Analytical Chemistry:

Key to Progress

on National Problems

Nafonai

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Standards


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IG6077
UNITED STATES DEPARTMENT OF COMMERCE

Peter G. Peterson ^ Secretary

NATIONAL BUREAU OF STANDARDS • Lawrence M. Kushner, Acting Director

ANALYTICAL CHEMISTRY: KEY TO

PROGRESS ON NATIONAL PROBLEMS

Proceedings of the 24th Annual Summer Symposium on Analytical Chemistry sponsored by ANALYTICAL CHEMISTRY, ACS Division of Analytical Chemistry, and NBS Analytical Chemistry Division, and held at the

National Bureau of Standards, Gaithersburg, Maryland, June 16-18, 1971.

W. Wayne Meinke and John K. Taylor, editors

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards Washington, D.C. 20234

National Bureau of Standards Special Publication 351

Nat. Bur. Stand. (U.S.), Spec. Publ. 351, 481 pages (August 1972)

CODEN: XNBSAV

ISSUED August 1972

For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402 (Order by SD Catalog No. Cl3.10:351),Price $3.50
ABSTRACT

This book is the formal report of the proceedings of the 1971 Summer

Symposium in Analytical Chemistry held at the National Bureau of Standards, June 16-18, 1971, and cosponsored by the Analytical Chemistry

Division of NBS, ANALYTICAL CHEMISTRY, and the American

Chemical Society's Division of Analytical Chemistry. It contains six in- vited papers by subject matter experts comprehensively reviewing urgent

research problems for which advanced analytical techniques need to be

developed in the important areas of agriculture, air pollution, clinical chemistry and biomedicine, oceanography, solid state research and elec- tronics, and water pollution. Summaries of related discussions by leading

analytical chemists serving as panel members or in attendance at the Symposium are included. Hundreds of important analytical problems retard- ing progress in these important areas are described and extensive

references are given to permit a deeper insight into the problem areas. Accordingly this volume should not only stimulate interest in important

problems but should provide a valuable guide for highly relevant analytical research for some time to come.

Key words: Agriculture; air pollution; analytical chemistry;

biomedicine; clinical chemistry; electronics; oceanography; sohd state; water pollution.

ii

Library of Congress Catalog Card Number: 78-189152
FOREWORD

The Analytical Chemistry Division of the NBS Institute for Materials

Research provides a major national focal point for analytical chemistry

through its continuing efforts to encourage meaningful analytical measurements, to exercise leadership in attacking analytical problems of the

Nation, and to fill in gaps in critical measurement competences. This

Division consists at present of about 100 technical personnel encompassing some 60 different analytical competences from activation analysis

and atomic absorption to vacuum fusion and x-ray spectroscopy. These

competences are charged with the responsibility for research at the

forefront of analysis, as well as for an awareness of the practical sample,

be it Standard Reference Material or service analysis.

One important mechanism by which the Division exercises leadership

in the "state of the art" of a competence is that of sponsoring special conferences and symposia. Experts from around the world are invited to these conferences to summarize the present status of the many facets of a particular competence. In addition, contributed papers as well as comments of rapporteurs or summaries of panel discussions are often used to help better define this status. The first such conference sponsored by the Division was a broad one on "Trace Characterization — Chemical and Physical" held October 3-7,

1966 at the new NBS Laboratories at Gaithersburg, Maryland. On June

12-13, 1967 a seminar on a more restricted topic, "Quantitative Electron

Probe Microanalysis," was held in these same facilities and on October 7-

11, 1968 the Division hosted the 1968 Internafional Conference on "Modem Trends in Activation Analysis." On January 30-31, 1969 a Symposium on Ion-Selective Electrodes was held. The hard-cover

proceedings of each of these meetings are available from the Superintendent of Documents, Government Printing Office as NBS Monograph

100, NBS Special Publication 298, NBS Special Publication 312, and

NBS Special Publication 314, respectively.

As a continuation of these definitive "state of the art" conferences, the

Analytical Chemistry Division was very pleased to join ANALYTICAL

CHEMISTRY and the American Chemical Society Division of Analytical Chemistry to host the 24th Annual Summer Symposium in Analytical

Chemistry. The theme of this Symposium was "Analytical Chemistry:

Key to Progress in National Problem Areas." Over 450 scientists representing a broad spectrum of industrial, governmental, and educational institutions attended, and many others expressed interest in the

iii
proceedings. The format of the symposium provided an excellent opportunity for dialogue between analytical chemists and experts in problem

areas of air and water pollution, health, agriculture, oceanography, and

solid state research.

This volume contains the six invited papers presented at the Symposium, together with summaries of selected segments of the discussion

sessions.

W. Wayne Meinke, Chief

Analytical Chemistry Division

iv
PREFACE

This book is the formal report of the proceedings of the 1971 Summer

Symposium in Analytical Chemistry held at the National Bureau of Stan- dards in Gaithersburg, Maryland, June 16-18. The Analytical Chemistry

Division of the NBS Institute for Materials Research joined ANALYTICAL CHEMISTRY and the American Chemical Society Division of

Analytical Chemistry in sponsoring this 24th Annual Summer

Symposium.

In keeping with its theme — Analytical Chemistry: Key to Progress in National Problem Areas — the Symposium focussed on six very important

areas where analytical chemists can play a vital role. These areas in- cluded Agriculture, Air Pollution, Clinical Chemistry and Biomedicine,

Oceanography, Solid State Research and Electronics, and Water

Pollution.

A half-day session of the Symposium was devoted to each of the six subject areas. Leading authorities with broad knowledge of the problem

areas were invited to present keynote lectures, summarizing the state-of- the-art of measurements in their fields, and to pinpoint urgent measure- ment needs for the future. Following the lectures, a panel of analytical

chemists, conversant with the measurement problems involved, discussed

possible approaches to some of the problems raised by the speakers.

Finally, each half-day session ended with a general discussion period in which audience participation was encouraged.

This volume includes the six invited lectures and summaries of the

discussion sessions. Many hundreds of important analytical problems re- tarding progress in important areas are described, with sufficient background information to assist in establishing priority and a rationale

for an orderly approach to provide optimum impact in problem solving.

Accordingly, we firmly believe that this volume will stimulate interest in many "real-world" problems and provide a valuable guide for highly rele- vant analytical research for some time to come.

Identification of commercial materials and equipment by the authors in

this book in no way implies recommendation or endorsement by the National Bureau of Standards.

An undertaking of the magnitude of the Symposium and this book

would not have been possible without the cooperation and assistance of

many individuals. The enthusiastic participation of the six invited

speakers, the six session chairmen, and the 24 analytical chemists who

V
served as discussion panel members is deeply appreciated. Many members of the staff of the Analytical Chemistry Division and the National

Bureau of Standards served on numerous committes and assisted during

the Symposium in various capacities. Particular thanks are given to Bourdon F. Scribner, Richard A. Durst and Philip D. LaFleur for their

dedicated efforts in conducting several aspects of the symposium.

The NBS Office of Technical Information and Publications under the

direction of W. R. Tilley, and special help from Robert T. Cook, Rubin

Wagner, and Mrs. Rebecca Morehouse gave invaluable assistance in many phases of the effort, varying from the initial publicity brochures and

program to this final computer-assisted printing and publication of the

proceedings.

Within the Analytical Chemistry Division, special thanks are given to Mrs. Barbara Turner and Mrs. Ellen Zimmerman for their untiring effort

in typing the coded manuscripts and other material and to Mrs. Joy

Shoemaker for preparing the many tables and figures. Particular appreciation is expressed to Mrs. Rosemary Maddock who provided coordination

and editorial assistance in the many phases of preparing this book and to James R. DeVoe for developing and providing appropriate interfacing

with the Office of Technical Information to expedite the computer

typesetdng.

November 29, 1971

W. Wayne Meinke

John K. Taylor

vi
24th ANNUAL SUMMER SYMPOSIUM

ANALYTICAL CHEMISTRY: Key to Progress

on National Problems

General Chairman — W. Wayne Meinke

Assistant General Chairman — Richard A. Durst

Arrangement Chairman — Bourdon F. Scribner

Program Chairman — John K. Taylor

Social Chairman — Philip D. LaFleur

Speakers

Robert A. Laudise

George N. Bowers, Jr., M.D.

George W. Irving, Jr.

A. P. AltshuUer

Khalil H. Mancy

James H. Carpenter

W. Wayne Meinke

Robert S. Melville

Philip D. LaFleur

Session Chairmen

James R. McNesby

John K. Taylor

Richard A. Durst

Panel Members

J. Paul Cali

Henry Frankel

George H. Morrison

James C. White

Henry M. Fales

George G. Guilbault

Barry L. Karger

Howard V. Malmstadt

David Firestone

Herbert A. Laitinen

Charles Merritt, Jr. Mario A. RoUier

Walter C. McCrone

Peter K. Mueller

Paul Urone

PhiHp W. West

James N. Butler

Vincent P. Guinn

David M. Hercules

Robert E. Sievers

Vaughan T. Bowen

Velmer A. Fassel

Richard S. Juvet, Jr. Theodore J. Kneip

CONTENTS

Page

Abstract ii Foreword iii Preface V

Session Chairmen vii CHAPTER 1 The Chemical Analysis of Things as They Are 1 G. E. F. Lundell

CHAPTER 2

Opportunities for Analytical Chemistry in Solid State Research

and Electronics 19

R. A. Laudise

Panel Discussion 65

CHAPTER 3

Analytical Problems in Biomedical Research and Clinical Chemistry 77

George N. Bowers, Jr., M.D.

Panel Discussion 147

CHAPTER 4

Analytical Problems in Agricultural Science 161

G. W. Irving, Jr., and W. C. Schaefer

Panel Discussion 233

CHAPTER 5

Analytical Problems in Air Pollution Control 245

A. P. Altshuller

Panel Discussion 287

CHAPTER 6

Analytical Problems in Water Pollution Control 297

Khalil H. Mancy

Panel Discussion 383

ix
CHAPTER 7

Problems in Applications of Analytical Chemistry to Oceanography 393

James H. Carpenter

Panel Discussion 421

Index 431

X
CHAPTER 1

EDITOR'S NOTE - -

It is almost forty years since Dr. G. E. F. Lundell presented the

following paper before the Division of Physical and Inorganic Chemistry

at the 85th Meeting of the American Chemical Society, in Washington,

D. C, March 26-31, 1933. The points he makes regarding sampling,

practical analysis, accuracy, etc. are as pertinent today as they were

then. Readers of this volume will find this paper to be of considerable

interest. ANALYTICAL EDITION

Industrial

AND ENGINEERING

Volume 5 July 15,

Number 4 Chemistry 1933

Published by the American Chemical Society

Harrison E. Howe, Editor

The Chemical Analysis of Things as

They Are

G. E. F. Lundell, Bureau of Standards, Washington, D. C. ATTHE outset we might ask the question "Why are chemical analyses made?'^ The crude analyses of the

earliest days were the explorations in an unknown

world, the charting of naturally occurring materials and of

the simple compounds that were then made by man. Next came the analyses upon which the present structure of chemistry, geology, and many of the other sciences is based.

Those were the happy days in which most research workers

made their own analyses, and the profession was in high

standing. Today, by far the greater number of chemical

analyses deal with materials that enter into commerce,

1
2 ANALYTICAL PROBLEMS

man-made materials for the most part, in which man has

outdone his Maker. Moreover, these analyses are usuallymade by second or third parties who are often entirely

divorced from the history of the material or the purpose of

the analysis, and who therefore are not only working in the

dark but also often have only a monetary interest in the

test. The need and importance of chemical analyses are not

dying out, for chemical analysis now serves the arts as well as the sciences. It therefore seems appropriate to record some observations on present trends, and on considerations

that should not be overlooked.

"The Chemical Analysis of Things as They Are" has been

chosen as a subject because so many talks and articles on

analytical subjects deal with "The Chemical Analysis of

Things as They Are Not.'' This has been brought about

because in the field of analytical chemistry, as in other fields of endeavor, there has been a constant drifting toward

specialization. As a consequence, there is an increasing

tendency to devote more and more time to determinations

which deal with the final act of a chemical analysis, and less and less time to chemical analysis itself—in other words, to consider chemical analysis as dealing with one or two variables instead of the dozen or more that are often involved.

This gradual loss of the analytical viewpoint is evident in

contemporary articles that purport to deal with chemical

analysis.

For example, in a recent article entitled "Determination of

Aluminum" it is claimed that good determinations of aluminum can be had by adding lithium chloride, treating with

ammonium hydroxide until phenolphthalein turns pink,

filtering, washing, igniting, and weighing as the compound

5AI2O3 2Li20. Not one word is said concerning the behavior

of the other elements. To the analyst, it is self-evident that

approximately one-half of the elements can be precipitated

under these conditions. His chief thoughts on encountering

such a reaction would therefore not concern its use for the

determination of aluminum, but rather what effect lithium

would have on determinations of aluminum and iron that

are made by precipitating with ammonium hydroxide in the

usual manner, and whether aluminum causes retention of

lithium in methods for separating lithium from accompanying elements.

Methods of the type described are about as helpful to the

analyst as the method for catching a bird which the old folks used to recommend to children—namely, to sprinkle salt on
CHEMICAL ANALYSIS 3

its tail. To do that, one obviously must have the bird in

hand, and in that case there is no need for the salt. So it is with much of the advice that the analyst receives. Minute

directions are given for the salting away of the quarry after

it has been £ jparated from its fellows. In this connection,

it is interesting to note that when the analyst does happen

to encounter a ''pure'^ compound his chief concern is with the

impurities rather than the major constituent. In other words,

the absolute purity of a pure compound is establislied by

determinations of its impurities.

A contributing factor to the state of affairs that has been

outlined was the decision some years back that the analyst was a benighted creature who was working in the dark and

sadly in need of enlightenment. And so the structure

of analytical chemistry was brought under the spotlight,

usually by nonanalysts. Appropriately, the ''foundations of

analytical chemistry" received first attention. Without

question, a study of the foundations of analytical chemistry

is eminently fit and proper, provided one finally leaves the

cellar. But too many never attempt to reach the upper

floors, and so their outlook is restricted and their viewpoint

narrow. And they never know the delights of browsing

in the attic, and dusting off some of the old antiques that

should either be renovated or discarded—heirlooms like the

methods for siHca and for alumina—not to mention many

others. Of course there is a good reason for confining analytical

discussions to determinations which involve one or two

variables. A system containing ten to twenty diverse

components can hardly be handled on a strictly scientific basis, and any handling of it requires actual experience in

analysis. The solubility product of hydrated aluminum

oxide can be brought into the picture if ammonium hydroxide

is added to a solution of pure aluminum sulfate. But what

about precipitations that are made in a solution containing

iron, titanium, phosphorus, vanadium, and a dozen other

constituents as well? The difference between the two

problems is illustrated in Table I. The first case, that of aluminum sulfate, represents chemical analysis as it is not. The second, that of bauxite, represents chemical analysis as it Table I. Determination of Aluminum

Material

AbCSOOs.lSHzO

Bauxite

AI2O3 SO3 H2O

AI2O3 Fe203 TiOa ZrOz P2O5 V2O3 Cr203 Si02 CuO MnO CaO MgO NaaO K2O SO3 H2O

Components-
4 ANALYTICAL PROBLEMS

is, and is no exaggeration, for thousands of tons of bauxite

are sold, and naturally one of the most important considerations in the sale is the alumina content. If aluminum sulfate

is pure enough to warrant precipitation by ammonium

hydroxide, followed by filtration, ignition, and weighing of the resultant AI2O3, the operations are a waste of time, for one would get the same result much more quickly by igniting

the weighed sample and then again weighing. In the case of bauxite, treatment with ammonium hydroxide yields all of the first eight constituents and more or less of the copper.

Obviously then, the analysis resolves itself, not to a simple

determination of aluminum, but to a complex analysis in

which, as a matter of fact, the aluminum ion is never precipitated by itself. So we have two classes of workers in the analytical field, the determinators and the analysts. The determinators, who

are by far the more numerous, may in turn be divided into

two general classes: first, the common determinators who

follow a method explicitly, without knowledge or concern

as to the reactions involved ; and second, the educated

determinators who can handle systems containing one or perhaps two variables, or who, like determinators of pH,

are chiefly interested in group effects and make no effort to go

beyond this. The first are the laborers. They need not be

chemists, and they require constant supervision. The second

are the white-coUar workers who exhibit the usual extremes

of workers in this classification. The determinator's salvation lies in the development of truly selective methods of

analysis, and his final resting place will be a heaven in which

he has a shelf containing 92 reagents, one for each element,

where No. 13 is the infallible specific for aluminum. No. 26

the sure shot for iron. No. 39 the unfailing relief for yttrium,

and so on to uranium.

As for the analyst, he is a comparatively rare bird and is often referred to as a disappearing species, like the old

family practitioner who does the best he can, guided by theory

and experience, of which the most comforting is experience.

It must be admitted that one of the reasons for the gradual

disappearance of the analyst is the anomaly that, in spite

of the fact that he works entirely with material things, his reward is chiefly spiritual. Who ever heard of a consulting

analyst, or of an analyst who patented a new method of analysis or incorporated a company to promote its use? Other reasons for the scarcity of analysts are perhaps the

peculiar characteristics, some inherent and some acquired,
CHEMICAL ANALYSIS 5

that he must possess. He must have the inquisitive habit

of a detective, for oftentimes the composition of the material

under test is absolutely unknown. He must be an expert

manufacturer of pure chemicals, for on this ability the

success of his gravimetric determinations depends. He must

be an efficient dispenser of liquids of which he knows the

exact effect. And, above all, he must serve a long apprenticeship, and keep in constant practice.

So much for the personnel and the general aspects of the

project. We come now to the most important details of the

job. These are (1) the sample on which the analysis is made,

(2) the method of analysis that is used, (3) the accuracy of

the result, and (4) the cost of the analysis.

The Sample on Which the Analysis Is Made

Most analysts are well aware of the difficulties that attend

the taking of a truly representative sample of the material

that is to be analyzed. Not so well understood are the pitfalls in the way of obtaining the smaller sample on which the

analysis is actually made, pitfalls such as (1) differences

between the compositions of fine and coarse particles; (2) the fixation of oxygen, water, or carbon dioxide during

grinding, sieving, or drying; (3) uncertainties as to the water

content after drying and weighing the sample; and (4) changes in the composition of the sample during storage.

The extent of errors that may be caused by the first and last are illustrated in Tables II and III. For examples of errors that may be caused by oxidation, we have but to turn to examples such as the oxide film formed on the particles obtained in machining samples of steel that

are to be used in determinations of oxides in steel, the oxidation of sulfur in samples of pyrite that are ground instead of crushed, or the more insidious oxidation of sulfide ores such

as sphalerite that takes place during drying or storage.

With regard to the fixation of water and carbon dioxide,

it is interesting to note that many fired materials and not a few rocks exhibit this property, particularly when in the finely

powdered state. For example, samples of a soda-lime glass

which had been dried at 105° to 110° C. showed no loss on

ignition in the solid state, 0.5 per cent when first ground

and sieved, and 1.0 per cent a few years later, even though

it had been stored in a can with a fairly close cover. Hydrated

materials present special problems, especially if they are to be

used as standard samples, for the expulsion of water may be so
6 ANALYTICAL PROBLEMS

Table II. Differences between the Compositions of

Particles in a Sample

Matebial

Cast iron

Journal bearing

Ferrotungsten

Sieve Size Compositionoarbon

14-20 2.45

20-30 2.27 30-40 2.04 Copper Lead Tin

14-20 70.0 24.4 4.9 20-30 70.2 24.1 4.9 30-80 67.6 27.9 4.7 >80 63.2 31.2 4.4 Tungsten

80-100 77.6

100-200 80.2 200-325 79.5 >325 70.4 Table III. Changes in Samples during Storage

Composition Composition

Matebial When Prepabed afteb Stobage

% %

Ferrovanadium .33.6 V 33.1V

Zinc ore 31.4 Zn 30.4 Zn

Soda-lime glass 74 . 1 Si02 73 . 4 Si02

gradual that portions of the same material dried for like periods at 105° to 110° C. retain quite different amounts of water. Finally comes the old question of hygroscopicity,

which presents no mean problem at times. Pyrolusite, for example, while not markedly hygroscopic, is sufficiently so to cause entirely erroneous results if two successive samples

are taken from the same dried portion. It may be remarked

that difficulties in sampling are not confined to commercial

materials. Many an analysis of a ''mineral" is useless and

misleading for lack of care in selecting the sample, and many

a determination of atomic weights has been in error because

the sample on which it was made was not worthy of the

analytical effort that was put on it. Method of Analysis

The second consideration in the chemical analysis of everyday materials is the method of analysis that is used. It is evident that the method should give the result that is desired. It is not so well understood that all methods of chemical determination are in reality based on reactions of

groups, and that they can give accurate results for a given

member of the group only so long as the other members of the

group are absent or their effects can be discounted. Thus,

determinations of the chloride ion by precipitation as silver
CHEMICAL ANALYSIS 7

chloride succeed, provided ions such as bromide, iodide,

monovalent thallium, or lead are absent; determinations of

iron by reduction with zinc and titration with permanganate

leave little to be desired, provided the solution is free from

elements such as titanium, chromium, vanadium, or molybdenum; and the electrodeposition of copper is almost perfect unless attempted in the presence of elements such as bismuth, antimony, mercury, silver, gold, or tellurium.

Moreover, many do not seem to appreciate that an element

in the presence of others may behave quite differently than

when it is by itself. There is no dearth of methods that are entirely satis- factory for the determination of elements when they occur

alone. The rub comes in because elements never occur alone,

for nature and man both frown on celibacy. Methods of determination must therefore be judged by their '^seiectiveness.'' It is in this respect that most methods are weak and

that improvements must come, improvements such as are shown in the recently discovered a-benzoin oxime method,

which not only provides a separation of molybdenum from

practically all the elements, but also yields a precipitate

that can be used for its quantitative determination as well. Think what a boon the discovery of a simple, selective

precipitation procedure for the determination of silica would

be. Or simple direct methods for the determination of everyday elements such as aluminum, calcium, magnesium,

sodium, and potassium, or important commercially used

elements like antimony, cobalt, beryllium, boron, and

tantalum.

We are also in need of more simple and accurate methods

for the determination of substances that are present in very

small amount. Most of the present-day methods require

entirely too much time, and results are none too satisfactory.

It is not uncommon for two determinators to report results which differ by as much as 500 per cent. Such errors are not

confined to chemical analysis alone. They apply equally

well to estimations made by physical methods, such as spectroscopic, the only difference being that they are not so well known. It might be observed in passing that spectroscopic tests of things as they are are by no means infallible. The term "spectroscopically pure" has a quantitative

limitation which is sometimes much more serious than is suspected. In quite a few applications, the spectroscope is not so sensitive as chemical methods. For example, the

chemist can easily discover amounts of molybdenum or anti428-999 O - LT - 72 - 2
8 ANALYTICAL PROBLEMS

mony in steel, or bismuth in copper that the spectroscopist

cannot detect by direct test. The spectroscopist can, however, always excel the chemist by combining spectroscopy

with chemical analysis—^in other words, by first making

separations in which the element that is sought is separated

from most of the accompanying elements, and then properly

concentrated before the spectroscopic test is attempted.

The sensitivity of such tests is bounded only by the analyst's

ability to control his blanks.

Because of their lack of selectivity, methods for the chemical analysis of things as they are do not stay put. Therein

lies the danger of standard methods of anal3''sis. As soon as the other group members creep in—^for example, through

changes in manufacture—the method must be changed.

We cannot determine tin in steels by the same method

that is used for tin in bronzes, not because of the presence of

the larger amounts of iron, but because steels are prone to carry group members such as molybdenum and vanadium,

whereas bronzes do not—as yet. The most excellent bis- muthate method which we use for a plain carbon steel must

be discarded when we encounter a steel containing cobalt;

the standard procedure for carbon in steel was entirely

satisfactory until special steels such as those of the highsulfur or 18-8 types were made; the evolution method for sulfur in steels fails with those containing titanium, zir- conium, or selenium; and the methods for silica and alumina

in glasses begin to waver when boron is encountered.

One criticism of present-day researchers in analytical

chemistry is that there are too many repairmen as compared

with builders. In other words, too many are tinkering on

old methods of determination that are scarcely worth the

attention, as compared with pioneers who blaze new trails. We have gone about as far as we can go along the old paths.

Worthwhile developments must come as a result of new

methods of attack, such as the utilization of complex ions, or a systematic study of the reactions of organic compounds.

How far we have advanced in the chemical analysis of things as they are during the past 25 years can be illustrated

by the data shown in Table IV. As for newly proposed methods of analysis, it might be

pleaded that the author (1) tell in what respects the method

is superior to established methods, including considerations

such as time required and accuracy of results ; (2) enumerate

the substances that prevent its use; (3) outline its desirable
CHEMICAL ANALYSIS

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ANALYTICAL PROBLEMS

applications; and (4) tell the whole story in one installment, or at least give a good synopsis of the whole story in the

first installment. There is no excuse for passing out information in the "continued in our next" style, which, for example,

characterized articles on the use of 8-hydroxyquinoline.

Accuracy of the Result

We have now arrived at the third consideration in the

chemical analysis of things as they are—namely, the accuracy

of the result obtained. There is a wide misconception, even among analysts themselves, as to the accuracy of the results obtained in analytical work, and many confuse precision

with accuracy. It is a comparatively simple matter to establish the precision that can be expected if a certain method of analysis is applied by an analyst or by a group of analysts

to a given material. On the other hand, it may be extremely

difficult to judge the probable accuracy of a result, for all results are matters of opinion rather than fact, and so the

true result is never known. This is one of the most unsatisfactory aspects of chemical analysis. When an analyst scans a bare result, he can never be sure whether the determinator

provided for all interfering compounds, hoped that they were absent, or was too ignorant to worry. How' difficult the task may be can be illustrated by the data shown in

Table V. These represent the results that were reported

by thirty different analysts situated here and abroad. It can be seen that 60 per cent of the analysts reported results that were essentially within the average deviation from the

general average, and that the average deviation (precision

that can be expected if the same analysts determine calcium

phosphate in this type of material) is in the neighborhood

of 0.33 per cent. But what about the most probable value?

Is it the general average, is it within ±0.33 of the general

average, or is it a value outside of this limit? Anyone who

chose the result (77.40) that shows the greatest deviation

from the general average would be regarded as taking a very

long shot, and yet this is the one which is nearest the correct

result. That such difficulties are not confined to everyday

analyses is illustrated by the history of the atomic weight

of antimony. This was given as 120.062 =±= 0.0037 by Clarke

in 1920, after most carefully reviewing and weighting the

determinations of the atomic weight of antimony that had

been made up to that time. He states that ''the figure

is not quite satisfactory'* and "that some more work should be
CHEMICAL ANALYSIS 11

done." Subsequent analyses bore this out, for 120.2 was

adopted in 1921, and the still higher weight 121.77 was chosen

in 1925. Note now that this weight is not very far off from

the value 121.82 which Clarke obtained by recalculating

analyses made by Dumas in 1859, and of which Clarke

stated '^The values, say all over 121, are almost certainly

in error, and ought to be rejected."

