chem. 72,1982 (1968).

[50] Parker, C. A., and Rees, W. T., Analyst 85, 587 (1960).

[51] Azumi, T., and McGlynn, S. P., J. Chem. Phys. 37, 2413 (1962).

[52] Chen, R. F., Anal. Lett. 4, 459 (1971).

[53] Weber, G., and Bablouzian, B., J. Biol. Chem. 241, 2558

(1966). [54] Deranleau, D. A., Anal. Biochem. 16 , 438 (1966).

[55] Rosen, C.-G., Acta Chem. Scand. 24, 1849 (1970).

[56] Weber, G., J. Opt. Soc. Am. 46, 962 (1956).

[57] Price, J. M., Kaihara, M., and Howerton, H. K., Appl. Opt. 1, 521 (1962).

195
[58] Szalay, L., Gati, L., and Sarkany, B., Acta Phys. Acad. Sci. (Hungary) 14, 217 (1962).

[59] Kaye, W., Appl. Spectr. 18, 9 (1964).

[60] Birks, J. B., and Munro, I. H. in G. Porter, ed., Progress in Reaction Kinetics 4, 239 (1967).

[61] Miiller, A., Lumry, R., and Kokubun, H., Rev. Sci. Instr. 36,

1214 (1965).

[62] Spencer, R. D., and Weber, G., Ann. N.Y. Acad. Sci. 158, 361

(1969).

[63] Chen, R. F., Arch. Biochem. Biophys. 133 , 263 (1969).

[64] Meserve, E., in R. F. Steiner and I. Weinryb, eds., Excited

States of Proteins and Nucleic Acids, Plenum Press, N.Y.,

1971, p. 57.

[65] Chen, R. F., Vurek, G. G., and Alexander, N., Science 156,949

(1967).

[66] Berlman, I. B., Handbook of Fluorescence Spectra of Aromatic

Molecules, Academic Press,' N.Y. 1965; 2d Ed., 1971.

[67] Weber, G., Methods in Enzymol. 16, 393 (1969).

[68] Szymanowski, W., Z. Physik 95 , 450 (1935).

[69] Metcalf, W. S., J. Chem. Soc. 3726 (1960).

[70] Bennett, R. G., Rev. Sci. Instr. 31, 1274 (1960).

[71] Bailey, E. A., and Rollefson, G. K., J. Chem. Phys. 21, 1315

(1953).

[72] Brewer, L., James, C. G., Brewer, R. G, Stafford, F. E., Berg,

R. A., and Rosenblatt, G. M., Rev. Sci. Instr. 33, 1450 (1962).

[73] Brody, S. S., Rev. Sci. Instr. 28, 1021 (1957).

[74] Kirchhoff, W., Z. Physik 116, 115 (1940).

[75] Maercks, O., Z. Physik 109 , 685 (1938).

[76] Amata, C. D., Burton, M., Helman, W. P., Ludwig, P. K., and Rodemeyer, S. A., J. Chem. Phys. 48, 2374 (1968).

[77] Forster, Th., and Rokos, K., Chem. Phys. Lett. 1,279 (1967).

(Paper 76A6-744)

196
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Newer Fluorometric Methods for the Analysis

of Biologically Important Compounds

G. G. Guilbault

Department of Chemistry, Louisiana State University, New Orleans, La. 70122

(June 15, 1972)

Newer fluorometric methods for the analysis of biologically important compounds will be discussed: enzymes such as LDH, alkaline phosphatase, lipase and cholinesterase, and substrates such as glucose, urea and uric acid. These methods are based on the production of fluorescence initiated by an enzymic

reaction. New reagentless fluorescence methods will be described for enzymes and substrates. These

methods are highly precise (1%), fast (less than 1 minute) and involve no preparation of reagents. These methods, as adapted to clinical laboratory procedures, will be discussed.

Key words: Enzymes, fluorometric analysis; fluorometry of enzymes; fluorometry of substrates;

silicone pad; solid surface fluorometry.

I. General

Enzymes are biological catalysts which enable the

many complex chemical reactions, necessary to the

existence of life, to take place at ordinary temperatures.

Because enzymes work in complex living systems, one

of their outstanding properties is specificity. Thus, an enzyme is capable of catalyzing a particular reaction

of a particular "substrate" to produce a characteristic

and measurable reaction end product. This specificity

of enzymes and their ability to catalyze reactions of

substrates at extremely low concentrations is of

significant use in biochemical analyses.

Enzyme catalyzed reactions have long been used for such analytical purposes for determining the presence

of specific substrates, enzyme activators, enzyme

inhibitors, and enzymes themselves, and in determining

the concentration of these substances. Numerous

methods have been described for the determination

of enzymic activity between an enzyme and a substrate. Until recently, however, the disadvantages associated

with the use of enzymes in these analytical techniques

have seriously limited their usefulness.

A detailed discussion of the use of enzymes for analysis can be found in review articles by Guilbault

[1-3] and books by Guilbault [4] and Bergmeyer [5]. The concentration of substrate participating in an enzyme reaction can be calculated in one of two

general ways. The first method measures, by chemical,

physical, or enzymatic analysis, either the total change

that occurs in the end product or the unreacted starting

material. In this method, large amounts of enzyme and

small amounts of substrate are used to insure a complete reaction. In the second method, which is a

kinetic method, the initial rate of reaction is measured,

in one of many conventional ways, by following the

production of product or the disappearance of the

substrate. In this method, the rate of reaction is a function of the concentration^ of substrate, enzyme,

inhibitor and activator. On the other hand, because enzymes are catalysts,

and as such affect the rate and not the equilibrium of

reactions, their concentration and activity must be

measured by the rate or kinetic method. Similarly,

activators and inhibitors that affect the enzyme's catalytic effect can be measured only by the rate method.

While, as pointed out above, the substrate can be measured either by a total change or a rate method, the

latter method is faster because the initial reaction can

be measured without waiting for equilibrium to be

established. The accuracy and precision of both methods are comparable.

In the past, manometric methods, pH procedures,

and spectrophotometry have been used for determining

enzyme activity. Spectrophotometry has been generally preferred because of its simplicity, its rapidity and

the capability of measuring lower enzyme and substrate

concentrations. Spectrophotometry embraces the use

of colorimetric methods where colored products are produced as a result of enzyme activity, and fluorescent

methods where fluorescent compounds are produced as a result of enzyme activity. Fluorescent procedures

are several orders of magnitude more sensitive than

colorimetric methods and thus have replaced the

colorimetric methods in numerous instances.

Previous fluorometric methods, although they have

197 _

502-591 O - 73 - 14
been improvements over other prior art methods of determining enzyme activity have not eliminated all of the problems associated with enzymic analyses.

Fluorometric analysis depends on the production of a fluorescent compound as a result of enzyme activity

between a substrate and enzyme. The rate of production of the fluorescent compound is related to both the

enzyme concentration and substrate concentration.

This rate can be quantitatively measured by exciting

the fluorescent compound as it is produced and by

recording the quantity of fluorescence emitted per unit of time with a fluorometer.

The prior methods for fluorometrically measuring

enzyme reaction rates, however, have been wet chemical methods and rely on reacting a substrate solution

with an enzyme solution. Wet chemical methods involve time-consuming and wasteful preparation of costly substrate solutions and enzyme solutions. For

example, when determining the presence and concentration of an enzyme, a substantial amount of substrate

must be accurately weighed and dissolved in a large

amount of buffer solution usually about 100 ml to pre- pare a stock solution. The enzyme reaction is then

usually carried out by measuring, for example, 3 ml

of stock substrate solution into an optical cuvet,

adding a measured amount of the enzyme solution to the substrate solution, and recording the change in fluorescence eminating from the resultant solution per

minute with a fluorometer. This standard wet chemical

method is costly and wasteful because laboratory

trained technicians' time and relatively large quantities of expensive substrate are required.

II. Solid Surface Fluorescence Monitoring System

Solid-surface fluorometric methods, using a reagentless system, have been developed for the assay of

clinically important enzymes, substrates, activators

and inhibitors. The method comprises forming a solid reactant film of one of the reactants on an inert silicone matrix pad, contacting the film of the first reactant with

a solution of the second reactant (substance to the

measured) to produce a fluorescent material, and

monitoring the change of fluorescence with time to determine the concentration of the second reactant. The reactant film may be formed by dissolving the

reagents in a solvent, depositing the reactant solution on the silicone pad so that the solution spreads evenly over the pad, and evaporating the solvent from solution. The reagent may be applied to the pad either from an

acetone solution if a substrate or in a polymeric film such as polyacrylamide if an enzyme. Either substrate

or enzyme and/or coenzyme can be deposited on the

pad in film form depending upon whether the substance

to be assayed is an enzyme or a substrate. After the pad is formed (fig. 1), it is attached to a slide made of a rigid material, such as a glass or metal slide,

by applying an adhesive to the slide and overlying the

pad on the adhesive. The pads are placed approximately 1 or 2 cm from the bottom of the slide (fig. 2).

1 mm 3 mm

8 mm

SLIDE (GLASS OR METAL)

Figure 2. Slide support. The fluorescent cell consists of a rectangular cell made of ordinary sheet metal, wood or plastic material

(fig. 3), having portholes to allow radiation to enter and

leave the cell cavity. The top of the cell is provided

with guides at opposite sides so that the slide containing the reagents can be reproducibly placed within the

cell. In order to obtain as low a background as possible,

the cell is painted with a black optical paint having a

dull finish.

GUIDES FOR SLIDES

7,5 cm

FIGURE 1. Pad support (all measurements are in mm). Figure 3. Fluorescence attachment used to hold pads. 198
Before the enzyme solution is added, the glass slide

is put into the fluorometer and a background reading is taken. A blank rate is taken using all components of the

system, except the unknown. The slide is then removed

from the fluorometer and 20 of a solution of the sub- stance to be assayed is applied to the slide. The recorder

(100 mV input) is started immediately, the slide is placed back into the fluorometer, and the rate is re- corded. A calibration plot of the change in fluores- cence units per minute versus concentration is prepared

and used for subsequent analyses of enzyme or

substrate. After a run is completed, the pad can be peeled from

the slide, and the slide cleaned and stored for subsequent use. The physical properties of the silicones and the

stability of the reactant films of enzyme and substrate

in the presence of silicones further make these materials

ideally suited for use. The silicone materials can retain a reactant film on their surfaces for an indefinite time

and permit the direct measurement of fluorescence

from its surface when an appropriate second reagent

solution is dropped onto the first reactant film. Background interference due to light scattering and

nonspecific fluorescence are minimal compared to other materials.

The number of enzyme systems that could be

monitored by this type of solid-surface device are as numerous as the number of enzyme systems known.

Some of the more clinically important systems that

will be studied and for which methods have been

developed are as follows.

III. Assay of Clinically Important Enzymes

A. Cholinesterase

Low levels of cholinesterase (ChE) are found in individuals with anemia, malnutrition, and pesticide

poisoning. High levels indicate a nephrotic syndrome.

Preliminary results have indicated that cholines- terase in solution can be monitored using a film of

iV-methyl indoxyl acetate on the surface of a silicone rubber pad:

N- Methyl indoxyl acetate ChE

yV-Methyl Indoxyl

(Fluorescent —

X

ex

:=495 nm, X em = 525 nm)

The rate of production of iV-methyl indoxyl is followed

and is proportional to the cholinesterase concentration.

Attempts have been made to develop methods for the direct assay of ChE in blood and body tissue. The

reproducibility and accuracy obtainable using several

configurations of pad and various experimental conditions have been compared and the optimum system

developed. A linear region of 10

4 to 1 unit has been

obtained [6]. Preliminary studies have indicated that pads made

from either method were stable for at least 60 days

(table 1), if kept in a cold dark place.

Table 1. Pad stability data 8

Day" Rate, AF/min c

0

24

103060

3.10

3.04

3.08 3.20

3.00

3.00

a A 500 /xg/ml solution of cholinesterase was freshly prepared for each day. Twenty microliters of a 0.1 M substrate solution were

applied to the pad and allowed to evaporate to dryness.

b Days after preparation of pad.

0 Rates were an average of at least three runs.

B. Alkaline Phosphatase

High levels of alkaline phosphatase are observed in rickets, Paget's disease, obstructive pandicea, and

metastatic carcinoma.

The fluorogenic substrate naphthol-AS-BI-phosphate was used for the assay of alkaline phosphatase in serum. Guilbault et al. [7, 8] found that this substrate is the best for the direct assay of alkaline phosphatase in serum. A solution method was used and an accuracy

and precision of about 2 percent was obtained. The

deep green fluorescent naphthol AS-BI formed is measured. Attempts have been made to monitor this reaction on a solid surface. A drop of serum to be assayed

has been added directly to the naphthol AS-BI-phosphate on the surface and the rate monitored. No

solutions are required. From 10 ~ 6 to 10 units are assayable [9].

M—P03 H, o

Br

hH2 0 - ONH

OH

CONH

o

OCH3 OCH3

Naphthol AS-BI-Phosphate

(Non-Fluorescent)

Naphthol AS-BI

(Fluorescent)

Further attempts have been made to extend the use- fulness of this method for a wide variety of biological

samples.

The color of the silicone rubber however, affects both

the background and reaction rates. With any of the

filter systems, the background and reaction rates increase in the order black<grey<clear< white. Each

possible combination of pad color and filter was ex- amined and it was found that the most accurate results could be obtained if a combination of 7-54 filter and

grey silicone rubber pads were used.

Table 2 shows the results obtained from a series of serum samples. Over 80 serum samples were analyzed

with a relative error of ± 5 percent.

199
Table 2. Determination of serum alkaline phosphatase

Units alkaline phosphatase

taken a

20

2224

26

2830

3133

3739

4143

48

53

6164

Units alkaline phosphatase found 3-0

21.5

1924.5 23

25

31

313734

40

39

42

49 "54

d 60

d 64

a Units expressed as /xmol phenolphthalein liberated min" 1 l"1 serum.

b Obtained by multiplying the rate by 31.5.

c Average from 3 or more different samples—each analysis in duplicate.

d One sample—average of 2 determinations.

Figure 4 shows the correlation between the serum

alkaline phosphatase values obtained from the "solid

surface" method and alkaline phosphatase values

obtained from a solution method employing phenolphthalein phosphate. The graph shows good agree- ment between the two methods over a wide range of serum values. If the serum values are expressed as /xmol phenolphthalein liberated min-1

1

1 serum, then

normal serum has 27-105 units.

50 100 NAPHTHOL AS-B1

Table 3 shows the alkaline phosphatase values

obtained from highly jaundiced serum compared to the phenolphthalein phosphate method. All the results from these samples were low and implied a limitation on the method. It is, however, possible to predict

which samples will give low results. Generally, it is those samples which are very dark yellow or orange- yellow in color. The light-yellow serum gave good

results as did haemolyzed serum. The low values

obtained from jaundiced serum is the main limitation

of the method, but it is possible to recognize these

solutions and carry out the analysis by an alternative

method.

Table 3. Analytical results obtained from jaundiced serum

samples

Units alkaline phosphatase

taken a

2734

5986

135

185 200

Units alkaline phosphatase found a - b

18

2130

2144

115 138

a Units expressed as /xmol phenolphthalein liberated min" 1 l" 1 serum.

b Average of 3 results.

C. Lipase

A low plasma lipase indicates vitamin A deficiency

and some malignancies. Lipase assay is most important

in diagnosis of pancreatitis.

Two fluorogenic substrates have been evaluated for the assay of lipase: iV-methyl indoxyl myristate, a

substrate developed by Guilbault and Hieserman [10], and 4-methyl umbelliferone myristate, a substrate

recently prepared in our labs. The production of the green fluorescent naphthol

AS-BI or the blue 4-methyl umbelliferone myristate can be followed on the surface.

O^ Xt

CH3 O

(Crt )i2CH3 O

0-|| !i! CCH3 A/v*-

o

OH

4-Methyl Umbelliferone Myristate

4-Methyl Umbelliferone = 340 nm Km — 450 nm

Figure 4. Correlation between results obtained with naphthol AS-BI method and phenolphthalein method. A direct assay of lipase (10~ 4 to 10 units) in serum and

pancreas can be effected. 200
D. Lactate Dehydrogenase

Levels of this enzyme are elevated in individuals

with acute and chronic leukemia in relapse, myocardial

infarctions and carcinomatosis.

Fluorometric analysis of dehydrogenases in the past

have been based on measurement of NADH fluores- cence. Guilbault and Kramer [11] have described a

fluorometric method of coupling reduced nicotinamide

adenine dinucliotide (NADH) to an electron acceptor

(resazurin) in the presence of the cofactor phenazine

methosulfate (PMS).

Lactate + NAD. LDH

NADH + Resazurin

(Non-Fluorescent)

PMS

NADH + pyruvate

Resorufin + NAD

(Fluorescent)

A semisolid state fluorescence method for the

analysis of lactic dehydrogenase (LDH) has been

developed [12]. The commercially available enzyme

in aqueous solution, and the enzyme in blood serum

were studied. The serum analysis is compared to a method described by Bergmeyer, et al. [13]. As little as 160 units of LDH per milliliter of blood serum can be

analyzed with an accuracy of better that 3 percent.

The reaction system used was:

Li Lactate + NAD LDH

(I) NADH + Pyruvic Acid.

The rate of production of the fluorescent product

NADH was monitored.

Some of the results obtained in the assay of LDH in serum using the solid surface method are shown in table 4. An average relative error of 2.3 percent was

obtained.

IV. Assay of a Clinically Important Substrate

High levels of glucose may indicate diabetes.

Guilbault et al. [14] have described a fluorometric

assay system for glucose using glucose oxidase, per- oxidase, and p-hydroxyphenylacetic acid. The rate of production of fluorescence is proportional to the

amount of glucose present.

Glucose . H2 02 CH2 COOH CH2 COOH

CH2COOH o

HO

-H2 02

Nonfluorescent

o

OH \/1 OH

Fluorescent

To develop a reagentless surface method for assay

of glucose, the enzymes glucose oxidase and peroxidase, and the substrate p-hydroxyphenylacetic acid were placed on the surface. The addition of glucose

initiates the reaction; fluorescence is produced and its rate of production will be proportional to the glucose

present in blood [15]. A range of 10 -5 to 10 _1 M glucose can be assayed.

Table 4. Data and results of the assay of serum LDH using the proposed method

ate, AF/min. Concentration u/ ml found Relative error taken (percent)

6.6 160 168 + 4.9

7.3 190 189 -0.3

7.2 200 186 -6.5

10.1 280 276 -1.3

13.2 360 372 + 3.3

13.9 380 394 + 3.4

15.3 440 437 -0.6

16.0 460 459 - .2 17.2 500 496 - .8 18.8 560 546 -2.5

21.4 620 626 + 1.0

Average relative error 2.3

B. Advantages of Solid Surface Method

The advantages of this radically new system for the

clinical laboratory are:

1. The time required for analysis by present

methods is 30 to 60 min to prepare reagents plus 10 to 20 min per assay. If a sample is available at 1 a.m., the technician must proceed through the regular

preparation and analysis routine. The solid surface

method requires 10 bottles of pads, one for each of 10

common tests. The technician would open the bottle marked "LDH test," pick out a pad, put it in the

instrument on the strip holder, add a 10 sample of

blood or urine, and read out the results directly in mg percent urea. Total time is about 2 min.

2. The accuracy of current methods (mostly spectrophotometric) are limited to an error of about 2 to 3

percent over a narrow range (0.15 to 0.85 Abs units).

Because fluorescence methods are based on the pro- duction of a signal over zero signal, the accuracy

of these methods is independent of scale reading, and

have an accuracy of 1 percent over a 3 to 4 fold linear

range of concentrations.

3. Temperature must be controlled in all enzyme

assays based on a kinetic approach. This is expensive

and bothersome. The solid surface fluorescence

method proposed is not temperature dependent, since

the silicone rubber pad used to support the sample is nonheat conductive. Thus, provided the sample of

blood is at the same temperature as used to prepare

the calibration plot (generally room temperature,

25 °C) the temperature of the environment does not

affect the results. For example, in the assay of alkaline

phosphatase, we obtained the same rate at 10, 15, 20,

25, 30, 35, and 40 °C external temperature, provided

201
we added the blood from a sample kept at 25 °C. This

represents a significant improvement.

4. The reagents used in present clinical procedures

are unstable, and new solutions must be prepared daily.

o-Toluidine and peroxidase in glucose assay, or NAD

in LDH assay are examples of this. Yet when the

reagents are placed in solid form on a surface, they can be kept for months with no deterioration. In our ChE, alkaline phosphatase and LDH assays, we found we could use our pads for 90 days, using the same

calibration plot everyday.

5. Many clinical methods are subject to interferences

because of (a) protein absorption in the near UV-visible

or (b) absorbing impurities in the reagents. By using

reagents that fluoresce in the visible, and reagents in a solid form, these two interferences are eliminated.

V. References

[1] Guilbault,G. G., Anal. Chem. 38,527R (1966).

[2] Guilbault, G. G, Anal. Chem. 40,459R (1968).

[3] Guilbault, G. G.,Anal. Chem. 42,334R (1970).

[4] Guilbault, G. G, Enzymatic Methods of Analysis, (Pergamon

Press, 1970).

[5] Bergmeyer, H., Methods of Enzymatic Analysis (Verlag Chemie,

Germany, 1965).

[6] Guilbault, G G., and Zimmerman, R., Anal. Letters 3, 133

(1970). [7] Guilbault, G. G., and Vaughn, A., Anal. Letters 3, 1 (1970).

[8] Guilbault, G. G, and Vaughn, A., Anal. Chem. 43, 721 (1971).

[9] Guilbault, G. G, and Vaughn, A., Anal. Chem. Acta 55, 107

(1971). [10] Guilbault, G. G. and Hieserman, J. Anal. Chem. 41,2006 (1969).

[11] Guilbault, G. G, and Kramer, D. N., AnaL Chem. 36, 2497

(1964).

[12] Guilbault, G. G., and Zimmerman, R., Anal. Chim. Acta, 58,

75 (1972).

[13] Bergmeyer, H., Bernt, E., and Hass, B., in Methods of Enzymatic

Analysis, H. Bergmeyer, Ed, (Academic Press, New York,

1963), p. 736.

[14] Guilbault, G. G, Brignac, P., and Zimmer, M., Anal. Chem.

40, 1256 (1968).

[15] Guilbault, G. G, and Tang, D., unpublished results.

(Paper 76A6-745)

202
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, Md., March 22-24, 1972 (Issued May 1973). Inorganic Ions in Glasses and Polycrystalline Pellets as

Fluorescence Standard Reference Materials*

R. Reisfeld

Department of Inorganic and Analytic Chemistry, Hebrew University of Jerusalem, Jerusalem, Israel

(June 15, 1972)

The absorption and fluorescence of inorganic glasses and polycrystalline disks doped by heavy

metal ions is discussed, and their use as fluorescence standards is evaluated. The advantages of the

glass standards over other media is summarized. The glass standards are divided into two groups (1) glasses doped by trivalent rare earths such as Gd3+

, Tb3+, Eu3+, Sm3+

, and Tm3+ which have narrow band optical spectra as a result of intraconfigurational transitions, and (2) glasses and polycrystalline disks doped by ions such as Tl+

, Pb2 +, Ce3 +

, and Cu+ which have broad spectral bands since the optical spectra originate from interconfigurationally allowed transitions. Optical and physical parameters, including matrix effects, quantum

efficiencies, decay characteristics, Stokes' shifts and spin-orbit versus orbit-lattice interactions due

to the different transitions will be discussed. Group (1) glasses are suitable for use as standards where a narrow well-defined fluore: -nee range

is required, and group (2) glasses are suitable for use as standards whenever a substance with a wide

range of fluorescence is measured. Special emphasis will be placed on energy transfer between donor and acceptor ions. Key words: Fluorescence standards, inorganic ions; glass standards in fluorescence; rare-earth- doped glasses.