Table V. Analysis of Phosphate Rock

(All analyses on dry basis) CaaCPOOi Reported by Thirty Laboratories throughout the World

% % % % %

77.40 78.07 78.33 78.61 78.77

77.73 78.16 78.33 78.62 78.79 77.77 78.19 78.40 78.62 78.81 77.84 78.21 78.42 78.63 78.84

77.85 78.24 78.42 78.72 78.86

77.96 78.28 78.60 78.72 79.10

General average, 78.38 Average deviation from general average, 0.33 (14 low and 16 high)

A fair question therefore is: "How can the most probable

value be established?" In classical researches, as in determinations of atomic weights, it is chosen by most carefully

examining the credentials of the candidate. These must be in

minute detail and, in the case of atomic weights, would

suffice except for one serious omission, the integrity of the

sample, for which no one on earth can vouch. In the analyses

of everyday things, the safeguards that attend the acceptance

of atomic weights are of course impossible, because cost and

time are prohibitive. When arguments arise, industries

usually employ an umpire, who often knows less about the

game than the disputants, or the results are checked against a standard sample of like composition. How the most

probable value is established for standard samples, such as

are furnished by the Bureau of Standards, is a long story

that can be summarized by the statement that it is based on

experience, on the work of others in the field, and usually on

determinations made by as many fundamentally different

methods as possible.

As for estimating the accuracy of his work, it can be said

that the determinator is usually an optimist in thought and

expression, while the analyst is a confirmed pessimist. The

determinator reports silica in glass as 71.61, if not 71.611.

The analyst, who knows that he is doing nicely to insure

results in the first decimal place, reports 71.6 and thus is honest with himseK and deceives no one as to his powers.

The percentage errors that have been made by representative
ANALYTICAL PROBLEMS

analysts who have done the best they could in analyses of materials with which they were familiar are shown in Tables

VI and VII and emphasize the fact that the ordinary analyst

of things as they are is not so much concerned with keeping

his errors below one part in one thousand as he is in keeping

them below one in one hundred. It should be observed that

the accuracy is high in determinations of the high-priced

constituents, and that, while the accuracy is not flattering

in many of the other determinations, it is still inside the limit at which the data can be used. Moreover, in most cases the

accuracy in terms of the material is quite satisfactory.

As regards the correlation of data, an error of one point

(0.01 per cent) in determinations of carbon in a 20-point

(0.20 per cent) carbon steel represents a 5 per cent error, and is yet within the accuracy of ordinary sampling and any

use that can be made of the analysis in the ordinary case. With respect to accuracy in terms of the material, we may

easily report 0.051 per cent for suKur in a steel containing

0.050 per cent. This error in terms of the steel is but 1 part

in 100,000, or equivalent to measuring a mile to an accuracy

of about 0.5 inch, and it has no more significance to the

metallurgist at present than the 0.5 inch has to a mile runner,

or the time that is recorded. It is also obvious that even

larger errors may not be fatal, provided approximately the same error is always made. For example, no harm is done if a

glass batch containing 70 per cent of Si02 shows 65 per cent by a method that gives very uniform results and is in daily use in checking its composition. If the actual content is of Table VI. First Reports of Analysis of Chrome Ore

AI2O3 FeO MgO Si02 Ti02 CaO MnO

36.62 17.15 14.18 15.95 7.82 0.91 0.32 0.19

36.62 19.37 14.24 15.99 7.86 0.91 0.38 0.22«

36.86 20.28 14.25 16.19 7.99 0.93« 0.44 0.23

36.91 20.55 14.35 16.21 8.05 0.93 0.49 0.35

36.93 20.68 14.36 16.33« 8.21 0.93 0.58

36.98" 20.82 14.40« 16.36 8.22 0.98 0.68

36.99 20.84 14.49 16.80 8.24 0.99 0.83«

37.05 20.84« 14.50 17.49 8.25« 1.06 1.12 37.05 20.86 14.56 8.28 1.12 1.69 37.37 14.82 8.59 GENERAL AVERAGES

36.94 20.15 14.42 16.42 8.15 0.97 0.73 0.25 AVERAGE DEVIATIONS FROM GENERAL AVERAGES

0.15 0.84 0.14 0.37 0.18 0.06 0.33 0.05 AVEEAGES OF RESULTS THAT FALL WITHIN AVERAGE DEVIATIONS

36.97 20.53 14.44 16.25 8.21 0.95 0.60 0.23 RESULTS OUTSIDE AVERAGE DEVIATIONS

3 1 4 4 4 4 4 2

<* Result nearest to most probable value.
CHEMICAL ANALYSIS 13

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ANALYTICAL PROBLEMS

moment, and the approximate error of a cheap, rapid method

is known, the latter is often used and the correction made,

as is done in evolution sulfurs on cast iron.

Cost op Chemical Analyses

When we enter the field of chemical analyses of things as they are, we step from the academic into the commercial

domain, and so it is proper to discuss costs. In this field, accuracy and costs usually go hand in hand.

So naturally the money that is spent on analyses should not exceed what is necessary to insure the accuracy that is needed.

For this reason, in applied analyses we often employ two sets of methods, the cheap routine methods which we use whenever we can, and the expensive umpire or referee methods

which we use only when necessary. For example, in determinations of phosphorus in plain carbon steels the rapid

alkalimetric method serves entirely satisfactorily for all ordinary purposes, and so the slower and more expensive

molybdate-magnesia method is brought out only in cases of serious disputes, or occasional checking of the alkalimetric

method.

In any discussion of costs of analyses one should consider

the producer as well as the consumer of chemical analyses.

If the consumer is at the same time the producer, as in steel works or research laboratories, the situation takes care of

itself, for it's all in the family. Consequently, the cost of

personnel and of the methods of analysis is, or should be,

that which will give the necessary results. In this case, the

ratio of analysts to the less expensive determinators depends on the type of work, the former predominating in

research laboratories and the latter in plant laboratories.

Methods of analysis are also carefully chosen on the basis

of speed, dependability, and cost. Purchasers of chemical analyses, like other buyers, usually

get what they pay for, and as a rule are unwilling to pay very

much. In purchases of clothing one can buy ready-made or custom-made suits. The former are relatively cheap, be- cause they can be made in quantity from a standard pattern,

and are quite satisfactory for the man whose figure follows

conventional lines. Similarly, in purchases of chemical

analyses we can buy ready-made or custom-made analyses.

The former are cheaper, for, like carbon in plain carbon steels,

they can be made by a standard operation. They are also
CHEMICAL ANALYSIS 15

perfectly satisfactory, so long as the composition of the material is not unusual and ordinary accuracy suffices. The

moment, however, that you depart from these specifications and enter the custom-made department, costs mount

rapidly.

This leaves the producer of analyses—which is no new

situation, for he usually gets left. It can be stated at the

start that he seldom receives a fair return, except possibly

in the case of commercial laboratories which sell ready-made

determinations in large volume. Under present conditions

there is little profit in custom-made analyses. One reason

for this state of affairs is that the analyst sells an intangible

commodity, an opinion. Under the circumstances, the

purchaser cannot see why he should pay five dollars if someone

else is quoting fifty cents. This might not trouble the analyst

if the opinion were the result of exclusively mental processes, such as an opinion at law, for then the analyst would not be

bothered much by overhead, except when talking through

his hat. The difficulty is that the opinion must be based on physical as well as mental effort, and if one discounts the

mental, he still has to take care of the physical. It should

also be remarked that the purchaser cannot check the correct- ness of +he opinion except by buying other opinions, which

is an expensive as well as a poor way to settle an argument.

The intention in this article has been to present a picture

of the chemical analysis of things as they are, and not to glorify

the analyst on the one hand or to depreciate the state of

analytical chemistry on the other. The work of and the demands on the analytical chemist are growing .more exacting

every day, and we cannot afford to lose the analytical viewpoint.

Received April 12, 1933. Presented before the Division of Physical and

Inorganic Chemistry at the 85th Meeting of the American Chemical Society,

Washington, D. C, March 26 to 31, 1933.

Robert A. Laudise, is Head of the Crystal Chemistry Research Department. He joined Bell Telephone Laboratories in 1956 after receiving a

B.S. in chemistry from Union College in 1952 and a Ph.D. in inorganic

chemistry from the Massachusetts Institute of Technology in 1956. While

at M.I.T. he was an Arthur D. Little Fellow. His interests at Bell Laboratories have included materials preparation and crystal growth. He has

been especially interested in hydrothermal crystallization and in the

preparation of piezoelectric, ferroelectric, nonlinear optical and related

materials. His society affiliations include the ACS, the American Ceramic

Society, the American Mineralogical Society, AAAS, and Sigma Xi. He

is Associate Editor of the Journal for Crystal Growth and a member of

the Crystal Growth Commission of the International Union of Crystallography. Dr. Laudise served as a Consultant to the President's Science Advisory Committee and the National Academy of Science. He is Editor of

"Crystal Syntheses" and author of the book, "The Growth of Single

Crystals."

17

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 35

L

Analytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

Held June 16-18, 1971, Gaithersburg, Md. (Issued August 1972). CHAPTER 2

OPPORTUNITIES FOR ANALYTICAL CHEMISTRY IN

SOLID STATE RESEARCH AND ELECTRONICS

R. A. Laudise

Bell Telephone Laboratories, Incorporated

Murray Hill, New Jersey 07974

Current practices and problems in analysis in solid state research and development are reviewed. A plea that the analyst assume a broadened

responsibility for characterization of materials is made. Examples of solid state problems which were caused by inadequate appreciation and application of

analysis are given. In particular, it is pointed out that trace analysis at and below the ppb level, the careful assessment and mapping of physical perfection

by x-ray and related techniques and the use of solid-state physical measure- ments such as conductivity and magnetic resonance will become more important in the future. Mapping techniques and perfection techniques are reviewed

in some detail. Recent new methods of stoichiometry assessment are described, and it is suggested that the determination of perfection and defects should become an orderly subdiscipline under the aegis of analytical scientists. The case is made that, insofar as both research and technological progress in solid state and electronics are rate limited by the availability of materials with

appropriate properties, it is limited in many cases by our ability to characterize

materials. It is concluded that with a modest reorientation of viewpoint and

disciplinary content, analytical science could become the core discipline of

materials characterization.

Keywords: Analytical chemistry; electronic materials; materials characterization; solid state research; stoichiometry; trace analysis.

I. Introduction

The electronics industry presently has gross sales of about $25 billion per year [1]. From a start usually dated from the transistor in the late 1940's, this is a staggering growth. This growth is not complete; competent estimates place sales at about $32 billion per year by 1974 [1].

Solid state materials, single crystals, ceramics, glasses, polymers and thin

19
20 ANALYTICAL PROBLEMS

films are the building blocks of the devices from which electronic systems

are constructed. Indeed it is axiomatic that the rate limiting process in the

progress of present day solid state research and electronic technology is the attainment of materials with desired properties. Several illustrations of technological progress which is materials limited are:

1. Bulk power transmission could be economic with superconducting transmission lines if super conductors with a transi- tion temperature permitting operation at liquid-nitrogen temperature were available. Many researchers even feel that superconductors at or only somewhat above liquid hydrogen would

suffice. Present materials come close to being usable in the

liquid H2 range.

2. Bulk information transmission could be economic with

laser carriers if a material for a transmission line with a loss at optical frequencies of ~ 20 db/km could be prepared economically.

3. Compact suitcase sized computers might be designed if magnetic materials with small stable magnetic domains whose

movement is rapid in modest magnetic fields could be prepared

cheaply.

Materials science, the interdisciplinary field which deals with the

preparation, characterization, and relationship of properties to bonding

and structure, is more and more coming to be the field which assumes the

responsibility for the discovery and control of properties of materials for

solid state research and electronics. The above examples of materialslimited problems have been chosen partly because they will provide strik- ing and exciting economic benefits if the materials science problems can be solved. The typical daily activities of most materials scientists are usually directed toward more modest ends. However, regardless of the

ends, materials scientists' goals are to prepare materials with desirable

properties. In order to do this they must attempt an understanding of the

connection between chemical composition, physical perfection, and other

properties of scientific or technological importance. Thus, the determination of chemical composition and physical perfection is central to progress

in materials science and hence to progress in the electronic and related

materials industries. The study of materials from this viewpoint has come

to be called materials characterization.

Frankly, the purpose of this paper is to proselytize for materials characterization. Its purpose is to convince you that the new field of characterization needs the analytical chemist and that he will be richly rewarded

by adopting its viewpoints. Clearly what is needed is a group of enthu-
SOLID STATE RESEARCH 21

siasts for characterization — and in our belief, analytical chemists constitute the logical nucleus for this group.

In the remainder of this paper we examine the problems and successes

of characterization, indicate where analytical chemistry presently contributes, and where with slight alteration in viewpoint and disciplinary

background it could play an increasingly important role. We ask forgive- ness for choosing most of our examples from our own field of electronic

single crystals. In addition to it being the field where characterization demands are perhaps most subtle, it is also where techniques of analysis

have often been honed to the highest resolution and accuracy.

We also recognize that characterization may not be an ideal word to emblazon upon the new analytical chemist's banner. As W. W. Meinke

[2] has pointed out, it does not connote the breadth of the whole of

analytical chemistry nor the concern with good sample statistics which is the hallmark of the analytical professional. Nevertheless, we recommend

it as a banner for the analytical chemists concerned with materials

science, and we particularly recommend that the viewpoints concerning

the importance of using a variety of nonchemical techniques be infused in the field. Following some examples of the problems which improper

characterization can produce, we will divide our discussion of the field of

characterization into the four main areas: Identity of Material; Determination of Major Constituents; Determination of Minor Constituents;

Determination of Physical Structure-Perfection and Defects.

Because of space considerations, exhaustive literature references to techniques cannot be given; however, we have tried to provide entry into

the literature through a few references on each of the less familiar

techniques. It is hoped that at least a few of the techniques will prove new

to analytical chemists. Similarly, elaborate documentation of the

techniques, materials, and phenomena of solid state electronics cannot be

made. Entry references are, however, provided for the less familiar subjects.

We shall begin by defining materials characterization. The most

complete study of national needs in the field of materials characterization was made by a special National Academy of Sciences and National

Academy of Engineering Committee of the Materials Advisory Board [3]

whose definition was:

"Characterization describes those features of the composition

and structure (including defects) of a material that are signifi- cant for a particular preparation, study of properties or use and

suffice for the reproduction of the material."
22 ANALYTICAL PROBLEMS

Very simply, a material is fully characterized when we know the identity,

concentration, and position of all its constituent atoms. Thus, no material can ever be fully characterized; however, many materials have been

characterized to a point where relationships between their properties and

the location and concentration of their constituent atoms could be made.

A whole spectrum of techniques from exotic physical methods to classical

analytical chemistry is employed in characterization; but the principal departure from conventionality is in viewpoint.

To understand this viewpoint we will begin by describing what characterization is not. Characterization is not the measurement of user oriented

properties alone. In electronic materials, user-oriented properties

(whether the user is a device designer or a solid state theorist) tend to be

measured to death on uncharacterized materials. For instance, no matter

how important the user may feel these properties to be, either for his theory or for his device, the measurement of conductivity and mobility of

a semiconductor single crystal do not characterize it.^ Such measurements

in conjunction with other measurements such as careful chemical analysis

may be used to determine the nature, concentration and location of those

atoms which effect conductivity and mobility. Indeed, physical measure- ments of semiconductor properties when used together with a sound

theoretical understanding of conductivity mechanisms and stringent controls on crystal preparation are among the most sensitive composition and

structure sensitive techniques in the characterizer's armory, and when

combined with other techniques, are the best means of characterizing

semiconductors. Once the number of atoms and their location are known

with appropriate accuracy, all of the other properties including conductivity and mobility are determined.

It is interesting to point out that electrical characterization will tell us nothing about electrically inactive impurities. This has been strongly

brought home to the semiconductor community by its successive characterization errors involving silicon. Silicon single crystals for some strin- gent applications are prepared starting with ultrahigh purity

trichlorosilane, (CH3)SiCl3.^ Many impurities are reduced or even essentially eliminated by careful fractional distillation. The trichlorosilane is then reduced with H2 and the resulting Si is further purified and grown

into single crystals by zone refining. To prevent contamination from the

container during zoning, a crucibleless technique, float zoning, is used. A

' Without belaboring the point, we point out that conversely chemical analysis alone does not characterize a semiconductor.

2 See Laudise [4] for a description of methods of crystal growth; and Kane and Larrabee [51 for a complete review of sil- icon characterization.
SOLID STATE RESEARCH 23

combination of electrical measurements and metallic cation analysis (principally by vacuum spark mass spectroscopy) showed that in Si produced

even with the techniques used over a decade ago, metallic impurities were

certainly below 10^-^ atoms/cm^ and probably below 10^^ atoms/cm*^ (20

ppb - 0.02 ppb atomic). This is indeed impressive and well may make Si among the purest materials man has made. Semiconductor materials people got in the habit of considering that float zone Si had less than 10^^ atoms/cm^ total impurities. However, this purity claim completely

neglects anions. A rude shock came when careful infrared measurements

[6,7] showed as much as 10^" atoms/cm^ of oxygen (2 ppm) in "high purity" Si. The detectability limit for very careful infrared absorbtion measurements at 9 fim (corresponding to an SiO stretch) is about 2X10^^

atoms/cm^, and very carefully prepared Si now has oxygen levels which

are below this level. Can we claim then that our present Si has less than 2X10^*^ impurities?

No! Recent work has shown that C contamination from the

trichlorosilane (C's distribution constant is unfortunately ^ 1 so that zone

refining does not remove it) is probably in the range 10^^ - 10^^ atoms/cm^.

C is usually detected by vacuum spark mass spectroscopy whose detection limit is about 500 ppb. More recently a-activation analysis shows

great promise for C analysis and infrared (IR) measurements at a C-O absorption band have provided increased sensitivity for C in samples of Si

deliberately grown with O.

We know less about N2 but enough to suspect it of being a worse con- taminant than C. To round out the picture, fortunately CI is volatilized

during the zone refining and has a low solubility in Si anyway so it is not an important contaminant. However, we must remember that H+ is a nonmetallic cation, very often ignored in semiconductor analysis and thought

by some to be as bad an actor in Si as C. The point for the characterizer to remember is that Si, the lynch pin of

solid state research and of the electronic industry, was thought for a long

time to have a few ppb impurities — it actually probably has a few ppm impurities. Errors of 10^ are disturbing. No one claims analysis in the ppb

range is easy. It can be claimed, however, that it's challenging and important. For instance, if enough O is present in Si (for instance, by growth in an Si02 crucible), the quality is so poor that useful devices for many applications cannot be fabricated. Mass demands for Si single crystals at the

edge of our ability to control purity and quality will soon be upon us for

the silicon single crystal vidicon tube in the PICTUREPHONE®.

Precise measurements on impure materials continue to abound in the

literature. When one leaves the Olympian heights of the semiconductor

428-999 O - LT - 72 - 3
24 ANALYTICAL PROBLEMS

researchers, impurity and casualness about purity grow apace. I will not

give literature citations so as to protect the guilty. Perhaps it is excusable

that 1970 papers on the electrical properties of nitrides and carbides do

not give information on impurities (even semiqualitative emission spec- troscopy) or stoichiometry. Carbides and nitrides are hard to prepare and

we may be forgiving. But in spite of the futility of such work, one still finds

researchers publishing (and referees accepting) color center studies on halides of unspecified or only vaguely specified purity e.g. "purchased from

Harshaw in 1968."

Data which W. G. Pfann collected from the literature [3] which are shown in Table 1 reinforce our point. The ratios of resistance at 273 and

4.2 K is often taken as a measure of purity and perfection of metals. Data

for 1961 and 1964 are given. Purity and perfection sensitive measure- ments made on, for instance, Al in 1964 may not agree with those made in

1 96 1 because Al ( 1 96 1 ) is not Al ( 1 964). We really ought to know how Al

(1961) differs from Al (1964) in order to begin to discuss discrepancies in physical measurements. Most of us usually forget to even recognize that Al(i961)^ Al(1964).

Table 1. Resistivity ratios for various metals in 1961 and 1964.

Element 1961 1964

Al 7,000 26,000

Cu 1,700 8,000

Mo 3,300 12,000

Ni 900 2,200

Re 4,000 55,000

W 6,700 80,000

Statements like 5-9's pure are, of course, almost always fraudulent. To

know something is 5-9's pure, one must analyze for all of the elements of

the periodic table with techniques having resolution of ~ 1 ppm and find

that the summation of all the impurities is < 10 ppm. I know of only a few

cases where this was actually done, although casual listings of 6-9's (or4-

9's, etc.) purity abound. The classic work of Ph. Albert [8] on zone

refined Al shows the way. We draw the veil of propriety over other examples and proceed to look at techniques of characterization.
SOLID STATE RESEARCH 25

II. Identity of Material

The grossest question we may pose about a material is "What is it?" For the solids of interest in solid state and electronics, surprisingly, we

sometimes answer even this question wrong. The principal reasons for er- rors of this sort are due to the problem of sampling and homogeneity. The

solids of interest in solid state are generally not powders where sampling

may be accomplished by variations of the classic methods like coning,

tabling, or riffling. The sample is generally a solid body — a glass sample,

a single crystal, a solid single piece of ceramic or a polymer. In the most

complicated case the analyst must choose a representative number of

such samples from either a commercial or research scale production facili- ty, must map individual samples for homogeneity and must choose

representative regions of the individual samples for analysis.

A. Mapping

Perhaps one of the greatest adjustments of viewpoint required is the

willingness to map and the willingness to consider the design and development of mapping techniques a part of the science of analysis. Among the

mapping techniques which have been used are:

7. Resistivity Probing [9, 10]

This technique generally uses small area probes (often four point

probes) with special precautions to assure ohmic contact between probe

and material. Figure 1 shows a map of a germanium single crystal. Resistivity probing is generally used to reveal impurity or dopant profiles in semiconductors. It can be made quantitative with considerable precision.

Spatial resolution of 0.05 in. (determined by the probe size and spacing)

is quite easy to achieve. Resistivity variations of ± 20 percent are quite

easy to detect. When mapping is the object only relative values of resistivity need be

compared. However, the real power of resistivity measurements is that in connection with mobility and Hall measurements they allow one to get at the charge carrier concentration in a semiconductor. Variations in resistivity can be caused by variations in impurity or dopant concentration

effecting carrier concentration, variations in stoichiometry, variations in
26 ANALYTICAL PROBLEMS

E

5 10 15 20 25

DISTANCE, mm

Figure 1. Electrical resistivity map of a Czochralski grown Ge crystal. The distances are measured along the growth axis. Resistivity variations correspond to carrier concentration variations caused by distribution constant fluctuations resulting from temperature and growth rate changes. After Slichter, W.P. and Burton, J. A., in Transistor Technology, Bridgers, H. E., Scaff, J. H. and Shive, J. N., Editors, Van Nostrand, Princeton

(1958). impurity content or perfection which affect the mobility of the charge carriers, or variations in impurity content which produce traps which in turn

alter effective carrier concentration. In typical semiconductor materials

resistivity changes are detectable at carrier concentration ranges down to about 10^^ - 10^^ atoms/cm^. Trapping centers in the range as low as lO^^ _ 10'-^ atoms/cm-^ can, however, effect carrier lifetime and mobility. Thus

lifetime and mobility measurements coupled with trace analysis (see

section on determination of minor constituents) are usually essential to make intelligent quantitative use of resistivity measurements.

2. Optical Mapping Methods

Optical mapping methods which depend upon index of refraction variations have been used mainly for glasses and crystalline laser and nonlinear

optical materials. Figure 2 shows a map of Verneuil grown ruby. Index

variation methods reveal impurity, stoichiometry, and strain variations.

They are difficult to calibrate perhaps mainly because little effort has been

expended, but they can be remarkably sensitive. For instance, it will be

shown (in the section on Determination of Major Constituents) that variations of stoichiometry in LiNbOs of less than 10~^ cause readily noticeable index of refraction variations. Materials with large stress-optic coeffi- cients will reveal small strains as observable birefringence variations.

Readily polarizable atoms or groups lead to large stress-optic coefficients

and such groups are required to produce materials with large electro-optic

and second harmonic generation coefficients. Thus, the materials of spe-
SOLID STATE RESEARCH 27

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60
28 ANALYTICAL PROBLEMS

cial interest for optical communication studies are also especially

susceptible to strain damage and especially susceptible to characterization by observation techniques dependent on index of refraction.

Another important variable which can easily be mapped optically (al- beit without much quantitative information being obtained unless considerable effort is expended) is the density of particulate matter. Tyndall

scattering of a laser beam is the quickest crude test for particulate matter. Obviously more subtle techniques like electron microscopy or careful an- gular and wavelength measurements of scattering should be used where

a serious evaluation of particulate inclusions is required. Clearly particulate matter whose size is much less than the wavelength of light will not

scatter. Indeed many kinds of non-Rayleigh scattering may be missed, but

as a quick diagnostic tool, laser light scattering applied as an adjunct of

other mapping techniques can often be very useful.

3. Etching Studies^

Etching studies reveal a variety of physical imperfections. Etching is especially useful for delineating grain boundaries, dislocations and stained

regions due to surface damage in sample preparation. Figure 3 shows an

etched specimen of magnesium-manganese ferrite. Usually etching

reveals the presence of discrete entities so that calibration is not appropriate.

4. Decoration^

Decoration provides a three dimensional map of imperfections. If one

diffuses an appropriate impurity into a crystal, it may precipitate at grain

boundaries, stained regions and dislocations.

5. X-ray Studies^

Techniques such as the Schulz technique provide a good means of

mapping low angle grain boundaries. More subtle topographic techniques

{i.e., Lang and double crystal topographs) are a means of detecting and

mapping strains and dislocations. Precision lattice parameter techniques

are a means of mapping the homogeneity of stoichiometry, impurities and

of detecting strains.

3 Discussed further in the section on Determination of Physical Structure. Laudise [4] gives an elementary discussion of these techniques, lists etches, decoration reagents, and describes the nature and genesis of physical imperfections.
SOLID STATE RESEARCH 29

Figure 3. Polycrystalline sample of Mgo.675Mno.525Fe1.8O4 etched 3 minutes at room temperature in HF-HNO3-H2O showing individual crystallite grains (500X). After Monforte,

F. R., in Laudise, R. A., Reference [4].
30 ANALYTICAL PROBLEMS

6. Electron Probe Analysis

The principle, of course, is that of x-ray fluorescence. In essence, a beam of electrons is brought to focus on a spot on the specimen of from

0.1 to 3 /Ltm. The electrons generate the characteristic x-ray spectra to depths of 1 to 3 fim below the surface. Commercial analytical instruments

are provided with optics to analyze the emitted x-rays according to wavelength and intensity and a viewing system such as an optical

microscope to aid in selecting the exact area to be analyzed. The fact that ixm sized areas can be analyzed and that the probe can be moved systematically, make it an ideal mapping tool. It is especially useful for measurement of particulate inclusions in a matrix, diffusion zones, corrosion

layers, and dust particles. Detectability limits are typically something like 100-500 ppm. Perhaps a lower limit is 30 ppm.^ By comparison, the

limits in ordinary x-ray fluorescence (albeit on large samples) can be 1 ppm; and in special cases perhaps as low as .01 ppm.

Thus the probe is very useful in what are by many solid state and elec- tronic research criteria relatively dirty samples. For those cases its spatial

resolution can be better than 1 ixm. It might be pointed out that expressed

as a detectability weight limit the probe can find lO-^^ g in a 10 cubic

micrometer volume. From this viewpoint it probably surpasses all other

instruments. Also a high ppm detectability limit should not be interpreted too literally or pessimistically. For instance, a local concentration of 100

ppm might correspond to an average concentration of 1 ppb in the whole

sample and be undetectable by bulk analysis procedures yet easily found

by the probe when it is employed in a mapping mode.