Introduction

The use of fluorescence and phosphorescence as experimental tools has increased immensely in the

last decade. The fluorescence technique is used now

as a common tool of research by physical chemists,

physicists, analytical chemists, biochemists and

biophysicists. The kinds of analyses which have been

performed to date using fluorescence have varied,

including such methods as trace metal determination,

analysis for traces of organic materials and particularly for determination of trace constituents of

biological systems. It provides some of the most

sensitive and selective methods of qualitative and

quantitative analysis. (Lower limits of detectability

using fluorescence analysis lie often in subpart per

million (ppm) and part per billion (ppb) range.) Fluo- resence analysis especially, has many practical applications in the field of organic, inorganic, agricultural,

biological and clinical chemistry.

The reader interested in the principles and applications of fluorescence in analysis is referred to two

recently published books [1, 2] 1 on this subject and to a very comprehensive review on measurements of

photoluminescence quantum yield in solutions written

by Demas and Crosby. [3]

* This work was performed under NBS contract G-103.

1 Figures in brackets indicate the literature references at the end of this paper.

The following are a few examples in which fluores- cence is used in health and environmental problems.

Organic Compounds

Many organic compounds fluoresce and/or phosphoresce and these properties have been widely used

for analysis. An understanding of the basic aspects of the luminescence of organic compounds can greatly

enhance its efficiency as an analytical tool. The ability of proteins to luminesce has now been

fairly thoroughly investigated and is mostly due to the presence of the fluorescent aromatic amino acids

tryptophan and tyrosine, [4, 5]. Any biological process can be inhibited, stimulated or altered through photochemical methods by inducing electronic excited

states in proteins and nucleic acids. In the investigation of these excited states by photoabsorption and

luminescence techniques, a great deal of information can be obtained, not only about the number of bio- polymeric molecules in the system, but also about their

environment.

Significant qualitative and quantitative information

concerning molecular structure can be obtained

through fluorescence. When one considers that in- formation of this kind can be obtained rapidly from the

interior of the undamaged, usually functioning cell, one realizes the special importance of spectral lumi203
nescence research techniques in biology. An example

of fluorimetric methods in this area is: the fluorescent

tags for labeling proteins [6] and amino acids [7-10]

which have proven useful in immunochemistry,

virology, bacteriology, parasitology, and mycology.

Agriculture

Determining factors in the degree of pest control

by chemicals are (1) efficiency of application, (2) the loss by spray particle drift to immediate areas not intended to be treated, and (3) the effective life of the

chemical on plants, animals and in soils after appli- cation. In this respect, fluorescence has made an

outstanding contribution in pesticide analysis. The

excitation and emission spectra, decay times, analytical curves, and limits of detection, have been obtained for many pesticides in media such as milk, animal feeds,

fruits and vegetables, tissues, etc. [11]. Fluorescent

tracer studies of spray and dust deposits on plants and

soils have been reported [12, 13].

Biomedical Research

In this area, fluorescence spectroscopy has been used

largely as an analytical tool for the assay of many materials of biological origin, [14] such as the examination

of carcinogenic materials in the environment, [15] in metabolic studies of aromatic hydrocarbon carcinogens

[16] and the determination of drugs, enzymes and

proteins.

Of great interest are the experimental observations

of the luminescence of cells and tissues in such pathological states as carcinogenesis, inflammation, psychic

diseases and muscle atrophy due to denervation.

Investigators have sought a correlation between

luminescence intensity and the pathological process. However, more information about the structural chemical organization of the protoplasm during the development of the pathological process will be obtained if the comparison involves, not merely the intensity of the fluorescence of various cells, but the magnitude of the changes in fluorescence intensity due to several

factors. The use of luminescence to a successfully

selected set of experimental treatments will prove to be a useful technique in the field of molecular and cell biology and primarily in the field of practical medicine

[17].The mechanism of cancer induction by aromatic

hydrocarbons is of particular interest and it is possible

that the initial interaction is a complex formation be- tween the hydrocarbon and a vital cell constituent,

such as the nucleic acids which may be determined by

changes in fluorescence spectra and parameters

[18—20]. Various aromatic hydrocarbons may fluoresce

at widely differing frequencies, a fact which is of considerable analytical importance. A history of fluores- cense work with carcinogenic hydrocarbons was

included in a lecture by Cook in 1950 [21]. Presently,

fluoroscence analysis is used in many studies to deter- mine qualitatively and quantitatively the presence of

polynuclear hydrocarbons in the environment, usually

after separation by thin-layer chromatography [22]. A method for binding dyes to nucleic acids is widely

used in staining biological tissues for fluorescence

microscopic examination and is used as a screening

procedure for cancerous tissue [23]. Fluorometric techniques have made great inroads

in the clinical laboratory as the basis of sensitive

analytical methods [24, 25].

A. General

In all research and measurements in which fluores- cence is involved, it is of utmost importance to measure

the true wavelength (energy) of excitation and emission, and the fluorescence efficiency. To determine

fluorescence efficiency directly, it is necessary to measure the rate of emission at all wavelengths and in

all directions. In practice, this is a difficult measure- ment to perform with accuracy. In view of the difficul- ties associated with absolute methods, and the ease

with which the fluorescence efficiency may be deter- mined by reference to a standard, a search for a suit- able standard is of great importance in spectrofluorimetry. The requirements for a good reference compound

to be used as a standard are stringent. It should have a

well-defined fluorescence and excitation spectrum, a

relatively high quantum efficiency, it should be of the same geometry as the samples to be measured and

should be stable. There should be little or no overlap

between the absorption and the emission spectrum.

The reference compound used in any instance should

have its absorption and fluorescence in the same general region as the compound being studied. This last restriction is needed because most spectrofluorimeters

record spectra which are not corrected for instrumental

parameters.

Fluorescence measurements are mainly made

on liquids in aqueous or organic solvents, using glass or quartz cuvettes, and in the solid phase with single

crystals, in pellets [26] and in solvent glasses [27]. It would therefore be desirable that the standard refer- ence material also be made of the same or similar

material having a similar refractive index, thus avoiding the refractive index correction (discussed later).

It is also essential that the standard have a small absorption, since a higher absorption introduces an

error in the efficiency determination. This arises from

the following consideration: The optically dilute measurement rests on Beer's Law IoB — /o(l — 10~ AL

) where B is the fraction of light absorbed by the sample,

Io (quanta/sec • cm2

) is the irradiance of incident

light, A is the absorbance/cm, and L(cm) is the path

length [3]. By expanding this expression in a power

series we obtain B= 1 - [ 1 -2.303 AL] + [2.303 AL] 2/2 +

— 2.303 AL. For solutions that are optically

dilute, (absorbance less than 0.05) we can truncate the

series and the following approximation is true: B —

2.303 AL. Thus at low solute concentrations, the

fluorescence intensity is proportional to concentration.

With these criteria in mind we have attempted to prepare glass and pellet standards. The results of 204
our work are described in this paper. For the latest references to liquid standards the reader is referred

to reference [3], for a sodium salicylate standard to reference [28], and for quantum yield determinations

of powder phosphors to reference [29]. In this paper the physical parameters of doped

glasses and alkali halides suggested as standards will be described. In order that such materials be used as standards, and the use be at optimal conditions for a fluorescence measurement, the following knowledge

is needed:

1. Absorption and/or excitation spectrum.

2. Fluorescence spectrum.

3. Oscillator strength or molar absorptivity at the

excitation wavelength.

4. Quantum efficiency for different excitation wavelengths.

5. Fluorescence output as a function of dopant

concentration.

6. Lifetimes of fluorescence.

7. Quenching of fluorescence by additional processes

such as energy transfer of radiation-induced color

centers.

8. Stability.

The principles upon which the determination of

quantum efficiencies is based will be discussed.

Quantum yields of glasses and pellets prepared in

this laboratory, for excitation to various electronic

levels and the oscillator strengths of absorption to these levels will also be presented. The concentration

dependence of quantum efficiency will be shown.

A short theory of the fluorescence of the material

is also outlined for each group of proposed standards

in order to simplify the choice of proper materials.

B. Determination of Quantum Yields

The luminescence quantum yield of a compound

is defined as the fraction of excited molecules that luminesce after direct excitation by a light source. The quantum yield, 17, which is actually the ratio of probabilities of radiative transitions, 1A'- to the sum of the probabilities of radiative and nonradiative

transitions, Af, is defined as:

number of photons emitted

number of photons absorbed

14

£4+24r

(i)

1. Comparative Method

Here, the fluorescence of an unknown is compared

with the fluorescence of a compound whose fluores- cence yield is well known. For dilute solutions, the

following equation has been used [30]:

nsFsA u

(2)

where tj u and rj s are quantum yields of unknown and

standard respectively, n the index of refraction, A the

absorbance at the wavelength of absorption and F the

integrated fluorescence output in quanta/unit wavelength. This equation is valid for equal numbers of photons incident on u and 5. More often the following

equation is used:

r)u = r) s Fu AJ(ks ) nl

FsA„I(Ku ),nf

(3)

In this equation, F is the integrated area under the

corrected emission spectrum, and /(A) is the relative irradiance of the exciting light at wavelength A.. A is the absorbance.

n n

2 Equations (2) and (3) differ in the ratio — and — As

ns n\ we see by comparison of the two formulae, refraction

corrections can be substantial, modifying quantum

yield results by factors of 2 or more. Therefore, one

should be especially careful when applying the refractive index correction. A comprehensive discussion on

this correction can be found in reference [3, p. 1018].

As mentioned in the introduction, when the fluores- cence of the unknown is compared with a standard of the same refractive index and geometry, the refractive

index correction is avoided.

2. Determination of Quantum Yields from Lifetime

Measurements

An alternative method is the determination of

quantum yields by measurements of the lifetime of

fluorescence and the natural lifetime of the state. Here we use the parameters appearing in eq (1) 24 1 Tnat 24+24T

__

\ji 7"exp ^ T na, (4)

The quantum yield would equal unity if there were

no nonradiative transitions. The terms "quantum yield," "luminescence

quantum efficiency," and "fluorescence yield" are used interchangeably.

Formula (1) serves as a basis of determination of

quantum efficiency.

The determination of absolute quantum yields by

this formula is difficult as mentioned in the introduction, hence a relative technique is often employed.

where Texp is the experimentally determined lifetime

from the level in question and 2r~.Jt is the sum of natural lifetimes, corresponding to the sum of the

inverse of the radiative transition rates from this

level. One term, A^, appearing in the sum ~SA r is

equal to and is obtained directly from the ab- sorbance spectra as shown below. The other terms

in the sum are then obtained from the relative areas 205
in the corrected emission spectra. A detailed example

of these calculations is presented below for Eu(III)

doped glasses.

The natural radiative lifetime is related to the

Einstein coefficient of spontaneous emission. Using

this relation, several authors developed expressions

relating the natural lifetime of fluorescence to the

absorption spectrum of the compound [31]. Here, a distinction can be made between two cases:

1. When the absorption and emission of a compound

occur at close wavelengths, that is, zero or very small

Stokes shifts are observed, and the spectrum is narrow

as in the cases of rare earths in various media, one can

use an approximation developed for atomic spectra.

Using this approximation [32], one obtains for the

natural lifetime the following expression, which is valid for a transition between two levels:

T-a\ = 2.880 x lO^nHgJgu) <v*>Se(v)dv (5) where n is the index of refraction, < v

2 > the average

squared wave number for the absorption maximum,

gu and gi the degeneracies in the upper and lower

states, respectively, and e(^) the molar absorptivity

as a function of wave number.

In the case where there is a considerable Stokes

shift between the absorption and emission as a

result of strong vibronic coupling, for example in transition metal ions, mercury-like ions and organic

compounds, a modified equation can be used for

Tnat [33], namely

t~\ = 2.880 x 10-V< vr^r1 — f e{v)dv

nat "/ lav

- m gu J v W

The quantity <vj3 >l~* is given by

\F(v) av <vj3 >ral = f (7)

I F (v)v~3 dv

where F(v) is the fluorescence output in photons as a function of wave number and v is the wave number

ofF(v).

3. Doped Glasses

The materials which we suggest as versatile standards for spectrofluorimetry and which were studied

in this laboratory will be now described. They are divided in the text into four major groups: I. The

rare-earth-doped glasses. II. Thallium-doped alkali halides. III. Glasses doped with thallium. IV. Glasses

doped with Ce3+

, Pb2+

, and Cu+.

I. The Rare-Earth-Doped Glasses

The characteristic spectra of the trivalent rare earths are attributable to the presence of deep lying

4/ shells in the ions. The electrons of these shells are screened by the outer shell electrons, and as a result

they give rise to a number of discrete energy levels. The presence of the surrounding lattice has little effect on the position of these levels; therefore, a close resemblance between the energy level diagram

of the free ion and that of the incorporated ion exists

(fig- 1).

. Considerable attention has been paid in the past

few years to the study of both the absorption and

emission spectra of the rare earths [34, 35]. Trivalent rare-earth ions incorporated in inorganic

solids exhibit luminescence owning to the intraconfigurational transitions of the 4/ electron shell. The

optical excitation of this luminescence can be governed

by one of three possible mechanisms:

(a) excitation in the narrow 4/ levels of the rare- earth ion;

(b) excitation in the broad levels due to 4/-5J*

transitions, or charge transfer processes;

(c) absorption of the host followed by energy transfer from the host to the rare earth activator. While the first mechanism is operative in all the rare earths showing fluorescence, the second is well known

mainly in europium and terbium. In the case of Eu3+

, this band is due to a charge transfer process between

the Eu3+ ion and oxygen center. In the case of Tb3+

, however, the band is due to a 4/-5/ transition in the

Tb3+ ion. (Samarium also was found in some cases to exhibit a charge transfer type of excitation at wavelengths shorter than 220 nm.)

The narrow band emission of the trivalent rare earths in various media is always due to an inner /

shell electronic transition. Intra- /-orbital transitions are parity forbidden in the free ion. In a solid, glass or

liquid in the absence of a center of symmetry, the

parity selection rule may be relaxed by the mixing of the 4/ configuration with appropriate levels of configuration of opposite symmetry using odd terms in the

static or dynamic crystal field expansion. There will also be an additional interaction owing to the influence

of the crystal field of the host which produces broadening and splitting of the levels. As in the transition metal

ions, which have been treated extensively by the ligand

field theory, an increase in separation of a field-split

degenerate level indicates an increase in the ligand

field seen by the rare earth ion. The crystal field splittings in the rare earths are, however, much smaller

than the transition ion splittings, and are not more than

a few hundred wave numbers. For transitions between

two nondegenerate levels, increasing the rare-earth- ligand interaction displaces both levels toward lower

energies. The displacement of the upper, less shielded,

level is usually greater, and thus the stronger perturbation causes shifts to longer wavelengths.

The electrostatic perturbation of a rare-earth ion

should be directly related to its binding energy in the

host medium. As a first approximation, this energy

may be derived from a hard-sphere ionic model by the

Born equation:

^ ZiZj b

206
421-

2fr s

/2

3 H4 "l 9/2

5 I46H

5/z

7 F0

8 S V66 Hl5/2 5

I B

4

I

l5/z

3 H6 V7/2 Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb

FIGURE 1. Energy level diagram of the rare-earth ions after G. H. Dieke, in Spectra and Energy Levels

of Rare Earth Ions, H. M. Crosswhite and H. Crosswhite, Eds. (John Wiley & Sons, New York,

N.Y., 1968), p. 142.

207
£" = coulombic potential of the active ion; Z,-= charge on the active ion; Zj= charge on the matrix ions: 6 = repulsion coefficient: r = distance between active

ion and nearest anion; rc = 4 to 10.

It was shown in a series of papers by Reisfeld et al. [36-40], that the behavior of rare earths in glasses is similar to that of rare earths in inorganic crystals of low

symmetry except for inhomogeneous broadening of the

spectra due to a multiplicity of rare-earth sites in glasses. According to our theory [39] a rare-earth

ion occupies a center of a distorted cube comprised

of four tetrahedra of borate, phosphate or silicate. Two oxygens belonging to such a tetrahedron pro- duce an edge of the cube (fig. 2). The coordination

O © oxygen

rare earth

boron or phosphorus

FIGURE 2. Proposed rare-earth site model.

of the rare earth in such an arrangement is with eight

oxygens. The observed broadening in the spectrum

can occur due to the existence of nonuniform, nonidentical ligand fields caused by slightly different values of rare-earth oxygen distances. An average

rare-earth oxygen distance was calculated by assuming

a rare-earth sits in a center of a regular cube. In a cube made of borate tetrahedra, in which the boronoxygen distance is 1.48 A and the oxygen-oxygen

distance 2.4 A, the rare-earth oxygen distance is 2.1 A. In a cube made of phosphate tetrahedra in which

the phosphorus-oxygen distance is 1.57 A and the

oxygen-oxygen distance is 2.56 A, the rare-earth oxygen distance is 2.22 A. It should be realized that in

reality the symmetry is lower than cubic, and for the

particular cases studied it was found to be of Cs symmetry Eu 3+ in phosphate glass, [40] and C2 for Tm3+ in phosphate and borate glasses [39]. As a

result of this lower symmetry, forced electric dipole

transitions become possible and absorption and

fluorescence is observed.

The fluorescence can, in some cases, be quenched

due to transfer of electronic excitation energy to the

vibrations of the surrounding medium. Such inter- actions reduce the fluorescence quantum yield. In general the larger the energy difference between the

two electronic states, the smaller the nonradiative

transition probability between them [41]. For rare- earths, the energy difference between the electronic

levels below which considerable quenching occurs is about four vibrational quanta (phonons). The nonradiative decay of the higher excited levels in the

glasses is generally assisted by the phonons of the

glass. The particular vibrational frequencies which

are responsible for the quenching are the following:

bond

silicate, Si — 0 metaphosphate, P —

0

borate, B-0

stretching

frequency

1010-1115 1140-1300

1310-1380

bond length

A

1.62 1.57

.39 (trigonal) 48 (tetrahedral)

from which we conclude that in borate glasses, quenching will occur between levels that are separated by

about 5000 cm -1

, while in silicate by about 4000 cm -1

. We shall now show how this general theory works

for specific cases.

A. Europium

The observed optical absorption and emission spectra

of Eu 3+ in glasses are characteristic of transitions

between the lower energy levels of the 4/B configuration of this ion. The oscillator strengths of the characteristic absorption bands in a phosphate glass to- gether with the assignments of the transitions are given

in table 1. The oscillator strength was calculated from the well- known equation

/=4.32X 10-9 e(v)dv i

(9)

where e is the molar absorptivity at the energy v (cm -1

). The values of / presented in table 1 were corrected

for population distribution of total concentration in To, Ti, and T2. Using a Boltzmann distribution

correction, the relative concentration for the three

levels at room temperature were Co = 0.792, C\ = 0.200 and C2 = 0.008, respectively.

208
Table 1. Oscillator strengths and quantum yields of fluorescence of Eu3+ in phosphate glass

Percent quantum

Transition Wave number Wavelength Oscillator yield of Do

assignment cm -1 nm strength X 107 fluorescence

7 F2 ^ 5 D0 16319 612.8 1.544

7 F,-* 5 D„ 16771 592.2 0.351

7 Fn-^ 5 D„ 17256 579.6 .006 0.95

7F,-+ 5D, 18700 534.7 .505

5 F0 ^ 5 D, 18993 526.4 .146 .91

7 F„-» 5 D2 21493 465.3 1.248 .88

7 F„^ 5 D 3 24009 416.5 .547 .87

7 F0 ^ 5 L 6 25380 394.0 8.981 .87

7 F, -^ 5 G5 , 5 G6 26041 384.0 6.019

7 F,,-» 5 G3 26507 377.2 2.726

7 F, -^ 5 LS 27285 366.5 .391

7 F«-* 5 D4 27567 362.7 1.926

7 F,-+ 5 H3 30464 328.2 1.242

7 F,-^ 5 H5 31152 321.0 3.001

7 F0 ^ 5 H6 31397 318.5 6.776

Fluorescence was observed from the 5 Do level and much weaker fluorescence (by a factor of three

orders of magnitude) from the 5 Di level. The corrected

5D0 fluorescence to the T ground state multiplet for Eu3+ in phosphate glass is given in figure 3. The cor- rected excitation spectrum of the fluorescence is also presented in this figure. The assignments, and

relative intensities of the transitions are presented

in table 2. (The transitions from the 5 Do excited level

to the 7 F5 and 7 Fe ground states are very weak, less than 1 percent of the total intensity, and are not

included.) The 5 D0 —

7Fi emission band for Eu3+ are magnetic dipole-allowed transitions; the other

fluorescence bands arise from forced electric-dipole

transitions. The large ratio of the fluorescence intensities for the 5 D0 —*

7 F2 and 5 D0 -*

7 Fi transitions

implies a low symmetry field at the europium sites. Quantum efficiencies of fluorescence from the 5 Do

level of Eu3+ in phosphate [41] and silicate glasses

[42] were obtained by two independent methods,

(a) lifetime measurements, and (b) comparison with a

liquid standard.

a. Lifetime measurements. The quantum yield

determination based on lifetime measurements is calculated from eq (4). The measured lifetime of the

5 D0 excited state in silicate glass was 2.69±0.03 ms

Figure 3. Corrected excitation (broken line) and emission (full line) spectrum of the

5 D0 fluorescence of Eu3+ in phosphate glass. 209

502-591 O - 73 - 15
and in phosphate glass was 2.83 ±0.03 ms. The natural radiative lifetime was calculated using

eq (5). In this equation, e was corrected for the rela- tive population of the 7Fo level, which was taken as 0.771 in silicate and 0.792 in phosphate. Using the

calculated value for /4( 7Fo^ 5 D0 ) the ratio of the intensities of fluorescence and the fact that A(7 F0 -^ 5 Do)=/f( 5 D„-» 7F0 ), the sum of radiative

transition probabilities is expressed by: ^ 1 ^ A ,,5n S( 5 Dq^ 7Fi) 2-= X^(5Do- 7Fo) [i+

S(5Do ^ 7Fo)

5( 5 Do-^ 7 F2 ) S( 5 Do^ 7F3 ) S( 5 Do^ 7F4 )

"

S

(

5 D0 -> 7 F0 ) S ( 5 D0 -* 7F0 ) S

(

5 D0 -> 7F0 ) . (10)

where S is the area of the relevant emission bands

obtained from the corrected emission spectrum.

Table 2. Emission wavelength and relative areas of the 5 D0 —*

7 F, transitions for Eu3+ in phosphate glass

Transition Relative assignments Wavelength fluorescence

(areas)

5 D„^ 7 F„ 578.5 0.036

5 D0 -> 7F, 591.9 1.000

5 D0 ^ 7 F2 612.1 3.000

5 D0 — 7 F3 654.7 0.219

5 D0 ^ 7 F4 692.3 1.026

the sample used for absorbance measurements to that used for fluorescence measurements: k is the excitation wavelength.

The quantum efficiency determined by direct excitation to the 5 Do level obtained by this method was

0.95 ±0.03 for the phosphate glass, [41] and 0.93 ±0.05

for the silicate glass [42]. The very similar values for the quantum yield ob- tained by the two independent methods suggests use of the phosphate glass as a reliable fluorescence

standard at wavelengths similar to those for the

fluorescence of europium, as summarized in table 2. To obtain this quantum efficiency, it is necessary to excite the 5 D0 level of europium from the 7Fo level at 580 nm, or from the 7 Fi level at 591 nm. Excitation to higher levels results in a decrease of fluorescence

yield from the 5 Do level, as additional paths for radia

tive and nonradiative transitions become possible.

The quantum yields of fluorescence upon excitation

to selected levels in phosphate glass are presented in table 1 [41]. The values of quantum efficiencies for fluorescence monitored by 5 Do emissions when excited

to higher levels, as listed in the table, were obtained

from the ratio of the area under the excitation spectrum

to the ratio of the area under the absorption band for a given level using eq (12). No corrections were made for the change of refractive index with wavelength.

Q.Y." = ^T^Q-Y- a d2)

r JA a A. e x 6

For Eu3+ in phosphate glass, the quantum yield

obtained by this method was 0.95 [41] and for Eu3+ in silicate, 0.90 [42]. Europium-doped glasses do not show concentration quenching of the 5 Do fluorescence

[43].b. Determination of Quantum Yield by Comparison Method. Here, the comparison was made

with a solution of Eu(N03 ) 3 with a known quantum

efficiency of 4 percent [4l]. The fluorescence spectra were taken on a Turner Model 210 spectrofluorimeter2 which gives corrected emission spectra in quanta/unit

bandwidth, and excitation spectra corrected to constant energy [44]. The glasses were of the same geometry

(standard cuvette form) as the cells in which the

fluorescence of the solution of europium nitrate was

measured.

Equation (11), which is a modification of eq (3) for use with a corrected spectrofluorimeter [45], was used:

r)u = ris [(FuA sdu k ex. sn%)l(FgAuKx uds " raf)] (11)

where all the symbols have the same meaning as in eq (3); subscripts u and s refer to the unknown and

standard, respectively; d is the dilution factor denoting

the ratio of the fluorescence species concentration in

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the best

available for that purpose.

Q.Y.ft Quantum efficiency of 5 D0 fluorescence by

excitation to a given level Q.Y.a Quantum efficiency of 5 D0 fluorescence by

excitation to the 5 Do level Fb peak area of a given level in excitation

spectrum

Ai, peak area of a given level in the absorption

spectrum

Fa peak area of 5 Do in excitation spectrum

Aa peak area of 5D0 is absorption spectrum

\exQ wavelength of 5 D0 absorption (581 nm)

kex b wavelength of absorption for given transition.

Therefore, europium glass is proposed as a standard

where the emission of the unknown is close to the

emission of Eu3+ (table 2) and the excitation wavelength is also near one of the excitation wavelengths

of europium (table 1). The fluorescence of europium shows a linear de- pendence on concentration up to 7 weight percent

and no quenching is observed. Therefore, the concentration of the standard used will be governed by the

desired wavelength of excitation because of different

oscillator strengths. For excitation at 580 or 591 nm,

1-2 weight percent of Eu3+ in glass is suggested. The

europium glass standard is suitable for compounds

having narrow fluorescence in the wavelength range

of 600-700 nm. 210
B. Gadolinium

Gadolinium has a comparatively simple optical

absorption and emission spectrum because the tri- valent gadolinium has a half-filled configuration

corresponding to 7-4/ electrons. The ground state is

8S7/2, which is not split in a crystal field. Its first excited state lies around 3200 cm-1

, which corre- sponds to the 312 nm band. Gadolinium is the only

rare-earth ion whose spectrum consists only of bands

lying in the UV region. The emission spectrum of gadolinium in borate glass [46] has a major band

with maximum at 312 nm due to the 3 P7/2_8S7/2 transi- tion (fig 4), which is essentially unchanged in other

300 350

A nm

Figure 4. Corrected excitation (broken line) and emission (full line) spectrum of the 6 P7/2 fluorescence of Gd3+ in borate glass.

matrices [47]. The oscillator strength of this transition

and of transitions to higher levels from 6 P7/ 2 are tabulated in table 3. The quantum yield of the 312 nm

fluorescence was computed by taking a ratio of the

measured lifetime of 6P?/2 fluorescence, which is 4.10 ±0.01 ms, to the natural lifetime of this transition

obtained by use of formula (5). The calculated lifetime was found to be 4.10 ms. The quantum yield thus

obtained equals 1 with an estimated error of ± 1 per- cent. The quantum yield of 6P7/2 fluorescence at excitation to higher levels was obtained relative to the

quantum yield of excitation at 6 Py/2 using eq (12). The quantum efficiencies of the 312 nm fluorescence

of gadolinium in borate glass are tabulated in table 3. Borate glass containing gadolinium can be used as a standard for fluorescent materials having emissions

around 312 nm and excitation peaks around 250 and

273 nm. Because of the narrowness of the band, it is suitable for materials with narrow fluorescence.

Gadolinium in borate glass shows a linear fluorescence

output as a function of concentration from the ppm

range [48] to 7 weight percent (the highest concentration introduced).

C. Terbium

The electronic configuration of Tb3+ is 4Z8

. The:

absorption spectrum of Tb3+ arises from the transi- tions from the ground Te state to higher levels of the

/ orbital giving rise to sharp bands of absorption due

to f-f* transitions. In glasses, an additional broad band

occurs in the UV due to the 4/8 —* 4f7 5d transition. The oscillator strengths of Tb3+ together with the

quantum efficiencies of the 5D4 level are presented in table 4. Terbium fluorescence is observed in the glass from

the 5 D4 and 5 D3 excited states. The 5 D3 fluorescence,

around 378-369 nm, is quenched at higher concentrations of Tb3+ due to Tb3

"

1" — Tb3+ interactions. The

5 D4 fluorescence consists of seven bands due to the

transitions: 5 D4 —>

7F6, 5,4, 3, 2, i,o- The wavelengths for maxima of these bands together with their relative peak

areas are presented in table 5. The corrected emission

Table 3. Oscillator strengths and quantum yields of fluorescence of Gd3+ in borate glass

Transition Wavelength Wave number Oscillator Quantum

assignment nm cm-1 strength X 106 yield of 6 P7/ 2 fluorescence

*S 7/2 ^ 6P7/2 313.0 31949 0.176 1

6P5/2 307.0 32573 .074

6

l7/2 280.25 35682 .117

<

6

I 17,2 277.0 36101 .781

(

6

I../2

6

Il3/2 274.3 36456 1.679 0.870

6

I.5/2

6D 9/2 253.6 39432 0.406 .735

6D7/2 247.6 40388 .385 .735

6D3,2 6D5/2 245.5 40733 .099 .590 211
Table 4. Oscillator strengths and quantum yields of 5 D4 fluorescence of Tb3+ in borate glass

Transition assignmentWavelength nm

Wavenumber cm-1 Oscillator strength X 107 Quantum

yield We -» 5 D4 485.5 20597 0.633 1

5 D3

] 5 G6 | 378.9 26392 3.812 0.60

5 L10; 368.7 26954 7.204

5 G5

5 G2 | 352.5 28369 7.622 .62

5 G4 i 5 U

5 G3

5 L8

5 L7 i 340.6 29360 5.764 .256

5 L6

5 Di 325.7 30 /U3 U.4U/

5 Do .197

5 H7 I 318.5 31397 4.405

303.4 32960 5.249 .06

5 H4V

5 H5j

5 H3 283.6 35261 1.787

5 Ie

5 F4 4/8 ^4/"2 5<f 220.5 45351 385X104

Table 5. Emission wavelength and relative areas of the 5 D4 level of Tb3+ in borate glass

Transition assignments

*D4

- *D4

- *D4

-

5 D4

-

7 F6

7 F5

7F4

7 F3

Wavelength nm

486

541 585 624

Relative fluorescence

(areas)

0.040

.150

.033

.019

spectrum from the 5 D4 level and the corrected excitation spectrum are presented in figure 5. No quenching of the 5D4 terbium fluorescence was

c' served in our work with borate glasses from the ppm

range to 6 weight percent [49]. The lifetime of the 5 D4 fluorescence of terbium

follows a simple exponential with an e

_1 decay time

of 2.80 ± 0.05 ms. The quantum yields of 5 D4 fluorescence on excitation to this level were obtained using the measured

08

07

t Q5

inz

Ld

I-Z 04 >

H 03 <

) -

i 1 1

n

! jl

! S

! In

i

i

j

j

i

- / i A -\i

l

i , «/

1

1

1 1 i

1 1 { ll t* 1

- i v^s —i UVu.1 1 i

X nm Figure 5. Corrected excitation (broken line) and emission (full line) spectrum of the

5 D4 fluorescence of Tb3+ in borate glass. /=the intensity decreased by a factor of 30. 212
lifetime and absorption and emission spectra in a

similar way as the quantum yields for europium, using

eqs (4), (5), and (10). The radiative probability for the 7F6 —*

5 D4 transi- tion was calculated using eq (5) and equals 57.6 s _1

.

The total radiative transition probability, from the

5 D4 level, \A[ [5 D4 ] is given by:

%Af [5D4 ]=£/f [»D4 ->T,].

i = 0 From the emission spectrum we found that ^Af[W4 ]=6A pD4 -^T6 ] ;

thus, the total radiative transition rate from the 5 D4 level is 345 s _1

, which is equivalent to

1 Tnat

2.9 ms. Hence the quantum yield

Q.Y. =

H=0.97

with an estimated error of about 3 percent.

The quantum yields of the 5 D4 level on excitation to higher levels were obtained as they were for Eu3+ and Gd3+

; i.e., by taking ratios of the areas under the

excitation spectrum to the areas in the absorption

spectra, and multiplying by the ratio of excitation

wavelength at 480 to the wavelength for the given

excitation. Equation (12) was used for this calculation.

Terbium can be used as a standard for compounds

which fluoresce at approximately 480, 543, 587, and

624 nm.

D. Samarium

The electronic configuration of Sm3+ is 4/5

. Its absorption spectrum arises from transitions from the

ground 6 H5/ 2 level to higher levels of the 4/5 configurations. The oscillator strengths of Sm3+ in phosphate

glass are presented in table 6. The excitation and emission spectra of Sm3+ in phosphate glass are presented in figure 6. The visible fluorescence of Sm3+ arises from the transitions

from the 4 Gs/2 level to the ground 6 H multiplet. The

relative fluorescence intensities to each level in the

multiplet are presented in table 7. The fluorescence

arising from these transitions is linear with concentration up to about 0.6 weight percent. At higher

concentrations an ion-ion energy transfer occurs. This

multipolar interaction can be represented systematically (see ref. [35], p. 97):

[4G5/2^ 6F9/2]^ [6H5/2 -> 6F9/2 ] or

(Sm3 + 4 G5 / 2 >*+ (Sm3 +

6 H5/2 )-* 2(Sm3 +6 F9/2 )*.

Therefore, if samarium glass is to be used as a fluorescence standard for the wavelength range pre- sented in table 7, a glass containing less than 0.6 weight percent of this element should be used.

The quantum yield of the 4 G5/ 2 fluorescence of

samarium in phosphate and borate glasses was calculated by use of eqs (4), (5), and (10). The radiative transi- tion probability from 6 H5/2 to 4 Gs/2 in phosphate

glass as obtained from the absorption spectrum,

using eq (5), is 41.3 s _1. The total radiative transition

probability from the 4 G5/2 level, 5^4f[

4 G5/ 2 ], is given by

Table 6. Oscillator strengths and quantum yields of 4G5/ 2 fluorescence of Sm3+ in phosphate glass

Energy level Wave number cm -1 Wavelength nm

Oscillator strength X 106 Quantum

yields

4G5/2

6F3/2 4G7/2 4I9/2 4M,5/2 , 4I,„2 4Il3/2 4F5/2 4M, 7/2 , 4G9/2, 4I, 5/ 2

( 6P, 4P)5/2

4 L17/2 , 6F7/2 , 6P3/2 4Kh/2 , 4 M2i , 2 , 4L15/ 2 4G„/2 , 4D1/2 , 6P7/2 4Li7/ 2 , 4Ki3/ 2 , 6F9/ 2 4D3/2 , ( 4D,«P) 5/2 4H7/2 4K15, 2 , 4H9/2 , 4D7, 2

( 4K, 4L) l7/2 , 4L19/2 4Hn/2

17467-

18779-

19800-

20181- 21267- 21978-

22321- 23255-

24242-

18348

19047 20100

-21621 21978 22346

23255 24630

25316

25252-26109

26109-27173 27173-28089

27972-28368

28490-29450

545.0-572.5

525.0-532.5 497.5-505.0

462.5-496.0

455.0-470.0 447.5-455.0

430.0-448.0

406.0-430.0

395.0-412.5

383.0-396.0

368.0^-383.0 356.0-368.0

352.5-357.0

340.0-351.0

0.0476

.0115

.0183 1.5370

0.1388

.0273 .2169 1.0039 4.0749

0.2298

1.2246 1.2230

0.0264

.7457

0.950

.653

.491

.213 .264

.249

.224

.226

.155

213
Figure 6. Corrected excitation (broken line) and emission (full line) of the 4 G5/ 2 fluorescence of Sm3+ in phosphate glass.

Table 7. Emission wavelength and relative areas of fluorescence from the 4 G5/ 2 level of Sm 3+ in phosphate glass

Transition Wavelength Relative assignments nm areas ^5/2-*^ 562 1.000 597 5.128

4G5,2 -» 6H,/2 645 4.856

4G5/2-* 6H„/2 707 0.574

4G5/2 -» 6H13,2* 8301 0.487

4G5/2^ 6H15/2 * 900 1

* These two transitions were obtained by calculation assuming

quantum yield of level 4 G5/ 2 to be 0.95.

15/2

i = 5/2 The measured lifetime of the fluorescence from the

4G5/2 level is 1.91 ms. By assuming the quantum

efficiency of fluorescence from this level to be 0.95, we can calculate the transition rate 4 G5/ 2 —

6Hi3,

6Hi5, which is out of the range of measurements, by

use of the formula 3

Q.Y. = re

(H +

^(6H5/ 2^ 4 G5/2 ) x

6H9/2 ) A ("FU/2 -» 4 G5/2 ) A («H5/2 -> 4 G5/2 )

+' A(4 G5/2" sH11/2 ) ^(6H5/2 -

0.95 = 0.00191[41.3(1.000+ 5.128+ 4.856-

4 +

G5/2 )

0.5744 + x)].

• 6 H,3/! ) +

A ( < GW->«H„<Z )

The numbers in parenthesis are the relative fluores- cence intensities of the transitions 4 G5/ 2 —>

6H, as given

in table 7. The relative areas of 4G5/ 2 —»

6Hi3/ 2 , 6Hi5/ 2 thus calculated amount to about 4 percent of the total emission of the 4 G5/2 , hence the assumption that the

quantum yield equal 0.95 is justified. The estimated

error in Q.Y. here is about 3 percent.

The quantum yields of the 4G5/ 2 fluorescence on

excitation to higher levels were calculated by eq

(12) using as an internal standard the fluorescence

yield on direct excitation to the 4G5/ 2 level. They were

independently calculated by use of Eu3+ glass as standard. The values of quantum yield are presented in table 6. Glasses containing small concentrations of Sm3+ can

be used as fluorescence standards for compounds

which fluoresce at around 560, 602, and 640 nm. By examining the quantum efficiencies of Eu3+

, Gd3+

, Tb3+

, and Sm+ in glasses, we see that the

fluorescence quantum yields from the lowest excited

fluorescing levels approach unity. This is because

the differences between these levels and the ground

levels (or multiplets) are more than five phonons.

In such cases, the electronic transitions are almost

totally radiative.

E. Thulium

The electronic configuration of trivalent thulium is 4/12

. It possesses more than one strongly emitting level and selective excitation of the various fluorescent levels have been achieved in both phosphate and borate

glasses [50]. Because part of the fluorescence occurs

in the infrared, we were unable to obtain the relative quantum efficiency of fluorescence of thulium for various levels. It was found, however, that an estimation of an "apparent quantum efficiency" of these

glasses could be of practical use if the materials are used as fluorescence standards. Therefore, these

parameters will be presented here. 214
Oscillator strengths of Tm3+ in phosphate and obtained by Gaussian analysis of the absorption

borate glasses are presented in table 8. They were spectrum.

Table 8. Oscillator strengths, /, of T,„ +3 in borate and phosphate glasses Borate Phosphate

Transition

assijinments Wave number Wavelength fx 106 Wave number Wavelength /x 10" cm -1 nm cm-1 nm

3 H6 -* 3 H4 5847 to 6134 1630.0 to 1710.0 3.94 5714 1750.0

3 H6 -» 3 H5 8257 1211.0 2.09 8250 1212.0 1.82

3 H6 -* 3 F4 12626 to 12903 792.0 to 775.0 4.39 12626 792.0 3.00

3 H6 -» 3 F3 14619 684.0 4.02 14550 687.2 2.53

3 H6 -* 3 F2 15100 662.0 0.70 15060 664.0 0.22

3 Hf -> 'G4 21231 to 21505 471.0 to 465.0 2.19 21052 475.0 1.58 21505 465.0

3 H6 -* >D2 27855 to 28050 356.5 to 355.0 3.75 27855 359.0 3.06

3 H6 -» »I. 34246 to 34662 292.0 to 288.5' 33057 302.5' 34364 291.0

4.14 31.87

3 H6 -» 3 Po 35082 285.0 35057 286.0

3 H6 -* 3 P, 36363 275.0, 36166 276.5-

3 H6 -> 3 P2 37950 to 38387 262.5 to 260.5 6.13 38095 262.5 14.46 The absorption occurs from the ground 3H« state. wavelengths are presented in table 9. The emission spectra of Tm3+ at various excitation The excitation spectrum of the 453 nm fluorescence

Table 9. Emission spectrum of Tm3+ at various excitation wavelengths

Emission

Excitation (nm) Assigned transition borate phos phate

X(nm) R.A.* A(nm) R.A.*

»&« > 3 H4 651.50 1.000

468.0('G 4 ) 'G 4 3 F 2

> 3 H5

3 H«

752.75 665.500.114

.083

3 F3

> 3 H6 690.00 .708 'D 2

> 3 H4 456.00 1.000 453.00 1.000 'D 2 3 H5 517.00 0.008 513.00 0.011

358.0('D 2 ) 'D 2 'G4

> 3 F4

> 3 Htl

665.00

478.00

.127

.118

663.50

478.00

.034

.063 'G4

* 3 H4 652.50 .180 651.50 .074

3 F2 » 3 H6 665.50 .014 665.50 .009

'Is » 3 H4 355.00 .446 350.00 1.000

'Is > 3 H 5 385.00 .125 383.00 0.094

3 Po

'Is

» 3 F4 |

> 3 F4 J 465.00 .155 463.00 .200

'Is > 3 F, 500.00 .110 498.00 .060

3 Po

'In

3P>

'Is

3 F3 | 530.00 .061 521.50 .094 288.0( 3P„)

> 3 F2 J

» 'G4 |

> »Gj

705.00 .754 701.00 1.000

'D 2 » 3H5 367.50 .275 365.00 0.133 D2 > 3 H4 456.00 1.000 453.00 .455 'D2 » 3 H5 517.00 0.067 513.00 .044 'D 2 » 3 F4 'G4 >

3 Hh 480.00 .190 478.00 .105 'G 4 ^ 3 H4 3 F2

> 3 Hb

3 F3

> 3 Hb

* Relative areas; for each excitation the areas are given relatively to the strongest emission band which is taken as unity. 215
(

1 D2 —* 3H4) and the emission spectrum with excitation

at 262 nm ( 3H6 —>

3Po) are presented in figure 7. The "apparent quantum yield," (A.Q.Y.), of 450-

478 nm fluorescence of Tm3+ was calculated by the

comparative method, using Tb3+ as a standard.

The A.Q.Y. of Tm3+ is defined by eq [12a]:

A.Q.Y. Tm3+ J F[('G4 -^ 3H6 )+('D2 -* 3 H4 )] [A ( 7Fn 5D4 ) ]

1 [f( 5D4 ^X 7 F() A(3Hb -^ l D2 )

(Q.Y.Tb3+

) (12a)

in which F[(1 G4 -»> 3 H6 ) + (>D 2 -> 3 H4 )] is the sum of

the corresponding fluorescence intensities (expressed

as areas under the emission spectrum) of Tm3+

; A ( 3Hk—» is the absorbance for the 358 nm band

of Tm3+

; F(5 D4 —>S 7 F,) is the sum of fluorescence

intensities of the 5D4 fluorescence of Tb; A (T« —

5D4 ) is the absorbance for the Tb3+ band at 358 nm; and

Q.Y. Tb3+ is the quantum efficiency of Tb3+ taken as

0.63. The apparent quantum efficiency for 10 weight

percent of Tm3+

, calculated in this way, is 0.037 in borate and 0.043 in phosphate glasses.

Here, not all the transitions of Tm3+ were taken into account, but since the relative transitions to each level are constant for a given host, Tm3+ doped glasses can

be used as standards for fluorescence around 450—480

nm and excitation around 358 nm. This small value of

A.Q.Y. is not surprising since it is known [50] that at one weight percent of Tm3+ the concentration quenching of fluorescence is about 50 percent due to multipolar transitions between neighboring thulium ions.

It should also be remembered that the fluorescence

around 455^475 nm is one of seven transitions from

! D2 and one of the six J G4 transitions. Because of its low A.Q.Y. value, use of Tm3+ in the region of 450-480 nm fluorescence has no obvious

advantage over Tb3+ as a standard. However, it can

be used as an internal standard for Tm3+. This fluo- rescence can be used to determine the apparent yield

at 355 nm (

T

6 -> 3H4 ) , 385 nm ( »I 6 -> 3 H5 ) and 367 nm

(*D 2 —>

3H6), upon excitation to 288 nm (3P0) or 265 nm

( 3 P2 ), which is of practical value as a standard in the

UV region.

II. Thallium-Doped Alkali Halides and Glasses

While the rare-earth ions have characteristic sharp

fluorescence bands largely independent of the host,

due to /-/* transitions, mercury-like ions in general and

thallium in particular have much broader fluorescence,

with the fluorescence maxima strongly dependent on

the composition of the host matrix. The quantum

efficiency also is dependent on the host as described

below. Monovalent thallium is a well-known fluores- cence emitter in the UV region and was used in our

studies in various matrices as a possible standard for UV fluorescence.

The absorption spectrum of thallium consists of

three main bands: A, B, and C. A review of the absorption and luminescence of this element can be found

in references [51] and [52]. The absorption band A,

which is discussed here, is assigned to the spinforbidden transition 1 A1 „^ 3 Ti« (Tj— 3r4°), equivalent to 1 So —*

3 P 1 in the free thallous ion (see fig. 8), which is triply degenerate. It can be split in a cubic

field by a dynamic Jahn-Teller effect [54], or the

degeneracy can be lifted when the symmetry is decreased in glasses [55] from cubic to tetragonal or

lower. The oscillator strengths, /, for the maxima of the A bands are presented in table 10. It was shown

by Scott and Hu [53] that when the chloride ligand is replaced by an oxygen ligand in aqueous solution, the

maxima of the band is shifted towards a shorter wave07 -

06 -

X nm FIGURE 7. Corrected excitation spectrum of the 'D2 fluorescence (broken line —II decreased by a factor of 2) and emission spectrum (full line) excited at 3 ?2 band of thulium in phosphate glass. 216
FREE THALLOUS ION CUBIC FIELD EXCITED STATES OF IONIC LATTICE

Figure 8. The energy of thallous ion in a cubic field.

length. The fluorescence occurs at a somewhat longer

wavelength due to the Stokes' shift. The relation between absorption and fluorescence can be most easily explained by the use of the configurational coordinate diagram, given in figure 9 [56]. The potential energy of the luminescent center in a

crystal lattice is plotted here as a function of the

configurational coordinate r. The quantity r represents

a mean distance between the luminescence center and

the surrounding ligands. The horizontal lines represent

the vibrational states of the metal-oxygen bond vibrations. At a temperature of absolute zero the luminescent

center will occupy the lowest vibrational level of the

ground state. The ions surrounding the central ion

vibrate about their equilibrium positions situated at a distance ro from the central ion. At higher temperaFigure 9. Configurational coordinate diagram of a luminescent

center. The potential energy E of the center in the lattice is plotted as a function of the configurational coordinate r for the ground state and the first excited state. In practice, r is identified with the dis- tance between the central cation and the surrounding anions. Vibrational states are represented schematically by horizontal

lines in the parabolae. The excitation and emission transitions correspond to vertical transitions between the two curves. Since Ar 5^ 0, the emission shows a Stokes' Shift (wavelength of the emission is longer than that of the excitation). Since the center can

be in various vibrational states both at the ground level and at the excited level, and since ArT^O, the transitions occur in a broad band of energies (schematically indicated by vertical dashed

lines). Nonradiative return from the excited state to the ground

state is possible via the point of intersection, S, of the two curves. This requires an activation energy A£, which can be supplied at higher temperatures (thermal quenching of the emission). In the region where the two parabolae intersect, the curve is marked by

dashes since the situation is actually more complicated than is indicated here. This is due to interaction between the ground and

the excited state at the situation of the intersection point. The

present treatment is not invalidated by this.