7. Laser Microprobe AnalysisU 1]

A very small area of a sample may be easily volatilized with a focused

laser pulse. The resulting vapor is excited by a spark or arc and the radiation analyzed by conventional emission spectroscopic techniques. The

trace analytical powers of emission spectroscopy are thus combined with

the spatial resolution inherent in laser optics. The laser produced vapors

might also be analyzed by mass spectroscopic techniques with much in- creased sensitivity.

* The use of cathodoluminescence can in selected cases improve the sensitivity to 1 ppm. We can expect increasing applications of this technique.
SOLID STATE RESEARCH 31

8. Autoradiography

If a material is grown, doped, diffused, or irradiated so as to contain a

radioactive species, the uniformity of dispersion of this species can be as- sessed by exposing photographic film to the sample. Detectability limits and resolution limits will be quite specific to the case at hand. The

technique has been used considerably to study diffusion profiles, segregation effects, and the shape of growing interfaces during crystal growth. It has been one of the principal means whereby nonuniformity in dopant dis- tribution in semiconductors has been recognized. Figure 4 is an autoradiograph of germanium.

9. X-irradiation

The production of color centers can sometimes be used to show in- homogeneities in the entities associated with their formation. For in- stance, Al+^ + Na+ are thought to be responsible for the smoky color center of natural quartz. X-irradiation of synthetic quartz produces

specimens like that of Figure 5 which reveals inhomogeneities of impurity

distribution.

10. Electron Micrographs

Electron micrographs are important in revealing particulate inclusions

in materials. Angstrom resolution may be achieved in favorable cases.

Identification of the inclusion is, however, much more difficult. The

scanning electron microscope has obvious advantages here.^

B. Phase Identification

The general question, "What phase do I have?" is often given short

shrift. X-ray diffraction is the classic method for phase identification and

the "Powder Diffraction File"^ is the phase identifier's bible. It should be

pointed out that the file has now been computerized [12] and that Guinier

powder patterns are now generally accepted as preferable to Debye-

^ Field ion microscopy, although more limited in applicability, is actually capable of observing atomic point defects in favorable cases.

* Published by the Joint Committee on Powder Diffraction Standards, 1845 Walnut Street, Philadelphia, Pa. 19103.
32 ANALYTICAL PROBLEMS

Figure 4. Autoradiogram of Au* doped Czochralski grown germanium crystal — striations correspond to distribution constant fluctuations and demark the growth interface. Courtesy of W. P. Slichter.
SOLID STATE RESEARCH 33

Figure 5. Inhomogeneities in X-irradiated hydrothermal quartz. Sample size is about 3 inches in maximum dimension. The variations in darkness correspond to growth on dif- ferent faces where the distribution constants for Na+ and A\+^ (which are responsible for the color center) are different. At higher magnification striae corresponding to variation in the distribution constants caused by growth rates and temperature fluctuation are visible. Courtesy of D. L. Wood, after Laudise. Reference [4]

.

Scherrer-Hull patterns because they provide higher resolution and greater

interplanar spacing accuracy. The further automation of diffractometry

will be expedited by the recent general availability of systems which

directly produces digital data. In ordinary diffraction, diffracting planes in a powder sample diffract monochromatic radiation through various angles. If the x-radiation is white, then at a given diffraction angle radiation

of discrete energies (dependent upon the atomic spacing of the diffracting

planes) will be detected. Thus, a quantum counter capable of energy dis- crimination placed at a given fixed diffraction angle can generate digital

information related to the spacings in the powder. Li-drifted detectors are appropriate and in use in a recently available system which should give

added impetus to automated phase identification [ 13]

.

Perhaps the most neglected instrument in the materials analysts' ar- mory is the microscope. If we could make one definite plea, it would be

that every instrumental methods course have at least adequate laboratory

work devoted to chemical microscopy. A specimen should always be ex-
34 ANALYTICAL PROBLEMS

amined first with a microscope before analysis of almost any sort begins.

For a powdered crystalline specimen this will often reveal that what was

supposed to be a single phase sample is actually a mixture. Examination

in crossed polarizers is helpful in this respect. Crystal habit, index and

birefringence often give clues to the identity of the material, simplifying or

permitting the omission of later, more time consuming, qualitative

analyses. The measurement of interfacial angles and the study of optical

interference figures are comparatively simple and rapid procedures which

every analyst should have at his command. Standard works on petrography or materials oriented microscopy texts like Bloss [14] should be

consulted for the details of technique. Standard compiladons [15] of the

optical properties of solids which are easily used in identification should

be at hand in the analytical laboratory.

C. Preliminary Qualitative Chemical Assessment

The classical chemical means of establishing material identity is qualitative analysis. Methods ordinarily thought of as quantitative can often be

used qualitatively or semiqualitatively as important aids to identification. Thus, several well known techniques which provide rapid nondestructive

analysis eminently suited to identification ought to be mentioned:

/ . Emission Spectroscopy

Perhaps the most generally used method of rapidly assessing a material

is emission spectroscopy. Altogether some 70 metallic and metalloid ele- ments can be determined rapidly, generally down to levels of a few ppm.

Although ordinarily thought of as a means of determining traces it often

turns out that in materials characterization the trace level of interest is considerably below its detection range. However, special procedures

have been developed which for certain elements can improve the sensitivity limits considerably. Addink [16,17] gives a good listing of sensitivity limits. Special procedures even allow the determination of anions

like C, N, O and the halogens in favorable circumstances at levels as low

as 10 ppm. Nevertheless, in modern characterization the real forte of

emission spectroscopy is rapid-survey-assessment, that is, establishing

identity and establishing what impurities in general are present above the

ppm level. Its other strength is for the careful quantitative analysis of
SOLID STATE RESEARCH 35

traces in the ppm-percent range. Obviously chemical preconcentration is a general way of improving the sensitivity of spectroscopy and most other

instrumental methods. The limits to which preconcentration can be

pushed are set by trace impurity introduction and losses of constituents

whose determination is desired during the preconcentration steps and

will, of course, be unique for each element and preconcentration

procedure.

2. X-ray Fluorescence

Ordinarily thought of as a tool for the quantitative analysis of major and

minor constituents, x-ray fluorescence spectrometry can also be very useful in qualitative identifications. Its precision is often competitive with

tedious quantitative wet analysis and even more often superior to wet

qualitative analysis. The fact that it is nondestructive is advantageous. Its sensitivity is from ~ 5-20 ppm for elements with atomic numbers above

Ti to ~ 50-200 ppm for elements with atomic numbers near C. More ef- fort on the effect of valence, coordination, electronegativity and structure on the shape, position and intensity of long wavelength x-ray spectral

lines and absorption edges could pay handsome dividends in turning x-ray fluorescence into a characterization tool of greater power and discern- ment.

3. Other Instrumental Techniques

Most of the techniques ordinarily used for the determination of major

phases and many of those used for trace analysis can be used in a survey

mode for assessing identity. Some of these methods are discussed in ap- propriate sections below, but for completeness flame emission and atomic

absorbtion spectroscopy, spectrophotometry and polarography, should be

mentioned here as often having very useful roles to play in preliminary

surveys of identity. The possibilities of automating wet inorganic qualitative analysis in a manner which has proven so successful to clinical analysis should be considered.

As a part of materials characterization the determination of phase dia- grams and the use of the phase rule viewpoint in considering homogeneity

ranges, stoichiometry problems, and defect formation should be

emphasized. The recent development of rapid, reliable, comparatively in-
36 ANALYTICAL PROBLEMS

expensive differential thermal analysis (DTA), thermalgravimetric analysis (TGA), and calorimetric apparatus should do much to expedite this ap- proach. In particular, the determination of polymorphic phase transitions

and exsolution regions is important. Because a physicist knows the identi- ty of his material at room temperature, he has no reason to assume

(without characterization studies) that it is similar, for instance, at liquid

helium temperatures. High and low temperature x-ray studies are thus an

important adjunct of characterization. Dilatometric studies, dielectric stu- dies and optical studies conducted as a function of temperature are also

often needed to establish whether a material undergoes a phase change.

To establish uniquely the order of a phase change, the most definite measurements, of course, are heat capacity determinations. In particular, we

would recommend the excellent series edited by Alper [18] as an in- troduction to the phase equilibria viewpoint and the techniques of diagram

determination from the materials science-characterization viewpoint.

Obviously one analyst cannot be a high temperature calorimeterist, a low temperature x-ray diffractionist, a dielectric studies expert and seven

other kinds of esoteric specialist. However, in addition to fulfilling the

role of analytical chemist the competent characterization group should

know when the services of some of these experts are needed, and un- derstand their strengths and limitations. Figure 9, the phase diagram of a

part of the Li20 - Nb205 system which will be discussed below, well illus- trates the sort of characterization insight the phase equilibria viewpoint can produce.

III. Determination of Major Constituents

Analytical chemistry has its center of gravity in this area. Most analytical techniques are more than good enough to establish an empirical formula with great confidence for a Daltonide compound where ± 1 percent is often acceptable. The principal problem in determining major constituents

in electronic materials analysis is in obtaining precision enough to deter- mine stoichiometry in Bertholide compounds. Thus, stoichiometry determinations will be the main subject discussed in this section. It is probably

true that conventional volumetric and gravimetric analyses are sufficient,

if only we would apply ourselves to determine elements and hence

stoichiometry within ~± 0.1 percent. After all, T. W. Richards Nobel

winning atomic weight determinations based on classic analytical

methods showed over sixty years ago that careful conventional
SOLID STATE RESEARCH 37

techniques, principally gravimetry and nephelometry, permit the deter- mination of chemical atomic weights to five significant figures. It might be

assumed that similar techniques carefully applied today could permit analysis to similar precision. If anyone is still interested in the lengths to which careful technique can push conventional methods, they might read

Richards' papers [19].

Kane and Larrabee [20] review the difficulties of determining

stoichiometry in compound semiconductors. Two examples taken from

recent work in Bell Laboratories will perhaps show the importance of

stoichiometry in electronics research and development and illustrate some novel methods of determining it. The importance of nonlinear optical materials for solid state research

and development has been reviewed elsewhere (Laudise, Carruthers and

Jackson, 1970) [21]. Suffice to say that optical communications hinges to a considerable degree on obtaining high quality modulator and parametric

oscillator crystals. Modulator crystals are needed to impress the desired

signal onto optical carriers and parametric oscillator crystals are required

to generate a variety of carrier frequencies. Modulator crystals must exhibit a large electro-optic effect, that is, a large change in index of refraction with applied electric field. Parametric oscillators require crystals with

a large coefficient of harmonic generation, that is, crystals which effi- ciently convert coherent laser light of a given frequency to a doubled

frequency.^ For both effects the polarization must depend in a nonlinear

fashion upon the field — hence, the pnenomena are called nonlinear optical

effects. Lithium niobate was one of the first practically useful nonlinear

materials discovered and it continues to be of high importance as a parametric material. The problems of stoichiometry observed in this material and the methods of observation and correction are probably

generally typical of most of the nonlinear niobates and tantalates [22]

.

For harmonic generation it is necessary that the index for the generating wave equal the index for the generated (doubled) wave throughout the

crystal. The dispersion in index with wavelength is compensated for by

the use of materials whose birefringence exceeds their dispersion. In such

materials an appropriate choice of direction and temperature leads to index and phase matching for generating wave and harmonic. It is thus important that the index and birefringence be extremely uniform throughout

the device crystal (typically several cm in length).

Crystals are grown by the Czochralski technique (pulling from the

melt). Figure 6 shows the dependence of indices (no - n^ = birefringence)

' The frequency is not always double the generating frequency, but for tutorial purposes, we will discuss this simplest case.
38 ANALYTICAL PROBLEMS

2.30

2.28

2.26 O ORDINARY

EXTRAORDINARY ^ 2.24

a: 2.2 2

2.20

2. 18

0.8 0.9 1.0 1.1 (Li/Nb) MOLE RATIO

1.2

Figure 6. Refractive index at 6,328 A of LiNbOa as a function of Li/Nb mole ratio in the melt from which the crystal was grown. After Bergman, J. G., Ashkin, A., Ballman, A. A., Dziedzic, J. M., Levistein, H. J., and Smith, R. G.,Appl. Phys. Letters 12,92 (1968); see

also Carruthers, J. R., Peterson, G. E., and Bridenbaugh, P. M., J. Appl. Phys. (in press)

for data on distribution constant and Bridenbaugh, P. M., J. Cryst. Gr. (in press) for most

recent data on growth.

Upon the Li20 - Nb205 ratio in the melt from which the single crystals are grown. From Figure 6 and the requirement that variations in birefringence

be less than ~ 10^^, the requirement that melt stoichiometry not vary more than ~ ±0.2 percent arises. If the phase diagram Nb205 — Li20 is such that for the compound LiNbOs the maximum melting point does not occur at Li20/Nb205= 1.00 {i.e., LiNbOs melts "non-congruently"), then

we can expect that Li20/Nb205 and An along a grown crystal will look

like Figure 7. Indeed Figure 7 is a superb example of careful mapping.

The linear variation is due to the fact that segregation takes place during

freezing, i.e., if for instance, the composition of the melt is such that the
SOLID STATE RESEARCH 39

\ \ 1 \ 1——I 1 1 1 1 1 1

•

• •

I

.

• •••••

• 10"

_

4 cm -

• •

• ••••

1 1 1 1 1 1 I 1 1 1 \ \—

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 POSITION ALONG X AXIS (mm)

Figure 7. Birefringence as a function of length in a Czochralski grown LiNbOa

crystal — the melt composition was Li/Nb = 1. The mapping wavelength is 0.63 /im. The

ordinate is the net phase length introduced by a compensator and is proportional to the

birefringence. After Nash, F. R., Boyd, G. D., Sargent, M. Ill, and Bridenbaugh, P. M.,

J.Appl. Phys. 41, 2564 (1970).

freezing crystal is richer in LioO than its melt, LizO will be rejected upon

freezing the melt and the crystal will thus grow richer in Li20 as it is pulled. Another way of stating the situation is to say that the effective dis- tribution constant keff= (Li^O concentration in the crystal)/(Li20 concentration in the melt) 1 . The random fluctuations superimposed upon the

linear variations are due to growth rate and temperature changes altering

keff. To further understand and control the phenomena one needs ex- tremely accurate characterization and mapping of stoichiometry variations in the crystal. Fortunately, G. E. Peterson [23] showed recently

that broad linewidth ^^Nb nuclear magnetic resonance is extremely sensitive to stoichiometry in LiNbOs. Figure 8 [23] shows that the linewidth can be used to distinguish LioO/NboOs to within ±0.2 percent. Figure 8 was made with powder samples of LiNbOs which are prepared by carefully sintering, grinding, and re-sintering of Li2C03 and Nb205 with

several re-firings and x-ray Guinier pictures used as tests for complete

reaction so as to insure sample integrity. Such procedures.combined with

very careful weighing of pure starting materials enabled the final composition of the LiNbOs powders and hence the accuracy of Figure 8 to be

established to ±0.2 percent. Single crystals give similar resonance

signals so that Figure 8 can be used to determine the composition of

428-999 O - LT - 72 - 4
40 ANALYTICAL PROBLEMS

30 I

1— 1 1 1 1 1 1 r

0 L I I I I I I I I I 46 47 48 49 50 51 52 53 54 55 MOLE PERCENT Li^O

Figure 8. Variation of half-width of low field derivative ^^Nb NMR peak as a function of composition for LiNbOa ceramic specimens. After Peterson, G. E. and Carruthers, J. R.,

Sol. St. C/zem. 1,98 (1969).

crystals grown from melts of various compositions. An additional powerful technique for deducing composition in ranges where nuclear magnetic resonance (NMR) sensitivity is unsatisfactory is the careful comparison

of ferroelectric Curie temperatures between ceramic samples of known

composition and unknown composition melt grown specimens [24].

These methods combined with conventional techniques enabled the

careful determination of the phase diagram which is shown in Figure 9

[25]. Birefringence mapping (Fig. 7) plus techniques based upon the careful determination of the temperature dependence of second harmonic

generation [26] were used together with resonance linewidth measure- ments of sections of grown crystals to determine the stoichiometry variation along a grown crystal. These techniques clearly demonstrated that as the phase diagram would predict crystals grown at any stoichiometry ex- cept 48.6 percent Li20 (the maximum in the melting point, where the

liquid and solid compositions are equal) showed extreme linear

stoichiometry and birefringence variations from end to end and large ran- dom fluctuations due to growth rate and temperature fluctuations. Such

crystals were not usable for devices. Crystals grown at 48.6 percent Li20

where the liquid and solid compositions were identical (ke//= 1.00) were

extremely homogeneous and gave good figures of merit in devices. Thus

stoichiometry characterization and control proved an important key to the

problem of securing homogeneous crystals for optical applications.
SOLID STATE RESEARCH 41

LIQUID

MOLE PERCENT U^O

Figure 9. Part of the Li20-Nb205 diagram. After Carruthers, J. R., Peterson, G. E., Grasso, M.,and Bridenbaugh, P. M. (to be published).

Figure 1 0 shows the results of a similar study of linewidth in gallium

phosphide single crystals [23]. Gallium phosphide is pulled from a melt

encapsulated under liquid B2O3 at high pressure to prevent P evaporation,

and is an important electroluminescent device material whose electrolu- minescent efficiency can vary by large factors for reasons considered by

most researchers to be obscure. Figure 10 strongly suggests a "stoichiometry problem" in GaP with growth being carried out presently

away from the congruent maximum in the Ga-P diagram. Confirmatory

experiments are underway.

Whenever nuclei of appropriate spin are present, we may expect

linewidth measurements to become an increasingly powerful tool in stoichiometry studies.

Less tractable cases will still require techniques such as the comparison

of x-ray and hydrostatic density. Even in cases where nonstoichiometry

is well established, density comparisons can shed light on the mechanism

of charge compensation in off-stoichiometric materials. Typical of this is
42 ANALYTICAL PROBLEMS

0 I I I I I I I I I 01 2345678

LOCATION IN INGOT

Figure 10. Peak-to-peak NMR derivative height vs ingot location in pulled GaP normalized so that P-P height = 100= starting material. After Peterson, G. E., Carnevale, A. and Verieur, H. , Mat. Res. Bull. 6,51 (1 970).

Barns' and Carruthers' study [28] of LiTaOs. The generalized defect

model formula which resulted from this study is

Li(l -(5-n)a:)Tai + a-nxe~03.

The existence region was found to be 46.0 to 50.4 mole percent Li20 at room temperature. Charge compensation by oxygen vacancies was deemed implausible on the basis of optical spectroscopy studies. Since the

pycnometric density was found to be larger than the x-ray density, it was

reasonable to assume that the excess tantalum atoms are compensated for
SOLID STATE RESEARCH 43

by lithium vacancies and by excess electrons. Depending upon the

stoichiometry the value of n varies between 0 and 2; the most probable

values suggesting that the formula is Lii_4jTai+j:xe~03. Presently scatter

in n values when data is fitted to the densities causes considerable concern. This is somewhat mitigated by the fact that n values calculated using

only the Li/Nb ratio are in agreement [29]. Improvements in pycnometric density and Li/Nb ratio determinations are required, especially if one

wishes to search for oxygen lattice defects. Coulometry is applicable both to major constituent and trace analysis.

It can be especially precise for the determination of major constituents.

Reliability at the 0.002 percent level can be obtained in favorable cases. Thus, it could find an important role in stoichiometry determinations.

Marinenko [30] at the National Bureau of Standards has recently shown

that constant current coulometric titrations with amperometric end point

determination can determine stoichiometry with great precision. One important advantage is that since the technique is essentially titration with

electrons, its accuracy can be independent of calibration of standards.

Provided the reaction actually going on is the reaction calculated and pro- vided no losses occur in preparing the material for reaction, the precision

obtained may in the limit be set by the precision to which we know the

value of the Faraday.

In nonstoichiometry above 0.1 percent, volumetric methods are conventionally employed. For instance, Bachelder and Sparrow [31] fused

InSb with Na2C03 + S and then dissolved in dilute HC 1. After oxidation

of any unreacted S with KCIO4, the antimony was titrated iodimetrically.

Polarography is applicable to trace, minor, and sometimes even major,

constituents in solution. In principle any element subject to a redox reaction can be determined. The behavior of some 80 elements has been

discussed. Sensitivities of from 0. 1 to 0.001 ppm can be obtained. The use

of this technique for trace analysis and minor constituent analysis is well

known. Its precision near the detectability limit is usually reported as ~

20 percent generally with precisions near 1 percent in favorable concentration ranges. Differential techniques have pushed precision to 0.02 - 0.05 percent in some cases. Serious application of the technique for

stoichiometry determination would now be appropriate.

Perhaps the most sensitive stoichiometry dependent property is lattice parameter. Calibration here is a problem. In the simpler case where one

considers substitutional impurities on a lattice, estimates of accuracy are

possible. If we assume Vegards law is obeyed, when two end members

have lattice parameters differing by 10 percent, then given a standard

deviation in the lattice spacing measurement of 1 ppm the concentration
44 ANALYTICAL PROBLEMS

of one in the other can be determined to 0.001 percent. Barns has shown

that these numbers can be realized in practice [32]

.

In addition to stoichiometry determination, the other main problem of

major constituent analysis in electronic materials is the determination of

oxidation state. The precise determination of oxidation state of an element in a com- pound is a recurrent problem. To a first approximation some difficulties of oxidation state determination are semantic rather than analytical. They

arise because of confusion between formal charge and real charge. The

chemist or materials scientist has every right to ask the analyst to help him

determine formal charge. The determination of real charge is much more

subtle and has only recently begun to be capable of measurement. Once

exact stoichiometry has been determined, it is conventional to round off

to the nearest whole numbers and to assign the formal charge {i.e.,

valence) so as to preserve electrical neutrality. This presents difficulties requiring analytical assistance when more than one multivalent element

is present in the same compound or where several valency assignments

are possible for a given element in the compound, e.g., iron in Prussian

blue. Classical wet chemical and electrochemical methods are applicable, if one can assure himself that dissolving doesn't alter valence. A precision

of perhaps 0.01 percent might be expected in fortunate cases. For ap- propriate nuclei (Fe, Sn, I, Au, and some rare earths) Mossbauer spec- troscopy is applicable with a precision of 0. 1 percent and no worries about

the need of dissolving. The detectability limit is somewhere around the

fraction of 1 percent level in favorable cases. Mossbauer spectroscopy

has shown that the oxidation states of the Fe in Prussian blue are Fe4"^ [Fe"(CN)6]3 [33] . Visual color observadon and absorption spectroscopy

is also sometimes useful in estabhshing oxidation state. At a higher level of subtlety we may ask what is the true charge on a

particular element, taking into account its actual environment, degree of

covalency of its bond, etc. nuclear quadrupole resonance (NQR) spec- troscopy can, in some instances, provide interesting insights into this question. Figure 1 1 shows the results of some NQR studies in which the

actual charges were calculated. These studies are especially interesting

because they provide one of the first direct measurements of one of the

chemist's favorite concepts — degree of covalency.
SOLID STATE RESEARCH 45

6.0

o

o

^4 I

2

Z

<zo

oo

z

2.0

2.2 2.3 2.4 2.5 2.6 ELECTRONEGATIVITY

Figure 11. Nuclear quadrupole coupling constant of ^^Cu in chalcopyrites plotted against the electronegativity of the chalcogen. After Peterson, G. E., in Magnetic Resonance,

Coogan, C. K.,et a/., editors, Plenum Press, New York (1970).

The analysis of impurity levels in the range of from 0. 1 percent to 1 ppb

received great impetus from the necessity of assaying and controlling

trace elements in semiconductors. There are three mainstays of trace

analysis:

This is probably the most widely used tool for assessing the quality of

pure materials. It has found its full glory in the metals industry, especially

in basic oxygen steel making where automatic spectrographic analysis

coupled with computer process control provides direct feedback of the

results of analysis into the process. The principal reason why emission

spectroscopy has found such wide acceptance in metals processing (other

than economies of scale which enable the results of one series of analyses

to be used to control the properties of many tons of steel) is that the concentration range of interest from ~ 100 ppm to 1 percent is generally in the range of good sensitivity.

For the technique to be generally useful in the impurity range of interest

in semiconductors either preconcentration or refinement of the source is

IV. Determination of Minor Constituents

A. Emission Spectroscopy
46 ANALYTICAL PROBLEMS

generally required. Preconcentration techniques have been used for elec- tronic materials spectroscopy, especially for Ge, Si, and GaAs. However,

preconcentration procedures all suffer from inaccuracies due to the necessity of introducing additional reagents (which are contaminant sources) in the chemical preconcentration steps and due to the unavoidable losses

which occur during those steps. Thus, direct methods are almost always

to be preferred if appropriate sensitivity can be achieved.

The principal problem for Si and Ge is that oxide bands obscure the

spectrum of most impurities. The conventional expedients are to strike the arc in argon or nitrogen or to use the so-called split-burn technique.

Since impurities are selectively volatilized in the arc, the background can be reduced with respect to any one impurity by moving the plate during

the burn. Because the oxide problem is less, the better sensitivity is obtained in compound semiconductors like GaAs. Generally the important

anions, especially oxygen, sulfur, and the halogens cannot be determined

with reasonable sensitivities by emission spectroscopy.

B. Flame Emission and Atomic Absorption Spectroscopy

These two methods are complementary in that often elements with poor

detection limits for one technique have good limits for the other. In favorable cases 0.01 ppm can be detected in solution; a few ppm is comparatively routine. Alkalis and alkaline earths have the best detection

limits, while metaloids and metals have poor detection limits. Matrix ef- fects, dilution effects by dissolving, and contaminant interferences are problems.

C. Vacuum Spark Mass Spectrometry

Mass spectroscopy is of broader application and of higher sensitivity

than emission spectroscopy. Modifications of Hannay and Ahearn's spec- trometer [35] have been commercially available for over ten years and

are now the work horses of trace analysis in most solid state laboratories.

The general rule is that sensitivity is 10^ better than emission spectroscopy

(Cu is an important exception, emission spectroscopy is more sensitive).

About 3 ppb atomic is possible and the Picker-Nuclear bulletins are recommended as an especially comprehensive listing of sensitivities for

various elements [36].
SOLID STATE RESEARCH 47

D. Activation Analysis

Emission and mass spectroscopy are probably most valuable because

they are comparatively rapid survey methods. Actually their sensitivity

does not reach to the limits of the levels where impurities can cause electrical effects. For instance, perhaps the best that can be expected from mass spectroscopy is a limit of 2 ppb atomic which corresponds to ~ 10^^ atoms/cm^ on Si. This is not an unusual doping range; dopings to 1/5, and

even occasionally 1/10 this level, are not unknown. Undesired impurities

which act as traps can have effects much below this level. When the

proper conditions are met, activation analysis can reach to these levels. Generally only the highest thermal neutron fluxes such as are obtainable

only in large nuclear reactors give sensitivities of interest for the impurity

levels found in semiconductors.^ Two classes of impurities are amenable:

1. Those with high enough neutron cross sections and long

enough half lives so that the induced activity can be dealt with

(at fairly low impurity concentrations) at sites removed from the

reactor. That is, half lives greater than ~ 24 h still permit

reasonable induced activities.