Table 10. Quantum effici

Matrix Max. absorption Max. excitation nm nm

Borax 230 248 Phosphate 212.5 230 KC1 247 247 KBr 261 261

(QE) of thallium in 4 matrices

Max. emission Oscillator QE Stokes' shift nm strength,/[35] (cm-1

)

325 0.072 0.19 12709 300 .080 .23 13726 300 .074 .5 7153 320 .097 .44 7064 217
tures, higher vibrational levels may be occupied. In

figure 9, the horizontal lines represent vibrational

states. Due to the absorption of radiation of the appropriate wavelength (in this case UV radiation) the center is raised to an excited state. Since the equilibrium distance re of the excited state will not in general

be equal to that of the ground state, and since the center may be at different vibrational levels, this transition will correspond to a fairly broad absorption

band. The fact that the optical absorption corresponds

to a vertical transition in figure 9 is attributable to the

rapid nature of electronic transitions as compared to vibrational transitions which involve the heavier

nuclei (Franck-Condon effect). Once in an excited state, the system will relax towards the equilibrium state (of the excited level) by

dissipation of heat. From this state or nearby levels the

system returns to the ground state by emitting radiation. The emission, therefore, also consists of a broad

band. Line emission is found only in the exceptional case where the configurational-coordinate curves are

identical in shape and have the same equilibrium dis- tance, as in the case of the rare-earth ions. Because

of the heat dissipation, the emission always lies at a lower energy than the absorption. This displacement of emission with respect to absorption is known as the

Stokes' shift. With the aid of the simple model in figure 9 (the MottSeitz model) one can explain:

(a) the broad-band character of the emission and

absorption of many centers;

(b) the shift of the emission to lower wavelengths;

and,

(c) the temperature dependence of the emission.

If the equilibrium configuration of the excited state

lies outside the curve of the ground state (fig. 10), then, after excitation, the intersection point of both

curves is reached before the above-mentioned equilibrium configuration is reached, and the system relaxes

nonradiatively to the ground state. No emission is then possible. The radiationless return to the ground

state is temperature-independent. This is the model

which Seitz proposed to explain the absence of

luminescence in certain cases [57]. In other words,

the condition for the absence of luminescence is a

large difference between the equilibrium distance of the excited state and that of the ground state. Dexter, Klick, and Russell later proposed a different model [58] which shows that even under less rigorous

circumstances than in figure 10, nonradiative transi- tions to the ground state may occur (fig. 11). The

characteristic feature of the situation in figure 11 is that the intersection point S of the two curves is lower

than the level reached after excitation. When, after excitation, the system now relaxes while vibrating,

to the. equilibrium position of the excited state, the

intersection point of the two curves is passed. Here too, a temperature-independent, radiationless return to the

ground state can take place.

We learn from these models that the difference, Ar,

between the equilibrium configuration of the excited

i FIGURE 10. The Seitz model for explaining the absence of lumines- cence; see figure 9. The minimum of the curve for the excited

state lies outside the curve for the ground state; luminescence is then not possible.

Figure 11. The Dexter-Klick-Russell model for explaining a low luminescence efficiency or the absence of luminescence. The

i intersection point S of the two curves lies below the vibrational

level reached after excitation. The nonradiative return to the ground state requires no activation energy (as it did in fig. 9).

218
state and that of the ground state must be small if luminescence is to occur. Because of the well-defined and broad fluorescence

of thallium in the UV region, we have determined the

optimum conditions for preparation of thallium doped

alkali halide pellets in phosphate and borate glasses.

Also, the dependence of fluorescence efficiency on

concentration of Tl + was measured. It is shown that the thallium doped pellets and glasses can be used as fluorescence standards. The detailed results of the

measurements are given.

A. Thallium Doped Potassium Chloride Disks

Polycrystalline pellets were used [52] as mentioned

in the introduction. Such pellets are used in biomedical

research for fluorescence measurements.

The optical absorption and fluorescence of alkali halides containing thallium has been widely studied;

references can be found in a review paper by Teegarden

[51]. The absorption band A with maximum around 247 nm

in potassium chloride, arises from the spin-forbidden

transition 'A,, -» 3 Ti« (corresponding to 'So ~* 3 Pi in a free thallous ion), and is accompanied by the fluores- cence with band maximum at 300 nm. Because of large half-widths, the absorption and fluorescence are

suitable for quantum-yield studies.

1. Preparation of Disks

Baker Analyzed KC1 was ground for 1 h with an

automatic mortar and pestle and dried for 48 h at 120 °C. T1C1 (BDH Analytical Grade) was then added

and the mixture was homogenized in an electric

vibrator. In order to obtain low concentrations, the

stock mixture was diluted twice with dry KC1 powder.

Prior to diffusion, the homogenized mixture was ground

in an agate mortar with a small amount of triple- distilled water, and redried at 120 °C for 24 h. As known from previous work [59, 60], only thallium

that has undergone diffusion has an absorption band

at 247 nm. The completeness of diffusion was checked

by heating the mixtures at different temperatures for various periods and measuring the absorbance. The

diffusion was then carried out by heating the mixtures

at 400 °C for 4 h. Under these conditions, all of the

thallium introduced diffuses into the KC1 powder.

The absorption measurements were made on a Cary 14 spectrophotometer, with a KC1 disk as blank.

The effect of the emission on the absorbance readings was negligible, mainly because the solid angle subtended by the photomultiplier was small (0.005 rad), as compared to the full sphere (4ir rad) over which

emission occurs.

2. Preparation of Quinine Sulfate

Fisher Reagent Grade quinine sulfate was recrystallized from water five times and dried in a vacuum

oven at 60 °C. Stock solutions were prepared by

dissolving the purified quinine sulfate in 0.1 A^H2 S04 ,

analytical grade. The water used for the stock solutions was distilled, passed through an ion-exchange column,

and then doubly distilled from a quartz still. Special

low-fluorescence 1 mm quartz cells were used to hold

the standard solutions.

3. Emission Measurements

The corrected emission was measured on a Turner

210 spectrofluorimeter in which the regular 1-cm cell was replaced by a special sample holder adapted to hold disks and cells of 1 mm thickness. The sample

holder could be rotated to permit continuous variation

of the angle of incidence of the exciting light. Because

of the optical arrangement of the spectrofluorimeter,

it was necessary to place a black-velvet screen between

the sample and the collecting mirror to reduce reflec- tions. All measurements were made at constant

excitation energy and at room temperature.

Some representative A-band absorption curves are shown in figure 12. A plot of thallium concentration versus absorbance is linear, as can be seen in figure 13. The oscillator strength, /, was calculated from the

generalized formula of Smakula, (13), given by Patek

(ref. [35, p. 72] ). /=0- 82xl017T^T272 ^

a(£) ^ (13)

/FY1 where n is the refractive index, I —

J the concentration of the ions per cm3

, a the absorbance in cm -1 and

E the energy in electron volts. The refractive index of

a KC1 crystal was taken from Landodt-Bornstein [61]

as 1.59. Multiplying 1.59 by the ratio of the pressed

disk density (1.690 glcm3

) to the crystal density (1.984

glcm3

) yields an index of refraction value for the

pressed disk of 1.57. The oscillator strength was 0.074 ± 0.002. Figure 14

shows the corrected emission spectra. The linear

concentration dependence of the emission-peak height

is shown in figure 15. The quantum yield was determined by use of eq (11)

using quinine sulfate as a standard [52]. The quantum yield of 0.508 for quinine sulfate in 0.1 N H2 S04 used as a standard was taken from the

data of Melhuish at A exc = 366 nm. Fletcher showed

that the quantum yield was constant to within 5 percent

over the wavelength range 240-290 nm, excluding a minimum at 270 nm. The refractive index ns was taken

as a weighted mean of the refractive indices of the

standard solution and the quartz windows of the cell. The excitation wavelength for the unknown and the

standard was 247.5 nm. The emission-peak height was

critically dependent on the angle of incidence of the

exciting light, so that the small but inevitable difference

of the angles of the sample and reference could cause

a considerable error. On the other hand, the shape

factor of the emission (ratio of emission-peak area to peak height) was independent of the angle. To elimi- nate the error, we found the maximum peak height by

219
0.7

FIGURE 12. A band absorption spectra. I. KC1:T1+ 20 ppm; II. KBr : T1+ , 20 ppm;

III. Phosphate :T1+, 50 ppm; IV. Borax :T1+, 100 ppm.

£ 04 -

Figure 13.

40 50 60 70 CONCENTRATION ppm

Absorbance versus concentration of Tl+

II. KBr; III. Phosphate; IV. Borax.

in I. KC1;

varying the angle of incidence and multiplied it by the

shape factor to obtain the emission-peak area, A. The quantum yield obtained in this way was 0.50

± 0.03. Pressed polycrystalline KC1:T1 disks differ only

slightly in their density from KC1:T1 crystals, and they

have the same optical characteristics. All of the

thallium diffuses into the polycrystalline disks, and they can therefore be prepared to the predetermined uni- form concentration. The linear concentration de- pendence of the absorbance (fig. 13) gives a check on

the reproducibility of the samples, and the agreement

of the oscillator strength of the disks with that of the

crystals permits their use as a substitute for crystals.

From the linear concentration dependence of the

emission, it is concluded that the quantum yield is constant over the concentration range 2—30 ppm.

Pressed polycrystalline KC1 disks doped by diffusion

with T1C1 can therefore be used as UV luminescence

standards at room temperature. The recommended

05

260 280 300 320 340 360 380 400 420 440 460 480 500 X nm

Figure 14. Corrected emission spectra I. KC1:T1+; II. KBr:Tl+; III. Phosphate : T1+ ; IV. Borax:TI 220
40 50 60 70 CONCENTRATION ppm

Figure 15. The dependence of fluorescence output on Tl+ concentration: I. KC1; II. KBr; III. Phosphate; IV. Borax.

concentration is 2 ppm, corresponding to 0.624 X

10 -6 mol/mol.

B. Thallium-Doped Potassium Bromide Disks

The absorption spectrum of KBr : Tl in polycrystalline disks and single crystals is well known [60, 62]

and is presented in figure 12. The concentration

dependence of the absorption maximum is presented

in figure 13. The corrected emission spectrum of the

A band is presented in figure 14. The dependence of

fluorescence intensity on concentration is presented

in figure 15. The oscillator strength was calculated by use

of Smakula's equation (13), and was found to be

0.097 ±0.003. The quantum yield of the A band in KBr : Tl was calculated using KC1:T1 as a standard.

The emission and excitation spectra of the disks were measured on a spectrofluorimeter built in this laboratory, the description of which may be found in reference [36]. The emission spectra were corrected

using standard compounds and their reported spectra.

To correct for the difference of excitation energy in the standard and the unknown, a correction factor was

introduced into eq (11). The refined formula for the use with this spectrofluorimeter is of the form:

FuAs ds ksK F

B

s Fl

FsAudu\un

2

s f%Fl

(14)

in which the parameters have the same meaning as

in (11). f| is the relative fluorescence output at peak

maximum in the excitation spectrum of the standard

measured with our apparatus; Fs 1S tne relative fluorescence output at peak maximum in the excitation

spectrum of the standard measured on the Turner

Model 210 spectrofluorimeter; Fu is the output at

peak maximum of the unknown in the excitation

spectrum of the standard measured in our apparatus;

F% is the output at peak maximum of the unknown

measured in the excitation spectrum of the standard on the Turner Model 210 spectrofluorimeter. The

density of the pressed KBr disk is 2.67 g/cm3

. We used as a standard a KC1:T1 disk with a quantum

yield of 0.50. The quantum yield obtained for the

KBr : Tl disk is 0.44 !± 0.03. All data concerning

KBr : Tl disks are presented in table 10. Because of the linear dependence of absorption and

emission on the T1+ concentration, it is concluded that the quantum yield is independent of the concentration

in the 0.5-25 ppm range. Pressed polycrystalline

KBnTl and KC1:T1 disks can easily be prepared to pre- determined concentrations for use as UV fluorescence

standards at room temperature. The recommended Tl

concentration is 2 ppm, corresponding to 0.837 X

10- 6 mol/mol KBnTl and 0.624 X 10~ 6 mol/mol in KC1.

ME. Thallium-Doped Glasses

To compare our results in glasses with the cor- responding data of alkali halides, we have prepared

and measured glasses doped with thallium.

Glasses were prepared from sodium phosphate

monobasic and from borax. Glasses, 1 mm thick and

12 mm in diameter were obtained by molding the melt on a tile. The absorption spectra were recorded on a Gary 14

spectrophotometer using undoped glasses or disks as blanks, and are presented in figure 12. The concentration dependence of the absorption is presented in figure 13.

1 . Excitation and Emission Spectra

Excitation and emission spectra were taken on a spectrofluorimeter built in this laboratory [36]. The

spectra were corrected with respect to light characteristics and spectral distribution of the photomultiplier

by comparison with a similar spectrum taken on the

Turner Model 210 spectrofluorimeter using standard

compounds (quinine sulfate, fluorescein, etc.)

.

The corrected emission spectra are presented in figure 14. The concentration dependence of the

emission is presented in figure 15. The quantum

efficiencies were obtained by comparison of the glasses

with alkali halide-thallium-doped disks using eq (14), the results of which are presented in table 10. When a pellet or a glass containing thallium is excited through absorption into the A band at room

temperature, the emission is composed of two time

components, one with a short lifetime of about 25

ns and one with a long component of 400 ns. We

have proposed an explanation for the two decay constants [55]. The proposed explanation is as follows: The absorption is from l S 0 ground state to 3 Pi excited state. The

emission occurs also between these two levels and is followed by a Stokes' shift (see table 10). It is an

allowed emission with a short lifetime. However, part

221
of the population of the 3 Pi level is rapidly depleted

by a radiationless process to the metastable 3 Po state (see fig. 16). This assumption is proven by an experiFlGURE 16. Configurational coordinate diagram of Tl+ in a tetragonal symmetry.

ment of Tomura [63], who had observed a decay of

2 — 1 ms at 20 K due to this transition. At an elevated

temperature, the vibronic modes promote tunnelling

from 3 Po back to 3 Pi. This process occurs with an

activation energy of 0.06 eV in KC1. The next process

is the delayed emission from 3 Pi. The rate of this process is the rate of tunnelling which controls the

population of 3 Pi. Since both the instant and delayed

emission occurs from the 3 Pi state, the wavelength of emission for both decays is the same. Also, the delayed

fluorescence is not polarized [63] because the memory

of polarization is lost during tunnelling. To determine

the quantum yield from the lifetime measurement,

additional calculations have to be made because of

this delayed fluorescence.

In this mechanism, for all four cases described here,

the broadening of the emission in KBr:Tl is due to emission from Px , Py and another emission from Pz , and not from P0 . Splitting of the various P orbitals by

the Jahn-Teller effect is stronger the heavier the anion

of the host lattice. The shift of the absorption band to longer wavelength in the order Tl + -phosphate, Tl+ -borax, Tl + - KC1 and Tl+ KBr (also T1+-K1) indicates that the

ligand field acting on T1+ increases in the same order. The stronger the ligand field the smaller the difference

between the excited and ground state and hence the

absorption occurs at longer wavelengths. This also indicates that, in the case of borax, the electrons

of the oxygen participating in the thallium-oxygen

bond are shifted more to the Tl (stronger polarization of the bond), which is due to stronger electron affinity of phosphorus (0.78 eV) than of boron (0.33 eV).

IV. Glasses Doped by Pb2 +

, Ce3 +, and Cu+

In a search for standards to cover the fluorescence

range from the UV region to the red region, it was

found that in addition to thallium doped glasses [64], glasses containing Ce3+ [65], Pb2+ [66], and Cu+ [67] are suitable for standards, considering the positions of

their emission wavelengths and half bandwidths.

The corrected emission spectra of various glasses

containing these ions are presented in figure 17. The

250 300 350 400

X nm

450 500 550

FIGURE 17. Matrix effects on position of emission band of T1+, Ce3+

, Pb2 *, and Cu+.

I. Phosphate: TP; II. Borax:Tl+; III. Phosphate : Ce3+

; IV. Borax:Ce3+

; V. Borax:Pb'2+

; VI. Germanate: Pb2 +

: VII Phosphate: Cu+. 222
absorption spectra of Ce3+ and Cu+ in phosphate

glass and Pb2+ in borax glass are presented in figure

18. The absorption is linear with concentration in the

range measured (1-1000 ppm). The dependence of

fluorescence on concentration is linear as shown in figure 19, and this range of concentration can be used

for preparation of the standards. The maximum

emission and excitation wave lengths of the glasses

and the half bandwidths of the emission are presented

in table 11. The determinations of quantum yields of these

glasses are now undergoing research. For these

determinations, doped potassium chloride disks are used as standard references [64-67]. For the quantum

200 220 240 260

X

Figure 18. Absorption spectrum of I. Ce3 * 100 ppm; III. Cu + in

280 300 320 340 nm

in phosphate, 400 ppm; II. Pb-' + in borax, phosphate, 50 ppm.

t i

1

1 r

0 200 400 600 800 1000 1200

ION CONCENTRATION ppm

Figure 19. The dependence of fluorescence intensity on concentration I. T1+ in phosphate; II. Ce3+ in borax; III. Pb2+ in borax.

yield determination of Tl in phosphate and borate

glasses, and Ce3+ in phosphate glasses, quinine sulfate was used as a relative standard [68].

Conclusions

Rare-earth-doped glasses are suggested as relative standards for use with compounds having narrow band

excitation and emission of fluorescence. Thalliumdoped alkali halides are standards suitable for determination of substances in alkali halide pellets, while

thallium doped glasses can be used as standards for glassy substances or liquids in quartz cells. Other

varieties of glass standards and pellets are now under

investigation. Energy transfer studies which are of

great importance in understanding quantum efficiencies

will be summarized in a separate review.

The assistance of E. Greenberg in preparation of

this paper is gratefully acknowledged. I wish also to ex- press my thanks to Y. Eckstein and L. Boehm for fruit- ful discussions, and E. Hoffman for providing relevant

literature on fluorescence in biomedical systems. I especially wish to express my gratitude to 0. Menis who

suggested the topic for this paper. 223
Table 11. Emission and excitation wavelength dependence on matrix for lead, cerium, thallium and copper

Wavelength, nm Emission

Ion Matrix half-bandwidths, nm

Excitation Emission

T1+ KC1 247.5 300 40

T1+ Phosphate 219.0 302 70

T1+ Borate 233.0 325 90 Ce3+ Phosphate 297.0 334 55 Ce3+ Borate 3 310.0 364 80 Ce3+ Borax" 311.0 365 65 Ce3 + Silicate 317.0 380 85 Pb2 + Phosphate 241.5 309 70

Pb- + Borate 3 261.0 424 140 Pb2+ Borax" 241.0 425 165 Pb2+ Germanate 295.0 440 125 Cu + Phosphate 270.0 445 120

a Mixture of boric acid and boric oxide. "Sodium borate only.

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Proc. 8th Rare Earth Conference 2, 743 (1970).

[38] Reisfeld, R., and Hormadaly, J., Proc. 9th Rare Earth Conference, 1, 123 (1971).

[39] Reisfeld, R. and Eckstein, Y., Solid State Chem. 5, 174 (1972).

[40] Reisfeld, R., Velapoldi, R. A., Boehm, L., and Ish-Shalom, M.,

J. Phys. Chem. 75,3981 (1971).

[41] Reisfeld, R., Velapoldi, R. A., and Boehm, L., J. Phys. Chem.

76, 1293 (1972).

[42] Velapoldi, R. A., Reisfeld, R., and Boehm., L., Proc. 9th Rare

Earth Res. Conf. 2,488(1971).

[43] Reisfeld, R., and Greenberg, E., Anal. Chem. 47, 155 (1969).

[44] Turner, G. K., Science 146, 183 (1964).

[45] Fletcher, A. N., J. Mol. Spectry. 23,221 (1967).

[46] Reisfeld, R., Greenberg, E., Velapoldi, R. A., and Barnett, B.,

J. Chem. Phys. 56, (4) 1698 (1972).

[47] Reisfeld, R., Glasner, A., and Mustaki, A., Proc. Int. Colloqu.

Optic and Spectroscopic Phenomena in Ionic Crystals, Bucharest, 1968; Mustachi, A.. Reisfeld, R.. and Glasner, A.,

Israel J. Chem. 7,627(1970).

[48] Reisfeld, R.. and Biron, E., Talanta 17, 105 (1970).

[49] Reisfeld, R., Arye, Z. Gur., and Greenberg, E., Anal. Chim. Acta 50, 249 (1970).

[50] Reisfeld, R., and Eckstein, Y., Anal. Chim. Acta 56, 461 (1971).

[51] Teegarden, T., in Luminescence of Inorganic Solids, Goldberg, P.. Ed. (Academic Press, New York, N.Y., 1966),

p. 83.

[52] Reisfeld, R., Honigbaum, A., and Velapoldi, R. A., J. Opt.

Soc. Am. 61, 1422 (1971).

[53] Scott, A. B.. and Hu, K. H., J. Chem. Phys. 23, 1830 (1955).

[54] Sturge,M. D.. Solid State Physics 20,91 (1967).

[55] Reisfeld, R.. Greenberg, E.. Deinum, T.. Werkhoven, C. J.. and van Voorst, J. D. W., to be published.

[56] Blasse G, and Bril, A., Philips Technical Review 31, 304 (1970).

[57] Seitz, F., Trans. Faraday Soc. 35, 74 (1939).

[58] Dexter, D. L, Klick, C. C, and Russell, G. A., Phys. Rev. 100, 603 (1955). 224
[59] Glasner A., and Reisfeld, R., J. Phys. Chem. Solids 18, 345

(1961).

[60] Glasner, A., Rejoan, A., and Reisfeld, R., J. Phys. Chem.

Solids 19,331 (1961).

[61] Landolt-Bornstein, Zahlenwerte und Funktionen, Bd. 2, Teil 8, Optische Konstanten (Springer, Berlin, 1962).

[62] Reisfeld, R., and Honigbaum, A., 1972, unpublished results.

[63] Tomura, M., and Nishimura, H., J. Phys. Soc. Japan, 21,

2081 (1966).

[64] Reisfeld, R., and Morag, S., J. Appl. Phys. Letters 21,57 (1972).

[65] Reisfeld, R., Hormodaly, J., and Barnett, B., Chem. Phys.

Letters.

[66] Reisfeld, R., and Lieblich, N., unpublished results.

[67] Reisfeld, R., and Kosloff, R., unpublished results.