2. Those whose cross sections are small but have short half

lives less than ~ 24 h so that reasonable activities, and hence

impurity concentrations, can be dealt with if measurements are made quickly following irradiation. Obviously when half life is long and cross section is small, the element

may not be detected. Interferences can be dealt with by chemical separation, by energy and particle dependent radiation detection, and by distin- guishing materials of different half lives. The ideal cases where no contamination or losses will occur are those where no chemical processing is necessary. Some work on the analysis of anions by activation analysis has

been published, but much remains to be done in this area. When specific

activity requirements are met, an important limitation in reproducibility

is often sample homogeneity. Perhaps the most important advantages of

activation analysis are that often chemical processing can be avoided and

usually the problem of standards is obviated because direct calculation of

concentrations from absolute activity is possible. One cannot claim to be

seriously involved in trace analysis at the levels of interest for electronic

materials unless he has activation analysis capabilities.

* Ion activation analysis {e.g., He^ for O in Si and GaP) and a-activation analysis {e.g., for C in Si) are important activation techniques, enabling the determination of materials where interferences or neutron cross section-half life considerations

limit the sensitivity of neutron activation.
48 ANALYTICAL PROBLEMS

E. Isotope Dilution

Isotope dilution is useful when cross section-life time constraints

prevent the attainment of the desired sensitivity by activation analysis.

The method is advantageous in that quantitative separation methods need

not be available for the system under study. All that need be measured is the specific activity of the isotope added, its weight, and the specific activity of an aliquot taken from the sample after one has assured that the

added isotope is uniformly dispersed. To assure uniform dispersion it is helpful if the isotope and the sample can be dissolved. Accuracy comparable to activation analysis is possible. The technique is limited by the

availability of isotopes of appropriate activity of the elements desired. It clearly should be used much more often for trace analysis.

F. CoPREx Technique

C. L. Luke and his coworkers [37] have used preconcentration in combination with x-ray fluorescence to achieve sensitivity levels in the ppbppm range. The unknown is dissolved and the desired element or ele- ments are precipitated with an appropriate group reagent and isolated by

filtration onto, for instance, a Milipore filter. Conventional x-ray fluorescence of the filter gives very good sensifivities and contamination

and losses in the preconcentration steps can be kept to a minimum. The

technique can rival radiochemical techniques for sensitivity and obviously

has advantages of convenience.

G. Other Methods

Nuclear magnetic resonance studies have been applied to a variety of

trace analysis problems. Sensitivity varies with the type of nucleus

present but is generally in the 10^^ nuclei/cm^ or 0. 1 atomic percent range. However, double resonance schemes can push sensitivity to the 10^^ range. Electron spin resonance is useful for detecting paramagnetic atoms

and electrons. The optimum cases are in the range 10^^ spins/cm^, which

can make it. fantastically sensitive for free radicals, color centers, and

transition metals. The usefulness of resonance as a general analytical tool depends to a large degree upon inventive modifications of existing

techniques and the willingness of resonance researchers to undertake

characterization.
SOLID STATE RESEARCH 49

Vacuum fusion is, of course, useful for determining minor constituents

when they are gaseous. It can also be a very sensitive method for analyzing for traces. Recently more rapid apparatus [38] allowing 60+ samples/day to be analyzed makes the technique considerably more attractive. Precision and accuracy depend to an unusual degree upon the

calibration situation. Other methods of trace analysis which have been applied with some

degree of success to semiconductor materials include fluorometric analysis, polarographic analysis, and infrared absorptometry. IR absorption

has proven especially useful in characterizing oxygen in Si and was

discussed in the Introduction. In quartz [39] it has been shown that protons cause an absorption

(determined by the OH stretch) at 3 ^tm and careful analysis of crystalline

quartz has proven correlations of H+ concentration with acoustic Q.

Production of high acoustic Q quartz was brought about to a considerable

degree because of this characterization discovery.

Trace analysis in inorganic electronic materials has not progressed

anywhere as far as that for semiconductors. Perhaps in part because in general properties are not so trace element sensitive, but also due in large

degree because matrix interference is often more severe. It is often easier

to get consistent relative results with a single method than any sort of

reasonable agreement with various methods so as to convince one that absolute accuracy has been obtained. Table 2 showing the results of careful

crystalline quartz analysis, brings this point home. An important problem

in trace analysis which is often overlooked is that when methods which

sample very small volumes of a sample give poor agreement in replicate

analysis, the problem may often be unrecognized short distance sample

inhomogeneities. An additional requirement for improving most methods

is the availability of better standards.

An especially important technological problem where progress is probably at least partly trace analysis limited, is the production of low loss glasses for laser communications. Requirements for losses at optical

frequencies of less than 20 db/km will necessitate extremely pure (2 ppb

for some transition metals) glasses.

V. Determination of Physical Structure — Perfection and Defects

The determination of physical structure and perfection should be one of

the foundation stones of characterization and begins with the accurate
ANALYTICAL PROBLEMS

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SOLID STATE RESEARCH 51

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52 ANALYTICAL PROBLEMS

determination of structure. The principal techniques used are those of x- ray diffraction. The x-ray techniques applied to perfection are quite dif- ferent from the more familiar methods used in structure determination.

Serious perfection studies almost always involve the use of a wide variety

of additional techniques often including etching, decoration, electron

microscopy and careful density measurement. Analytical chemists in characterization will find it necessary to be aware of the power and limita- tions of these techniques. Analytical groups concerned with characterization will often find it worthwhile to include experts with a mastery of one

or more of these techniques at their command, and university training pro- grams for analytical chemists may include perfection determination options (especially x-ray techniques). Following some discussions concerning structure, we can do hardly more here than to recite a brief litany of

perfection techniques, give some indication of their applicability and pro- vide a few entry references, so as to give the reader a feel for the scope of

this often neglected field. Lattice parameters and symmetry determinations from x-ray powder

pictures were discussed above. However, it is important to point out that

at the conclusion of a symmetry determination it is only fair to say

"Presently, this material has no higher symmetry than that found. Subsequent more subtle tests may reveal lower symmetry." Many x-ray

crystallographers fail to realize that for some materials physical tests other than x-ray diffraction are more subtle at revealing symmetry. Optical tests such as birefringence, interference figures, piezoelectric tests with a Geibe-Schiebe circuit and powder second harmonic generation

tests [40] ought to be mandatory checks for a lack of a center of symmetry used in conjunction with x-ray investigations. Complete structural determinations are now, of course, greatly accelerated by general availability of automatic diffractometers [41]. In many electronic materials it is important to determine the sense of

the electric axis. Two techniques are in general use. The first technique

[42] involves the measurement of the integrated intensity of Friedel pairs

in transmission through thin plates of the material. A preferable method

has been given recently [43] which makes use of the scattering close to an

absorption edge of a constituent element where differences in the anomalous scattering are a maximum.

Determination of the sign of an electric axis is an especially ulcer

producing occupation. One cannot take refuge in statements or estimates

of accuracy of precision. You are either 100 percent right or 100 percent

wrong.
SOLID STATE RESEARCH 53

Once orientation and structure are established, the next question which

often arises is "Is the material a single crystal?", that is, does it contain

grain boundaries large enough to limit its usefulness. Grain boundaries

and twins may be revealed by optical examination (often between crossed

polarizers) by re-entrant angles in single crystals, by etching studies, or by

x-ray techniques (commonly the doubling of Laue spots). Grain boundaries are revealed conveniently by Schulz x-ray pictures [44] . The Schulz

technique is very much neglected and provides such a straightforward

evaluation technique that it should be more widely practiced. Figure 12

shows the geometry and Figure 1 3 a typical picture. The single crystal is

Figure 1 2. Schulz technique — schematic of apparatus arrangement.

placed at an angle of about 25° to a nearly parallel x-ray beam. White

radiation is used. The several spots produced on the film are merely Laue

spots, about the same size and shape as the specimen. Each of the spots

will be made up of spots of slightly different orientation, each corresponding to a region in the crystal oriented slightly. Resolution is of the order of

0.1°.In magnetic and ferroelectric materials it is often important to carry out many studies with crystals which contain only one magnetic or electric domain. Magnetic domains are revealed in transparent specimens by

microscopic observation in crossed polarizers (Faraday rotation of the

polarization depends upon the sense of the magnetic axis). In opaque

specimens one is forced to resort to magnetic colloid techniques (Bitter

patterns) to observe domains. In these techniques the domains orient

small particles of colloidally dispersed magnetic oxides which are smeared on the surface to be examined (or placed close to the surface in
54 ANALYTICAL PROBLEMS

Figure 13. Schulz picture of Verneuil grown sapphire. Spot size and shape correspond to sample size and shape. Approximately rectangular region in spot is misoriented region in sample. Courtesy of R. L. Barns, after Laudise, Reference [4].

a container arranged for easy viewing, i.e., a commercial magnetic tape

"reader"). The ultimate method for examining magnetic structure is, of

course, neutron diffraction. (See, for instance, Schieber [45] for a review

of these techniques.)

The problem of detecting domains in a ferroelectric is analogous. However, birefringence differences brought on by electric dipole reversal are sometimes not observable between crossed polarizers and electric dipoles

tend to be easily neutralized by surface charges so that colloid techniques

are unreliable. The direct measurement of the pyroelectric constant, a, is the best way to assure that a given ferroelectric specimen consists of a sin- gle electric domain. It will be remembered that a = dPsldT where Ps is the

spontaneous polarization which is related to the magnitude and direction

of the electric dipoles. Etching and optical studies can sometimes be used

to reveal electrical twinning in piezoelectrics.
SOLID STATE RESEARCH 55

At a considerably higher level of perfection, {i.e., ^10^"^ dislocations/

cm^) dislocations can be studied by:

1. X-ray Techniques

X-ray techniques include the back reflection Berg-Barrett technique;

transmission (diffraction) Berg-Barrett technique, Bormann anomalous

transmission technique, Lang topographs, double crystal rocking curve

photographs, and others. All of these techniques are quite specialized and

have been described in the literature so that no detailed remarks are required here. (See Newkirk and Mallett [46] for a review of x-ray techniques used in perfection studies.)

For illustrative purposes we will consider the Lang topograph

technique. In the Lang technique a highly collimated narrow beam of x- rays is diffracted through the crystal and the Bragg reflection from the

parallel planes approximately normal to the fact of the specimen is recorded on the film. The film and specimen are moved simultaneously

and a complete "map" of the dislocation network can be obtained. The

dislocations are observed as regions of intensified reflecting power

through their action in altering the distribution of energy between multiple

reflected primary and diffracted beams. The result is that dislocations

produce dark regions on the film. Figure 14 shows a typical Lang topograph. The hardest thing for a neophyte to glean from the literature is what can be expected of each x- ray technique. In Table 3 we have tried to assemble this information.

References [4] and [46] should be consulted for "entry" references to each technique.

2. Etching Studies

Etch pits delineate regions of strain and often there is a one-to-one cor- respondence between etch pits and dislocations. The correspondence

between etch pits and dislocations can be shown by a combination of x- ray studies and etch studies by correlation with plastic deformation [47]

or a count of etch pits along a low angle grain boundary and experimental

correlation of the relationship between pits in the various regions [47].

Etching studies also reveal damage due to surface preparation. Since

etching is so easy to perform, it should be considered a standard tool in

428-999 O - LT - 72 - 5

SOLID STATE RESEARCH 57

Table 3. X-ray techniques for studying perfection.

1. Double crystal

spectrometer^'^

2. Schulz technique

3. Back reflection,

Berg—Barrett technique^

4. Transmission diffraction,

Berg—Barrett technique^

5. Bormann Anomaloustransmission technique^'^

6. Lang technique^

7. Lattice parameter

measurements

8. X—ray interferometer^''^

measures misorientations as small as 0.01 sec. of arc; maps dislocation

networks; investigates individual

dislocations. Measures lattice spacing variations as small as

0.01 A. measures misorientations of a few

minutes to several degrees.

maps dislocation networks,

investigates individual dislocations.

maps dislocation networks,

investigates individual dislocations.

maps dislocation networks,

investigates individual dislocations.

maps dislocation networks,

investigates individual dislocations. measures absolute and changes in lattice parameter and expansion

coefficient to 1 ppm. Critical tests of symmetry. measures misorientations of 0.01

sec. of arc; maps dislocation

networks, investigates individual

dislocations; effective only with

rather perfect crystals.

Best spatial resolution

Best sensitivity to small strains
58 ANALYTICAL PROBLEMS

the materials characterizer's repertoire (see Laudise [4] for a review of

etching and a listing of etches).

3. Characterizing Vacancies

Characterizing vacancies involves the most careful technique and most

successful studies have involved correlation of x-ray and pycnometric

density. If the vacancy serves as a trap, for instance, the resulting center may be susceptible to optical or electron paramagnetic resonance (EPR) studies.

4. Decoration

Decoration, the selective precipitation of a diffusant along low angle

grain boundaries and at dislocations is especially useful because it can

provide three dimensional maps of perfection (see Lark-Horovitz and

Johnson [48] for detailed discussion).

Ion probes, especially when used in channeling studies, show great

promise in allowing the direct determination of whether ions are substitutional or interstitial. Determining whether an impurity is substitutional or interstitial is also

sometimes possible by careful density comparisons.^ Its electrical NMR,

EPR or NQR behavior will often be revealing. In many cases the delib- erate doping with a particular impurity combined with a careful study

of the effect of co-doping with other impurities on its distribution constant may be a convenient means of establishing whether the impurity

is substitutional or interstitial. For instance, in the lattice A+C , if we dope with D+ + , which can go either (a) substitutionally at the A+

site or (b) interstitially, the number of C" vacancies will be one for case

(a) and two for case (b). If we co-dope with E= which is known to go

to the C" site by the law of mass action for the two cases, we have

(for melt growth):

(a) (D++)n,elt+ (E=)„,elt - [D++]A+site+ [E=]c-site

(b) (D++)n,elt+ 2(E=)„,elt ^ interstitial + 2[E+]c-sites

" Bearden and Smakula have both considered this method. A more accurate recent value of Avogadro's number, together with Bowman's careful work, on density measurements, have considerably increased recent accuracy. In addition, one must be sure that tabulated isotopic abundances actually apply to the sample at hand [49,50].
SOLID STATE RESEARCH 59

For the two cases

(a) C,SD meltD(E )melt

(b) CsD=C:meltD melt where Csd is the concentration of D in the solid and Cmeit d its concentration in the melt. This relationship can be easily cast into conventional dis- tribution constant terms.

Another way of surveying characterization, particularly those aspects

which are conventionally within the domain of analytical chemistry, is to

list the techniques and state what they can do. Table 4 taken from

Reference [3] is a convenient summary arranged in this fashion.

Reference [3] also gives a consensus as to limitations of existing

techniques. Many strengths and weaknesses of present techniques

become apparent from a survey of Table 4.

It is clear that solid state research and electronics has great needs for more and better characterization activity and that analytical chemistry

could serve as the core discipline for this activity. At the risk of being at best parochial and at worst outright wrong we will identify those areas which we think are crucially in need of additional support together with some predictions of future directions in characterization:

1. The development of mapping techniques for sample

homogeneity determination should be fostered. Perhaps it would

be more realistic to admit that many crucial problems could be

dealt with by present mapping techniques if only researchers

and analysts would be sensitized to the need for mapping and

willing to apply known techniques. The importance of mapping

is readily apparent when we realize that the vitally important

economic matter of yield {i.e., percentage of usable devices

emerging for a multi-step processing line) in integrated circuits can be related to homogeneity and defect mapping. We can ex- pect here that subtle x-ray techniques will more and more be

used as process controls.

2. The more widespread use of the optical microscope in analytical work is appropriate. No new techniques are required

here. An increased emphasis on the microscope in instrumental

VI. Conclusion
60 ANALYTICAL PROBLEMS

Table 4. Precision and sensitivity of analytical techniques.

Technique

Wet chemistry — titrimetry

Wet chemistry — gravimetry

Wet chemistry

Coulometry

Mossbauer

spectroscopy

Electron probe

microanalysis

Emission

spectroscopy

Spark source mass

spectrometry

Atomic absorption

Flame emission

Spectrophotometry

Polarography

Applications

Major and minor

phase —

concentration;

also impurities

Major and minor

phase —

concentration

Major phase —

valence

Major phase —

concentration

Major phase —

valence also

impurities —

valence

Homogeneity of

major phase

minor phases

Impurities

( survey)

Impurities

( survey)

Impurities

Impurities

Impurities

Impurities

Sensitivity

10" ^M in solution

10~5 m in solution

10-6 _ 10-7 M in solution

1000 ppm/1 g

sample

Neutron activation

Vacuum fusion —

mass spectrometry

X—ray fluorescence

spectrometry

Impurities

Impurities —

02,N2,H2

Major and minor

constituents

Down to 0. 1% -

0.0001% depending

upon density of

matrix

Down to 0.1% over

a 1 — 5 fi m scan

diameter

0.1-100 ppm

0.01-0.1 ppm

0.005-0.1 ppm in solution 0.1—10

ppm in solution

0.002-0.1 ppm in solution 0.1-10

ppm in solution

0.0005-0.1 ppm

in solution

All in solution:

0.1—1 ppm

10-100 ppm

.005 ppm

0.001 ppm (with

anodic stripping

preconcentration)

0.001-0.01 ppm

0.7 ppm

100 ppm

20 - 200 ppm generally; 0.1 at best

Precision (%)

0.01

0.1

0.2-1.0

0.01

0.01

0.001-0.005

Semiquantitative now

0.5

5-10

Semiquantitative now 5-10

1-5

5-10

1-5

5-10

2-10

0.1-0.2

20

5-10

2-10

20

5

0.1
SOLID STATE RESEARCH

analysis courses would do much to make microscope

techniques more widely used. Their use would not solve any

unique solid state problems; it would, however, greatly expedite

many routine identifications.

3. Development of new techniques and the more widespread

application of known techniques to stoichiometry determination

are required. In solid state technology the 50's and 60's were to a large degree the era of the elemental semiconductors. Binary

compound semiconductors are now well established and ternary

materials are of increasing interest. The 1970's will see increasing emphasis on binary and ternary compounds, not only for

semiconductor research and development but also in nonlinear

optical and magnetic applications. As the interest in and commercial importance of compounds grows, so will the need for

the accurate assessment of compound stoichiometry. X-ray and

hydrostatic density studies, specialized resonance determinations and coulometric analysis should all become of increasing

importance.

4. Reliable methods for trace analysis at the ppb level and

below should be developed. In the 1970's we may expect that

electroluminescent lamps will begin to find wider practical application. Electroluminescent efficiency is often dependent

upon trap concentration well below the detectability limits of mass spectroscopy. Low loss laser transmission glasses require

impurity levels to be controlled in the ppb level. The techniques

showing most promise at these levels are activation analysis,

coprex and isotope dilution. Increasing attention should and

will be paid to these techniques. We may even have to grow ac- customed to thinking of mass spectroscopy as a survey

technique for relatively dirty materials.

5. The determination of perfection and defects should become

an orderly sub-discipline with its own practitioners. Presently

there are isolated individuals with great expertise in, for in- stance, the use of particular x-ray perfection techniques. It would be most salutary if some researchers developed a broad

interest in perfection assessment. Steps to encourage this development might be taken by means of emphasis on x-ray

perfection studies in characterization training, emphasis on per- fection studies amongst professional x-ray researchers, and in- creasing movement of analytical chemists into the area. This

field is every bit as big in scope as the chemical analysis part of
62 ANALYTICAL PROBLEMS

characterization and just as essential. It is critically un- dermanned and should receive every encouragement.

To conclude, what materials characterization truly needs is the active

support and encouragement of a cadre of trained professionals. It is my

belief that this group of research and applied scientists should nucleate

around the analytical chemists. These are the scientists who take pride in sample statistics, to whom ingenuity in deriving new methods is coupled

with pride in the painstaking application of tried and true procedures.

Couple these attitudes with:

1 . An appreciation and knowledge of x-ray techniques which

I believe are extremely powerful in elucidating the physical per- fection of materials.

2. A familiarity with and willingness to make use of solid

state physical measurements such as conductivity and

resonance, and

3. A willingness to push reliable trace analysis techniques,

especially radiochemistry well into the ppb range. With this mix of attitudes and techniques I have little doubt that many

of the next important developments in solid state-materials science will be

catalyzed by the analytical chemist-materials characterizer.

VII. Acknowledgments

I would like to acknowledge many discussions concerning various

aspects of analysis and characterization with R. L. Barns, J. R. Carruthers, G. E. Peterson and H. D. Keith, and to thank K. Nassau for

comments on the manuscript.

VIII. References

[1] Electronics, January 4, 1971, p. 35 ff- [2] Meinke, W. y/.,Anal. Chem. 42, 26A (1970).

[3] MAB Report 229-M, 1967, Washington, D. C.

[4] Laudise, R. A., The Growth of Single Crystals, Prentice Hail, Englewood Cliffs, New Jersey (1970).

[5] Kane, P. F. and Larrabee, G. B., Characterization of Semiconductor Materials,

McGraw-Hill, New York (1970).
SOLID STATE RESEARCH 63

Kaiser, W., Keck, P. H. and Lange,C. F.,Phys. Rev. 101, 1264 (1956). Kaiser, W. and Keck, P. H.J. Appl Phys. 38, 882 (1957). Albert, Ph., Modern Trends in Activation Analysis, Proceedings of 1961 International Conference, College Station, Texas, December 15-16, 1961 , p. 86 (1962). Valdes, L. B., Proceedings ofthe IRD, 42, 420 (1954). Lark-Horovitz, K. and Johnson, V., Editors, Solid State Physics, Vol. 6 Part B, Electrical, Magnetic and Optical Properties, Academic Press, New York (1959). Adams, M.D. and Tong, S. C.,Anal. Chem 40, 1762 (1968). Vand, V. and Johnson, J., Fortran IV Program - Version Xfor the Identification of Multiphase Powder Diffraction Patterns, American Society for Testing Materials, Philadelphia (1969). Gressen, B. C. and Gordon, G. E. , Norelco Reporter 17,19(1 970). Bloss, F. D., Editor,/4A2 Introduction to the Methods ofOptical Crystallography, Holt

Reinholdt and Winston, New York (1961). Winchel, A. and Winchel, H., The Microscopical Character ofArtificial Inorganic

Substances, Academic Press, New York (1964). Addink, N. W. U.,Spectrochem. Acta 11, 168 (1957). Addink, N. W. H.,Z.Anal. Chem. 208,81 (1964). Alper, A. M., Editor, Phase Diagrams, Materials Science and Technology (three volumes). Academic Press, New York (1970). Richards, T. W. and Forbes, G. S., Carnegie Inst. Wash. Publ. 69, 47 (1907); Z. Anorg. Chem. 55, 34 (1907). Kane, P. F. and Larrabee, G. B., Characterization of Semiconductor Materials,

McGraw Hill, New York (1970). Laudise, R. A., Carruthers, J. R., and Jackson, K. A., Annual Reviews of Materials

Science, 1971, in press; see also Bergman, J. G. and Kurtz, S. K., Materials Science and Engineering, 5, 235 (1970); Laudise, R. A., Bell Labs Record (January 1968); Laudise, R. A., Proceedings of the International Conference on Preparation and

Characterization of Electronic Materials, Chania, Crete (July 1969), to be published.

Ballman, A. A., J. Am. Ceram. Soc. 48, 1 12 (1965); see also Reference [21]. Peterson, G. E. and Carruthers, J. R. , 7. Sol. St. Chem. 1,98(1 969). See Reference of Figure 9. Bridenbaugh, P. M.,J. Cryst. Gr., in press; see also Reference [23]. See Nash et al. , Reference of Figure 7. Peterson, G. E. and Verleur, H., to be published. Barns, R. L. and Carruthers, J. R.,J. Appl. Cryst. 3, 395 (1970). Laudise, R. A., to be published.

Marinenko, G., to be published; see also Marinenko, G. and Taylor, J. K., Anal. Chem. 39, 1569(1967).

Bachelder, M. C. and Sparrow, P. M.,Anal. Chem. 29, 149 (1957). Barns, R. L., Mats. Res. Bull. 2,273 (1967). Robin, M. B., Ivory Chem. 1,337 (1962). See Reference [20] , p. 3 1. Hannay, N. B. and Ahearn, A. J., Anal. Chem. 26, 1956 (1954). Picker-Nuclear, 1275 Marnaroneck Ave., White Plains, New York. Luke, C. L.,Anal. Chem. Acta 41, 237 (1968); also Private Communication.

Von Thaddaus, A., Arc. Eisenhuttenw. 33, 527 (1962). King, J. C, Wood, D. L. and Dodd, D. M.,Phys. Rev. Lett. 4, 500 (1960); Ballman, A.

A., Dodd, D. M., Keubler, N.A. Laudise, R. A., Wood, D. L., and Rudd, D. W.,

Appl. Opt.l, 1387 (1968). [40] Kurtz, S. K. and Perry, T. T., J. Appl. Phys. 39,3798 (1968).
64 ANALYTICAL PROBLEMS

[41] See for instance, Abrahams, S. C, Rev. Sci. Instr. 33, 973 (1962); Cetlin, B. B. and Abrahams, S. CActa Cryst. 16, 943 (1963). [42] Abrahams, S. C, Reddy, J. M. and Bernstein, J. L., J. Phys. Chem. Solids 27, 997

(1966). [43] Bams, R. L., Keve, E. T. and Abrahams, S. C.,J. Appl. Cryst. 3, 27 (1970). [44] Schulz, L. G., Trans. AIME 200, 1082 (1954). [45] Schieber, M. M., Experimental Magnetochemistry, North Holland, Amsterdam

(1967). [46] Newkirk, J. B. and Mallett, G. R., Editors, Advances in X-ray Analysis, Vol. 10, Plenum Press, New York (1967); see also Reference [4]. [47] Gilman, J. J., Johnston, W. G. and Sears, G. W., J. Appl. Phys. 29,1Al (1958); Pfann, W. G. and Lovell, L. C, Acta Met. 3, 512 (1955); Vogel, F. L.,Acta Met. 3, 245

(1955). [48] Lark-Horovitz, K. and Johnson, V. A., Editors, Solid State Physics, Vol. 6, Part A,

Preparation, Structure, Mechanical and Thermal Properties, Academic Press, New

York (1959). [49] Taylor, B. N., Parker, W. H. and Langenberg, D. N.,^^v. Mod. Phys. 41, 375 (1969). [50] Bowman, H. A. and Schoonover, R. M.,NBSJ. ofRes, 71C, 179 (1969).
PANEL DISCUSSION

Analytical Problems in Solid State

Research and Electronics

CHAIRMAN

W. Wayne Meinke, Chief, Analytical Chemistry Division, National Bureau

of Standards

PANEL

James C. White, Associate Director, Analytical Chemistry Division,

ORNL, Oak Ridge, Tennessee

George H. Morrison, Professor of Chemistry, Cornell University, Ithaca,

New York

J. Paul Call, Chief, Office of Standard Reference Materials, National

Bureau of Standards, Washington, D.C.

Henry Frankel, Project Engineer, Environmental Technology, International Business Machines, Essex Junction, Vermont

Robert A. Laudise, Head, Crystal Chemistry Research Laboratory, Bell Telephone Laboratories (Speaker)

65
66 ANALYTICAL PROBLEMS

Cali — A large portion of Dr. Laudise's paper was directed toward areas of research in the solid-state that are properly classified as "state-of-theart." I would like to direct my comments toward the problems faced

within the industry itself— the "life on the production line" problems.

Over the past several years two major trends in semi-conductor materials

analysis have developed.