[68] Reisfeld, R., Velapoldi, R. A., and Greenberg, E., unpublished

results. (Paper 76A6-746)

502-591 O - 73 - 16

225

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburg, AAd., March 22-24, 1972 (Issued May 1973). Development of a New Fluorescent Reagent and Its Application to the Automated Assay of Amino Acids and

Peptides at the Picomole Level *

Sidney Udenfriend

Roche Institute of Molecular Biology, Nutley, New Jersey 071 10

(June 15, 1972)

Methods for the assay of amino acids and peptides are most important in elucidating the struc- ture of proteins and peptides. In many important areas of research such as in endocrinology, neurobiology, and genetics, methods are needed with sensitivity higher than is available with the widely used colorimetric ninhydrin procedure. A short while ago, we noted that all primary amines react with ninhydrin and phenylacetaldehyde to give a ternary product which is highly fluorescent. The

chemistry of that reaction has now been elucidated and the conditions have been modified and im- proved so that essentially quantitative yields of fluorescent products are formed with all primary

amines. The reaction has been automated and is being used as the detecting system for chromatography of amino acids, peptides and amines in the 10 to 100 picomole range. Problems concerning

the fluorescence instrumentation and the isolation and chromatography of these compounds in the picomole range will be discussed.

Key words: Fluorometry, amino acids; fluorometry, peptides; picomole fluorometry.

Collaborative studies by biologists and chemists at Nutley have led to the development of a new fluoro- metric reagent for amino acids and peptides and its application to their automated assay at the picomole

level. The origin of these studies, as well as the

chemistry and the analytical applications, are pre- sented in this report.

A number of years ago McCaman and Robins

[l]1 reported a highly specific and sensitive fluoro- metric method for blood phenylalanine assay. The

procedure is now generally used for the diagnosis

of phenylketonuria. In this method, fluorescence de- velops when phenylalanine is treated with ninhydrin

and a peptide. The other amino acids which are nor- mally found in plasma and protein hydrolyzates give no fluorescence. On the other hand, almost any peptide can be used in the phenylalanine assay. These very unusual requirements led us to investi- gate the reaction with the possibility of reversing the

conditions to assay peptides fluorometrically by adding

ninhydrin plus phenylalanine. When this was tried it worked. Other than phenylalanine, the only a-amino

acid which yielded fluorescence (although of far less intensity) was a-aminobutyric acid. This amino

acid is not normally present in tissues. On further investigation, it was found that phenylalanine itself was not the active agent, but was first

* This article is based on studies carried out in collaboration with Wallace Dairman,

Keijiro Samejima, Manfred Weigele, Willy Leimgruber, Stanley Stein, and Peter Bohlen.

1 Figures in brackets indicate the literature references at the end of this paper.

oxidized and decarboxylated by ninhydrin to phenylacetaldehyde. The latter, in the presence of additional

ninhydrin, combined with primary aliphatic amines to yield highly fluorescent derivatives. It was found that several other aldehydes yielded fluorophors with

ninhydrin and amines but that the most intense fluores- cense by far was obtained with phenylacetaldehyde.

The products obtained on reacting amines, amino

acids and peptides with ninhydrin and phenylacetaldehyde all exhibit the same excitation and emission

spectra (fig. 1). Excitation maxima are at 275 nm and

390 nm with emission at 480 nm. To investigate the mechanism of the reaction further,

the fluorophor formed on reacting phenylacetaldehyde

and ninhydrin with a simple amine, such as ethyl- amine, was prepared. The purified product could not be

obtained in crystalline form, but was shown to be in equilibrium with a nonfluorescent congener which was

apparently a lactone. The latter was , crystallized and

yielded a molecular ion with the highest mass at 305. This and other findings suggested that the product was

the result of a ternary reaction involving one molecule

each of amine, phenylacetaldehyde and ninhydrin.

The purified fluorophor obtained with ethylamine was used as a standard to show that under the conditions of the reaction the yield of fluorescent product was about 50 percent. There was considerable varia- tion in the fluorescence obtained with various other

amino acids. Much of this was due to differences in quantum yield as well as to completeness of reaction.

227
EXCITATION EMISSION

200 250 300 350 400 450 500 550 600

WAVELENGTH (nm) FIGURE 1. Spectral properties of fluorophor obtained on reacting an amino acid with ninhydrin and phenylacetaldehyde.

The procedure was automated with the use of appropriate pumps and a flow cell cuvette in an Aminco

Microfluorometer.2 Application of this automated

procedure was made for amino acid analysis. The

sensitivity at this stage with most amino acids was

found to be below the nanomole level. This compares to a limit of 10 nanomoles for most commercial amino

acid analyzers which utilize the colorimetric ninhydrin

procedure.

Details of the procedure utilizing ninhydrin and

phenylacetaldehyde have been published [2, 3].

It possesses the sensitivity and many other qualities

which could have made it a useful analytical procedure.

However, elucidation of the mechanism of the reaction

[4] led to the development of a new reagent and made

it obsolete. Weigele and Leimgruber and their colleagues at Hoffmann-La Roche in Nutley had been collaborating

with us in our studies. They concentrated on the

mechanism of the ninhydrin phenylacetaldehyde reaction utilizing mass spectrometry, x-ray crystallography and other procedures on a number of crystalline

derivatives. The structure of the fluorophor obtained

with ethylamine was proven to be as shown in figure 2. The condensation with ninhydrin and phenylacetaldehyde also yielded two nonfluorescent products which

could be converted to the fluorophor by treatment

with base.

Brilliant deduction led Weigele and Leimgruber3 to synthesize the compound, 4-phenylspiro [furan-2(3H),

H-phthalanJ-S^-dione, RO 20-7234. This compound

was found to react with amines to form the identical

2 In order to adequately describe materials and experimental procedures, it was occasionally necessary to identify commercial products by manufacturer's name or label. In no instances does such identification imply endorsement by the National Bureau of Standards, nor does it imply that the particular product or equipment is necessarily the best

available for that purpose.

3 Weigle, M., and Leimgruber, W., to be published.

0

"MINOR FLUOROPHOR" "MAJOR FLUOROPHOR" "NONFLUORESCENT" R-NH2 — PRIMARY AMINES R=-C„H2„+ 1

. etc. AMINO ACIDS -CH(R')-C02 H

PEPTIDES -CH(R' )-C0NH-CH(R" )-C0 . .

.

FIGURE 2. Formation of fluorophor from ninhydrin, phenylacetaldehyde and ethylamine.

fluorophor obtained with ninhydrin and phenylacetaldehyde (fig. 3). With this new reagent the reaction is a

RO 20- 7234

OHC

"MAJOR FLUOROPHOR"

Figure 3. Comparison of reactions of amines with RO 20- 7234 and with ninhydrin and phenylacetaldehyde.

binary one rather than a ternary one. The resulting

advantages are many-fold. The ternary reaction with

ninhydrin does not go to completion; about 45 percent

with ethylamine and much less for aspartic acid. Yields

with the new reagent are almost quantitative with all amines, amino acids and peptides. The ternary reaction

is run at 65° for 25 min. The binary reaction is instan- taneous at room temperature. In both cases the rea- gents are nonfluorescent. In the ternary reaction,

ammonia does not react at all. With the new reagent,

ammonia reacts to a slight extent (one thousandth as 228
much as a typical amino acid). The conditions of the

binary reaction lead to several times more intense

fluorescence for the same amount of fluorophor.

The fluorophor obtained with peptides (fig. 4) are

5 NANOMOLE/ML OF LEU-ALA

0.5 U.M0LE/ML OF Ro 20-7234 (100 FOLD EXCESS) 25% ACETONITRILE

75% AQUEOUS BUFFEP (pH &,

FIGURE 4. Formation of fluorophor with a peptide and the reagent RO 20-7234. more intensely fluorescent than the fluorophors

formed from the corresponding amino acids. This may

be due to a greater quantum yield. The free carboxyl

group close to the fluorophoric group may quench the

fluorescence. Whatever the reason, this is a decided

advantage for peptide assay. The new reagent is ideally suited to detect amino

acids and peptides in the automated assay of column

effluents. Such assays have now been set up and even

with relatively unsophisticated pumping systems and

fluorescence instrumentation, it has been possible to carry out analyses in the picomole range. Figure 5 shows the chromatographic separation of an amino

acid mixture (neutral and acidic), 250 picomoles of

each. Figure 6 shows the chromatographic separation

of 50 picomoles each of the basic amino acids.4 A number of applications have already been made

which make use of this great sensitivity. It has been

possible to measure the peptides oxytocin and vaso- pressin in an aliquot of an extract of a single rat pituitary (fig. 7). Thus, the method will be of great

SEPARATION OF AMINO ACIDS

ON A BASICS COLUMN

80 -

Column: 7.5 x 0.28 cm g Resin: DURRUM DC-4A 2 Sample: 50 pMOLES OF EACH

ii 60- AMINO ACID O

3

0 10 20 30 40 50 60

TIME (minutes)

Figure 6. Chromatography of basic amino acids in the picomole

range.

importance in the isolation and characterization of

physiologically active peptides and their metabolites.

Another important application has been to the assay of

spermine and spermidine. The sensitivity in this case

is so great and the tissue levels so high that microgram

quantities of brain tissue are sufficient for assay. Most

important, the simplicity of the apparatus required

to operate in the picomole region using fluorescence

should make it possible to devise relatively inexpensive

commercial equipment. It is hoped that commercial

instruments, which make use of this reagent, will soon be available.

CHROMATOGRAPHY OF ACIDIC AND NEUTRAL AMINO ACIDS

RESIN: DURRUM DC-4A COLUMN: ,50*0.28 cm

TEMPERATURE: 60°

_l I I I

1 1 1

30 60 . 90 120 150 180 200

TIME (MINUTES)

Figure 5. Chromatography of neutral and acidic amino acids in the picomole range.

4 Details of these procedures will be published in a subsequent paper.

229
RAT PITUITARY

PROFILE OF 500 - 10,000 M.W. ON CM CELLULOSE 2-17-71

STD: 1/3 OF COLUMN ALIQUOT COLUMN ALIQUOT: EQUIVALENT TO 1/2 OF I RAT PITUITARY

STD START

FIGURE 7. Chromatographic separation of the oxytocin and vasopressin in an extract

of one rat pituitary.

References P] Samejima, K., Dairman, W., Stone, J., and Udenfriend, S., Anal. Biochem. 42, 237 (1971).

[1] McCaman, M. W., and Robins, E., J. Lab. Clin. Med. 59 , 885 W Weigele, M., Blount J F. Tengi J. P Czajkowski, R. C, and

(1962) Leimgruber, W., J. Am. Chem. Soc. 94, 4052 (1972).

[2] Samejima, K., Dairman, W., and Udenfriend, S., Anal. Biochem. 42, 222 (1971). (Paper 76A6-747)

230
NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 378, Accuracy in Spectrophotometry and Luminescence Measurements, Proceedings

of a Conference Held at NBS, Gaithersburq, Md., March 22-24, 1972 (Issued May 1973). Considerations on Organic Compounds in Solution and

Inorganic Ions in Glasses as Fluorescent Standard Reference

Materials

R. A. Velapoldi

Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234

(June 30, 1972)

The use of various organic compounds in solution and inorganic ions in glasses has been investi- gated as possible fluorescence Standard Reference Materials. Emphasis was placed on measuring

physical and chemical parameters such as stability, reproducibilities of absorbance and fluorescence measurements, relative quantum efficiencies as a function of excitation wavelength, etc., for quinine

derivatives and selected organic compounds. A brief discussion is included on the use of rare earth and non-rare earth inorganic ions in glasses as standards.

Key words: Emission spectra; excitation spectra; fluorescence; fluorescence standards; glass standards; quinine derivatives; rare earths; relative quantum efficiencies; solution standards.

I. Introduction

The rapid growth of publications in fluorimetric

research and applications during the last two decades,

and the variability of data and results in those publications, underline the need for standardization of

fluorescence nomenclature and data presentation.

These needs were discussed previously by investigators [1—3]

1

, who collectively presented a proposal for fluorescence standardization [4]. The main objective

of this proposal was to supply a firm basis for reporting

fluorimetric results so that comparisons of data among

laboratories would be meaningful.

There are basically two methods which can be used

for the presentation of data: (a) in absolute radiative or energy units, and (b) by comparing fluorescence

data of the compound under study with data obtained

for an accepted standard. Very few laboratories are equipped to make absolute radiant energy measurements; however, comparative spectra of standards run under the same experimental conditions on the same instrument can be easily done. This latter method, which effectively corrects for instrumentally

dependent perturbations on the true fluorescence

spectra is most widely used and applicable provided

the users know the limitations, not only of their instru- mentation, but also of the standard.

Not included here are fluorimetric measurements

obtained on "uncorrected" instruments which are presented without units of intensity or benefit of

comparative measurements. Many of these types of

published results have led to confusion and are often

of no quantitative value.

Figures in brackets indicate the literature references at the end of this paper.

Fluorescence standards can be used to (a) correct spectra for instrumental parameters such as the

wavelength dependence of exciting lamp intensity,

monochromator transmission and detector response,

(b) determine comparative quantum efficiencies,

(c) compare data among laboratories, (d) determine

periodically "in house" instrumental stabilities,

(e) calibrate fluorescent lifetime determinations, and

(f) calibrate polarization value measurements.

The requirements for a "general" fluorescence

standard are quite specific. It should:

(a) have broad fluorescence spectra,

(b) be easily purifiable,

(c) be stable in solution or as the solid,

(d) have as little overlap as possible between the

excitation (absorbance) and emission spectra,

(e) not be subject to oxygen quenching,

(f ) have a constant quantum efficiency as a function

of exciting wavelength,

(g) have isotropic emission,

(h) have the same emission spectrum shape independent of exciting wavelength,

(i) be soluble in aqueous and organic solvents, and

(j) absorb and emit in the same general regions as the compound under study.

No single compound exhibits all these desirable

characteristics. In fact, the last requirement precludes

the use of a single standard, for although absorbance

may occur over a wide range (e.g. rhodamine B absorbs

from 200 nm to about 560 nm), emission is usually

limited to one small wavelength interval (for rhodamine

B, 550 nm to 650 nm with maximum at 573 nm).

For this reason, in addition to the fact that fluores- 231
cence spectra are composites of true spectra and

instrumental parameters dependent on wavelength, it is necessary to have available a series of fluorescence

standards with emission maxima that covers the wavelength range of current interest. With the wide use of

lasers and interest in radiative measurements in the

near-infrared, this range now extends from 250 nm

to 1100 nm. One of the major objectives of the fluorescence pro- gram at the National Bureau of Standards is the selection, production, and certification of a series of

fluorescence standards (Standard Reference Materials) which can be used for the standardization and

comparison of data and methodologies among laboratories [5, 6]. It is essential that these Standard Refer- ence Materials (SRM's) be certified with absolute

values. Data reported here, however, are in relative units and not absolute since instrumentation for absolute measurements is not available at the present

time. Absolute measurements will be made and

values assigned to the SRM's as soon as instrumentation becomes available. The work at NBS is following two parallel investigative paths: (a) the use of solutions of organic molecules

and metal chelates in various solvents, and (b) in close

collaboration with Reisfeld, The Hebrew University,

Jerusalem, the use of inorganic ions in solid matrices.

Group (b) may be further split into rare earth and nonrare earth inorganic ions. The investigative work on

the inorganic ions has been covered extensively earlier

in this series of papers by Reisfeld [7]. Discussion

here, then, will be restricted primarily to the use of comparative standards in solution and some of the

factors which affect quantum yields and spectra.

Since quinine and its derivatives are probably the

most widely used comparative standards [13], and

since a great deal of controversy exists as to their usefulness as standards, the emphasis of this paper is on the reproducibility, stability, and effects of wavelength excitation and acid concentration on the spectra

and quantum efficiencies of the quinines.

I would like to digress briefly and discuss some of

the equations and assumptions which have to be made

in using comparative techniques and standards. Two

types of solutions may be employed: optically dense

and optically dilute. With optically dense solutions and

front surface excitation and emission, Vavilov's ex- perimental set up with some modifications is generally

used [1, 8-12]. This method is not as widely utilized as the optically dilute method since stray light is high,

compounds with large emission and excitation overlap

give erroneous results, and only compounds with a high

molar absorptivity can be used easily [13]. The optically dilute method is based on the Beer- Lambert Law and several assumptions. Combining

the definition of quantum efficiency [14], eq (1),

l A

where Q — luminescence quantum efficiency

/f = rate of fluorescence emission in quanta/s. Ia— rate of light absorption in quanta/s.

with the Beer-Lambert Law, eq (2),

It

l o

eel (2)

where /0 is the radiative flux of the exciting fight in quanta/s, It is the radiative flux of the transmitted

light in quanta/s.

e is the molar absorptivity in liter, mole -1

, cm -1

, c is the concentration in moles/liter,

/ is the path length of the cell in cm,

and expanding the exponential term in a power series,

the expression for the rate of light emission becomes

[3, 13, 15]:

/F = /„(2.3ec/)

' 2.3ec/ (2.3ec/) 2 Q. (3)

For dilute solutions (i.e. absorbances less than 0.05),

eq (3) may be approximated with only slight error by

eq (4) [15],

If = /o(2.3ec/) Q. (4)

In determining comparative quantum efficiencies, a term for the index of refraction of the solvent is added

[16—18], resulting in the final form of the applicable

equation:

(5)

(1)

where Q is the quantum efficiency,

FA is the integrated fluorescence area (2

quanta/unit bandwidth),

Io is the radiative flux of the exciting light in quanta/s,

17 is the average refractive index of the sol- vent over the emission wavelength range, and

u and 5 refer to the unknown and standard,

respectively. In the studies reported here, comparative quantum

efficiencies were calculated using eq (6) [20]:

where \exs lKxu replaces h and converts from an in- strumentally produced constant energy to quanta/s.

The assumptions have been made, however, that (a) instrumental geometries are the same for the unknown

and standard, (b) emission is isotropic, (c) the exciting

fight is monochromatic if excitation for both samples is 232
at different wavelengths, (d) no reabsorption and re- emission occur, and (e) the absorption is less than 0.04. (Recent work permits solutions with absorbances of

0.2 to 1.0 to be measured by the sideview technique

[21, 22].) In addition, use of dilute solutions minimizes factors (d) and (e), and use of the same instru- ment and cuvette minimizes factor (a). Anisotropic

emissions (polarization effects) may cause large meas- urement errors which may be corrected [13, 23]; but

the easiest way to avoid polarization problems is to use

a standard which emits isotropically.

Assuming then that corrected spectra may be obtained [11, 12, 15], the fundamental problem in spectrofluorimetry is the lack of well-characterized standards.

Table 1 lists various organic compounds which have

been suggested for possible inclusion in a series of

Table 1. Compounds proposed as fluorescence standards

[1, 3, 8-10] Compound Emission maxima

(nm)

j3-Naphthol 354,402

2-Aminopyridine 368 Anthracene 383,404,428,454

Pyrene 390

Quinine sulfate 454

Quinidine sulfate 454

3-Aminophthalimide 510 a Fluorescein 518

/V,/V-Dimethylamino-m-nitrobenzene 542 Rhodamine 6G 557 Aluminum(III)-PBBR-chelate 635

4- Dimethylaminonitrostilbene 742

a Value is average.

fluorescence standards [1, 3, 10, 24, 25]. Some may not be suitable as general standards. Fluorescein undergoes degradation in sodium hydroxide, and solutions

should be prepared and used within 12 hours. The

purification of /3-naphthol by recrystallization in air results in some discoloration due probably to oxidation. Aromatics such as anthracene and pyrene

have narrow emission and/or excitation spectra (25

nm or less) and are not suitable as standards for fluorimeters which use wide slits since fluorescence

j spectra, as absorption spectra [26], are bandpass de- pendent [27, 28]. Other factors, such as acid concentration or excitation wavelength, are reported to affect the fluorescence of quinine and quinidine sulfates [21,

28-37] and 3-aminophthalimide [38]. The presentation

and discussion of some of these factors and their

effects, follow. The experimental section is presented

as addendum A. The following abbreviations are used in this paper:

Q- quinine; QS- quinine sulfate; QDS- quinidine

'j sulfate; 3-APT-3-aminophthalimide; N,N-DMAMNBj iV,iV-dimethylamino-m-nitrobenzene. Quinine samples

I are designated A, B, C, etc. and are listed in addendum

A.

II. Results and Discussion

A. Quinine Sulfate and Related Compounds

1 . Physical and Chemical State

It has been reported that the history of the quinine

determines its optical characteristics [24, 35]. A

report by Fletcher [28], however, suggests this is not the case, and that the observed differences were

probably due to the presence of varying amounts of hydrated water. Although quinine sulfate is obtained

Table 2. Water weight losses of various quinine derivatives as a function of pretreatment

Percent Calculated Sample 3 weight molecular

loss formula F No drying 4.24 QS b 5.4H 2 0

C No drying 4.% OS 5.7H2 0

E No drying c4.37 QS 5.0H,O E Vacuum dried, in 0.18 QS 2.1H2 0

desiccator for 6 months E Vacuum dried within 24 .03 QS 2.0H2 O

hours H No drying 4.36 QDSd 5.4H 2 0

H Vacuum dried within 24 0.02 QDS 2.0H 2 O

hours

J No drying .00 Q

e 2.0H 2 O

"See experimental section for designation.

b QS= quinine sulfate. 'Average of two determinations.

d QDS = quinidine sulfate.

e Q = quinine.

as the dihydrate, varying amounts of water are

definitely present. Table 2 summarizes the weight

losses as determined by thermogravimetric (TGA)

measurements for various quinine derivatives before

and after vacuum drying. It can be seen that with no

1.1

1 1

1 11 1 1

1 I

I I | -

2 .

3 — .1.1,1 1.1,1- 40 60 80 100 120 140 TEMPERATURE, °C

Figure 1. Thermogravimetric analyses of quinine sulfate (Sample

E). 1. undried after recrystallization; 2. dried in vacuo at 55 °C

for 18 hours, placed in desiccator, removed periodically to obtain sample; 3. freshly dried as 2. Heating rate was 5.0 °C/min in dry

air. Weight scale offset 10 mg. 233
drying, approximately 5 to 6 waters of hydration per

molecule rather than 2 are actually present. Figure 1 shows three TGA curves obtained for QS-2H2 0,

sample E. There is a slight loss of water for the vacuum

dried sample that had been kept in a desiccator for

six months and removed periodically to obtain samples

which shows the hygroscopic nature of QS-2H20.

As pointed out by Fletcher [28], insufficient drying can

cause variations in absorbance values, leading investi- gators to propose the inequivalence of quinine derivatives. For this reason, samples used for the following

studies were predried as outlined in the experimental

section.

2. Absorbance Measurements

Average weight absorptivities for various QS-2H20

samples from different suppliers with varying re- crystallizations were calculated from eq (7), and the

results are summarized in table 3.

WA=~

cl

(7)

where WA is the weight absorptivity.

A is the absorbance.

c is the sample concentration in grams

solute/gram solvent.

/ is the path length of the cell in cm.

Sample F gave consistently high weight absorptivities, but was included in the statistical analyses.

The average weight absorptivities at 317.0, 347.5,

and 365.0 nm for samples A to F differ from previous

data [28] by —1.2 percent, +0.5 percent, and —2.3

.percent in that order. Values for the average of all samples, including E, differ from previous data by — 1.0 percent, —0.5 percent, and — 1.0 percent in that

order.

Table 3. Determination of weight absorptivities for quinine sulfate with varying recrystallizations in 0.1 /V HUSO.) from different suppliers

Sample"'6 Rxc Weight absorptivities X 104 365.0 nm 347.5 nm 317.0 nm 250.0 nm

A 0 0.922 1.430 1.148 7.720 B 0 .916 1.418 1.146 7.700 C 0 .918 1.428 1.145 7.710 D 3 .922 1.438 1.140 7.820 F 4 .941 1.454 1.162 7.920 E 6 .925 1.436 1.152 7.820

E' d 6 .947 1.445 1.146 7.717

Average e 0.924 1.434 1.149 7.782 a .009 0.012 0.008 0.086 Percent <x .97 .84 .66 1.12

" All samples vacuum dried at 50-60 °C for 24 hours.