The first is a marked trend toward topographical analysis. With the advent of large scale integrated devices having device densities approaching

10^ devices per slice, resolution of structure at the micrometer level has

become an increasingly more important problem requiring an analytical

solution. Three techniques having large potential, especially if used in combination, are in use or in various stages of development. The first is electron microprobe analysis which is, in fact, now widely used, having as

it does the ability to determine semi-quantitatively a wide range of ele- ments and structure at the micrometer level of resolution. Its greatest suc- cess to the present lies in the determination of defects produced during

various stages of the metalization process. The second technique involves the use of radioactive isotopes to show

uniformity of doping. This well-established procedure involves doping the

bulk material with the radioisotope of interest, or producing the

radioisotope in situ by activation, lapping and polishing various subsections, and finally, through radioautography, making visible the spatial dis- tribution of the various dopants in the bulk material. The greatest potential for the determination of topographical features lies in the recently

developed ion-probe scanning analyzer. Although the capital investment

is very large, the benefits arising from its use warrant its serious consideration for controlling the quality of semi-conductor materials and

devices.

The second marked trend in industry is less emphasis on the analysis of

the bulk materials than was the case a few years ago. In the early 1960's,

major efforts were expended to develop fast, accurate methods adaptable

to the quality control of impurities and dopants in the bulk. These efforts,

although reasonably successful, have been eased because of the expense

involved, and because the reaction time is much too slow for production

line quality control. However, the most cogent reason for by-passing

these procedures is simply that the "pragmatic approach" works. By this

is meant the statistical sampling of representative slices of material measured electrically to provide very fast information as to whether the

material is within specifications and should therefore be further

processed.

However, this trend may have to be reversed and highly sophisticated
PANEL DISCUSSION 67

instrumental techniques brought again to the fore. Many of the multisandwich layer devices are now produced with yields approaching a few

percent or less. Obviously, better quality control of the starting materials,

the bulk base materials, and topographical control is required. Three

techniques are now widely used. Optical emission spectroscopy using the

split-burn technique, developed in part by Professor George Morrison

and his co-workers, allows sensitivities approaching the part per billion range. Spark source mass spectrography is a very sensitive method, but one where accuracy is lacking. Accuracy will come about as a wider range

of standard reference materials are made available. Radio-activation for many elements is still the most powerful technique, but problems still remain, especially the development and introduction of fast, quantitative

procedures for the determination of important light elements, such as H,

C, O, and N at the ppb level. In many of the above mentioned areas, the lack of a Standard

Reference Material (SRM) to provide accurate calibration is a serious

deficiency. NBS has not been unaware of this need. Unfortunately,

SRM's for these purposes are extremely difficult to produce in quantity;

the cost of measurement and certification is very high, typically running

to $100,000 or more for one material — say for a completely characterized

doped silicon; the market potential is relatively small; and NBS resources

for work in this area are limited. There are, however, several SRM's which fulfill in part some of the

needs in solid-state research and technology. There is now available a series of steel SRM's (SRM 661-668) that is extremely homogeneous and

can be helpful in the calibration of the spark-source mass spectrograph.

Also available is a series of glasses containing 61 added elements in the

range from 0.02 to 500 ppm (SRM 610-619). The high purity

metals-gold (SRM 685), platinum (SRM 680 and 681), and zinc (SRM

682 and 683) have many elements certified in the ppm to ppb levels and

find applicability in the calibration of isotope dilution techniques. Of use

for the calibration of the electron microprobe are the Au-Ag wires (SRM

481), the Ag-Cu wires (SRM 482) and the W-Mo alloy (SRM 480). In preparation is a high purity palladium metal SRM for magnetic

susceptibility measurements, and GaAs of known and certified stochiometry.

Frankel — Dr. Laudise cites as an area requiring additional support the development of mapping techniques for sample homogeneity determination which he believes should be fostered. He makes the point that the importance of mapping is readily apparent when we realize that the vitally
68 ANALYTICAL PROBLEMS

important economic matter of yield, that is, the percentage of usable

devices emerging from a multi-step processing line and integrated circuits can be related to homogeneity in defect mapping.

Although I agree with the potential benefit which can be obtained from

mapping techniques, I believe that there are two formidable obstacles to be overcome. Both obstacles arise because of the complexity of electronic

processing steps and manufacture.

To illustrate the problem I would like to go back to the statement that "yield . . . can be related to homogeneity mapping." Device production in general is not based upon perfect crystals. This stems from empirical tests conducted some time ago which indicated that dislocation-free material

does not yield more transistors or diodes than silicon with a moderate

dislocation density. The reason why dislocation-free material did not yield more good devices than material of moderate dislocation density is process induced defects. So that mapping is but a starting point in a long

complex search.

So now we come to our first obstacle — the difference in philosophy and

approach of characterization in electronic manufacturing process control

as contrasted to the solid state research approach. Research desires to study the relationship of structure and composition with properties.

Whereas, in manufacture, the desired properties are known— pre- dominately, if you will, by measuring electrical properties which in- cidentally also provide a handy comparison scale. Characterization

in manufacture is minimal and concerned almost fully with line processing problems. Characterization is thus a court of last resort — when

all else fails — try analysis and characterization.

The second obstacle to the characterization analyst arises from the

parochial interests of his analytical chemistry past. The range and scope

of his work require him to steer a course between the Scylla of studying

and characterizing defect for defects sake and the Charybdis of perfecting

instrumentation for instrumentation's sake. He can no longer indulge — for long — a pursuit in either direction without suffering in effectiveness to those dependent on his skill. The instrumentation is simply too numerous,

the process and physics too complex to permit such unrestrained indulgence.

If we want to reap the benefit of higher yield through characterization,

we must intimately incorporate characterization into the process itself. This requires involvement of characterization chemists with the process

itself since he must concentrate on line processing steps not only

processing problems. He must plan so that he gets most effective information from a sample since most samples are often in no condition to be
PANEL DISCUSSION 69

tested after electrical test. Incidentally, the "non-destructive" high energy

electrons as well as emitted x-rays can and do affect the electrical characteristics of certain critical areas of a device. Planning is also required to

select the most relevant test techniques and insure the best use of characterization specialists' time. Lastly, he must foster communication

between processing people and characterization specialists in terms of

using characterization as a stethoscope to monitor the health of the

product.

While I agree with Dr. Laudise that a tremendous amount of information can be achieved through proper use of the optical microscope, I can- not agree that no new techniques are necessary.

As we achieve increasing integration of electronic devices, the working

areas available for characterization — now in the micrometer range — will become even smaller. The tools which can yield analysis of these areas — notably Transmission Electron Microscopy, Scanning Electron Microscopy (SEM) (with x-ray analysis attachment), and Electron Microprobe — will become fhe

most important tools of any device characterization laboratory.

Additionally, surface contamination also becomes of very critical concern. Surface techniques such as Auger Spectroscopy and the Ion

Microprobe will become equally important within the next few years. Now combine all of these "mapping" techniques — spectroscopy, x-ray and microscopy— and we have a formidable armory. In fact, too formidable, since they can and do inundate us with reams of data and

information.

1 would point out that a single scan at one wave length for a wafer may

provide as much as 2^^ bits of information and a single scan in this case

resulting at or near the limits of the optical microscopy in covering only a

fifty by fifty micrometer square on the specimen. A ten by ten specimen

would require 40,000 such scans at each wave length of interest to acquire

all of the data.

It is clearly evident that the computer in the form of image analyzing

systems must be included as an analytical tool to decipher, compare,

categorize, correlate and recognize defects and patterns. Image analyzing

systems with automatic traversals, now primarily encompassing

microscopy, should soon be extended to process any raster input including SEM and microprobe.

Lastly, permit me to indulge in what may seem to be a commercial. It seems to me that we analytical chemists while fascinated with the data

processing that can be done by computers tied into our GC and x-ray dif- fraction are but on the first steps of the full use of computers. Consider the
70 ANALYTICAL PROBLEMS

potential of pattern recognition and the use of learning machine concepts

to determine relevance of each part of the characterization map. If analytical science is to become the core discipline of materials characterization, we must master these techniques to permit the analytical use of

analytical chemists.

Morrison — The importance of analytical chemistry to the advancement

of materials science over the past two decades is well documented. And

conversely analytical chemistry has profited tremendously from its as- sociation with solid-state research as evidenced by the dramatic development of new and refined techniques for trace determinations to fulfill the

demanding requirements of the study of ultra-pure materials. For in- stance, spark-source mass spectroscopy was born because of the need for determining trace impurities in silicon and germanium. Activation analysis really got its push because of the need for determining trace impurities

at the part per billion level. Certainly, then the challenges in the past of solid-state research have

done well for analytical chemistry. And as Dr. Laudise has pointed out so very clearly, this is only the beginning. There are many more challenges

for the analytical chemist in the solving of more sophisticated and complicated solid-state problems.

As has been mentioned, materials science is an interdisciplinary field which deals with the preparation, characterization and relationship of properties to bonding and structure. The three aspects are intimately related. There is one unfortunate thing, however. A problem arises when the

respective specialists must interface with each other. This problem exists

in all interdisciplinary efforts where the analytical chemist who is technique oriented must interact with colleagues who are problem

oriented. And that is what I would like to spend my remaining few

minutes on. Of the various national problem areas being discussed at this symposium, the interfacing on a large scale of analytical chemistry with solid state research has had the benefit of more than twenty years experience. Some

of the lessons learned could be of considerable help in developing efficient coupling of analytical chemistry with some of the new national problem

areas. For the past twenty years I have been intimately involved in the

analytical aspects of solid state research; ten years in industry and ten years in the university. From my experience the problems of interdisciplinary interaction are the same in both situations. As Dr. Laudise has pointed out, analytical chemists could serve as the
PANEL DISCUSSION 71

nucleus for the broader field of materials characterization encompassing

identification, determination of major and minor constituents, and the

determination of physical structure. The degree of involvement with these

many diverse specialties will depend upon the personnel available; however, broadening of the scope should not be done at the expense of

diminishing the efforts of the analytical chemist in doing the things he

does best, i.e. to keep working in the areas of major and minor composition analysis. In addition to providing service analysis on routine problems, the

materials analyst is constantly called upon to develop new quantitative

methods for new situations — new matrices, new chemicals, etc. Equally

important, the analytical chemist must constantly do research on new

techniques not directly related to specific materials if he is to continue to be of value in solving new and more complicated materials problems.

Because analysis is a specialty in its own right, requiring considerable

expertise, it has been found most expedient in materials research to establish central analytical facilities to handle the many diverse materials

prepared by other specialists and whose properties may be studied by still another group of scientists. At the Cornell Materials Science Center we have a number of central

facilities of which analytical chemistry is the largest which provide service

to professors, postdoctorals, and graduate students in solid state physics,

solid state chemistry, metallurgy, applied physics, electrical engineering

and other disciplines engaged in materials research. Different facilities are used to a greater or lesser extent depending upon the materials problem,

but analytical chemistry is involved in almost every case.

It has been our experience that the greater the degree of interaction

between the various groups, the faster and greater is the chance for suc- cess of the programs. We have also found that the analytical chemist,

because of his broad scope of assignments, must take the initiative in in- teracting with the other groups. Thus the analyst must be consulted in the

design of a preparation scheme so that sampling for analysis can be op- timized thereby leading to more useful data. He must also screen the start- ing materials. Similarly, close association with those studying the electrical, mechanical, and other properties of the materials is most essential if meaningful correlations with composition are to be achieved.

Although these points may seem obvious to the casual observer, in practice many problems arise because of the lack of communication

between the groups. Therefore, those analytical chemists embarking on

interdisciplinary programs in the newer national problem areas would do

well to heed this lesson.

428-999 O - LT - 72 - 6
72 ANALYTICAL PROBLEMS

White — I think it is important to look at this problem from the standpoint of the administrator who decides what capabilities existing within

his analytical organization can be used to meet the challenges that are presented in solid-state research and electronics. Another point of consideration is which capabilities does he not have that can have a considerable impact in this area. In simple language, we're saying — "What do

I have available for this problem, and what do I need?" By capabilities I should quickly add that I include manpower as well as instrumentation

and equipment. I'd like to pose a few questions that I hope will result in some feedback from you, and offer a few opinions of my own as to what

might be a solution to this overall problem of how the analytical chemist can best serve in this area. What are the characteristics of the analytical chemist who will be of

most value to the researcher? What kind of a man are we looking for? He

should be a Ph.D. or the equivalent. (Whenever I hear that phrase "or

equivalent" I wonderjust what is meant by it — other than the fact that this person is probably expected to do everything a Ph.D. does, and know

everything a Ph.D. does, but he probably doesn't get paid what a Ph.D.

gets paid.) Facetious remarks aside, I do feel that it is highly important

that the analytical component of the materials research team be a full- fledged partner with equal status. He must be considered so and of course he has to perform so. Since this field abounds with highly educated, highly

trained scientists, he has got to have his credentials. Also he must "speak

the language." I think this is vitally important. The successful analytical

participant in almost any multi-disciplinary venture always has a good

talking understanding of what the overall problem is. In this case he is going to be a materials researcher himself to a certain degree. Fortunately

for the analytical chemist, the "language problem" is not as difficult to surmount in this particular area as it is in the biomedical and clinical chemistry area that we will be talking about this afternoon.

Much of the typical training for the typical analytical chemist entails studies that are directly applicable e.g. physics, metallurgy, mathematics.

So we do know some of the language before we begin. I think it is true to say that we have accepted these disciplines in physics and metallurgy, as proper adjuncts to analytical chemistry. For some unexplained reason, I don't think we feel the same about biomedicine and biology, and I hope

we get that changed fast. To recapitulate then, our analytical input has to be backed by the in- dividual's credentials and an understanding of what the field entails. I think these are qualities that are rather readily come by, but now we face

the problem. Dr. Laudise has mentioned the great variety of analytical
PANEL DISCUSSION 73

techniques, all of which have some useful, and in some cases, even in- dispensable applications. The analytical man who makes the most significant, visible contribution by necessity will be a specialist — a mass spectroscopist or an activation analyst, or an electron probe microscopist. Unfortunately he can't be a specialist in all of these categories. Thus, we face

the Hkely possibility of overstaffing in a specific category to the detriment

of other categories, and create a real imbalance in analytical effort. The solution obviously is to have several specialists wearing the

analytical chemist colors. This is fine if you have these capabilities and

can afford to use them, and if your customer can afford to pay you for

them. I strongly suspect that all of the required analytical expertise is not going to be available in most laboratories, so the materials research is quite likely to suffer from this imbalance. The mass spectrometer might be

used because it's available or because there happens to be a mass spectroscopist "on the team," when activation analysis or polarography might

better be employed.

I would venture to say that we as analytical chemists have probably not given the materials field the best possible analytical chemistry available

for this particular reason. Nevertheless I think that we will also have to admit that the field has done remarkably well with what has been applied.

Much of this credit goes to the materials researchers themselves, who

have by necessity become analytical experts in certain specialties. Again,

Dr. Laudise has spelled out the opportunity that exists in this field for

analytical chemists.

How do we take advantage of this opening? In these times of too many

chemists and too few jobs I venture to say that there are not enough

analytical chemists who have been trained in performing in the materials

science business for the needs of the materials people. I refer to specific

type people of course, those people who can assume responsibility for the

analytical problems that make a particular input. My proposal of a solu- tion to this problem of specialists centers around what I would term the

analytical broker.

I think the analytical partner in this partnership should serve the function of advising and probably even more forcefully selecting the particular

method to be used for the problem at hand. As was pointed out earlier he

cannot be a specialist in all of these techniques. But he is going to have to possess these qualifications I mentioned before and credentials and

background about what a technique can do. And he will in turn then communicate the complexity of the analysis to the expert or the specialist who

might be employed. I think this man must be an analytical chemist.

George N. Bowers, Jr., M.D., is Director of Clinical Chemistry at Hartford

Hospital, Hartford, Connecticut. Dr. Bowers majored in chemistry at Colby College in Maine before attending Yale School of Medicine. In- ternship and medical residency at Hartford Hospital were followed by a fellowship in clinical chemistry with Dr. John Reinhold, Pepper Laboratory, University of Pennsylvania. For the last ten years he has been in hospital service chemistry at the 900-bed Hartford Hospital. Special

research areas of interest to Dr. Bowers have included clinical enzymology, ultraviolet and visible spectrophotometry, atomic absorption spec- trophotometry, high-purity standards, quality control systems, and highaccuracy referee materials for clinical laboratories. Dr. Bowers is Chairman of the Standards Committee and a member of the Editorial Board of

Clinical Chemistry of the American Association of Clinical Chemists, and

is a member of several groups concerned with the clinical laboratory and

medical science fields including the Advisory Panel (NAS-NRC) to the

Analytical Chemistry Division of NBS. Dr. Bowers is also actively con- cerned with environmental issues in Connecticut.

75

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 351,

Analytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

HeldJune 16-18, 1971, Gaithersburg, Md. (Issued August 1972). CHAPTER 3

ANALYTICAL PROBLEMS IN BIOMEDICAL

RESEARCH AND CLINICAL CHEMISTRY

George N. Bowers, Jr., M.D.

Clinical Chemistry Laboratory

Hartford Hospital

Hartford, Connecticut 061 15

With Drawings By

John Meyer, II, M.D.

Pathology Laboratory

Day Kimball Hospital

Putnam, Connecticut 06260

The key to progress in national problem areas in health rests over the long term with the success of our country's biomedical research efforts. Analytical

chemistry can contribute significantly to these vital efforts by providing the sound base of quantitative chemical measurements required by these health in- vestigations. But health research must not be construed too narrowly. The

responsibility for the chemistry related to our national health needs cannot be

assigned exclusively to any one scientific group. Indeed, some of the most

profound insights may well originate far beyond the usual confines of the medical research community. This will be especially true in the environmental

health areas where analytical chemists from many fields will make important

contributions. The elucidation and cure of disease processes are becoming ever more

dependent upon chemistry. As a direct result of successful recent past bio- medical research, the demand for diagnostic chemistry tests has increased

faster than almost any other health service. Service chemistry is thus closely

related to medical research and also medical education activities, as will be demonstrated by experience with transaminase assays in heart attacks and

calcium determinations in hyperparathyroidism.
78 ANALYTICAL PROBLEMS

To support the rational development and utilization of automated analytical

systems in health service and research laboratories, there is a clearly defined need for high purity standard reference materials and certified reagents. Clinical laboratories urgently require the development of high accuracy referee methods to improve the specificity and systematic bias of routine methods.

In short, life is a chemical system. Meaningful quantitative chemical measurements are essential to our understanding of its complex internal interac- tions as well as our profound interdependency upon other life forms in the environment. Keywords: Biomedical research; clinical chemistry; clinical enzymology;

enzyme standardization; health science; hospital laboratory; medical research.

I. Health, Biomedical Research and Education

"We are determined that the vital link between pure research

and practical achievement will never be broken. We are determined that research and discovery yield results which not only

increase man's knowledge but the strength of his body and the

length of his life. We do this because we have no choice. And

we do it because we believe in Thomas Jefferson's words, that the care of human life and happiness is the first and only legitimate object of good government."

Lyndon B. Johnson [ 1 ]

In this presentation I will attempt to explore the important relationship

which exists between medicine and analytical chemistry. My familiarity

with the quantitative chemical needs of medicine is both as a physician

trained in internal medicine and as one of three chemists now responsible

for chemistry services in a large general hospital. Although these vocational activities tie me closely to the delivery of health care services, I strongly support biomedical research as the most logical long-term solution to our nation's health problems. Thus, in terms of this Symposium's

theme, "the key to progress in national problem areas," the key to this country's future health rests squarely upon the success of our medical

research programs. Analytical chemists can and must contribute substantially to these vital medical research efforts by providing the sound base

of knowledge about the complex quantitative chemical measurements

which are required in these biological systems.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 79

A. Analytical Chemistry and the Health Field

The health field has many exciting roles for the analytical chemist who

enjoys making meaningful measurements. Each day millions of quantitative chemical analyses are required in direct support of individual patient care needs. These service measurements are now one of the mainstays of

medicine in the diagnosis and treatment of many human ills. As each year

passes, I see more and more chemists finding their way into these vital chemistry service laboratories all across the country. With rare exception,

I find these chemists deeply committed individuals who have found great

meaning and satisfaction in the chemistry services they provide to others. Table 1 lists the types and numbers of clinical laboratories to show where

clinical chemists should be or are now serving in the United States. Table 1. Clinical laboratories in the United States.^

7,000 Hospital laboratories

4,000 Independent clinical laboratories

615 Federal laboratories

400 State and local laboratories

? Unknown (nursing homes, unregistered, industrial and

insurance)

40,000 Physician office laboratories (each serving less than 3 M.D.'s)

^ Estimates by the Center for Disease Control staff in May 1971 during

discussions on the need for a National Laboratory Census. Total

clinical examinations in 1971 =1.4 billion for a cost estimated at $7 billion.

But health, like chemistry, should not be given too narrow or restrictive a meaning, nor can the responsibility for the chemistry related to our total national health needs be assigned to any one scientific or professional group. Indeed, some of our most significant health related chemical measurements and new medical insights will originate beyond the usual confines of the medical community. As an example, I am reminded that the

question of the health effects of environmental contamination by lead was

reopened in 1965 by a research geochemist, not by a physician, a toxicologist, a public health scientist, or a biomedical researc^r worker.
80 ANALYTICAL PROBLEMS

In a paper entitled "Contaminated and Natural Lead Environments of

Man" [2] Dr. Clair Patterson of the California Institute of Technology

presented his reasons for believing that our present-day urban environment increases man's atmospheric exposure to lead by factors of 100 to 1000 times and he speculated about the potential ill effects from this in- creased exposure on the American people. The point I would make is that

this question about health did not arise as the result of the direct study of

lead's toxicity to human subjects. Patterson's insight came from a background of years of experience in measuring the relative abundance of

nanogram to picogram amounts of stable lead isotopes by mass spectroscopy. His original geochemical research interests established the

earth's age as 4.6 billion years; however, later work increasingly centered on the unexpectedly large buildup of lead in ocean surface layers and glacial snows of Greenland [3] as shown in Figure 1.

800B.C. 1750 A.O. 1800 1850 1900 1950 AGE OF SAMPLES

Figure 1 . The buildup of lead in Greenland glacier snows correlates well with the industrial use of lead, particularly in recent years with the marked increase in atmospheric lead from

gasolines in the United States and Canada.

These studies clearly documented an ever increasing buildup of lead in the northern hemisphere from industrial uses of lead, predominantly from

alkyl lead in gasolines. Although many questioned Patterson's arguments

about potential health effects, it is interesting to note that Hunt, et al. [4]

after studying atmospheric cadmium, zinc and lead in 77 mid-western ci- ties in 1968 suggested, "preliminary analysis has shown a positive rela-
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 81

tionship between lead dustfall in residential areas of the 77 cities and car- diovascular mortality."

Thus in numerous ways, observations and meaningful quantitative

chemical measurements in each of the major subject areas discussed in this current NBS Symposium will have an important impact upon the

health of each of us. Environmental health considerations are coming to the forefront almost daily as we become cognizant that man and his technology are an inseparable part of, neither above nor outside, the natural world. Increasingly we recognize the folly of damaging this complex

world on which our existence depends. What is now needed, as Dr. Branscomb, the present NBS Director, has recently pointed out, is that we

react with wisdom to these problems created by our technology, using the

full power of science to bring about governmental policy "based upon the

rational consideration of the scientific facts in their social context" [5].

Can any analytical chemist working in food, water, air, or soil chemistry

fail to see the intellectual challenge and opportunities he must address

himself to in these complex environmental issues? Certainly in the health

field, be it in environmental health, in biomedical research activities or in the medical service areas, our national need for meaningful chemical measurements offers the analytical chemist a remarkable opportunity, in Dr. Meinke's recent words, "to make his contribution and to gain his satisfac- tion" [6].

B. Biomedical Research

Biomedical research is not a new and novel venture of recent origin. In- deed, from the beginning of recorded history, men have by the rational

processes of study and experimentation attempted to control the diseases

which crippled and killed them. The pictorial records of ancient Egyptians

relieving intercranial pressure by trephining operations or the dietary laws

of the ancient Israelites are two examples which come readily to mind.

With the systematic application of scientific knowledge to the study of

human ills over the past few centuries, man has progressively freed himself from the ravages of disease. In the last century chemistry has played an important role in our understanding of the fundamental processes of

life. For example, while ancient physicians diagnosed diabetes mellitus by

the symptom triad of polyuria, polydipsia, and polyphagia and confirmed

this diagnosis by tasting urine and/or blood for sweetness, today's physi-
82 ANALYTICAL PROBLEMS

Figure 2. The ancient physician suspected diabetes because of polyuria and confirmed the diagnosis by tasting urine for sweetness. The modern physician makes this diagnosis on

evidence from chemical instruments which can detect slight elevations of blood glucose from normal.

cian rarely sees patients so far advanced as to have excessive urination,

thirst, or food intake.

Present-day diabetics are often diagnosed on evidence of a slightly

elevated level of blood glucose. If there is any doubt, the diagnosis is usually confirmed by obtaining levels of glucose (also insulin in some

cases) after carbohydrate loading following careful dietary preparation of

the patient for several days. Thus the diagnosis of diabetes mellitus is no

longer based on symptoms caused by a disease which is out of control but

is now more a differential diagnostic problem to elucidate several chemical aberrations each of which may have altered the normal pathways of

energy metabolism. As a result of our increasing knowledge of the abnormal biochemical processes such as the diabetic story just cited, even the

layman now recognizes the fundamental chemical nature of life. In short,

life is a chemical process.

It is not surprising, therefore, to find that each new advance in organic

and inorganic chemistry, when applied to the study of man himself, has

produced many further insights into the basic chemical nature of health
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 83

and disease. By 1900 the introduction of safe systems of public water dis- tribution and the removal of sewerage and refuse coupled with inoculations for smallpox and vaccinations for tetanus had made human existence much less precarious. Also by the turn of the century, the accumulated knowledge of mammalian physiologic chemistry and pathologic

biochemistry was already considerable. Steadily over the first several

decades of the 1900's biomedical research in chemistry had made important contributions to health with the discovery of cures for or partial control of many nutritional deficiencies and metabolic disturbances, i.e., pellagra, pernicious anemia and diabetes mellitus. However, the chemist and

his chemical achievements attracted little public notice since scientists in pathology and bacteriology together with engineers in the public health

sciences, were the main contributors to controlling the devastating

epidemics of infectious diseases.

By the late 1950's and early 1960's, the record of chemistry related

research accomplishments was very impressive. Sulfa drugs had been in- troduced to control lobar pneumonia in the late thirties, penicillin successfully combated the ravages of syphillis and many other infections in the

early forties, powerful anti-inflammatory steroids became available in the

late forties, effective drugs against tuberculosis were introduced during

the fifties and poliomyelitis vaccines had conquered this dreadful disease

by 1960. These accumulated biomedical research and public health ef- forts had either eradicated or brought under control the impressive

number of human ills given in Table 2. However, in stark contrast with the diseases now successfully

managed, the causative factors of those childhood, infectious, degenerative and neoplastic diseases listed in Table 3 are still very poorly understood. Lacking knowledge of their causes they are also not prevented,

cured or rationally controlled by human actions. Despite this basic failure, our efforts have not been in vain since the diagnosis and the management

of these problem diseases is far more successful today than only a few

years ago. These improvements are the direct result of numerous in- vestigations which have increased our comprehension of the specific

biochemical functions of each organ and the complex yet measurable

chemical interrelationships between the various organs. Medical research also has been introducing many new anesthetic

agents, blood products and immunologic techniques which permit the

amazingly intricate new surgical operations of today. Direct surgical at- tack is now possible for nearly every organ in the human body. Several or- gans can now be totally transplanted due to the powerful support devices

such as heart and lung machines which have been perfected over the past
84 ANALYTICAL PROBLEMS

Table 2. Diseases which biomedical research and public health measures have prevented or controlled by 1970.^

Infectious Diseases

Bacterial Endocarditis Poliomyelitis

Cholera Puerperal Sepsis

Diphtheria

Epidemic Meningitis

Gonorrhea

Infantile Diarrhea

Lobar Pneumonia

Measles

Pellagra

Rickets

Adrenal Malfunctions

Trachoma

Tuberculosis

Typhoid Fever

Typhus

Whooping Cough

Yellow Fever

Rabies

Rubella

Scarlet Fever

Smallpox

Syphilis

Tetanus

Nutritional Diseases

Iron Deficiency Anemia Scurvy

Pernicious Anemia Dietary Deficiencies

Metabolic Diseases

Pituitary Problems

Thyroid Disturbances Diabetic Mellitus

Erythroblastosis Fetalis

Plumbism

Adopted from an article by P. Handler in Science 171, 144 (1971).

twenty years. In like manner, medical therapy has gained many powerful

new chemo-therapeutic substances such as the anti-inflammatory ster- oids, diuretics, hormones, numerous antibiotics, anti-neoplastic drugs

and potent psychopharmacologic agents. Altogether too few of us appreciate how profoundly public health engineering and biomedical

research have increased our nation's overall health. There can be no

doubt that the quality and longevity of each individual's life in America

has improved as a direct result of our past success in medical research.

Today the major efforts of biomedical research in the United States are directed towards developing a comprehensive knowledge of the

pathogenesis of these complex genetic, degenerative and neo-plastic

processes listed in Table 3. Dr. James Shannon, the former director of the
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 85

Table 3. Diseases only partially understood lacking definitive cures and

preventative measures in 1970.^

Birth Defects or Genetic Origin Diseases

Birth Injury Cystic Fibrosis Muscular Dystrophy

Cerebral Palsy Hemoglobinopathies Mental Retardation

Epilepsy

Arteriosclerosis

Myocardial Infarction

Rheumatic Arthritis Systemic Lupus

Erythematosus

Metabolic Enzyme

Deficiencies

Degenerative-Chronic Diseases

Bronchial Asthma Osteoarthritis

Multiple Sclerosis

Senile Psychoses

Emphysema and other

COPD

Peptic Ulcer

Regional Enteritis

Ulcerative Colitis

Infectious Diseases

Viral Diseases Without Hepatitis

Specific Immunizations

Acute Rheumatic Fever Pyelonephritis

Neoplastic Diseases

Cancer Polycythemia

Leukemia Dysproteinemia

Others

Schizophrenia Depressions

Suicide Mania

Mycosis

Prostatic Hypertrophy

Uterine Fibromata

Alcoholism

Drug Addiction

Adopted from an article by P. Handler in Science 171, 144 (1971).

National Institutes of Health, has clearly pointed out that real progress

toward prevention, control and ameHoration of these chronic diseases

rests with the efforts to understand their basic molecular mechanisms [7].
86 ANALYTICAL PROBLEMS

This will require more intensive support for research programs, some of

middle range (extending over 5 to 10 years) and others of much longer duration (15 to 25 years in length). These programs must be very fundamental, centering upon the molecular and cellular level of events in normal as well as in pathological processes. If these long-term programs are to be

successful, many of the investigators must be young, and dedicated to a long career of inquiry.

It is encouraging to hear our President call for a redoubled effort ($100,000,000) in cancer research in his 1971 annual fiscal message to Congress. However, the confusion and disruption caused by the drastic cuts in chemistry funding by the National Institutes of General Medical

Sciences (NIG MS) as reported in Science [8] does not encourage young

scientists to race into a life-long commitment to biomedical-chemistry

research.

As individuals and collectively as a nation, our future health interests are very closely tied to these long-term research programs. Diverting al- ready limited health research funds into short-term immediate health care

services will only delay the time when we obtain the fundamental

knowledge needed to solve these chronic disease problems. Rapidly

changing priorities in national health goals can only reduce the number of

young investigators entering into basic research. Our very real and proper concern for the inadequacies of the present American health care system

(or non-system as some are prone to call it) should not rob us of the

chance which biomedical research provides of obtaining the knowledge

which will ultimately prevent these chronic diseases.

The conclusions of the recent National Academy of Sciences report entitled "Life Sciences" pertaining to the relationship between biomedical

research and the development of health care services wisely states ... "We

warmly support an enlarged program of research directed at the improvement of our social instruments for the prevention of disease and the

delivery of health care. But, equally strongly, we urge that such a program

be funded in its own right, without injury to the national biomedical education and research capability" [9].

C. Research, Education and Service—A Medical Triad

To speak of biomedical research as an isolated scientific enterprise

divorced from medical education and health care services is unrealistic.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 87

This triad of research, education and service activities constantly interact

with one another strengthening the whole body of medical knowledge and

practice. Thus, for years it has been recognized that the "best" medicine

is practiced when an individual or institution has the benefit of a balanced

interaction between all three components as suggested in Figure 3. Medical education together with biomedical research has expanded rapidly over the past twenty-five years. During this time, the National Institutes

of Health (NIH) budget which goes predominantly for research and education increased from $2.5 million in fiscal year 1945 to a recommendation for $ 1 .9 billion in fiscal year 1 972.

While most people associate NIH only with research, the programs to

train medical scientists through clinical traineeships and extended fellowships for young medical and doctoral scientists has been a major reason we have had adequate research and education personnel for medical in- stitutions all across this nation. Thus public support of education and

research in America has made a long cherished medical ideal of a balanced and dynamic interchange between teaching, research and ser- vice, a reality more so today than ever before in history. Despite the recent severe funding cuts in chemistry which have already been menFigure 3. The "best" medicine is practised when there is a balanced interaction between

research, teaching and service.

428-999 O - LT - 72 - 7
88 ANALYTICAL PROBLEMS

tioned, the nation's present biomedical research and medical education

community is the most powerful scientific force dedicated to the advancement of positive human values that mankind has ever known. Or again in Dr. Shannon's words "... the United States now has no peer in the scope

and excellence of its medical research activities" [7]

.

One result of this federal support over the past twenty-five years has

been the discovery of literally hundreds of biochemical alterations. A

quick review of medical progress reports or special articles from the New

England Journal of Medicine, or any other broad-coverage medical journal reveals the vast amount of chemical knowledge and its correlation

with disease processes which has characterized the past two decades. As

a result of the dynamic interchange between research and teaching activities in numerous centers across the country, important biochemical

findings rapidly influence medical education. Research-oriented clinicians

in medical centers are continuously testing to verify or reject the value of

measuring these newly-found biochemical alterations in their patients.

Once a measurement has proven to be useful, the information is quickly

incorporated into the diagnostic and/or treatment regimen of these clinical investigators. Subsequently through journal publications, lectures and

teaching of medical students and house officers, knowledge of the pros

and cons of the new measurement diffuses out into the daily practice of

medicine.

As a chemist with the responsibility for providing chemistry services

for an acute-care one-thousand bed hospital, I can speak with certainty to the pressure for new determinations which medical research and education is constantly creating. Perhaps the success of our past research activities and the effectiveness of medical education which must help translate the new findings into meaningful patient care services can be judged,

in part, by the heavy daily usage of these objective scientific measurements.

D. Quantitative Chemical Measurements—A Resource Essential

TO BOTH Biomedical Research and Health Care Services

Biomedical research and health care activities not only require a tre- mendous volume of quantitative chemical measurements but also contribute heavily to the development of new methods of analysis. While a few investigators and clinicians may view quantitative chemical measure-
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 89

ments merely as "tools," a surprisingly large number recognize how poor

measurements limit their work. Many of these research scientists and

physicians share with the analytical chemist a deep concern for precision

and accuracy. In parallel with the general trends throughout analytical laboratories

today, health scientists utilize instrumental systems of analysis in preference to the traditional "wet" manual methods. Often sophisticated

electronic data processing is employed to further increase the speed and

reliability of the measurement. As in other areas, limitations of existing

analytical methods have repeatedly stimulated the health investigators to improve the accuracy of the measurement and to innovate. Both research

and service needs have resulted in unique innovations in measurement

systems to solve specific problems. An outstanding example of such a recent innovation is the development at Brookhaven and also simultaneously in England of whole-body neutron activation analysis which permits

the in vivo study of skeletal calcium metabolism in patients suffering with

osteoporosis [10]. Likewise in the clinical service area, the original AutoAnalyzer® developed by Skeggs represented an inventive means to solve

the problems of heavy daily workloads in the face of inadequate numbers

of skilled personnel [t 1]. This relationship between instrumental measuring capability and improved biomedical research or health care services is easily seen in a review of equipment found in either investigative or clinical laboratories

today in comparison with twenty years ago. In 1950 a few filter colorimeters, a simple pH meter and a spectrophotometer were all that was

required to be a reasonably well equipped laboratory. However, in 1 97

1

capital equipment plays a far greater role in the plans of any research or

service laboratory. While the instrument needs in research will vary

greatly depending upon the project, many of the vital services a clinical chemistry laboratory provides today would be utterly impossible without

the significant advances in analytical instruments which have occurred.

One might cite the contributions of the flame photometer for rapid and ac- curate sodium, potassium or lithium measurements. Certainly the advent

of atomic absorption spectrophotometers has improved measurements for

calcium and magnesium while also introducing the ability of testing for trace metals such as zinc, chromium and copper or the toxic ele- ments—lead, mercury, cadmium and arsenic. Although we could mention

several other important instrumental contributions in this section, I would

now like to show how the advent of one modern instrument, the pH, pC02

and PO2 meter with its capillary type electrodes and reliable electrometer,

has had a profound impact upon improving teaching and patient care.
90 ANALYTICAL PROBLEMS

E. Acid-base and Blood Gas Measurements

Until 10 years ago, the only test available to judge the body's acid-base

balance, even in the largest medical centers, was the plasma bicarbonate

or the slightly more refined but qualitatively similar CO2 content. Since it is possible for a high blood CO2 to be found associated with either alkalosis or acidosis much confusion was possible as illustrated so vividly by

Dr. Meyer in Figure 4. Biomedical researchers such as Henderson [12]

and Van Slyke [13] had almost completely described the physiologic

relationships and controlling mechanisms involved in acid-base balance

as well as alterations due to pathologic processes many years ago during

the 1920's and 1930's. However, only a few clinicians were able to obtain

pH and PCO2 measurements on patients. For example, during my fellowship training in clinical chemistry in 1958 at a major university medical

center, these measurements were usually made only by the most senior

doctorate chemist. Reliable measurements by technical people were un- heard of because the instrumentation was so difficult to operate correctly.

Figure 4. An elevated blood CO2 alone without pH and/or PCO2 measurement could mean

either acidosis or alkalosis. The clinician was forced to use "clinical-judgement."
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 91

In medical schools the subject of acid-base balance was usually taught

mainly by renal physiologists. The major emphasis was often on the in- terpretation of bicarbonate measurements using equations which involved

assumptions of a fixed respiratory response. The kidneys' regulatory role by virtue of its excretion of hydrogen ion by various exchange mechanisms was stressed (see Equation 1). H2C03^HC03- + H+ (1)

Few students or clinicians fully appreciated how rapidly the excretion of

Coo by the lungs could affect acid-base balance although this was well

known by respiratory physiologists and anesthesiologists.

The ability to measure pH, PCO2 and PO2 quickly and reliably on patients with respiratory and/ or circulatory problems has now shown how

rapidly alterations in alveolar gas exchange will alter the body's acid-base

homeostasis. The lungs' role in acid-base balance by ventilatory control

of CO2 is expressed by Equation (2). H2C03^H20 + C02. (2)

Thus the regulation of acid-base balance is the result of two distinct and

separate control mechanisms, one slow process of renal hydrogen ion

excretion and another rapidly responding process of respiratory CO2

excretion. Together these provide hydrogen ion homeostasis by Equation

(3). CO2 + H2O ^ H2CO3 ^ HCO3- + H+. (3)

Today good laboratory support allows even the medical student or young house officer to make more accurate evaluations of a patient's acidbase status than the most senior consultant in metabolism was capable of

doing 10 years ago. Despite the inherent complexities of whole blood pH

measurements, modem instruments perform remarkedly well and the

chemist rarely needs to confuse his clinical colleague with highly technical

problems as suggested in Figure 5. The following two cases illustrate what

a difference pH. pCOo and pOo measurements can make [14].

Case Number 1 A 63 -year-old woman with a long history of

pulmonary disease characterized by frequent infections, pneumonia, chronic productive cough and chest cage deformity was

admitted to the hospital due to one week of crampy abdominal
92 ANALYTICAL PROBLEMS

RESIDUAL LIQUID-JUNCTION POTENTIALS, ^

BUFFER ACCURACy ELECTRODE ^BUFFER ) TEMPERATURE COEFFICIENTS, ELECTRODE \

IMPEDANCES AND I R -DROP ARE ALL

| AMONG THE Ff\CTORS THAT CAN PRODUCEJ

UNDETECTED ERRORS IN MEASUREMBNT3 VVHOSE^ MAGMITUDE,,

Figure 5. Modern pH instruments perform remarkably well. The clinical chemist rarely needs to confuse his medical colleagues with the inherent complexities of whole blood pH

measurements.

pain, nausea, vomiting and mild diarrhea. Physical examination

showed only diffuse abdominal tenderness and marked

kyphoscoliosis. All laboratory tests were normal except CO2

content of 34 mmol/1 (normal 18-24 mmol/1). No pH or PCO2

measurements were made. Progress: The surgeon felt that CO2

elevation and history of vomiting suggested metabolic alkalosis

(loss of HCl in vomited gastric fluid). The patient was treated

with ammonium chloride as preparation for surgery and four

hours later was unresponsive, cyanotic, hypotensive and air hungry. At this time pH was 7. 1 and PCO2 65 mm Hg and CO2

content had dropped to 21 mmol/1. The patient died several

hours later! Interpretation: This patient's initial acid-base

problem was respiratory failure with carbon dioxide retention.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 93

The administration of ammonium chloride converted a metabolic compensation into a severe metabolic acidosis on top of an

underlying problem of respiratory insufficiency. If either the

pH or the PCO2 in addition to the CO2 had been obtained, this lady's precarious respiratory status would have been recognized and not compounded.

Case Number 2 A 40-year-old fireman was stabbed in the

chest with injury to pulmonary artery, aorta and right ventricle

of the heart. In the Emergency Room his heart stopped but re- sponded to external massage. The heart and great vessels were

repaired and the patient responded well after blood transfusion.

During the ER resuscitative efforts gastric contents was aspirated into the lungs with resulting severe bilateral pneumonitis.

For several days the patient's condition was critical; despite

oxygen and steroids he remained deeply cyanotic and air hungry. On the fifth day he was much worse. Respiratory acidosis was clinically suspected since the CO2 content was 40 mmol/1.

However, before alkali was administered a venous pH was obtained which was 7.61 (severe alkalosis) while his PCO2 was

only 45 mm Hg. In retrospect, this severe metabolic alkalosis was due to the constant removal of HCl from the patient's stomach by a Levin tube. Treatment with intravenous fluids containing potassium, resulted in prompt clinical improvement and

return to normal acid-base balance in 48 hours. His subsequent

course was one of continued improvement. Interpretation: This

case might well have had a fatal outcome had the pH and

pCOo not been obtained prior to giving treatment.

Both of these patients were seen during the very early days of the in- troduction of blood gases and pH measurements to our hospital when

clinicians did not always recognize the valuable help such tests gave. Today Case 1 would never be evaluated by CO2 alone and the

happier outcome depicted in Figure 6 might have occurred. Our present

goal is to perform and report back to the clinician all pH, PCO2 and PO2

measurements within 20 minutes of the time it reaches the laboratory. In

the near future this needs to be reduced to 5-10 minutes; however, membrane problems seem to limit the long- term electrode stability and restan- dardization must precede each whole blood measurement at present.
94 ANALYTICAL PROBLEMS

Figure 6. Properly applied pH, PCO2 and PO2 measurements can restore the smile-of- health to many a patient.

II. Clinical Chemistry— Analytical Chemistry in Medicine

"The clinical chemistry laboratory a few decades ago was a modest handmaiden supplying a few chemical tests that ap- peared serviceable in diagnostic procedures ... A great change was inaugurated when a few progressive hospitals appointed to

their laboratories highly trained professional chemists, men

who were qualified to carry on investigative work in addition to supplying reliable data for diagnostic use. These chemists faced

great challenges in refining existing methods and in the development of a rapidly increasing number of new tests and

techniques."

Somogyi[15]

No part of clinical medicine, even the much publicized hospital emer- gency room services, has grown and changed more in the last decade

than clinical chemistry. This rapidly changing nature of clinical chemistry

services in any large institution today can best be understood by contrast with the same laboratory in 1950.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 95

A. Quantitative and Qualitative Considerations

Quantitatively, the total volume and types of tests performed have

steadily increased each year requiring more space, instruments and per- sonnel. Qualitatively, to meet articulate clinical demands, the services

have had to be more rapid with an ever greater degree of proven reliability. The following description drawn from my past twelve years of ex- perience at Hartford Hospital will attempt to provide the chemist who is unfamiliar with the practice of clinical chemistry (analytical chemistry in a medical environment) some idea of its functions and services. One will readily recognize that manual manipulations of test tubes, Bunsen burners, and visual comparative colorimeters now belong to a past era.

1. The Volume and Types of Tests Performed

The total volume of objective measurements requested of the entire

laboratory at Hartford Hospital has risen progressively since 1950 as shown in Figure 7 on the top curve. Chemistry services, as seen by the

lower curve of Figure 7 have increased at an even greater rate (going from

100,000 tests in 1959, to 190,000 in 1964, 377,000 in 1969, 430,000 in

1 970 and to an estimated 485 ,000 in 1971 ). This doubling in volume every

five years is typical of the demands made upon clinical chemistry laboratories in nearly all hospitals in North America and Western Europe.

Although it is difficult to judge just what the future growth trends will be,

there is at present no reason to expect this twenty-year pattern to suddenly reverse itself Therefore, we currently are making our plans on the

basis that this laboratory shortly after 1975 but before 1980 must provide

1 ,000,000 service chemical determinations yearly.

Only part of the increase in test volumes can be ascribed to greater use

of the thirty tests available in 1950 as listed in Table 4. The effect on total volume of introducing fourteen new tests while only dropping four old

tests between 1950 and 1960 was especially marked because of the addition of the two transaminase enzymes which will be discussed in greater

detail later. Between 1960 and 1970 thirty-five additional tests were of- fered but in just the last eighteen months approximately thirty new types

of tests have been added. Today, some one hundred discrete types of

chemical tests are available routinely whenever the physicians wish to initiate an order for them. Appendage A lists a breakdown by laboratory

sections of the 1969-70 volume and the observed precision (expressed as
96 ANALYTICAL PROBLEMS

1950 I960 1970 Figure 7. The growth in cHnical chemistry (bottom) is contrasted to the total laboratory

tests for the period 1 950 to 1 970. Note that chemistry services have gone from 20 percent

of the total tests in 1950 to 44 percent in 1 970.

the Percent Relative Standard Deviation (% RSD) for n = 80 to 120

days). Appendage B lists the tests not performed in our laboratory as of

June 1971; these are sent out to several outside reference type laboratories.

2. Organization and Personnel

Until July 1959, the scientific direction of clinical chemistry at Hartford

Hospital was shared by five general pathologists with no single individual

specifically assigned to manage the section. A medical technologist-supervisor guided the day-to-day operations of nine medical technologists who

easily handled the total yearly workload of approximately 100,000 tests by forty different methods. Emergency tests were limited to blood sugars,
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 97

Table 4. Tests added and revised in clinical chemistry

(Hartford Hospital experience 1950-1971).

OFFERED PRIOR TO 1950

(30 tests)

Blood sugai'*"^"^

(Non- protein nitrogen)

Uric acid"^ Creatinine*

Creatine

Cholesterol*

(Icteric index)

Bilirubin*

Total protein*

Albumin*

C.S.F. protein*

Acetone

Calcium*

Phosphorous*

Sodium***

Potassium***

Chloride**

Carbon dioxide

Cephalin flocculation

Thymol turbidity*

BSP retention*

PSP excretion*

(Sulfa levels)

Salicylates*

Bromide

Protein bound iodine*'

Amylase

Lipase*

Acid phosphatase*

Alkaline phosphatase"

(Sulkowitz)

Renal calculi**

ADDED PRIOR TO I960

( 14 new - 4 dropped + 30 = 38 tests)

Serum electrophoresis '52**

Hemoglobin S

'52**

Sweat test '53* Choline sterase '56*

At\-pical choline sterase 17—ketosteroids '57 Serum Fe ^ TIBC '58* SCOT Transaminase '58*

Di

SGPT Transaminase '58*

Tubulai' PO4 Reabsorption '58 Urea '58* Quality control '58* Microbilirubin '59* Methemoglobin '59* Plasma Hemoglobin '59

ADDED PRIOR TO 1970

(35 new + 38 = 73 tests)

Lactic dehydrogenase '60* Magnesium '60* Barbiturates '60* Ethanol '60**

Blood ammonia '61* Ceruloplasmin '61

Carotene '61 17-Hvdroxvsteroids '61

C.S.F. electrophoresis '65 Prostatic acid phosphatase '66 Macroglobulin '66 Amino acids '66 Urine sugai' — TLC '66 Plasma Cortisol

Blood pH '67* Blood PCO2 '67*
98 ANALYTICAL PROBLEMS

Table 4. Tests added and revised in clinical chemistry (continued).

VMA '62-*

Hemoglobin electrophoresis '62"^ A2 Hemoglobin '62**

Bicarbonate *63 5 Hydroxy— indole acetic acid *63* Osmolality '63*

Xylose '64 Doriden '64*

Urine electrophoresis '65*

Blood PO2 '67* Amniotic fluid '67* Lithium '67 Triglyceride '68 Galactose '68 Tolbutamide Top. '68

Urine Lead '69 Sperm Fructose '69 Creatine Kinase '69 ADDED TO PRESENT (June 1971)

(30+ new + 73 = 100+ tests)

Metanephrine ' 70

Blood lead '70*

Tissue lead '70 Copper *70

Zinc '70

Lipoprotein electrophoresis '70 Immunoelectrophoresis '70 Immunodiffusion for IgA, IgM,

IgG and fetoprotein '70 10 Microchemistry tests '70

6 Drugs by GLC '70 Complement '71 Placental Estriol '71 Placental (heat stable)

Alkaline phosphatase '71

Cadmium '71

Mercury '71 Blood volatiles'71

Drugs by TLC '71 Dilantin '71 Ionized calcium '71

Uroporphyrin '71

Coproporphyrin ' 7

1

Porphobilinogen '71 Urobilinogen '71

Melanin '71 Quant, urine proteins '71 Pregnanetriol '71 Lactic dehydrogenase

Heat stable '71 Glucose — enzyme specific '71

NOTE: Asterisks indicate number of revisions.

Parentheses indicate tests which were discontinued.

urea, acetone and salicylates which were performed, when regular personnel were unavailable, by residents and college students who also covered

the entire laboratory including the blood bank. The space allocation to chemistry to provide these services in 1959 was 1500 ft^. In contrast to that small, loosely organized but well-functioning laboratory of July 1959 mentioned above, the clinical chemistry laboratory of

July 197 1 has evolved into a highly-structured grouping of five sections as shown in the organizational chart in Figure 8. The former single produc-
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 99

CLINICAL CHEMISTRY LABORATORY — ORGANIZATION CHART

(Hartford Hospital, June 1971)

Shared Departmental

Admi nistrative and

Clerical Support

PATHOLOGY DEPARTMENT

(Chairman)

Visitors, Fellows

Residents

CLINICAL CHEMISTRY

DIVISION

(Directors)

Automation Section

(Clinical Chemist)

Supervisor +

8 MT's

I Part Time

Electrolyte Section |__. (Clinical Chemist)

Supervisor

8 MT's

I Part Time

Administrative

Section

(Clinical Chemist)

Supervisor +

2 MT's

I Clerical-Secretary

Teaching Supervisor

Instrumentation and Consultant Support

Special Students

Protein Section

(Clinical Chemist)

Supervisor +

6 MT's

1 Part Time

Endocrine Section

(Clinical Chemist)

2 R&D Chemists Supervisor +

5 MT's

I Part Time

Figure 8. Organizational graph showing the structure of clinical chemistry in a 1000 bed

acute care community hospital.

tion section has become four (automation, electrolytes, proteins and en- docrines) and a fifth off-line administrative section combines the functions

of clerical-secretarial support, standards and controls, research development, education and overall division management. The personnel number

49 individuals as follows: one M.D. chemist, one Ph.D. biochemist, one

Ph.D. analytical chemist, one Ph.D. post-doctoral fellow, one instrument

specialist, two developmental chemists, five supervising technologists,
100 ANALYTICAL PROBLEMS

one teaching supervisor, 36 medical technologists, two clerk-secretaries

and six full-time technologist equivalents (20 part-time persons) covering

nights, weekends and holidays. In addition, some 15 clerical and management persons are shared in common with the other clinical pathology divi- sions and four doctorate persons (two M.D.'s, one biochemist and one

physicist-instrument designer) provide various consultative support ser- vices.

To give another way of viewing the diverse skills and talents of the or- ganization, the technical, chemical, and medical competences of the

present chemistry organization are listed in Table 5. Chemistry space

requirements have also changed with approximately 4,000 ft^ in 1969-70

and within the 1972-73 hospital fiscal year new construction will increase

this to approximately 12,000 ft^, which should be adequate until 1980.

Table 5. Competencies in Clinical Chemistry (Hartford Hospital

Laboratory, June 1971).

I. Optical Methods

Spectrophotometry

Spectrofluorimetry

Flame photometry

Atomic absorption

11. Electroanalytical Methods

pH, acidity and blood gases

Potentiometry

Coulometry

Conductance

Ion specific electrodes

III. Chromatographic Methods

Paper chromatography

Thin layer chromatography

Gas chromatography

Ion exchange

Electrophoresis

IV. Other Instrumental Methods

Refractometry

Osmometry

V. Classical and Other Methods

Gravimetry

Solvent Extractions

Titrimetry

Ultramicrochemistry

Standards

Quality Control Materials

VI. Skills

Analytical — electrochemistry

Analytical — biochemistry

Enzymology - kinetics

Toxicology

Internal Medicine

Environmental Health

Protein chemistry
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 101

Table 5. Competencies in Clinical Chemistry, (Hartford Hospital

Laboratory, June 1971) (continued).

VI. Skills (continued)

Spectroscopy (AAS)

Computer programming

Statistical - control methods

(Pathology-Gross and Micro) Instrument fabrication

Poisonous mushroom

identification

Teaching skills Administrative techniques

Medical technology skills Immunology and genetics

Editorial and Publication

services

Reagent water purity

3. Instrumentation

Without any doubt, the most profound change which has occurred since

1950 in hospital laboratories of this country is the use of instrumental, to replace manual methods of analysis. In 1958 the chemistry laboratory

acquired its first single channel semi-automated blood analyzer

(Technicon's AutoAnalyzer ®). At that time the major dependency on in- struments rested on either Klett double beam filter colorimeters or the

Coleman single beam simple grating spectrophotometers of wide band

pass. Although, as shown in Table 6, absorption spectrophotometry (now

of significantly better photometric performance) is still the mainstay of the

clinical chemistry laboratory, the steady introduction of other types of in- struments is striking. The percentage of total tests performed by each

class of instrumental methods can be expected to change in future years

from those given in Table 6, especially as electroanalytical and gas chromatographic methods become more firmly established in hospital service

laboratories. I would also predict that Hquid-Hquid chromatographic

methods of analysis will be utilized more fully to resolve complex mixtures into readily measurable fractions hopefully without extensive

preparatory scrub-up procedures [16, 17].