6 See experimental section for sample designation.

c Times recrystallized.

d Absorbances measured on Cary 16, others on Cary 14, see addendum A.

e For samples A—F only (excluding E').

Determination of the weight absorptivities at 347.5 a value of 1.436 ±0.005. Weight absorptivities and

nm for three individual weighings of sample E yielded molar absorptivities are also given for QDS-2H2 0 in

Table 4. Molar and weight absorptivities (X 104

) of quinine and quinidine sulfates in 0.1 H2 S04

Compound

Wavelength

Concentration Sample

M X lO" 5 365.0 nm 347.5 nm 317.0 nm 250.0 nm

WA" e

c WA" WA" ec WA"

Quinine sulfate" 2.554 E 0.7235 (0.924) 1.1228 (1.434) 0.89% (1.149) 6.0930 (7.782)

Quinidine sulfate6 3.269 H .7189 (0.918) 1.1257 (1.438) .8902 (1.138) 6.0110 (7.676)

" Average for 6 determinations, samples A-F, table 2.

6 Average for two determinations.

c l,mol- 1 ,cm-' X 104

. d gsolvents.fgsolute)"

1

, Cm" 1 X 104

. 234
table 4. As expected, the molar and weight absorptivities for QS-2H2 0 agree with those for QDS-2H2 0

since they are stereoisomers (structures for quinine

and quinidine are I and II, respectively).

Table 5. Absorbance ratios for quinine sulfate dihydrate in 0.1 /V sulfuric acid

H3 CO

CH=CH2 ^CH=CH2

HQ. A i)

H3CO

I. Quinine II. Quinidine

A single determination for the molar or weight

absorptivities for quinine dihydrate gave results which, as a function of wavelength, were consistently

3 to 5 percent low as compared to the values for QS and

QDS after correction for the difference in molecular

weights. Plotting relative absorbances, i.e. A\IA25onm

versus wavelength, however, resulted in good agree- ment among the three spectra, figure 2, indicating the

probable presence of a systematic error in the deter- mination of the absorptivities for quinine dihydrate.

These values are being redetermined.

Values of the ratios for several absorbance peaks of

QS in 0.1 N sulfuric acid have been calculated from the

literature [28-30, 33, 36, 46-48], and these values

and those from the present work are reported in table 5. Although systematic errors exist since various spectrophotometers were used, comparison of the average of

the literature values and the present work show dif- ferences to be +2.77, +0.25 and +0.62 percent. The

first value is for the ^250/^347 ratio, and measurements

in the ultraviolet are usually not as accurate or precise

as those in the visible. Statistical evaluation (again

z/xio' 1.0 3,0 2.5

1.0 -

0.6

« O.'l

0.2

0,0

1 r 1

| 1 1 1 r j D

°

8

7 1 1 r | 1

1 1 1 1 | -

Q, CARY W

OS, CARY W

a

•

QS, CARY 16

g 0 QDS, CARY 11

0

1

0

• B 0

0

1 ' ' Do

> °m • 250 300 350 i)00

WAVELENGTH, nm

Figure 2. Normalized absorbance spectra of quinine (Q), quinine

sulphate (QS), and quinidine sulfate (QDS) in 0.1 N H2 S04 using absorbance at 250.0 nm as 1.0. Temperature, 25.0 °C.

Spectrophotometer A2 TMIA 346 A317IA346 A3e5IA34S Ref.

Cary 16 5.340 0.793 0.655 this work

Cary 14 5.43 .80 .64 this work

Cary 14 5.38 .78 .65 [33] Cary 14 5.36 .80 .62 [30] Beckman D.U 82 .63 [47] BeckmanD.U 5.43 .80 .59 [29] Bausch and Lomb 5.23 .80 .63 [30] Turner 210 5.4 .82 .66 [28] 5.0 .75 [46] 5.25 .80 .65 [48] 5.10 .82 .69 [36]

including systematic errors) of all the ratios shows,

however, that any individual ratio may be as much as 4 or 5 percent high or low at only the 68 percent con- fidence level. It is essential that accurate data be ob- tained for absorbance measurements, especially since

the determinations of relative quantum efficiencies depend on these measurements, eqs (5) and (6). The instruments used for the absorbance measure- ments in the present work were checked for accuracy

by comparison against optically neutral filters supplied

by Mavrodineanu [26]. Absorbance measurements

were also made on solutions of QS and 3-APT in 0.1 N sulfuric acid at peak maxima using various

instruments. The results are summarized in table 6. Good agreement was observed for these solutions. No

Table 6. The determination of absorbances of quinine sulfate and 3-aminophthalimide in 0.1 N H2 S04 using various spectro- photometers

QS 3-APT

A, 250.0 nm A, 385.0 nm Instrument

0.9164 NBS [26] 0.7506 .9158 Cary 16

.751 .919 Cary 14

.749 .914 Turner 210

apparent fluorescence artifact was observed as shown

by comparing absorbances from the first three instru- ments with those of the last instrument which has a monochromator between the sample and the detector

[20, 23]. Differences as large as 8 percent, however, were

observed when absorbances were measured on slopes

in absorbance spectra. Care must therefore be used

when absorbance measurements are made on slopes

of peaks since small errors in wavelength (and also large bandwidths if the slope is steep) result in in- accurate absorbance values.

3. Fluorescence Measurements

Studies were made to determine fluorescence

reproducibility of measurements on quinine sulfate dihydrate (sample E) in 0.1 N sulfuric acid. Table 7 summarizes results of repetitive measurements on five different quinine sulfate solutions. 235
Table 7. Reproducibility of repetitive fluorescence measurements

on different solutions of quinine sulfate dihydrate

(Sample E) in 0.1 N sulfuric acidd

centration Average emission Percent (ppm) peak area cr cr

u.o U.UOo u.uuz 9 83 Z.OO

l.vr^ 1 Q9

l Ob l.U .104 .(Jul i.uy

2.5a .260 .002 0.85

5.0a .706 .003 .42 5.0" .518 .001 .22

10.

0

a .915 .007 .78 10.0b .911 .003 .32 5.0C .517 .003 .59

a Dilution by volume, four sample weighings.

b Dilution by weight, three sample weighings.

c Quinidine sulfate dihydrate (Sample H) dilution by weight.

d \ex, 350 nm.

Fairly large standard deviations were obtained with

the 0.5 and 1.0 ppm solutions, probably due to instru- mental noise resulting from increased instrumental

sensitivity. As can be seen, dilution by weight yields

results for which standard deviations are approximately two times lower than those for dilution by vol- ume. Similar results were obtained for solutions of quinidine sulfate [38]. All weighings of the vacuumdried solid samples were made in air. Weighing in a dry nitrogen atmosphere should result in lower percent standard deviations since the dried material is somewhat hygroscopic.

Similar fluorescence measurements made on solu- tions of quinine sulfate samples from two different sources (samples A, B, D, and F), results summarized

in table 8, show that the relative integrated area under

Table 8. Measurement of fluorescence peak areas using quinine

sulfate dihydrate from two sources 3

Emission peak areas

Sample

A

BD

F

Average

crPercent cr

One day Three day

average average

0.555 0.554

.542 .544

.552 .556

.564 .558

.553 .553

.009 .006 1.64 1.12

a Xex , 350.0 nm; °C = 25.0.

the fluorescence peak may be considered to be the same if percent standard deviations of 1 to 3.0 percent

are acceptable.

Studies were also initiated to determine the stability of quinine sulfate (sample E) in 0.1 TV sulfuric acid

since little definitive data exist concerning the staTable 9. Statistical analyses for fluorescence determinations" of quinine sulfate dihydrate (Sample E) in 0.1 /V H2 S04 during two "short" time intervals

Concentration (ppm) 1.0a 5.0a 10.0 a 1.0* 2.0 b 2.0" H2 S04 , N 0.1 0.1 0.1 0.1 0.1 0.01 A, peak area .105 .557 1.116 .411 .816 .848

cr .004 .003 .005 .001 .002 .001 Percent cr 4.34 .59 .49 .21 .24 .14

a Eleven determinations during a 10 hour span (same solution).

b Four determinations during a 1 hour span (same solution). \ex = 350.0 nm; °C = 25.0.

Table 10. Fluorescence peak areas of quinine sulfate and a uranium doped glass as a function of time on corrected and uncorrected

instruments Instrument A,

corrected [20] Instrument B, uncorrected QS, Sample QS, Sample Uranium E

a - b E

a- C glass 1

Average peak area 0.739 0.398 0.383

cr .011 .027 .018 Percent cr 1.49 6.89 4.80

a 2.0 ppm in 0.1 N H2 S04 . b Fifteen measurements covering 26 days.

c Fourteen measurements covering 37 days. The uranium glass was too concentrated for use in the corrected instrument.

bility of QS in solution [43, 44, 49]. Table 9 summarizes

results for "stabilities" during two "short time"

(1 to 10 h) tests, while table 10 and figure 3 summarize

results for longer periods of time (25 to 35 days).

As might be expected, the results in table 9 show

stabilities are better for the 1 hour determinations than

for those stretching over a 10 hour period. From table

10, it is observed that glasses give more repeatable

values and thus lower percent standard deviations.

It is evident from the data in tables 9 and 10 and

figure 3 that the results are actually composites of quinine sulfate solution stability and instrumental

stability. The latter was maximized by using the over- lapping solution method, and the results from this study are graphically illustrated in figure 4. 236
-•-QS SOLUTION

-o- VOl* GLASS

INSTRUMENT A

ELAPSED TIRE, days

Figure 3. Instrumental and compound stability determinations

using 2 ppm Oxg solute/g solvent, 2.55 X lO"6 M) quinine sulfate

in 0.1 N H2SO4 and a uranyl glass on corrected (instrument A) and uncorrected (instrument B) spectrofluorimeters.

1.0

£ 0.1

"

0.2 -

0.0

o

OLD SOL'N/NEW SOLUTIOI!

120 ELAPSED TIME,

180

FIGURE 4. 2 ppm (2.55 X 10-6 M) quinine sulfate stability test in 0.1 TV H2SO4 using relative emission peak areas as a function

of time, designated by solid line and overlapping solution method

(peak area original solution/peak area fresh solution), designated

by broken line. A 37 percent decrease in relative peak area as a function of time was observed (solid line) which, on

face value, would indicate instability of the quinine

sulfate in solution. Preparing new solutions, however,

and comparing the peak areas to those of the "aged"

solution gave ratios of one (designated by dashed line)

proving quinine sulfate stability in solution over this period of time. The assumption was made that dried

QS in a desiccator in the dark was stable over the

6-month period of time. Absorbance measurements for the freshly prepared QS solutions agreed within the

standard deviations presented in table 3. The observed

37 percent decrease in peak area was due therefore to an instrumental factor and not QS instability.

No long term data as to the stability of quinine sul- fate in a 0.1 N sulfuric acid solution in light have been

established; however, preliminary results indicate

that a 2 ppm solution is stable for 2 weeks if irradiated

by typical laboratory fluorescent lighting [38]. Excitation in this study was done at 350 nm and emission

peak areas were measured in the usual manner. However, Melhuish [50] reports a 25 percent decrease in fluorescence intensity upon irradiation of the QS

solution with 313 nm light (and excitation at 313 nm).

Excitation at this wavelength also resulted in a deviation of quantum efficiency as a function of excitation

wavelength [13, 21]. The problems which arise when

this wavelength is used should be investigated.

4. Corrected Excitation and Emission Spectra

To insure consistency in relative quantum efficiency

measurements with instruments that give corrected

spectra, the excitation spectrum should be compared

with the absorbance spectrum and the emission

spectrum compared with corrected spectra obtained

by other investigators.

z/xio4

300 320 310 WAVELENGTH, nm

Figure 5. Normalized intensities which show equivalency of absorbance spectrum (solid line) and excitation spectrum (circles). Quinine sulfate solution 20 ppm (2.55 X 1Q-5 M) for absorbance spectrum and 2 ppm (2.55 X 10-6 M) for excitation spectrum, both

in 0.1 TV H2SO4; excitation bandwidth, 25 A, emission bandwidth, 100 A; temperature, 25.0 °C.

V xioM

530 570 610 WAVELENGTH, nm

Figure 6. Relative EQ (K) versus wavelength for quinine sulfate

[2 ppm (2.55X10-6 M) in 0.1 TV H2S04], 3-aminophthalimide

[5.64 ppm (2.68XIO-5 M) in 0.1 N H2 S04], and N,N-dimethylamino-77i-nitrobenzene [2.76 ppm (9.34 X 10"6 M) in 70 percent

re-hexane: 30 percent benzene, V/V] •= this study, 0=Lippert,

et al.,[l] corrected from Eq (v) toE^X.) [50]. 237
The equivalency of the excitation spectrum and

absorbance spectrum is shown in figure 5, which has,

as a direct consequence, the constancy of quantum

efficiency as a function of excitation wavelength (dis- cussed later). Some deviation is seen at wavelengths

> 350 nm as might be expected with the reported

shift in emission maximum as a function of excitation

wavelength [29, 31]. The emission spectra of QS and

3-APT in 0.1 N sulfuric acid and N,N-DMAMNB are given in figure 6 as Eq (k) versus k in which Eq (k) max=l.

Data show good agreement with those taken from

Lippert, et al. [1] which were corrected to Eg(k) from

Eg (v) by multiplying Eq {v) by A"2 [50].

5. Relative Quantum Efficiencies

a. Intercomparative Study. — To verify the validity of the relative quantum efficiency (QE) measurements, an intercomparative study was undertaken using

QS, fluorescein, pyrene, rhodamine B, and 3-APT in various solvents as comparison "standards." Table 11 summarizes the results of this study. The values of the

quantum efficiencies for quinine or its derivatives in sulfuric acid have been reported to be in the range

0.40 to 0.70 [12, 21, 32, 34, 35, 37, 43-45]. Melhuish [43] and Dawson and Windsor [21] have measured the absolute quantum efficiency for QS in 1.0 N sulfuric acid

and obtained excellent agreement (0.546 and 0.54 ±

0.02, respectively, Aex = 365 nm). Measurements by

Eastman [35] tend to support these findings. Systematic errors [13] have been suggested to explain low val- ues of 0.46 or less by Rusakowicz and Testa [24] and

Drobnik and Yeargers [34]. The quantum efficiencies chosen for this comparative study are the widely used

and accepted values of 0.55 and 0.51 in 1.0 and 0.1 N

sulfuric acid, respectively [43].

It is difficult to reconcile the results of this inter- comparative study and many others with those of

Scott, et al. [37], who recently reported the quantum

efficiency of QS in 0.1 sulfuric acid, based on life- time measurements, as 0.70. It has been reported that QS has an anomalous lifetime [45, 51], and thus basing

quantum efficiencies on this measurement alone is questionable. In addition, Scott, et al., suggest that the

discrepancies in quantum efficiencies may be ex- plained if previous investigators had ignored the red

"tail" (> 550 nm) of the corrected emission spectrum.

An examination of the corrected emission spectra,

however, figure 6 [1], shows that the spectra extend

quite far into the red (> 650 nm).

Quantum efficiencies for fluorescein in sodium

Table 11. Results of the intercomparative quantum efficiency (QE) study e

Solvent QE QE (lit.) QSa 0.17VH2 SO4 0.51 ±0.01 0.508[43] QDSb iyVH2 S04 .52 ±0.01

Fluorescein0 1 N NaOH .87±0.03 ".88 Pyrene" Toluene .57 ± 0.04 .60[21], 0.32[51] Rhodamine B

b Ethanol .72±0.02 .69[3], 0.71[13] 3-Aminophthalimide b 0.1 N H2 S04 .47 ±0.02

a Determined by intercomparison study with fluorescein, pyrene and rhodamine B.

b Duplicate determinations, duplicate measurements.

c Quadruplicate determinations, duplicate measurements.

d Average of 16 literature values, see text.

e Temperature, 25.0 ±0.1 °C.

Table 12. Dependence of the relative quantum efficiencies of quinine and quinidine sulfates on sulfuric acid concentrations a

Quantum efficiencies QS • 2H2 0 QDS • 2H2 0

[H + ],;v This work [21] Percent 0 Percent [32] This work

0.01 0.52 ±0.01 + 1.97

.02 .52 + 1.57

.10 b.51 0.50 0.52

.50 .54 + 5.71 1.02 .55 ±0.01 .54 + 7.68 6 .55 2.00 .57 + 9.84 3.56 .60 ±0.01 .60 + 17.72 13 .59

a Temperature, 25.0 ±.1 °C, Aex = 350.0 nm.

b Reference.

c Using 0.51 as 100 percent. 238
hydroxide have been reported to cover the range

0.76 to 0.96 [3, 9, 21, 23, 52-61]; however, the most

widely accepted value is 0.90 plus or minus five percent [13]. The values quoted for pyrene and

rhodamine B are less widely accepted: e.g. values of

0.97 and 0.92 have been reported for rhodamine

B [23, 57], although it was reported the measurements

in these cases were not made with a red-sensitive

photomultiplier [13]. b. Acid Concentration. — The variability of

quantum efficiency of quinine sulfate as a function

of three sulfuric acid concentrations has been reported

[21, 32]. These studies were extended to cover eight

acid concentrations, as summarized in table 12. The results for the 0.1, 1.0, and 3.5 N sulfuric acid

concentrations agree well with those reported by

Dawson and Windsor [21] and Eisenbrand [32], although the latter reported a constant quantum

efficiency for sulfuric acid concentrations from 0.01

to 0.2 N. The increases for acid concentrations less than 0.1 N are small, but apparently real, since Chen

also observed this same phenomenon and suggested

that this was due to less quenching by the sulfate ion

at low acid concentrations [62]. Although the weight

absorptivities of QS and QDS in three different acid

concentrations agree within experimental error, table 13, a slight trend towards lower absorbances

Table 13. Weight absorptivities a of quinine and quinidine sulfates as a function of H2 S04 concentration b WA a X 104 at 347.5 nm

[H2SO4] N QS QDS

0.10 1.43 1.44 1.02 1.40 1.41 3.55 1.39 1.40

a See equation (7). b Temperature 25.0 °C. may be noted. Coupled with the increased fluorescence area as [H+

] increases, the observed increase in quantum efficiency may be explained simply by ionization

changes; however, discussions by Chen [29, 62]

appear to negate this explanation. Further work is being done to resolve these discrepancies.

The increase in quantum yield of QS as a function

of acid concentration is paralleled by QDS as might

be expected due to their similarities in spectra,

figure 2 [30, 47] and molar absorptivities, tables 4

and 13. c. Excitation Wavelength.—The quantum effi- ciencies of quinine sulfate, figure 7, rhodamine B,

and rhodamine 6G, figure 8 (others discussed later) were found to be independent of excitation wavelength (±5 percent). The largest deviations for QS

were found from 260 to 280 nm where the absorbance is at a minimum aiid larger errors would be expected.

Several investigators [21, 29, 36] reported a variation

in quantum efficiency of fluorescence intensity as a

250 300 350

Wavelength, nm 400

Figure 7. Relative quantum efficiencies of quinine sulfate as a function of excitation wavelength (• = this work), quinine sulfate concentrations: Xex = 240-290 nm, 0.1 ppm (1.28 XlO"7 M) and

0.5 ppm in 0.1 N H2 S04 ; Xex= 260-390 nm, 2.0 and 2.5 ppm

(6.39 XlO"7 M) in 0.1 N H2 S04 ; excitation bandwidth, 25 A;

emission bandwidth, 100 A; temperature, 25.0 °C; 0=Fletcher

[28].

§1.05 1 1.00 °0.95

1.0 T

,'.5

QS » 3-APT * BIPY ..d— R.6G AA— R 'B

T"

3.0

R.6G S R.B only

500 550

• ** •* __«_.

350 WAVELENGTH, nm

1400

FIGURE 8. Relative quantum efficiencies as a function of excitation wavelength: quinine sulfate — same conditions as figure 7;3-amino- phthalimide, 5.64 ppm (2.68 X 10

5 M) in 0.1 N H2 S04 ; bipyrenyl

(BIPY), 0.14 ppm (2.98 XlO"7 M) in toluene; rhodamine 6Gi

(R.6G), 0.12 ppm (2.08 X 10-7 M) and 0.025 ppm (4.43 X 10"8 M)

in ethanol; rhodamine B (R.B.), 0.13 ppm (2. 15 XlO-7 M) in ethanol. Emission bandwidth, 100 A; excitation bandwidth, 25 A,

temperature, 25.0 °C.

function of excitation wavelength. Fletcher [28], however, found no more than ±5 percent quantum yield

deviation for excitation wavelengths from 240 to 400 nm (except for 272 nm region, selected values in

fig. 7), and other investigators also report essentially no variation in quantum yield at two excitation wavelengths [33-35].

B. Aromatic Hydrocarbons

Another large group of compounds with relatively

high quantum yields are the aromatic hydrocarbons.

Series of possible standards may be made by simply

adding aromatic rings to the base compound, e.g. benzene and naphthalene fluoresce in the UV,

anthracene exhibits blue fluorescence, naphthacene

fluoresces in the green, and pentacene in the red. Anthracene has been studied quite extensively in ethanol and benzene, and the quantum efficiencies 239
reported from these studies vary from 0.25 to 0.33

[3, 18, 21, 23, 43, 57, 63-67].

We have studied pyrene derivatives [42] to determine

if these compounds are suitable as fluorescence

standards and to determine if substituents on more

condensed systems exhibit the same trends in emission

wavelengths, intensities and quantum efficiencies as the substituted naphthalenes. Corrected excitation

and emission spectra for the pyrene derivatives are presented in figure 9. The 1-chloro-, 1-bromo-, and

1-nitropyrene compounds showed little, if any, fluores- cence but would be expected to phosphoresce at low

temperatures if the substituent "heavy atom" effect

is followed for the substituted pyrenes as in the case

of the naphthalenes [68]. The results for the quantum

efficiency calculations and data taken from Berl- man [51] for the naphthalenes and pyrenes are summarized in table 14. As can be seen, fairly good

4.0 3.0 |"l l| I . .

I

I

I VXIO

2.0

cza>

B

CD or

Pyrene

Bipyrene

Aminopyrene

excitation

250 350 450

j i i i_ 550

Wavelength, nm

FIGURE 9. Relative intensities (emission spectra: quanta/unit

bandwidth) versus wavelength for 0.13 ppm (5.69 X 10-7 M)

pyrene, 0.47 ppm (1.84 X 10~6 M) 1-aminopyrene and 0.14 ppm

(2.98 X 10-7 M) bipyrenyl in toluene. Excitation bandwidth, 25 A, emission bandwidth, 100 A; temperature, 25.0 °C.

agreement was obtained with the quantum efficiency

values from Berlman for the bipyrenyl. Better agree- ment for pyrene, however, was obtained with values

from Fbrster and Seidel [69], probably due to the use

of the same solvent, toluene, rather than benzene. The

value for the quantum efficiency of pyrene by Dawson

and Windsor [21] was obtained using ethanol as the

solvent. The differences observed here may be due to some of the problems associated in the general use of aromatic hydrocarbons as standards since aromatics

(a) usually show oxygen quenching, (b) have fairly

large emission-excitation spectra overlap, (c) are very sensitive to extremely small amounts of impurities, (d) have narrow emission bandwidths,

necessitating use of narrow slits, and (e) must have

large index of refraction corrections made (as in this work) when quantum efficiencies are compared to compounds dissolved in aqueous media.