By the criteria of some chnical laboratory scientists, an inspection of

Table 6 would suggest that our laboratory at Hartford lags in the use of

highly automated instrument systems and "on-Une" computer equipment.

Both systems have become very common in hospital and private clinical laboratories all across the country and will certainly play an ever increas-
102 ANALYTICAL PROBLEMS

Table 6. Methods and instrumentation (Hartford Hospital, June 1971).

I. Optical Methods and Instruments - 91% of total service testing

A. Ultraviolet—visible absorption 68%

1. Colorimetry (interference filter photometers) — 42%

a. 9 single channel autoanalyzers (181,000 testS/

2. Simple spectrophotometers (wide band pass) — 8%

a. 3 B & L Spec 20' s (service)

b. 4 Coleman Jrs. (teaching)

c. 2 Kletts (teaching)

3. Small spectrophotometers (narrow band pass) — 2%

a a. 3 Gilford 300 N's

b. 1 Beckman B

4. Enzyme — kinetic instruments (narrow band pass) — 16%

a. 2 Gilford 2000' s with Beckman DU monochromators

(71,000 tests)

5. Recording spectrophotometers — 1%

a. 2 Perkin-Elmer 202

b. 1 Beckman DK-1

6. Jligh performance spectrophotometer — 0. 1%

a. Gary 16 for standardization work

B. Flame Photometry (79,000 tests) 18%

1. Types of instruments

a. Instruments Laboratories Inc. Model 143 b (service)

b. 2 Baird KY-1 (teaching) C. Atomic absorption spectrophotometry (14,000) 3%

1. Types of instruments

a. 1 Instrumentation Laboratories Inc. Model 153

b. OCLI Instrument AAA-3 (loan) D. Fluorimetry < 1%

1. Turner Model No. 110

2. Aminco—fluromicrophotometer

E. Infrared spectrophotometry 0. 1%

1. Perkin-Elmer-700
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 103

Table 6. Methods and instrumentation (continued)

II. Electroanalytical Methods and Instruments - 4%

A. Blood pH, PCO2. systems

1. 3 Corning blood gas Model 160

2. 2 Radiometer PMQ 4

B. Other pH equipment

1. 2 Orion Model 401

2. Beckman Model G

C. Coulometers

1. 2—Cotlove chloride titrators D. Conductivity Meters for Reagent Grade Water Testing

1. Radiometer, Model CDM 2

2. Constant recording unit from Foxboro—Model 9460 AC/PC

Dynaloy Recorder C with Beckman conductivity

cell (cell constant = 0.001)

E. Specific Ion Electrodes

1. Chloride - Orion sweat testing Model #417

2. Calcium — Orion calcium ion— specific electrode

Model 99-20 and Model 801 meter

ni. Electrophoresis Chromatography Methods and Instruments — 3%

A. Protein electrophoresis units and densitometers

1. 2 Beckman Microzone Systems RlOl with densitometers

2. Starch gel apparatus - Buckler

3. Immunoelectrophoresis - Shandon

4. Lipoprotein electrophoresis — Shandon

B. Thin Layer Chromatography Apparatus — Buckler

C. Gas Liquid Chromatograph with flame ionization - Hewlett

Packard Co., Model 5750B

IV. Other Instrumental Methods ^ 1%

A. Protein by refractometry

1. American Optical Co. TS Meter Model 10400

B. Osmometer

1. 2—Advanced Instruments Inc. Systems

a. Model # 3D

b. Model #64-31

428-999 O - LT - 72 - 8
104 ANALYTICAL PROBLEMS

Table 6. Methods and instrumentation (continued)

V. Manual Methods <1%

A. Flocculations

B. Immunodiffusion quantitation C. Titrimetry

VI. Ancillary Equipment

A. Electronic Data Processing Calculators

1. Olivetti Underwood Programma 101

2. Monroe Desk Top Calculator Model #820

B. Computers — (limited access use)

1. Digital Equipment Companies - PDP 15 (quality control

data)

2. IBM 360 (hospital based) - (billing, inventories,

budgets)

ing role in providing chemistry services in the future. We have deliberately waited on the use of highly-automated equipment because of reservations about flexibility, standardization and accuracy. Yet at times one must admit to feeling somewhat confused by the swift changes in both the

economics of laboratory medicine and the growing technological complexity of the alternate instrumental choices. My pathologist friend Dr. Meyer has again caught something of this in his cartoon in Figure 9 which was used to illustrate an article by Alpert entitled, "Automated Instru- ments for Clinical Chemistry: Review and Preview" [18].

4. The Clinical Chemist's Role in the Medical Community

The central scientific responsibility of a hospital clinical chemist is to bring the full power of reliable quantitative measurements to the patient.

Although he must always provide his analytical work within the constraints of clinically determined time limits, valid measurements, not rapid answers, must be the goal. Precision and accuracy in clinical chemistry

are dependent upon (1) the standards and (2) the method. As in any

analytical technique, aqueous standards must be used concurrently and

span the entire range of values found in the patient's specimen if error is to be minimized. Just as the former practice of using factory precalibrated
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 105

Figure 9. One must admit to feeling perplexed at times by the swift changes and growing

technological complexity of analytical instruments for clinical laboratories. photometers without frequent calibration violated sound analytical practices, so does single point calibration of instruments with normal levels of serum reference materials which bypass the long-proven standardization

concepts upon which accurate measurements must rest. As always, ac- ceptable methods, be they manual or instrumental, must be judged in terms of sensitivity, precision, specificity and accuracy.

Once the accuracy of the analytical measurements has been assured by

proper use of standards and precise methods of low systematic bias, the

clinical chemist must then pay attention to the special "people problems"

given below.

a. Sampling. — Although the preparation of patients, their diets and the

timing of service requests are primarily the physician's responsibility, the

chemist must do everything he can to be sure a correct sampling is made

under conditions which later permit the physician to interpret the test. Life is a dynamic process with many involuntary changes (respiration,

diurnal rhythms, etc.) and voluntary changes (i.e., meals and activity).

Although completely physiologic, these normal processes can nevertheless invahdate the finest measurement if ignored. Dr. Meyer in Figure 10

has strongly suggested that one must be sure that all laboratory personnel

act with intelligence and concern in obtaining specimens. Sampling and

handling are as important as any of the subsequent phases of separation,

isolation, measurement and calculation. It has been my experience that sampling errors during specimen procurement, labelling or transporting,

coupled with mistakes in reporting have been far more frequent than measurement errors since the introduction of quality control programs as sug- gested by Figure 1 1

.
106 ANALYTICAL PROBLEMS

Figure 10. Laboratory personnel must act with intelligence and concern in obtaining

specimens from patients.

Figure 1 1 . More errors occur during collection, transporting and data handling than within

the laboratory since the introduction of quality control programs.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 107

b. Method and Personnel Interactions. — Senior medical technologists

who have achieved great manual manipulatory skills often resent and resist the introduction of new methods; especially instrumental methods

which tend, in their eyes, to reduce the importance of their contributions.

There is, therefore, an understandable reluctance by many clinical chemists to incur the displeasure of the technical staff by making method

changes too frequently. Yet when analytical upgrading is not constantly

taking place in a laboratory, the technological "shock" of later transitions

may be overwhelming as suggested in Figure 12. Technological obsolescence can occur very rapidly today for both the clinical laboratory

and its personnel.

Figure 1 2. Analytical upgrading of methods and instruments must constantly take place or

the "technological shock" of later transitions may be overwhelming.

Our own experience with new instrumental methods and revision of

older manual methods since 1959 reveals (from Table 4) that only seven

of the thirty-eight methods employed prior to 1 960 have not changed. Per- haps more importantly, however, has been the chemistry division personnel changes which have brought in the senior scientific chemical and en- gineering skills needed to permit the orderly incorporation and heavy dependence upon instruments. Thus each year clinical chemistry has

become more and more a team effort. No individual can any longer bring

to the task all of the developmental, technical, chemical, engineering and

management skills required. One needs only scan the "1971 Annual

Reviews of Clinical Chemistry" by Kingsley in this past April's issue of

Analytical Chemistry [19] to appreciate how impossible a job one faces

if he decides to "go-it-alone".
108 ANALYTICAL PROBLEMS

c. Communications with physicians and others. — It is very important that

the chemist communicate actively and effectively with the physician who

must order the clinical chemistry tests. "Laboratory Notes" represent our

annual effort each June to publish for the medical staff a comprehensive

listing of tests available, the normal values, analytical precision, special

information and precautions in sampling as well as the method reference

[20]. As noted in its introduction, more detailed instructions are available

in the "Nursing Procedure Book" or "Rapid Rolodex Files" which are

available at each of the eighteen nursing stations throughout the hospital

where physicians initiate written orders on their patients. Alternatively,

laboratory phones are always manned with personnel who can answer

routine queries. To further assure that tests will be available when

needed, a senior clinical chemist is always "on-call" by phone, and physicians and laboratory staff are urged to contact him directly. Therefore the

physician's dilemma shown in Figure 1 3 can often be resolved by a few

simple tests which are or can be easily obtained— if the physician knows

of their availability or by conversation with the on-call chemist, orders

them. By many informal routes, as well as those described, effective communications with physicians must be accomplished.

Figure 13. The physicians dilemma in diagnosis of coma can often be resolved by a few

relatively simple chemistry tests. Moral- when perplexed, talk with your friendly clinical chemist.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 109

When special tests are not performed within our laboratory, we also

take the responsibility after consultation with the physician to send the

specimen to an outside laboratory. Appendage B lists those 32 types of

tests which were referred out in 1970 (a total of 1,080 tests). Because of

frequent conversations with those requesting these outside services, the

rationale and priorities for adding new tests to our laboratory are more

easily determined. Since outside referral tests are generally fairly complex, the average cost to the patient is $25/test versus our average

charge for chemistry tests of approximately $3 /test.

5. Chemistry Costs vs. Charges—The Hospitals' Dilemma

The annual costs for operating chemistry services at Hartford Hospital

presently are calculated to be about $1,000,000. Since the test volume is approaching 500,000, the basic cost is approximately $2/test. However,

due to the fact that we, like most hospitals, cost account the entire laboratory as a unit and do not split out chemistry costs, the charge to the patient

is nearer $3/test in order to defray the entire pathology department costs. With the advent of automation, venture capital and aggressive laborato- ry management, many private clinical laboratories are now providing

limited chemistry services {i.e., screening profiles) at lower charges than

do hospitals. This is due primarily to the fact that hospital laboratories

have the extra costs of (1) teaching programs (2) autopsy expenses (3) round the clock fast-response services for emergencies and (4) costly ser- vices like microchemistry which averages not $2/test but closer to $10

while charges average $5 /test. To date neither hospital administrators nor

pathology laboratory directors have been willing to say that outpatients

are exempt from these costly inpatient hospital service charges. The

larger private non-hospital clinical laboratories have, therefore, had much

more latitude to lower charges and gain in volume.

As a result all across the nation we are seeing the rapid emergence of

two types of clinical chemistry laboratories. One, typically a unit within a hospital dedicated primarily to inpatient services, fast-response and 24

hour emergency testing and the second, generally private and detached

from the medical center, providing high volume out-patient testing for

physicians' offices with heavy dependency upon automated technology

mainly the Technicon SMA 12/60 units. Ultimately regional chemistry laboratories incorporating more of the

flexible management and the efficiency of the private enterprises, yet

providing all of the services of the present hospital laboratories, will
110 ANALYTICAL PROBLEMS

evolve. The most logical way to assure both the quality and quantity of

services under the direction of skilled laboratory scientists is to have

these laboratories be an integral part of the medical community they support. These regional service organizations will be of great scientific complexity and to maintain their excellence must be centers of education, of

original research and of innovative instrument developmental activities.

B. Special Considerations in Clinical Enzymology

"It is, I think, difficult to exaggerate the importance to biology, and I venture to say to chemistry no less, of extended studies

of enzymes and their action."

Hopkins [21]

As a specific illustration of the rapid technological changes which now

characterized clinical chemistry laboratories and their responsiveness to biomedical research events, let me review with you the phenomenal

growth of clinical enzymology in the past fifteen years. In 1950 the Hartford Hospital Clinical Chemistry Laboratory, in common with most

hospital laboratories, offered only four enzyme assays: amylase and lipase to help detect pancreatic disease, acid phosphatase as an aid in diagnosis

of malignant prostatic tumors and alkaline phosphatase for the diagnosis

of diseases of bone, of liver or of neoplastic origin. By 1955 the total volume of clinical enzyme tests by these four assays was approximately

6,000 or 9 percent of the chemistry tests performed in that year. Now 15 years later in 1970-71, 12 separate enzyme assays are offered routinely

and another 5 are available when required for special needs. ^ Last year

the total volume of enzyme assays was 9 1 ,000 or 2 1 percent of the present

total chemistry workload. Figure 1 4 shows how rapidly some of the newer enzymes used in diagnosing heart disease have caught on in the last few

years.

' With the advent of isoenzyme assays and a multitude of newer serum, urine and RBC enzyme assays it would be possible

to list 50 or more separate determinations that might be performed. For example, one might assay the red blood cells activity of delta aminolevulinic acid dehydrase activity since this enzyme has shown good correlations with blood lead levels, however, the direct measurement of blood lead is already available.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 111

1. Transaminase—Introduction and Research

The great impetus to clinical enzymology began in 1954-55 when serum

glutamic oxaloacetic transaminase (SGOT)^ was introduced as an aid in diagnosing patients suspected of having an acute myocardial infarction

(heart attack). At first clinicians were amazed and openly skeptical that the very minute amount of this enzymatic protein circulating in serum

could offer help as claimed in the differential diagnosis of chest pain.

However, in patient after patient and in study after study conducted

between 1955 and 1957 this SGOT assay gave information which correlated very well with the history and the highly esteemed traditional elec- trocardiographic evidence of infarction of the heart. Within months

doubts were replaced with enthusiastic acceptance in every institution in

- Enzyme classification (EC) No. 2.6. 1.1- L-Aspartic 2-oxo-glutarate aminotransferase. The popular name of this enzyme

in the United States is serum glutamic oxaloacetic transaminase (SGOT) or "the heart transaminase." Some publications use the term aspartic aminotransferase.
112 ANALYTICAL PROBLEMS

which the test became available. Today one seriously questions the diagnosis of acute myocardial infarction if enzyme changes are absent. Figure

14 also shows that the subsequent introduction of lactic dehydrogenase

(LDH)^ and creatine kinase (CK)^ has also resulted in heavy utilization of

these two enzymes in patients with suspected myocardial infarctions.

The original research investigations and published reports on the

transaminase enzymes can be traced back to 1937 to the work on pigeon

breast muscle by two Russian workers, Braunstein and Kritzmanon [22].

A few years later in 1940, the Americans Cohen and Hekhuis [23] showed

the presence of two distinct transaminase enzymes in rat tissues. The

glutamic oxaloacetic transaminase (GOT) gave activities as follows:

heart > muscle > brain > liver > kidney. In contrast, the glutamic pyruvate transaminase (GPT)^ activity was

highest in liver but very low in heart. In 1954-55 three New York

biomedical investigators, LaDue, Wroblewski and Karmen [24], tested

to see if these differences in organ activity were actually reflected in the serum after injury to an organ in cancer patients. Fortuitously they noted

marked elevations in the SOOT enzyme of several post-operative patients suffering from transmural myocardial infarctions. The diagnostic

significance of these observations was not missed and their findings were communicated quickly in widely read publications [25]. By 1957 the

value of the SOOT assays in the clinical practice of cardiology was amply

confirmed and had become firmly established in numerous medical centers all over the world. The pressure to supply SOOT assays routinely in

all hospitals receiving patients suffering from heart attacks was tremendous. However, by the early 1960's SGOT assays were generally avail- able and as one direct consequence of the rapid growth in clinical enzymology the need for chemists in clinical laboratories increased

considerably.

2. SGOT Methodological Considerations

The original assays of serum SGOT were performed by indirect

techniques requiring laborious paper chromatographic separations of the

3 EC No. 1.1.1.27, L-lactate: NAD oxidoreductase.

* EC No. 2.7.3.2, ATP: creatine phosphotransferase.

^ EC No. 2.6.1.2, L-Alanine 2-oxo-glutarate aminotransferase. The popular name of this enzyme in the United States is serum glutamic pyruvate transaminase (SGPT) or "the liver transaminase." Some publications use the term alanine aminotransferase.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 1 13

products. Karmen [26] soon introduced a unique and relatively simple ul- traviolet assay for SGOT determinations which permitted clinical laboratories throughout the world to measure this important enzyme in serum or any tissue with great ease. By coupling a product of the GOT reaction,

oxaloacetate, to the coenzyme dependent malic dehydrogenase (MDH)

(see Equation 4 and 5) the continuous detection of GOT activity was

possible. The rate of change at A340 nm under carefully controlled conditions is directly proportional to GOT activity.

aspartate + a - ketoglutamate^^ glutamate + oxaloacetate (4)

oxaloacetate -f reduced NAD :^malate + NAD~ + H+ (5)

The spectral absorbance curves of reduced NAD and NAD which are associated with these redox changes are shown in Figure 15. The continu- ous ultraviolet spectrophotometric technique for measuring the activity of

coenzyme dependent enzymes (or any components of these systems) had

NH

250 300 350 400

WAVELENGTH NM

Figure 15. The spectral absorbance curves of NAD and reduced NAD. The change in ab- sorbance at 340 nm permits the activity of many enzymes to be measured continuously

{e.g. transaminase).
114 ANALYTICAL PROBLEMS

been described and developed extensively by Warburg and Christian [27]

in the mid 1930's. Since there are nearly one-hundred coenzyme dependent systems known to biochemistry today, the importance of thoroughly

characterizing these coenzyme materials and having them available in ultra-high purity is obvious. Fortunately the need for coenzyme standard

reference materials has been recognized by those in charge of the NBS

Standard Reference Materials (SRMs) Program and I am told that work

is to begin on NAD in the Analytical Chemistry Division at NBS.

Over the last decade many people have suggested modifications in Karmen's malic dehydrogenase coenzyme linked system for GOT assays as follows:

a. changes in concentrations of reduced NAD, of aspartate,

and of a-ketoglutarate.

b. higher activity and purity of MDH enzyme to speed the

oxaloacetate conversion and exclude GOT contamination,

respectively.

c. inclusion of LDH to destroy pyruvate endogenous to serum (avoids preincubation).

d. closer control of kinetic and instrumental sources of variability (e.g., temperature).

e. changes in the buffer from phosphate to 2-amino-2-hydroxymethyl-1 ,3 propandiol (Tris).

These modifications were generally aimed at reducing the causes of variability in GOT results seen in both intra and inter-laboratory comparisons.

Although alternative chemical systems based upon single or double-point

sampling colorimetric techniques have been introduced, none has

received the widespread national and international study and endorsement of Karmen's continuous spectrophotometric procedure.

3. The Clinical Utility ofEnzyme Measurements

The time-activity pattern of serum glutamic-oxaloacetic transaminase

(SGOT) changes during the course of an uncomplicated myocardial in- farction is given in Figure 16. About six to twelve hours after onset of

chest pain, abnormal serum activity can be detected for the first time.

Subsequently, a peak activity four to five times the upper limit of normal

(4N-5N) is reached near 36 hours followed by return to normal levels by

the fourth day. Note that the serum glutamic-pyruvic transaminase

(SGPT) remains within or close to normal limits throughout this period.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 115

6N^

PAIN 2 4 10 12 DAYS

Figure 16. The daily activity pattern of SGOT and SGPT following the onset of an acute myocardial infarction. To more fully appreciate the dynamic shifts between intracellular and extracellular fluids which these serum enzyme patterns of activity reflect,

the article by Posen entitled, "Turnover of Circulating Enzymes" should

be reviewed [28].

As enumerated in Table 7, there are many other biological changes

which also occur during damage to cardiac muscle which are reflected in serum. The time-activity patterns of some of the other helpful serum en- zyme changes in comparison to the SGOT are given in Figure 17. The

very short-lived rapid elevation of the isocitric dehydrogenase^ (IDC) of

cardiac origin is seen within one to three hours after infarction. This heart

isoenzyme of ICD is very heat labile and has usually disappeared well

within the first twenty-four hours. The serum ICD isoenzyme of liver origin is heat stable and will often rise during the later course of an infarction of the heart if there is a complication causing liver damage. This ICD

assay [29] is rarely used today and is little needed since the introduction

of creatine kinase.

^ EC No. 1.1.1.42 L-isocitrate: NADP oxidoreductase.
116 ANALYTICAL PROBLEMS

Table 7. Biochemical changes reflecting cardiac muscle damage.

Enzymes

SGOTransaminase

Lactic dehydrogenase

LDH fraction one

Hydroxybutyric dehydrogenase

Creatine kinase

Other

Serum copper (ceruloplasmin)

Serum iron

Serum nickel

Erythrocyte sedimentation rate White blood count

Aldolase

Phosphohexosisomerase

Malic dehydrogenase

Isocitric dehydrogenase

(heat labile fraction)

Complement

C—reactive protein

Pyruvate

Lactate
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 1 17

The serum creatine kinase (CK) enzyme has a time-activity course which is very similar to that of SGOT except that the rise, peak, and

return to normal all occur a few hours eadier. Although the assay is much

more complex than SGOT, serum CK assays off^rthe distinct advantage

of greater clinical specificity as damage to other body tissues, especially

liver, does not release this enzyme into serum. Only skeletal muscle injury causes alterations and this is usually easily excluded.

Serum total lactic dehydrogenase (LDH) and its component isoenzyme

fraction found predominantly in heart (LDHi) have also been utilized extensively to diagnose myocardial infarctions. The more sensitive and more clinically specific LDHi, as well as the total LDH, does not return

to normal levels for 10 to 14 days making the assay very useful in patients

admitted to the hospital several days after the onset of their infarction.

Between 95 and 100 percent of patients proven to have an acute myocardial infarction at autopsy have been shown to have these SGOT, LDH or CK changes when studied properly.

Alterations from the typical time-activity patterns seen in myocardial

infarction often suggests that the patient has another cause underlying the

chest pain. The time-activity patterns shown in Figure 1 8 represent the

enzyme changes seen during the hospital course of a 50-year-old woman

admitted in shock to Hartford Hospital with a history of chest pain. The

tentative diagnosis of myocardial infarction was supported by electrocardiographic abnormalities compatible with, but not diagnostic of, acute

heart damage. Both transaminase enzymes were markedly elevated and

the exceedingly high abnormal SGPT level suggested a tissue source

other than or in addition to heart muscle. The high serum amylase obtained on the next day as a result of this high SGPT level lead to the correct diagnosis of acute pancreatitis which was later found to be secondary

to cholecystitis with cholelithiasis. There would be little difficulty to present numerous similar enzyme

time-activity patterns involving transaminase and many other serum en- zymes offered daily in the laboratory which have helped physicians to reach a correct diagnosis. Without the enzymes many of these diagnoses

would have been missed or not made at all until a subsequent admission

when the disease process was far more advanced. Although clinical en- zymology is already a very meaningful area to clinical medicine, it is obvi- ous that there lie ahead opportunities for innovative approaches with

enzymes to the rapid diagnosis of common diseases seen in daily practice

as well as the solution to some maladies which have plagued man for

centuries.
118 ANALYTICAL PROBLEMS

SGPT

PAIN 2 4 6 10 DAYS

Figure 1 8. The daily activity pattern of serum enzymes during an acute bout of pancreatitis

which was clinically thought to be a heart attack.

III. Current Analytical Problems and Opportunities

"The history of science shows that even during the phase of

her progress in which she devotes herself to improving the ac- curacy of the numerical measurement of the qualities with

which she has long been familiar, she is preparing the materials

for the subjugation of new regions, which would have remained

unknown if she had been contented with the rough methods of

her early pioneers. I might bring forward instances gathered

from every branch of science showing how the labour of careful

measurement has been rewarded by the discovery of new fields of research, and by the development of new ideas."

Maxwell [30]

Several analytical problems, and therefore opportunities, found in the

health areas will be discussed in this section. The order and weighting of
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 119

the following is strictly my own and again reflects a deep concern for meaningful quantitative measurements within medicine.

A. Standardization

Until very recently only a few medical researchers and clinical chemists

have come into this field with a background of analytical chemistry.

Therefore, the skepticism about chemical measurements which is the

traditional hallmark of the analytical chemist has not necessarily always

characterized medical research or clinical chemistry.

Standardization efforts in service laboratories have increased in recent years partly as a result of more chemists with analytical training entering

medical laboratories, but mainly due to the surveys which have clearly

demonstrated the need for better performance. Congress officially recognized this national problem by passage of the Interstate Clinical Laboratory Improvement Act of 1967 (CLIA 1967). The government proficiency testing under this CLIA '67 and also the Medicare inspections by state personnel are increasing the emphasis on standards.

The need for improved clinical standards and standardization

procedures received special attention by analytical chemists in 1968 in a paper entitled, "Standard Reference Materials as Reviewed by a Laboratory Supervisor— A Status Report" published in Analytical Chemistry

[31] by the Subcommittee on Reference Materials, Committee on

Analytical Chemistry, Division of Chemistry and Chemical Technology,

National Academy of Sciences — National Research Council. Today, only

three years later, in 1971 the availability of high purity certified organic

and inorganic standards has improved considerably as shown in Table 8 taken from Dr. Meinke's recent article, "Standard Reference Materials

for Clinical Measurements" [32]. I recommend the reading of this

article by anyone concerned with quality measurements in the medical

laboratory. In the protein area. Dr. Theodore Peters and a Protein Subcommittee

of the Standards Committee, American Association of Clinical Chemists,

have already proposed specifications for a primary protein standard [33].

Standard material, bovine serum albumin, meeting these specifications

has now been achieved by several manufacturers and the National Committee for Clinical Laboratory Standards (NCCLS) has accepted these

specifications as the first "Tentative Standard" in clinical chemistry. I should point out that there is a great opportunity for anyone interested in

428-999 O - LT - 72 - 9
120 ANALYTICAL PROBLEMS

Table 8. Standard reference materials for clinical measurements. SRM No.

1571

2201

2202

Name Purity

(%)

Property certified Amount

(g) Date issued

40h Sodium oxalate 99.95 Reductometric

standard

60 April 24, 1969

83c Arsenic trioxide 99.99 Reductometric

standard

75 Feb. 6, 1962

84h Acid potassium

phthalate

99.993 Acidimetric

standard

60 July 9, 1969

136c Potassium

dichromate

99.98 Oxidation

standard

60 March 24, 1970

1861c Potassium dihydrogen phosphate

99.9 pH 30 July 29, 1966

18611c Disodium hydrogen

r»nr»Gr»nflf f

99.9 pH 30 Sept. 1, 1970

350 Benzoic acid 99.98 Acidimetric

standard

30 April 15, 1958

911 Cholesterol 99.4 Identity and purity 0.5 Oct. 20, 1967

912 Urea 99.7 Identity and purity 25 Sept. 24, 1968

913 Uric acid 99.7 Identity and purity 10 Sept. 24, 1968

914 Creatinine 99.8 Identity and purity 10 Sept. 24, 1968

915 Calcium carbonate 99.9 Identity and purity 20 March 4, 1969

916 Bilirubin 99 Identity and purity 0.1 March 10, 1971

917 D—Glucose 99.9 Identity and purity 25 Nov. 18, 1970

918 Potassium

chloride

99.9 Identity and purity 20 Jan. 22, 1971

922 tris(HydroxymethyDaminomethane99.9 pH 25 May 1, 1971

923 tris(HydroxymethyDaminomethane

hydrochloride

99.7 pH 35 May 1, 1971

930 Glass filters for Absorbance 3 filters Feb. 24, 1971

spectrophotometry

Orchard leaves

NaCl

KCl

99.9

99.9

Major and trace constituents

pNa

pCl

PK

pCl

75

120

160

Jan. 28, 1971

April 15, 1971

May 1, 1971

Taken from Meinke, W. W., Standard Reference Materials for Clinical

Measurements, Anal Chem., 43, 28A (1971)
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 121

protein chemistry and its standardization problems to join Dr. Peters in developing standards for human serum albumin and various globulins.