Similar trends in quantum efficiencies are noted

upon comparing pyrene and naphthalene to bipyrenyl

and binaphthalenyl. The dimeric species have quantum

efficiencies that are higher than the monomeric species

which may be directly attributable to the lifetimes —

i.e. 2 to 3 ns for the dimers and 100 to 600 ns for the monomers [51, 70]. The dimeric species have more

asymmetry than the monomeric species and thus

possess higher transition probabilities.

C. 3-Aminophthalimide

Although the compound 3-APT in 0.1 N sulfuric acid

has been suggested as a fluorescence standard [1], care should be taken in its use. We have found that the absorption (excitation) spectrum, figure 10, and

the quantum efficiency, table 15, are dependent on the

acid concentration [38]. The decrease in the weight

absorptivities at 385 nm with increasing acid concentration, coincides with the increased absorbance of a peak at 296 nm. The quantum efficiency of 3-APT in ethanol reported

by Alentsov [52] was 0.6. A quantum efficiency of 0.47

in 0.1 N sulfuric acid is not unreasonable in view of the

difference in solvent. The observed decreases in the

quantum efficiencies as a function of acid concentra- ! tion are consistent with the presence of more than one

species, allowing additional radiationless transitions

such as energy transfer between the species to occur.

Table 14. Quantum efficiencies (QE) of some pyrene derivatives compared to similar naphthalene derivatives

Compound Solvent

Pyrene Toluene

1 -Aminopyrene Toluene

1,1 '-Bipyrenyl Toluene Naphthalene 3 Cyclohexane

1-Aminonaphthalene a Cyclohexane

l,l'-Binaphthyla Cyclohexane

^ex ^-emdnax) QE

338.5 394 0.48, a0.52, "0.32, c 0.53 401 413 .43 349 428 .78, "0.84 276 322 .23 319 376 .46 284, 294 361 .77

a Ref. [70].

b Ref. [51].

c Ref. [21]. 240
fxicr

4.0 3.0 20 15

1 1 1 1 —-0.IN HgSC^

i 1

5ppm

!

1.0 5

" }

, --- 1.8

" 5

"

| 3.5 40 "

i Gxcitotion

M /

' i i

' \

smission

1 \ \\ •

! \\' /

\ \v /A / . /

\ 1 \

\

1 \

* ' '/" ^ \

i T^- r -' r'' . , 300 400 500 600 700

Wavelength, nm

FIGURE 10. Relative intensities (emission spectra: quanta/unit

bandwidth) versus wavelength for 5 ppm (2.38 X 10~5 M) and

40 ppm (1.90 X 10-4 M) 3-aminophthalimide in 0.1 N~~, 1.0 /V , 1.87V ----- and 3.5 N—

H

2 S04 . Excitation bandwidth. 25 A; emission bandwidth, 100 A; temperature, 25.0 °C.

Table 15. Dependence of absorbance at 385 nm and quantum

efficiency for 3-aminophthalimide as a function of sulfuric acid concentration WAa X 10"

[H2SD4], N 385 nm 296 nm Quantum efficiencies'"

0.10 2.36 0.39 0.45 ±0.01

1.02 1.29 .78 .31 ±0.02

1.75 0.82 .94 .23 ±0.02

3.55 .32 1.02 .15 ±0.01

a See equation (7). b \ex=385 nm; °C = 25.0; reference = QS • 2H2 0 in 0.1 N H2 S04 .

This work is in progress and will be reported later in more detail. The quantum efficiency for 3-APT in

0.1 N sulfuric acid is constant witl in ±5 percent over

the excitation wavelength range of 310 to 425 nm,

figure 8.

D. Other Fluorescence Standards

^V,A^-dimethylamino-m-nitrobenzene has also been

suggested as a standard [1] and, although the quantum

efficiency appears to be relatively constant as a function of excitation wavelength [38], it is quite low and

not very suitable as a standard. In addition, as mentioned before, a solvent system of 70:30, V:V of

hexane : benzene is not desirable since fluorescence

analyses in the biochemical areas [71] require use of

compounds which are soluble in aqueous media.

Chen [62] has suggested standards which can be

used in aqueous solutions and are suitable for bio- chemical applications. Other compounds which might

be added to this fist that cover the "red" region,

include rhodamine B and rhodamine 6G, both used as quantum counters and both having relatively constant quantum efficiencies over a wide excitation wavelength range (250 to 600 nm), figure 8 [13, 72]. Studies

i should be made of these compounds in aqueous

rather than alcoholic media, although aggregation in water has been reported [73].

E. Inorganic Ions in Glass or Polycrystalline Matrices

I The use of inorganic ions in glass matrices as cuvette

or disc shaped samples, figure 11, is attractive, es- pecially when one considers ease of handling which

may result in decreased methodological errors [74,

75]. The inorganic ions may be split into two classes

determined by their absorption and emission spectra.

FIGURE 11. Cuvette and disc shaped cerium, lead, and copper doped silicate glasses.

The non-rare earths and cerium(III) have broad spectra with little structure due to interconfigurational

electronic transitions, making them prime candidates

for fluorescence standards. The rare earths in the

+3 oxidation states, on the other hand, have sharp

spectra due to intraconfigurational transitions (less than

25 nm bandwidths), and may be used for wavelength

calibration, spectral resolution of instruments, and, in special cases, as quantum yield standards. The non-rare earth inorganic ions interact to a greater extent with the matrix than the rare earth

ions. Thus changing the glass matrix from borate to

silicate results in fairly large shifts of emission maxima.

241

502-591 O - 73 - 17
Figure 12 shows these shifts for lead-doped phosphate,

borate, and silicate glasses.

V XlO

4.0 3.0

"i""r " i excitationi

1 1 •

i

1r emission

2.0

250 350 450 550

Wavelength, nm

Figure 12. Relative intensity (emission spectra: quanta/ unit bandwidth) versus wavelength for lead doped phosphate, 1; borate,

2; and silicate, 3; discs. Emission and excitation bandwidths,

100 A; temperature, 25.0 °C; angle of disc with exciting beam,

15° [74, 75].

Table 16 summarizes ranges of emission maxima for the non-rare earth ions in glass or polycrystalline

matrices. For an in-depth discussion of these effects and information concerning the rare earths, see

reference [7] and the references listed therein.

Table 16. Ranges of emission maxima for cerium and non-rare

earth inorganic ions in various solid matrices 3

Inorganic Ion Emission Range (nm)

T1+ Ce3 * Pb2+

Cu+ Mn2+

290-325 325-400

350-450 475-550

475-550

500-600

a For emission spectra, see reference [7].

F. Summary

In summary then, substantiative data are being

accumulated as to the applicability of various com- pounds for use as fluorescence standards. The next major step would appear to be interlaboratory testing

and comparison of data leading to the acceptance of either a single or a series of standards which are widely

available for general use. Quinine sulfate, due to its

desirable fluorescence characteristics, overall applicability, and widespread use and study, should

probably be the first material selected for an inter- laboratory comparison study.

III. Addendum A. Materials and

Instrumentation

In order to adequately describe materials and ex- perimental procedures, it was occasionally necessary

to identify commercial products by manufacturer's name or label. In no instances does such identifica- tion imply endorsement by the National Bureau of

Standards, nor does it imply that the particular product

or equipment is necessarily the best available for that purpose.

A. Materials

The following reagents were used in this study:

Perchloric Acid: National Bureau of Standards,

purified reagent, double distilled, lot 111103 [39]. Sulfuric Acid: Ultrex Grade, J. T. Baker Chemical

Co., Phillipsburg, N.J. Used as obtained.

Sodium Hydroxide: Reagent Grade, J. T. Baker

Chemical Co., address above. Used as obtained.

Distilled Water: Distilled water was passed through an ion exchange column (IWT Research Column,

Rockford, Illinois), followed by double distillation from a quartz still. Toluene: Certified, Fisher Chemical Co., Fairlawn,

N.J., was shaken with cold concentrated sulfuric acid, water, aqueous 5 percent sodium hydroxide,

water, and dried over calcium sulfate in that order.

It was then distilled from sodium under nitrogen.

Benzene and n-Hexane: Spectrograde, Eastman Chemical Co., Rochester, N.Y. Used_as obtained.

Ethanol, 95 and 100 percent: Pharmco Distributing

Co., Publicker Industries, Philadelphia, Pa. Used as obtained. # Quinine Sulfate Dihydrate: Sample A — Aldrich Chemical Co., lot 032007; sample B-N. F. Grade, Fisher

Chemical Co.; sample C — Pfalz and Bauer, 31-20

College Point Causeway, Flushing, N.Y.; sample

D — sample A recrystallized three times from warm

water; sample E — sample A recrystallized six times

from warm water; sample F — sample B recrystallized

four times from warm water. Quinidine Sulfate Dihydrate: Sample G— Aldrich

Chemical Co., lots 032781 and 062607; sample Hsample G recrystallized four times from warm water. Quinine Dihydrate: Sample J — Aldrich Chemical Co.,

lot 071491 dissolved in dilute sulfuric acid, precipitated by neutralizing with dilute ammonium hydroxide. Washed with water twice.

All quinine derivatives were dried at 55-60 °C under vacuum for a minimum of 18 h before use [28]. Fluorescein: Aldrich Chemical Co., purified three

times by the method of Koch [40]. Rhodamine B and Rhodamine 6G: Samples obtained

from R. A. Keller [41]. Rhodamine 6G was purified

by repeated recrystallizations, using ethyl acetate: 242
ethanol followed in one case by elution from a silica column (purification by R. A. Keller).

3-Aminophthalimide: Eastman Organic Chemicals.

Recrystallized three times from ethanol, mp-260 °C

[11- AVV-Dimethylamino-m-nitrobenzene: Eastman Organic

Chemicals. Recrystallized twice from chloroform and

once from acetone-benzene [1]. Pyrene, bipyrenyl, l-aminopj rene, 1-chloropyrene, and

1-nitropyrene prepared as designated [42].

B. Instrumentation

Absorbance spectra were obtained using Cary 14

and Cary 16 spectrophotometers, and a Turner

Model 210 "Spectro" in the absorbance mode [20]. The spectrophotometers (except Model 210) were

equipped with constant temperature cell blocks. Spectra were recorded or read at 25.0 ±0.1 °C. Matched

quartz cuvettes of 0.1, 1.0, and 5.0 cm were used.

Fluorescence spectra were obtained using a Turner

Model 210 "Spectro" which gives corrected excita- tion and emission spectra [20], and a Farrand spectrofluorimeter which gives uncorrected spectra. Unblackened quartz spectrofluorimeter cells were used.

Solvents were always run to determine baselines.

Before quantum efficiencies or band positions were

measured, the accuracy of the wavelength scales for the emission and excitation monochromators were

checked using a mercury lamp. A polished aluminum

block cut at an angle of 45° was placed in the sample

compartment. The monochromators were calibrated

at 253.6, 435.8, and 546.1 nm. If necessary, the wavelength scales were adjusted so that accuracies of

±0.2 nm over the 253.6 to 546.1 nm range were obtained. A special Schlenk type [76] quartz cell was

constructed, figure 13, by H. Deleonibus, Glassblowing Section, 129.06, National Bureau of Standards.

Stopcock and stopper were of polytetrafluoroethyl- ene to avoid contamination. The solution was placed

in the cylindrical sidearm and alternately frozen

using liquid nitrogen and evacuated and thawed at room temperature at least five times to deoxygenate

the solution. A test which repeated this cycle ten times resulted in a solution weight loss of less than 0.1 percent. Fluorescence studies, unless noted, were made on the corrected instrument. Quantum ef- ficiencies were determined in duplicate, except where

j noted. All fluorescence measurements were made with

25 A excitation bandwidth, 100 A emission bandwidth, and at 25.0 ± 0. 1 °C , except where noted.

Thermogravimetric measurements were run using

dry nitrogen on a Model 950 TGA, E. I. DuPont de

Nemours & Co., Wilmington, Delaware, by T. Sterling

or S. Wicks, Section 310.04, Analytical Chemistry

Division, National Bureau of Standards.

The glasses mentioned were prepared by the InIj organic Glass Section, 313.02, W. Haller, head, NaI' tional Bureau of Standards. They were cut to size and polished by the Optical Shop, Section 129.05,

S. Gerner, National Bureau of Standards.

Figure 13. Schlenk type [76] spectrofluorimeter cell used with

nitrogen manifold for degassing solutions.

The author would like to acknowledge the useful

discussions in fluorescence with G. H. Atkinson,

NBS, and the encouragement and support of O.

Menis and J. I. Shultz, NBS.

IV. References

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(Paper 76A6-748)

244
Author Index

Adamson,A. W., 153,160

Akins, D. L., 140

Al-Ani,Kh., 152

Alentsev, M.A., 161,162,239,240

Alexander, N., 194,195

Allison, J.B., 171

Allison, R., 152 Almgren.M., 156,188

Amador, E., 121,122

Amata.C.D., 195

Ames, I.J., 141,143

Anderson, S., 184

Anderson, W.T., 143

Argauer, R.F., 186,203,233

Aronson, J., 39

Arye, Z.G.,212

ASTM, 42

Atkinson, J. R., 32,35

Ayres,G.H., 121 Azumi.T., 181,191

B

Bablouzian, B., 192

Bailey, E. A., 195

Baldwin, B.A., 189,195

Bannister. T.T., 239

Barber, C.R., 56,57

Barbrow, L.A., 32,35

Barnett.B., 21 1,222

Barnett, R.N., 126,127,130,131,132,133

Bartholomew, R.J., 235,240

Bauer, G., 61 Bauman,R.P.,8

Becker, R.S., 169,203

Beckman, A.O., 98.104,1 1 1.132

Belk, W.P., 117,118

Benford, F., 142

Bennett, H.F., 139

Bennett, R.G., 195

Berdahl, CM., 60

Berg, R.A., 152,189,195,206

Bergmeyer, H., 197,201

Berlman, I.B., 194.195,203,238,240

Bernt, E. 201

Bessey.O.A., 123

Bhattacharjee, H.R., 152

Billmeyer. F.W., 2 Bird, L.F., 143

Birks, J.B., 137,152,189,193,194,195,205,238,240

Biron.E., 211

Bischoff, K., 32,35,60,70,71

Bixon, M., 152

Blackwell, L., 171

Blanchi,J.P., 153

Blasse,G., 217

Blevin, W.R.. 60,72

Blount, J.F., 228

Blum, A., 126 Blume, H., 152 Boehm, L., 208,209,210

Bojarski.C, 159

Bonnier, J.M., 153

Booth, J., 204

Borisevich.N.A.. 152

Born, M., 65,84,90

B^rreson, H.C., 142,147,148,186,189,233,235,239

Boutry,G.A.,64,66

Bowen,E.J., 143,156

Bowers, G.N., 123

Bowers, P.G., 153 Bowman, E.J., 236 Bowman, R.L., 185,191,192

Bradley-Moore, C, 140

Brand, L., 184

Braun, W., 171 Braunsberg, H., 127,130

Breeding, H.A., 32,35

Bressani, T., 73,74

Brewer, L., 195

Brewer, R.G., 195

Bridges, J. W., 189,239,240

Briegleb.G., 152

Bright, D.S., 109

Brignoc, P., 201

Bril, A., 204,217

Brinkman,G.A., 2,99 Brock, M.J., 123

Broderson,S., 96,111, 112

Brody.S.S., 195

Broughton, P.M.G., 127,130

Brovetto, P., 73,74

Brown, H.D., 161,163

Brown, W.J. ,60

Brownfield, R.L., 125

Bruening, R.J., 87 Bruner.E.C, 152

Buchmiiller, F., 32,35

Buckley, J.L., 55

Bures, J., 65

Burg, W.R., 110

Burke, R.W., 106,109

Burland, D.M., 152

Burns, J., 152

Burton, M., 195

Buzzell, A., 164

245
c

Cali, J.P., 123

Callis, J., 152,153,155,161,162,164,206,239

Calvet,E., 163 Campbell, D.G., 126,127

Campbell, N.R., 32,35

Carruthers, G.H., 21

Caulfield, P.A., 185 Cavanaugh, E.L., 126,130 Ceccon,H.L., 157,158

Cetorelli, J.J., 176 ChapmanJ.H., 137,185,231

Chastel,R., 163,164

Chfn.R.F., 183,184,185.186,187,188,189.

191,192,193,194,195.204,233,235,238,239,241

Childress, C.C., 122

Christiansen, R.L., 141,143

Clar, E., 169

Clarke, F.J.J., 9,20,27,32,35,44,49,64,72,78.82

Clifford, H.J. , 155 D

Dacol, F.H., 157,158

Dairman, W., 228

Dalle, J.D., 152

Danielson, J.D.S., 153,162,164

Davies,G.W., 99,100,102

Davies, W.E.R., 78 Dawson, W.R., 153,188,189,233,237,238,239,240 Dean, J.A., 121

Deardorff, E.R., 106,109

Deinum,T., 216,221

Delisle, C, 65 DeMaria, A.J., 158 Demas, J.N., 137,138,151.152, 153,154,155,156.160.

161,188,190,203,204,205,232,233,237,238,239,241

Deranleau, D.A., 192

DeSantis, F., 204

Coburn.N.H., 102

Cole, E.B., 114

Connick, R.E., 105 Cook, I.G.H., 127,130

Cook, J.M., 204

Copeland, B.E., 120

Cordle, L.C., 32,35

Cotlove, E., 126 Cox.R.C, 240

Crawford, B.H., 6 Crosbv,G.A., 137,138,151,152,153,154,155,156,160,161,

188,190,203,204,205,232,233,237,238,239,241

Crosswhite, H., 207

Crosswhite, H.M., 207

Crouch, S.R., 113 Cuckow. F.W., 4,32,35

Cundall, R.B., 140,141,147

C/.ajkowski, R.C., 228

Desmond, F.B., 118

Desvignes, F., 32,35

DeVoe, J.,39 De Vos, J.C., 54,55,56

Dexter, D.L.. 218

Deyl, Z„ 204

Dieke,G.H.,207

Dobrow.D.A., 122 Donaldson, R., 4 Douglas, A.E., 152

Drobnik,J., 188,233,238,239

Druding, L.F., 155

Drushel, H.V., 169,240

Dubois, J.T., 153

Dudley, D., 239

Dyson, D.J. , 152,189,195,205,238

Eastman, J.W., 142,147,152,188,233,238,239

Eberhardt,W.H., 96,111

Eckerle, K., 44,45,65,82,91

Eckstein, Y., 208,214,216

Edelhoch,H., 183,186

Edelman, G.M., 140,143,147,153,203,233,235,239

Edisbury,J.R., 2,26,47,103,104,119

Edwards, J., 74

Edwards, J.G., 206,213,217,219

Eisenbrand, J., 233,238,239

Eisinger, J., 189

Ejder.E., 138,237,238

Ei-Bayoumi,M.A., 152

Elbert, W.C., 169

Ellis,S.C.,4 Elster,J.,32,35 Englman, R., 152 Erb,W.,61

Erminy.D.E., 63,64,74

Ermolaev, V.L., 153 Evans, G.B., 140,141,147

Evans, W.J., 162,163

Ewing.G.W., 104

Eyring, H., 106

Fahrenfort. J., 2,99

Fatiadi, A., 240,243

Faulhaber, M.E., 64,67,78

Feinstein, A.R., 126

Finch, D.I., 55

Finckh, B., 147

Fischer, E., 152

Fischer, G., 152

Fisher, N.P., 176,177,178,179

Fisher, R., 140

Fletcher. A.N. ,186,187,188,210,232,

233,234.235.238,239,240,241

Fleury, P., 32,35

Flor.R.V., 122

Forester, L.S., 239

F6rster,Th., 137,153,156,184,185,189,195,231,232,240

Forsyth, J., 99

Franey,E.J., 122

Frankel, H., 239

Freed.S., 169,171

Freemire, R., 39

Frosch, R.P., 152

Fulton, E.R., 9 Furumoto, H.W., 157,158

Fussell, W.B., 59

246
G

Gailey, I., 2 Galley, W.C., 187

Garfinkel, S.B., 45

Garrett, A., 99

Gati, L., 192 Gaw, W.,67

Geitel,H., 32,35

Gibson, G.E., 156,232

Gibson, G.L., 32,35

Gibson, K.S., 31,42,95,98,104

Gill, J.E., 153,155.186,233,235.237

Gillham, E.J., 60

Gillod,P.,64,66

Gilmore, E.H., 156,232

Glasner, A., 211,219,221

Gleason, I.O., 122

Glenn, W.H., 158

Glick, D., 169

Haas, C, 2,99 Habell.K.J., 32,35

Haecker, W., 159 Hager.G.D., 152,155

Haight.G.P., 102

Hall.H.T., 106

Hallbach.E.W., 141 Hammer, A., 152 Hammond, H.K., III, 32,35

Hanna.D.C, 158 Hansen, G., 32,35,64

Harding, H.G.W., 4,6,32,35 Hardv.A.C, 2,96,1 11 Hare, G.H., 98,104,1 11,132

Harel.L., 208,221

Harris, E.K., 126 Harrigan.R.W., 152,153,155

Harrison, G.R., 95,1 43

Harrison, T.H., 21

Hartley, B.S., 204

Hass,B.,201

Hass,G., 49,142

Hatchard.G.G., 144,171

Haugen,C.R., 140,141

Haupt,G.W.,99,119

Hawes, R.C., 63,71,79,82,98.104,1 1 1,132

Hellstrom, H., 239 Helman, E.Z., 122 Helman, W.P., 195 Hendry. P.I.A., 118

Ingle, J.D., 113

Jackson, J. K., 59

Jaitly, J.N., 162

James, C.G., 195

Jardon.P., 153

Jeffries, R.J., 74

Johnson, E.A., 103

Johnson, H., 169

Johnston, R.G., 141

Goedicke, Ch., 152

Goldberg, P., 216,219

Goldring, L.S., 98,104,1 11.132

Goldstein, A., 157,158

Gordan, G.S., 126

Gordon-Smith, G.W., 58

Gouffe, A., 54

Gouterman, M., 1 52, 1 53, 1 55, 1 6 1 , 1 62, 1 64,206,239

Gowenlock, A.H., 118,122,129,131

Gradyushko, A.T., 153

Grant, H.S., 235,239

Gray, W.T.,55

Greenberg, E., 208,210,21 1,212,216,221,223

Gridgeman, N.T., 2,99 Gruezinger, O., 159 Gudmundson, R.A., 164

Guilbault.G.G., 197,199,200,201

H

Henry, B.R., 152

Hercules, D.M., 155,203,239,240

Hermann, W., 32,35

Herre, W., 152

Her7X>g, G., 171 Hess, W., 138,147

Hieserman, J., 200

Hildebrand, F.B., 12

Hill, A.V., 162 Himel.C.M., 240

Hirt.R.C, 239,240

Ho,M., 141,143.144

Hbfert. H.J. ,32,35

Hogness, T.R., 97,99

Holford. W.L., 32,35

Holland, L., 48,49

Hollifield.H.C, 169,171,172

Honada,K., 171 Honigbaum, A., 150,208,216,219,221,241,242

Hood, L.V.S., 169,180

Hoppmann, H., 32,35

Horecker, A.L., 114,120

Hormadaly, J., 208,222,223

Horrocks, A.R., 153 Howard, J.R., 99

Howarth, J.V., 162 Howerton, H.K.. 192 Hu, K.H., 216

Hughes, E., Jr., 187

Hurtabise, R.J., 169

Ish-Shalom, M., 208,221

J

Jones, J.H.. 235,239

Jones, O.C., 6,20,32,35,58,64,78

Jones, R., 140

Jortner, J., 152

Joyce, T., 240 Judd,D.B.,72

Jung, H.J., 64,72,73,74,75,76,77,78,79

247
K

Kaihara,M., 192

Kaiser, H., 32,35

Kajigaeshi, S., 171 Kanda,Y., 171 Karmen, A., 123

Kasha, M., 206

Kashima.T., 32,35,69

Kauffman, G.B., 155 Kaufman, J.H., 122

Kaye, W., 192

Kearvell, A., 153 Keegan, H.J., 72

Keller, R.A., 242,243

Kelley.M.L., 123

Kendall, J.M., 60

Kenigsberg, D.R., 233,236,237,240,241

Kernohan, J.C., 184

Ketelaar, J.A.A., 2,99 King, E.J., 118

King, E.L., 99,102

King, J.W., 120,123

Kingdon.H., 153

Kinoshita,M., 181

Kirchoff, W., 195

Klasen.H.A., 171

Klein, B., 122

KleugerJ., 152 Klick,C.C, 218

Kirshenbaum, L., 208

Kobayashi, T., 152 Koch, L., 242 Kok,C.J.,59

Kokubun,H., 194,195

Koller, L.R., 32,35 Konev, S.V., 204

Konig, H., 32,35

Kornberg, A., 114,120

Kortiim, G., 31,32,35,137,138,139,147,171,185,231

Kosloff, R., 222,223

Kostkowski, H.J., 32,35

Kovalev, 1. 1., 55 Kramer, D.N., 201

Krefft, H., 59

Krijgsman, C, 61

Kristianpoller, N., 152

Kropp.J.L., 153,233

Ku,H.H., 113 Kubelka,D., 171 Kunz, H., 32,35,56,64,72,73,74,75,77,78,79 Kusba, J., 159

L

Laighton,P.A., 143 LaMer, V., 239 Lampton, M., 140

Land, P.S., 77 Landon, D.O., 140

Larsen, N.T., 162

Latimer, P., 239

Latz, H.W., 169

Leach, S.J., 183

Lee, J., 186

Lee, R.D., 32,74

Leimgruber, W., 228

Leng, M., 153

Lentz,P., 152

Leonard, N.J., 186,189,233,238

LePecqJ.B., 188 Lerman, L.S., 184

Levshin, V.I., 183

Lewis, G.N., 206

Lewis, J.E., 110

Lieblich, N., 222,223

Lim,E.C, 152,155

Lin,S.H., 152

Linn.J.W., 157,158

Lippert, E., 138,141,146,147,148,149,

185,231,232,233,237,238,240,243

Lipsett, F.R., 141,152

Liquori, A.M., 204

Livingston, R., 239

Lloyd, G.P., 142 Loken,H.F., 126

Loof, H., 32,35

Lowry.O.H., 123 Ludwig, P.K., 195

Lukasiewicz, R.J., 171,172,173,174,176.179,180,181,194,195

Lumry,R., 194,195

Lush, H.J., 69,77

McBride, W.R., 139 McCaman, M.W., 227 McCarthy, W.J., 169,171,177

McClure, D.S., 153,156,232

McClure, W.O., 203

Mc

McComb.R.B., 123 McCullough, J.P., 161 McDermott, L.H., 32,35,64

McGlynn, S.P., 171,181,191

Mclntyre, R.J., 159 M

Mack, M.E., 158 Madsen.B.C, 169 Maecker, H., 58

Maercks.O., 195 Magdenburg, H., 58

Maier.H., 32,35

Mallick, E., 65 Mann, W.B.,45

Marcus, R.J., 140,141

Marsh, O.J., 164

Massey, V., 204 Massod.M.F., 122 Mathur, D.P., 159

Matovich, E., 164 Matwey, J., 110 Mavrodineanu, R., 32,45,46,49,81,82,85.99,233,235

Mayer, R.T., 240

Mears, T.W., 122

Medinger.T., 153,240

Meehan,E.J., 96,97,1 10,1 12 Meinke, W.W., 122

Meites, I., 162

Melhuish, W.H., 137,138,142,143,145,147,148,149,156.

159,185,186,188,231,232,233,236,237,238,240

Menis, O., 32,99,106,1 14,133,231,233,241

Merritt, L.L., 121 Meserve, E., 194

Metcalf, W.F., 195

Michaeli.G., 208,221

Michelotti, F.W., 122

Michelson, A.M., 153

Mielenz, K., 32,44,45,65,82.91

248
M

Mitchell, F.L., 118

MitteldorU.J., 140

Moatti, P., 69,73,77 Moore, G.E., 34

Morgenstern, S., 122 Morren, L., 61 Mosag.S., 222,223 Moss, D.W., 235

Mousa.J.J., 171,172,176,179,180,181

Moye, H.A., 169,<204 Muchnik, G.F., 55

Miiller, A., 194,195

Munk,F., 171 Munro.I.H., 193,194

Muray, J.J., 138 Murphy, T.J. ,242

Mustachi, A., 21

1

Muszkat, K.A., 152 N

Nagakura,S., 152

Nagele, W., 138,141,146,147,148,149,231,

232,233,237,238,240,243

Nair, V.S.K.,99

Nakamura, K., 152

Nancollas, G.H., 99

Nanjo, M., 55 Nauman, R.V., 187 Nebbia.G., 137,185,231

Obermuller, G., 159 O'Dwver.M.F., 152 OHaver.T.C, 169,171,1' Ohnet.J.. 32.35

Ness, S., 155

Neuss, J.D., 99,103

Nicodemus, F.E., 138

Nimeroff, I., 32,35

Nishimura, H., 222 Nonaka.M., 32,35,69

Nygaard,K.J., 152

o

Oliver. C.J. . 140

Opler, A., 112 Owen. J.A., 126.127 Owen, L., 34

Palmer, E.H., 32

Paoletti, J.. 188

Pardee, A.B., 189

Parker. C.A.. 1 37. 1 39. 1 40. 142,143. 144.147,1 69.

1 7 1 . 185. 190,203.23 1 .232.233.238.239.240

Parsons, M.L., 169.171

Parsons, T., 104

Pataki, G.. 204

Patek, K., 206,213,217.219 Penchina.C.M.. 140

Perrin.F.. 183

Perry, J.W.. 97

Petrash.C.G.. 146

Pfaff, J.D., 169

Phillips, D., 152

Quinn.T.J., 56.57

Rabinowitch, E., 239

Radin.N., 122

Rains, T.C.. 103

Ralph, G.E., 159 Rand, R.N., 114,119,129,130

Rees, W.T., 147,190,231,233,238,240

Reich, E., 153

Reingold, I.M., 122

Reisfeld,R., 150,208,209.210,211,212,214,

216,219,221,222,223,231,241,242

Rejoan, A., 219,221

Rense, W.A., 152

Reule, A., 32,35,49,64,65,119

Ricci,R.W., 152

Richardson, D.C., 102 Rieman, W., 99,103

Riley, C., 127,130

Rinder-Knecht, H., 204 Ringbom, A., 99,104,121

Roberts, B.G., 239,240

Robertson, A. R., 2,72

Phillips. R.E.. 204

Pike, E.R., 140 Pilkuhn.M.H., 159 Pochon, F., 1 53

Porro.T.J.. 18

Porter, G., 153 Poulson.R.E.. 97.1 12,1 19

Prat. H., 163

Preston. J. S.. 4.6,26,32,35,60.64 Pribram. J. K., 140

Price, J.M., 192 Primbsch. J.H., 140

Pringsheim, P., 153

Prue, J. E., 99,100,102

Purkey,R.M., 187 Q

R

Robins, E., 227

Robinson, G.W., 152 Rodemeyer, S.A., 195 Rodomakina, G.M., 169

Rogers, L.B., 240

Rokos, K., 195

Rollefson, G.K., 195

Rosen, C.G., 192

Rosen, P., 140,143,147,233,235,239

Rosenblatt, G.M., 195 Rosmus, J., 204

Rbssler, F., 59

Rotter, F., 64,69, 70

Rozynes, P.A., 171,172,173,174,179

Rubin, M., 204

Rucci,A.,73,74

Ruegg, F., 39

Rusakowicz, R., 146,149,150,152,189,233,238

Russell, G.A., 218

Rutgers, G.A.W., 55,60

Riittenauer, A., 59

249
St. John, P.A., 169,171,177,204

Saka,Y., 171

Salmre, W., 169 Samejima, K., 228

Samson, J.A.R., 205 Samways, P.R., 7,9,27 Sanders, C.L., 32,35,54,61,64,65,66,67,70,78,91

Sanders, L.B., 171,172,173,174,175,176,179

Sanders, P.G., 127,130

Srfrka'ny, B., 192

Sauerbrey, G., 78,79

Sawicki,E., 169,204

Sawtell.J.W., 156,169

Saxena.V., 152 Schanda, J.,78

Schlenk, W., 243

Schleter, J.C.,72

Schneider, W.E., 59

Schulman.S.G., 169,171

Schulte-Frohlinde, D., 152

Schurer, K., 55,57

Schwartz, S.E., 140,142

Scott, A.B., 216

Scott, CD., 114

Scott, D.R., 171

Scott, D.W., 161

Scott, T.G., 186,189,233,238

Seely.G.R., 143

Seibold-Blankenstein, I .,1 38, 1 4 1 . 1 46, 1 47, 1 48, 149,231,232.233.237.238,240.243

Seidel, H.P., 240

Seitz.F., 218

Seliger, H.H., 186

Selwyn, J.E., 241

Sevchenko, A.N., 153

Seybold, P.G., 152,153,155,161,162,164,206,239

Sharafy,S., 152

Shideler, R., 39

Shimada, R., 171

Shore, V.G., 189

Shultz, J.I., 32,99,106,1 14,133,231,233,241

Sidorov, N.K., 169

Sidwell, A.E.. 97,99

Siebrand, W., 152

Singleterry,C.R., 188,192

Slavin, W., 18,97,112,120

Smith, F.J., 171

Smith, J.K., 171

Smith, R.C., 158

Smith, R.G., 158

Snavely, B.B., 157,158

Soffer.B.H., 157,158

Solovyov, K.N., 153 Sommers, A.L., 169,240

Sorokin,P., 158

Spencer, R.D., 155,186,189,194,233,238

Sponer, H., 171

Spurlock, C.H., 99

Staiger, W., 1 38, 1 4 1 , 1 46, 1 47,1 48, 1 49, 231,232,233,237,238,240,243

Stair, R., 59,141

Stafford, F.E., 195

Staniland, L.N., 204

Stanley, T.W., 169

Stegemeyer, H., 152

Steiner, R.F., 183,186,194

Steinfeld, J.I., 241

Sterling, J.T., 34

Stickney, M.E., 98,104,1 1 1,132

Stone, J., 228

Straski, E., 204

Strayer, L., 203

Strickler, S.J., 152,189,195,206

Strong. J., 8

Stryer, L., 153

Sturge, M.D., 216

Sturtevant, J.M., 164 Sumner, J.B., 132 Sunderman, F.W., 117,118

Suzuki, M., 55

Sveshnikova, E.B., 153

Swift, T.J., 105

Szalay, L., 192

Szigeti, C, 78 Szymanowski, W., 195

Tachoire, H., 163,164

Tang, D.. 201

Tansley,T.L., 159

Taylor, D.J., 83

Teal,G.S., 126

Teale, F.W.J., 153,156,188,189,233,235,239,240

Teegarden,T., 216,219

Tengi, J.P., 228

Testa, A.C., 146,149,150,152,155,189,233,238

Thai, A., 243 Thomas, T.R., 152 Thomson, L.C., 105

Thornton, W.A., 152

Thun, R.E., 49

Tickle, K., 153

Tietz,N.W., 125,131

Tin,M., 169,175

Tolstorozhev.G.B., 152 Tomura, M., 222

Tong, J., 99,102

Tonks, D.B., 1 18

Trencseni, J., 152

Tsvirko, M.P., 153

Turnbull, J.H., 169

Turner, G.K., 140,210,232,235,236,238,243

Tuzzolino, A.J., 152 u

Udenfriend, S., 169,171,185,204,228,241 Umberger, J., 239

Van Bertalanffy, L., 204

Vandenbelt, J.M., 26,99,103

Vanderlinde, R., 127,128,129 Van de Stadt,W., 77 Van Duuren,B.L., 204

van Voorst. J.D.W., 216,221

Vasicek, A., 85 Vaughan, W.M., 155 Vaughn, A., 199

Vavilov, S.I., 141,143,155,156,232,233,239

V

Veith,D.A., 110

Velapoldi, R.A., 150,208,209,210,21 1,216,219. 223,231,233,236,237,240,241,242,243

Velick,S.F., 184

Vise, M.H., 169,171 Von Foerster, G., 171

Voss, W., 138,141,146,147,148,149,

231,232,233,237,238,240,243

Vurek,G.G., 194,195

250
Wacker, W.E.C., 121 Wadso, I., 161 Ward,D.C, 153 Ware, W.R., 189,195

Watts, R.J., 152,153,154,155,156

Waylonis, J.E., 142 Webb, P.P., 159 Weber, G., 153,155,156.183,184,186,188,189,

192.194,195,233,235.238,239,240

Wegner, E.E., 160 Wehry.E.L., 240

Weigele, M., 228

Weimer,E., 141,143,144

Weinberger, L.A., 188,192

Weinberger, R.G., 140 Weinryb, I., 194

Weissberger, A., 161 Weisstuch, A., 189

Weller, A., 160 Wende, B., 58 Werkhoven, C.J. .216,221

Wharton, J. H., 187

White, C.E., 141,143,144,186,203,233

Yeargers, E„ 188,233,238.239

Yee, K.W.. 37,38

YguerabideJ., 143,241

Yoshie', K., 55

Wilkinson, F., 153,240

Willard,H.H., 121

Williams, R.T., 189,239,240

Williams, G.Z., 126

Willis, C.E., 120,123

Wilson, E.B., 164

Winch, G.T., 32

Windsor, M.W., 153,188,189,233,237,238,239,240

Winefordner,J.D., 169,171,172,173,174,175,176,

177,178,179,180,181,204

Wing, J., 39

Winkler, M., 153

Witherell, P.G., 64,67,78

Witholdt.B., 184 Wokes, F., 236

Wolf, E., 65,84,90

Wolf,H.C., 152

Woolf, L.A., 110 Wootton, I.D.P., 118 Wouda.J., 59,60

Wright, W.D., 2,72 Wunderman, I., 138 Wybourne, B.G., 206

Yoshihara, K., 152 Youden, W.J.,45

Young, D.S., 114,122

Young, F.M., 96,1 11

z

Zachariasse, K., 160 Zimmer, 201 Zamyatnin, A.A., 162 Zimmerman, R., 199,201

Zander, M., 169,171,180 Zscheile, F.B., 97,99

Zief,M., 122 Zweidinger, R.W., 171,172,173,174,175,176

251

Subject Index

This brief Subject Index should be considered as a complement to the Table of Contents located at the beginning of this publication. The reader should use the Table and the Subject Index to find the information of

interest.

A

Absolute

luminescence, see accuracy

spectroradiometric measurements, 53-62

Absorptivities molar, 101-105,109-115

Accuracy

in luminescence, 137-150,173-175,185-190,231-233

in spectrophotometry, 2,3,31,35,84,95,112,114,119,126,130

Actinometers, 144,160-166

Amino acids assay, 227

automation in, 228

Aminophthalimide, 148,240

Aminopyridine, 149

Aperture, see also linearity accuracy conditions, 15,16

dual, 14-17,36,43-45

multiple, 14,35,65-71

optical interference, 16

Automation

in luminescence, 228

in reflectance measurements, 8-13

in spectrophotometry, 8-13,38,39,129,132

B

Bandwidth, spectral, 3 1 ,96,1 1 1 , 1 27, 1 28, 1 30, 1 3

1

Beam alignment errors, 86,87,89,97,110

geometry, 32,86-90

Biochemical measurements, 183-196,197-202,227-230

Black-body radiator, 54,58

"Blooming," glass, 48

Bolometer, 59 c

Calorimeters, 161-165

conduction, 163

flash, 165

heat flow, 164

Tian-Calvet, 163

Carbon arc in spectroradiometry, 57

Clinical chemistry measurements, 109,114,117,118,122,125-

134,201

Computerization, 8-13,38,39,83

Corrected spectra, 146,185,186,237,238

Counter, quantum, 143,144,156

Cuvettes, 4,97-99,110,133

D

Detection limits in phosphorescence, 175,176

Detectors, see photodetectors

in luminescence, 140,142,158,159,176-178,186

in spectrophotometry, 6,7,13,23,24,37,38,40,63-79,83

m-Dimethylaminonitrobenzene, 149

Errors beam alignment, 86,87,89,97,110

polarization, 20,45,89,90,144,190-192

propagation of, 113 random, 113

reflection, 86,87,97,98,111

systematic, 3,14-20,110,130-132

wavelength, 110,112

Excitation sources luminescence, 139,141,185,186

F

Filters liquid, 26,63,99-1 07, 1 47- 1 50,23 1 -243

optically neutral, 105

solid, 25,46-49,63,70,72,81,84.85,120,133,203-224,231-243

optically neutral, 49

Fluorescence, see also luminescence, 137-244

absolute, 137-150,151-167, 183-1 96

biochemical, 183-196

clinical, 201 enzyme studies, 197,199-201

glucose studies, 201

lifetime, 193-195,209

picomole, 227-230

reabsorption, 145

solid surface methods, 198,201

standards, 146- 150, 155, 188-190. 192, 193, 206-

216,219,239,240,241,242

G

Gillham radiometer, 60

Glass standards, see also filters in luminescence, 203-224,231-243

in spectrophotometry, 25,46-49,63,70,72,81.120.133

Glass surface, "blooming", 48H

High accuracy, see accuracy

I Instruments

National Bureau of Standards spectrophotometers, 31-46,82-84

National Physical Laboratory spectrophotometer, 3-8

Integrating sphere, 4,9,10,33,37,82

Interferences, optical, 16,90,91

Internal standardization, 159,160

Interreflection errors, 86,87,97,98,111

253
L

Lasers, 156-158,165,166

Lifetime

in fluorescence, 193-195,209

Light sources, 5,32,33,83, 139,141, 165,170,174-179,186

standard, 54-59

Linearity, see also aperture; non-linearity photometric, 14-18,35,43-45,63-79,1 19,130

Liquid filters, 26,63,99-107,147-150,231-243

optically neutral, 105

Luminescence, see also fluorescence

absolute, 137-244

in biochemistry, 183-196,197-202,227-230

corrected spectra, 146,185,237-238

filters liquid, 231-243

solid, 203-224,231-243

quantum efficiencies, 151-167,187-190,205,210,217,238-241

M

Maecker's arc source, 58

Molar absorptivities, 101-105,109,115

Monochromators, 5,8,9,33,83,127,139,141,142,191

stray radiation in, 18-20,42 N

2-Naphthol, 147

Narrow-band instrumentation, 127,128,130,131, see also wide- band

National Bureau of Standards spectrophotometers, 31-46,82-84

National Physical Laboratory spectrophotometer, 3-8

Neutral glass filters, see filters

Nonlinearity, 64-74,91-93 o

Optical filters, see filters

Optical interferences, 16,90,91 P

Path length, 98,110, see also cuvettes

Peptides, 229

Phosphorimeters, 170-173

precision, 173-175

rotating cell, 172

solvent selection, 171

Phosphorimetry, 169-182

accuracy in, 173-175

detection limits, 175,176

Photodetectors, 6,7,13,23,24,37,38,40,83,186, see also detectors

linearity of, 14-18,43-45,63-79,119,130

Photometry, time-ratio, 20-25

Picomole fluorometry, 227-230

Polarization

in luminescence, 144,188,190-193

in spectrophotometry, 20,45,89,90

Predisperser, 33,83

Q

Quantum counters, 140,143,144

Quantum efficiencies, 151-167,187-190,205,206,210,217,238- 241 Quantum yield, see also quantum efficiencies

Quinine and Quinine Sulfate, 147,187-189,233-239

Quinn-Barber, black-body radiator, 58 R

Radiometer, 63

absolute, 59-62

Radiometer units, 138

Radiation sources

in luminescence, 54-59,139,141,156-158,176-179,186,187

in spectrophotometry, 4,5,32,33,54-59

Reabsorption of fluorescence, 145

Recording spectrophotometer, 8-11

Reflectance, 9-14,26-28,111

Reflection errors, 86,87,97,98,111 Rhodamine B, 143

s

Sample

carrier, 34

position, 45,87,110

Slitwidth, see bandwidth and spectral bandwidth

Source calibration, 61,141

Spectra

corrected, 146,185,186,237,238

Spectral bandwidth, 3 1 ,96, 111,127,128,130,131

emissivity, 56

Spectrof1 uorometers

design of, 140,141

Spectrofluorometry, 137

Spectrophotometers, high accuracy National Bureau of Standards, 31-46,82-84

National Physical Laboratory, 3-8 Spectrophotometry, accurate, 1-136

Spectroradiometric measurements, 53-62 Standards

luminescence

liquids, 146-150,155,188-190,192-193,219,239-241

solids, 155,206-216,219,241-242

spectrophotometry

liquids, 95-108,122

solids, 3,25,26,45-49,132,133,135,136

spectroradiometry, 54-59

Stray radiations, 18,19,42,88,89,96-98,112,119,139

254
T

Temperature effect, 102,103,113

Thermopiles, 142,143

Tian-Calvet calorimeter, 163

Time-ratio photometry, 20-25

Transmittance comparison of data, 45-47,81

Tungsten strip lamp, 55-57

u

Ultraviolet filters liquid, 99,100

solid, 49 w

Wavelength calibration.41,42,83,95,112,1 13,119

Wide band instrumentation, 127,130

Y

Yield, quantum, see quantum efficiencies

255
FORM NBS-114A (1-71) u.s. dept. of comm. 1. PUBLICATION OR REPORT NO. 2. Gov't Accession

BIBLIOGRAPHIC DATA „ „_„

'No. SHEET NBS SP-378

3. Recipient's Accession No.

4. TITLE AND SUBTITLE

Accuracy in Spectrophotometry and Luminescence

Measurements Proceedings of the Conference Held at

NBS, Gaithersburg, Md. , March 22-24, 1972.

5. Publication Date May 1973

6. Performing Organization Code

7. AUTHOR(S)

R. Mavrodineanu , J. I. Shultz, and 0. Menis , eds

.

8. Performing Organization

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No.

11. Contract/Grant No.

12. Sponsoring Organization Name and Address

Same as 9.

13. Type of Report & Period Covered

Final

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summatypf most significant information. If document includes a significant

bibliography or literature survey, mention it here.)

This volume contains the 18 papers presented at the Conference on

Ami-rnrv -in ^nprtrnnhntniiiptrv anrl T.iiTTli TlPSrpnCP MpasilTementS organized bv

the Analytical Chemistry Division of the National Bureau of Standards

and held from March 22 to 24, 1972.

These papers discuss the problems encountered in accurate spectrophotometry and luminescence measurements of materials. They also define

the status of these competences from the standpoint of basic principles,

critical factors involved in the measurements, and the instrumental

conditions which must be fullfilled to assure accurate measurements.

Particular attention is given to the selection, production and use of

Standard Reference Materials in spectrophotometry and spectrofluorometry

.

Problems related to health are also covered in six of the papers,

two of which include original contributions to the application of

luminescence techniques to specific biochemical problems.

17. KEY WORDS (Alphabetical order, separated by semicolons)

Accuracy; critical parameters; instrumentation; linearity; quantum

efficiency; spectrofluorometry ; spectrophotometry; standards.

18. AVAILABILITY STATEMENT

[X| UNLIMITED.

1 I FOR OFFICIAL DISTRIBUTION. DO NOT RELEASE

TO NTIS.

19. SECURITY CLASS

(THIS REPORT)

UNCL ASSIFIED

21. NO. OF PAGES

268

20. SECURITY CLASS

(THIS PAGE)

UNCLASSIFIED

22. Price

4.85, domestic postpaid

4.50, GPO Bookstore

tfrll.S. GOVERNMENT PRINTING OFFICE : 1973 O-502-591 USCOMM-DC 66244-P71