7. pH Standardization—A Model to Avoid Chaos

The importance of NBS-SRMs to the clinical field is easily demonstrated by comparing the uniformity in standardization which exists in whole blood pH measurements. Because of the early leadership by NBS

personnel over the years in providing a centralized authoritative base for

measurements involving hydrogen ion activity with various electrodes,

the chaos in standardization which exists in many other areas of complex

measurements [e.g., proteins-enzymes) has been almost totally avoided.

Essentially all manufacturers and users of blood pH meters now employ

the same standards to evaluate the performance of their instruments. The

introduction in 1961 by Bower, Paabo and Bates [34] of the extremely

useful 0.04 M physiologic dual salt phosphate buffer (SRMs Numbers

186 Ib-KH2P04 and 186 IIb-Na2HP04) giving 7.381 ±0.001 pH units at 37 °C was very timely and is today the central reference point for clinical blood pH measurements in this country. Dr. Richard Durst tells me of yet another addition to the NBS buffers

intended for use in standardizing clinical pH measurements on whole

blood — this one a Tris-Tris HCl buffer matrixed in physiological concentrations of sodium and potassium to more closely simulate the ionic

matrix of whole blood. Certainly as we move to use ion selective elec- trodes other than for pH measurements, 1 would expect that the reference

standards would contain more than the one element under study in order

to more fully parallel the physiological specimens. This standardization of

ion selective electrodes is a critical area requiring much more intensive in- vestigation by chemists before widespread clinical applications occur. The principal problems are the limits of specificity and the little un- derstood binding of the ion of interest to various proteins.

2. Standardization Problems Peculiar to Enzymology

It is common knowledge among enzymologists that there are absolutely no accepted benchmarks to help one standardize enzyme activity measurements. However, the need for standardization in enzyme measure-
122 ANALYTICAL PROBLEMS

ments has been apparent for many years. For example, in 1956 when an

International Enzyme Commission of ten distinguished enzymologists^ was appointed by the International Union of Biochemistry, they were

charged *'To consider the classification and nomenclature of enzymes and

coenzymes, their units of activity and standard methods of assay (italics

mine), together with the symbols used in the description of enzyme

kinetics" [35]. It is interesting to note that the final Committee recommendations in 1961 and those endorsed by International Union of

Biochemistry in 1964 deliberately avoided specific recommendations

concerning standard methods of assay except to give general advice on

the conditions of measurement. Despite this lack of enzyme method stan- dardization, the lUB 1964 Enzyme Commission Recommendations have

been exceedingly successful in standardizing symbols and the activity

units. However, the need for method standardization still exists and is particularly acute in clinical enzymology. I personally believe standardization must include not only the methods of assay, but also the stable human

source enzyme reference materials which must validate the method.

While some people fear that selection of standard methods for enzyme as- says by nafional or international organizations will stagnate enzyme

method development, I firmly believe that the very existence of a stan- dard method of assay is required to unify methods research by giving an agreed-upon starting point of reference.

a. Enzyme Standardization Activities. — In the United States a Subcommittee on Enzymes of the Standard Committee of the American Association of Clinical Chemists (AACC) is developing recommendations for

standardized methods to assay serum enzymes. At the same time there is a separate effort within the Subcommittee on Enzymes of the Area Committee in Clinical Chemistry-National Committee for Clinical Laboratory

Standards (NCCLS), to develop reference enzyme materials of human

origin. Both groups recognize that enzyme standardization is very much

like the two faces on a coin; each face can be viewed separately but it is really not possible to detach one face from the other. Thus, the AACC Committee on methods recognizes the need for suita- ble human source enzyme materials to adequately define the operational

characteristics of the methods they choose. Conversely, the NCCLS Enzyme Subcommittee members cannot really choose the most likely human

source of stable enzyme reference material without evaluating them with

^ M. Dixon (England), E. F. Gale (England), S. P. Colowick (U.S.A.), A. L. Lehninger (U.S.A.), A. E. Braunstein

(U.S.S.R.), W. A. Engelhardt (U.S.S.R.), K. Linderstrom-Land (Denmark), P. A. E. Desnuelle (France), F. Lynen (Ger- many), O. Hoffmann-Ostenhof (Austria).
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 123

validated methods. Hopefully within 1972, these joint efforts of AACC

and NCCLS should result in almost simultaneous recommendations on enzyme methods and materials for at least one or more of the following:

(1) alkaUne phosphatase — human purified enzyme from placenta (2) aspartate aminotransferase — human purified enzyme from red blood cells

(3) lactic dehydrogenase — human purified enzyme fraction LDHi from

red blood cells and (4) prostatic acid phosphatase — human crude enzyme

from prostate and/or semen. There are several groups in Europe concerned with enzyme stan- dardization. There was a national symposium in Italy in 1969 [36] and I have just received correspondence from Professor A. Burlina telling of

another international symposium which is scheduled for November 27-

28, 1971 in Conegliano Vento, Italy. Correspondence from Professor R. Keiding reports of the formation of a committee of the Scandinavian Association of Clinical Chemists concerned with increasing the precision

and accuracy of clinical chemical enzyme assays. In England a working party of the Association of Clinical Biochemists

composed of well known clinical enzymologists^ who provide overlap

through individual memberships with the Association of Clinical Pathologists and The Royal College of Pathologists, has issued its first report

concerning standardization of the alkaline phosphatase King-Armstrong

Unit. This report gives a manual and an automated reference method for serum assay of alkaline phosphatase complete with detailed notes [37].

A second report concerning the serum GOT assay is due soon. There has

recently appeared a report from Germany which bears further discussion,

but first it would be remiss not to note that the Standards Committee of

the International Federation of Chemists has appointed an Expert PaneP

to bring together recommendations on standardization of enzyme assays

and reference material.

The German Society of Clinical Chemistry through its Commission for

Enzyme Diagnostics and Standardization has published recommendations on eight serum assays^^ under the title "Standardization of Methods

for the Estimation of Enzyme Activity in Biological Fluids" [38]. This

German Committee recognized the major importance of "optimized conditions" established by appropriate experimental evidence in specifically

recommending the need for the following conditions: (1) The continuous

monitoring of enzyme kinetics. (2) The reaction rate should be constant

» D. W. Moss - Chairman, D. N. Baron, P. G. Walker, and J, H. Wilkinson. "G. IN. Bowers, Jr., Chairman (L'nited States). H. U. Bergmeyer (W. Germany) and H. V. Moss (United Kingdom). "Lactic dehydrogenase, lactic dehydrogenase-fraction one, glutamatic dehydrogenase, glutamatic oxaloacetate

transaminase, glutamatic pyruvate transaminase, creatine kinase, alkaline phosphatase and leucine arylamindase.
124 ANALYTICAL PROBLEMS

during the whole period of observation. (3) The kinds and concentrations

of substrates, cofactors, activators and buffers should result in optimal

reaction conditions. This relates to substances as well as to concentrations. (4) The reagents should be free of inhibitors. (5) The measurement

temperature should be 25 °C; measurements at 25 °C are easier to stan- dardize than at 30 °C; a temperature of 25 °C can be kept constant and

conforms with the international temperature for physio-chemical data. This German Committee has made a significant contribution in being

the first national clinical chemistry society to publish specific recommendations on standard assays. The difficulties to be overcome in reaching

other national and subsequently international agreements on standardized

enzyme methods and/or standardized reference enzyme materials should

not be underestimated. However, reducing the tremendous chaos which

presently exists in the absence of such agreements will undoubtedly make

the task somewhat more rewarding.

I would now like to discuss in greater detail some of the problems as- sociated with developing enzyme materials and measurement systems in the light of some of my personal experiences with alkaline phosphatase over the last decade.

b. Standardized Reference Enzyme Materials — Experience with Alkaline

Phosphatase. — The need to develop a human alkaline phosphatase stan- dard reference enzyme material is of greater importance today than people not working in this area would believe. As an example, in 1967 we

published on the "Variability of Analytical Results in a Survey Reference

Sample Related to the Use of a Non-human Serum Alkaline

Phosphatase" [39]. This paper documented the marked differences seen between the human pooled serum enzymes and other materials containing

alkaline phosphatase enzymes from unknown sources. Studies were per- formed on human serum pool material, an unknown survey material, four

commercial control materials, and four crude enzymes from (a) chicken

intestinal mucosa, (b) bovine intestinal mucosa, (c) hog kidney and (d) the

E. Coli enzyme of dog feces. Each source of enzyme showed markedly

different kinetic characteristics when tested in relationship to heat stabili- ty, stability to alkaline denaturation, and inhibition by phosphate or phenylalanine. In my judgement, none was similar to the serum enzyme(s) to permit its use without warning users of the differences.

I am always amazed to see the simplistic assumptions and frank compromises people glibly accept in using reference materials containing en- zymes. After spending months to perfect a method or instrument to per- form assays on the serum of patients, they frequently seek to validate the
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 125

enterprise by using non-human enzyme materials of which little is known

beyond the name on the bottle. Several queries will help one to evaluate reference materials containing

enzymes. First, what is the base protein of the pool? Not all preparations

of pools start with native human serum but instead many use reprocessed

outdated blood bank plasma or even animal plasma or serum as the

protein matrix. Second, what is the species and tissue source as well as the

method of preparation of the enzymes added to the base pool prior to lypholization? What kinetic differences are introduced by non-human

source enzymes versus the enzyme(s) in human serum? Third, how has

the labelled activity value been assayed? If the exact details of the assay

methods are not made available it is impossible to verify by independent means any given activity value. Often activity is measured by one method

and factors used to calculate activity to be expected by another. And,

fourth, what is the stability of the materials in the dry or frozen state and

then after reconstitution or thawing?

In general, experience over the years suggests that relatively few commercial manufacturers of quality control or reference materials containing

enzymes have the factual data to give one an answer to all these

questions. Happily, one or two companies have recognized that open and

free scientific communication and cooperation is as important as momentary proprietary advantages. These companies have published the sources

of added enzymes and their assay methods. Some have even supported ef- forts to develop human source reference enzyme materials via the

NCCLS Committee. It is fortunate that mechanisms like NCCLS committees now exist which can obtain a consensus among users, manufacturers, and government on standardization problems in the U.S.A.

Since at this time neither the added enzyme materials nor the assay

method can possibly be considered to be "standard" (implying a broad

consensus), it is ridiculous to suggest as has been done frequently in the

past that some commercial products are "enzyme standards in serum."

Moreover, it is almost impossible with the present lack of agreement in standardization in clinical enzymology for any manufacturer or individual

to certify the enzyme activity of a serum without providing complete in- formation about the four questions asked above. Yet, each day thousands

upon thousands of patients have their serum alkaline phosphatase activity

determined on instruments which have been calibrated solely by accepting labelled values.

From many conversations with manufacturers of quality control

materials I am sure that most commercial concerns would welcome stan- dardization efforts in clinical enzymology by authoritative competent
126 ANALYTICAL PROBLEMS

committees. The manufacturers recognize better than anyone else the

utter chaos which exists at present. In Figure 19, Dr. Meyer has tried to capture the frustrations of the analyst who relies too heavily on the label values of enzyme activity from various preparations of control materials

each containing a different source of enzyme. Needless to say, help from

persons who can bring order to clinical enzymology will be welcomed.

Figure 19. SRMs for enzymes are as yet only a dream. The barnyard is full of non-human

sources of enzymes but they may be troublesome substitutes.

c. Standardized Enzyme Methods—Experience with Alkaline Phosphatase.—Table 9 lists just a few of the numerous methods that have

been used to measure serum alkaline phosphatase over the last 40 to 50

years. It is obvious that trying to choose any one method arbitrarily as a

standard assay method would be difficult. However, the conditions set forth by the lUB Enzyme Committee in 1964 would immediately

eliminate a considerable number of these methods since they are not optimal. The German Standardization Committee has specifically recom- mended continuous monitoring of enzyme kinetics and the experience in our laboratory would endorse this choice over sampling techniques. In 1963 my biochemist-colleague. Dr. Robert B. McComb, and I were

convinced by preliminary investigations that a continuous spec- trophotometric assay for serum alkaline phosphatase could be perfected.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 127

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As shown in the spectral absorbance curves given in Figure 20, the rate of

enzymatic activity of alkaline phosphatases is easily monitored from the

changing absorbance near 400 nm as the colorless substrate p-nitrophenyl

phosphate is hydrolized at the alkaline pH of 10.15 to the highly colored

(self-indicating) product, p-nitrophenol. This substrate was first in- troduced by Ohmori in 1937 [40]. It has been utilized in the well-known

sampling type assay for serum alkaline phosphatase introduced by Bessey, Lowry and Brock in 1946 [41]. Lowry and coworkers in a prior

paper had remarked about the increased activity found in propanolamine

buffers [42]. These buffers were little used until Garren and Leventhal

employed tris in a study of the alkaline phosphatase of E. Coli in 1967

[43].

404 NM

i

• I 1 —'

'

I

300 3 50 400 450

WAVELENGTH NM

Figure 20. The spectral absorbance curves of one alkaline phosphatase colorless substrate and self-indicating product which permits direct continuous spectrophotometric assays. Subsequently, Amador, Zimmerman and Wacker employed a continu- ous type kinetic assay to measure the low levels of alkaline phosphatase

found in human urine using the buffer 2-amino-2-methyl-l-propanol [44].

These authors did not apply the continuous type assay to serum choosing

instead the traditional Bessey, Lowry and Brock procedure. During the

August 1964 Annual Meeting of the American Association of Clinical
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 131

Chemists our paper and one read by Klein [45] described almost identical

experiences in measuring serum alkaline phosphatase in 0.75 mol/liter of

2-amino-2-methyl-l-propanol (2A2M1P) at pH 10.15 at 30 °C. This

buffer accentuated the molecular activity 4 to 5 times over the glycine

buffer used by Bessey, et al., as was shown in our subsequent publication

[46].

After extensive experience over several years in both routine and ex- perimental work it was believed that this continuous alkaline phosphatase

assay system had been sufficiently described and tested to qualify as a candidate for a standard enzyme method. It successfully met the exacting

conditions set forth by the lUB Commission of Enzymes in the 1964 re- port as well as the conditions of the recent German publication. However,

in 1967 Hausman, et ah, [47] described another propanolamine buffer,

diethanolamine (DEA), which gave twice the molecular activity with p- nitrophenyl phosphate. Subsequently, this "DEA" method has received

the German Commission's endorsement. Continued examination of nu- merous other buffers in our laboratory has revealed yet another

propanolamine buffer, ethyl-amino-ethanol (EAE), with superior buffer- ing capacity and 50 percent higher molecular activity than DEA as shown

in Figure 21 [48].

IM EAE

9.8 10.0 tO.2 10.4 10.6 PH

Figure 2L Serum alkaline phosphatase activity as a function of pH and differing buffers. 2A2M1P = 2-amino-2-methyl-l-propanol, DEA = diethanolamine, and EAp is ethylaminoethanol.
132 ANALYTICAL PROBLEMS

This experience with several different buffers, all quite satisfactory, il- lustrates how difficult it will be to select a standard assay method even when all conditions are in agreement with the recommendations of

learned bodies. There is agreement at present, however, that continuous

monitoring of enzyme kinetic activity is a prime consideration. The details of other conditions, i.e., buffer type, must be resolved by further intensive

bench work and an open review of all data by the various official committees. It is my present belief that standard enzyme methods after fulfilling

the criteria previously listed must also be tested extensively with the sta- ble human source enzyme materials which are now currently under

development. In addition it will be necessary to test sera from numerous

patients with diseases associated with the major isoenzyme changes

(bone, liver, intestine and placental) before making final decision on any

standard method for serum alkaline phosphatase assay or for that matter

any other serum enzyme.

The job will be difficult since there are no absolute goals—only empirical observation and many strong, sometimes conflicting, opinions. Even

tentative choices of expert panels and other official committees will at best be arbitrary and subject to much debate. However, once national and

subsequently international agreement has been reached, critical scientific examinations of new systems versus the established standard enzyme

assay method are sure to create a continued pressure towards revision

with methods proven to be superior. We need not fear ossifications with

standardized assay methods for once a goal is set it is the very nature of

man that there will be many who will attempt to surpass it (the goal of a few years ago of a four-minute mile is now the three-minute fifty-second

mile!).

B. Referee Method Development

"Believe those who seek the truth; distrust those who claim to have found it." Andre Gide [49]

For many years the American Association of Clinical Chemists has

published a series of volumes entitled, "Standard Methods of Clinical

Chemistry" [50] which has attempted to identify methods which have

performed well in a submitter's laboratory and have been proven by test to be satisfactory in several checker's laboratories. The editorial board of
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 133

senior clinical chemists and the eaitor of each volume review all data to assure that the methods published are constantly updated to reflect the

daily working experience in clinical laboratories. Although due concern

is given for precision and accuracy, the primary aim is to describe in detail methods which will reliably handle the daily workload within a time span

which is realistic for service laboratories.

There has been a growing recognition of the need for methods which

are designed primarily to give excellent precision and low systematic bias

without the limitations imposed by time, efficiency and technical skill. These referee methods would seek to establish the "true" content of a serum constituent more closely than is possible with present routine daily

production methods. A referee method would be utilized, therefore, to establish the bias of the more commonly employed methods. Once ac- cepted across the country, referee methods could also prove extremely

useful in uniformly setting the label value for the serum control materials

required to calibrate automated instruments which do not accept primary

standard solutions.

Obviously the precision required of a referee method cannot be set by

routine daily quality control data which have given the "allowable error" of Tonks [51] or the subjectively chosen "medically significant values"

of Barnett [52]. I believe the objective criteria set by Cotlove, et aL, as the "tolerable analytical variability" [53] and by Young, et aL, in the

"average intra-individual biological variation" [54] must be utilized to help set the overall precision plus accuracy goals for these referee

methods. Table 10 gives the "average intra-individual biological variation" from Young's paper in comparison to the long-term precision data^^ observed in the Clinical Chemistry Laboratory at Hartford Hospital for

the period October 1969 to the end of September 1970. I would certainly

expect that the relatively short-term batch to batch analytical precision of

a properly functioning referee method to be ± 1 percent RSD, or less,- ex- cept for the serum enzymes, cholesterol and creatinine.

The accuracy to be expected of referee methods is yet to be established.

While accuracy cannot exceed the purity of the standard (see Table 8), ideally it should approach this limit subject only to the uncertainty of the

random analytical variability. Since to my knowledge there is no factual

information yet available on systematic bias of extremely precise referee

type methods, the accuracy goals listed in Table 10 are admittedly subjective and arbitrary. However, our rather intensive and now extensive work

in attempting to determine the systematic bias associated with the mea-

" Based on 80 to 1 20 days of a "blind" serum pool at multiple levels.
134 ANALYTICAL PROBLEMS

Table 10. Referee method goals.

GOALS

Average

intraindividual Long-term

biological laboratory Preci- Accuracy Uncertaint Toe* 1 est VariaLlOn variation sion 1./0 irom x/o irom (moan iinif cl % RSD % RSD '0 iXxJlJ 100% ixjyj /o 100% wjyj /o Sodium \A 1.2 0.5 0.5 L5

(144 mmol/1)

Potassium 4.6 2.3 1.0 0.5 2.5 (108 mmol/1)

Chloride 2.7 2.1 l.U 0.5 2.5 (25 mmol/1)

Carbon dioxide 5.3

-

4.0 l.V 0.5 2.5 (25 mmol/1)

Calcium L6 L3 0.5 0.3 L5

(2.5 mmol/1)

Magnesium 2.4 2.4 LO 0.3 2.5 (0.85 mmol/1)

Phosphate 9.9 4.3 LO LO 3.0

(3.8 mmol/1)

Total protein 2.2 L5 LO LO 3.0 0

(7.4 g/dl)

Albumin 3.0 4.5 LO LO 3.0

(4.9 g/dl) Uric Acid 8.8 2.9 1.0 LO 3.0

(6.1 g/dl) Urea nitrogen 13.5 3.2 LO LO 3.0 (18mg/dl)

Glucose 6.5 3.0 LO LO 3.5

(94 mg/dl)

Cholesterol 4.2 4.5 2 2 6

(204 mg/dl)

Creatinine 4.4 5.3 2 1 5

(1.1 mg/dl)

SCOT transaminase 11 3

(6.4 U/1) 17 3 SGPT transaminase 17 3

(5 U/1)

Lactic dehydrogenase 19 8 3

(126 U/1)

Alk. phosphatase 5.8 8 3

(45 U/1)

^ See: Young, D. S., Harris, E. K., and Cotlove, E., Biological and Analytic

Components of Variation in Long-Term Studies of Serum Constituents in Normal Subjects: IV Results of a Study Designed to Eliminate Long-Term

Anal.vsis Deviations, Clin. Chem., 17, 403 (1971).

^ Hartford Hospital, '69-70. See also Appendage A.

^ These represent the opinion of the author on what is achievable for Referee Methods only. The uncertainty was calculated as (2 x precision + accuracy).
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 135

surement of total serum calcium by atomic absorption spectrophotometry

(AAS), suggests to me that it is presently unrealistic to expect accuracy to exceed 100 ±0.5 percent [55]. Yet to suggest that inaccuracy exceed

100± 1 percent except for enzymes and certain complex unstable biological substances would be to expect too little of our measuring capabilities.

With the availability of certified high purity standards from NBS as SRMs (Table 8) many chemists have asked, and quite properly so, how

the NBS standard would help to improve the daily measurements on patients' samples. While it is obvious that SRMs in solution can be used to

calibrate instruments or can be directly incorporated into the method, it is much less apparent to many that the very existence of the "certified"

SRM, in and of itself, establishes a new, higher level of purity for clinical standards than ever before. For example. Witter and coworkers in 1965

demonstrated (and again in 1970) conclusively that cholesterol standards

available on the market were mixtures of several compounds [56, 57].

Furthermore, even after following the recommended purification

procedures, special storage conditions must be maintained if deterioration

is to be prevented. Presently to assure against deterioration in cholesterol

certified at the 99.4 percent level of purity, the NBS-SRM 911 cholesterol

(plus at least one commercial supplier of cholesterol standards) package

and store their highly purified materials under inert gases. Thus the

specifications and analytical work behind the certification of an NBSSRM effectively act to upgrade the standards sold commercially.

The second answer as to how an NBS-SRM will affect the accuracy of

clinical measurements involves the development of these precise and ac- curate methods, these referee methods. An NBS-SRM or a comparable

high purity material must be available as the logical starting point and central focus of any referee method. Without such a high purity certified stan- dard even comparative method evaluations become rather meaningless.

The high purity chemical standards are used in all calibrations of instruments both in simple solutions and in as nearly an identical matrix as possible to the matrix of the unknown. The method is then tied to multiple

standards closely bracketing the level of the unknown. By proper statisti- cal design and experimentation, the random error components from each

procedural step are evaluated and reduced.

As an example of the way the accuracy of a referee method must be

evaluated, I will again draw on our recent experience in trying to determine the systematic bias associated with measuring calcium by AAS. The

same highly skilled analytical chemist^^ performed all the steps of six dif-

'- Mr. John Pybus, Visiting Clinical Chemist- Hartford Hospital. June 1969 to June 1971. (Senior Scientific Officer, on

leave, Auckland Hospital, Auckland, New Zealand).

428-999 O - LT - 72 - 10
136 ANALYTICAL PROBLEMS

ferent AAS methods and two minor variations for measuring total calcium

in serum. The Pybus, Feldman and Bowers method [55] which is now

being proposed as a referee method gave an identical result (within

±0.7 percent except for modification No. 8) to all the five other AAS

methods as shown in Table 11. Perhaps this simply means that all AAS

methods have a similar systematic bias! However, as shown in Table 12, an entirely different chemical isolation and separation route, that of

oxalate precipitation with monitoring of supernatant losses of calcium,

again gave an identical answer. It is important to note that a solution of

NBS-SRM 915, calcium standard, went through each and every step of

these methods. Our conclusion about the method was that it "appears to have a systematic bias of less than 1 percent."

Further, very recent experience in eight laboratories seeking to evaluate this proposed referee method for total serum calcium by AAS versus measurements by isotope dilution mass spectrometry (IDMS) at NBS

suggests, that the systematic bias is between 0.5 and 1.0 percent; however,

the work on serum comparisons between AAS and IDMS has not progressed far enough at this time (July 1 97 1 ) to suggest a closer figure.

The present studies of the NBS-Clinical Interaction Plan involving

NBS personnel and several clinical laboratory scientists has clearly

shown that precise and accurate measurements can be made in hospital

and other clinical laboratories when sufficient time and skilled personnel

are committed to the task. Unfortunately, all too few physicians demand

and actively support the need for accuracy in clinical work which reports

from the NIH group have shown are necessary [53, 54]. Most clinical judgements of a laboratory are based upon the repeatability of a split

specimen or in a crude comparison of one or two results sent to two

laboratories which may even use completely different methods. The cost of a determination seems to be far more important to many than any

questions concerning the analytical credence. In many institutions today

economic, not chemistry or service considerations seem to be the only

criteria for making decisions. There is little or no concern over standards,

proper standardization practices or meaningful quantitative measurements. When we fully recognize the superb ability of our body cells to react to only one specific chemical configuration while apparently rejecting the in- fluence of thousands of other chemicals in the fluids bathing our tissues,

I am sure that the ultimate analytical goal for chemical measurements in

'3 Joint NBS-Clinical Chemistry Interaction Plan.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 137
138 ANALYTICAL PROBLEMS

Table 12. Ca in serum as measured by oxalate precipitation and

the proposed method.

Serum calcium, mmol/liter Oxalate precipitation

Ca in ppt

Ca in supernatant

fluid Total

Present

method^

Serum A 1.99 0.03 2.02 2.035

B 2.14 0.035 2.175 2.18

C I.8O5 0.03 1.835 1.84

D 2.44 0.035 2.475 2.46g

E 2.585 0.03 2.6I5 2.625

F 2.17 0.03 2.20 2.I65

Mean 2.19 0.03 2.22 2.22

Percent 98.7 1.3 100 100

^ La, 10 mmol, in HCl, 50 mmol/liter.

medicine will require degrees of sensitivity, precision, specificity and ac- curacy as yet rarely practiced. There is indeed a place for meaningful

chemical measurements in medicine!

IV. References

[1] "Health Research Facilities Amendments of 1965," President's remarks at the signing of the bill, 9 August 1965.

[2] Patterson, C. C, Contaminated and Natural Lead Environments of Man, Arch. Environ. Health, 11,344(1965).

[3] Murozumi, M., Chow, T. J. and Patterson, C. C, Chemical Concentrations of Pollutant Lead Aerosols, Terrestrial Dusts, and Sea Salts in Greenland and Antarctic Snow Strata, Geochemica et Cosmochim Acta 33, 1247 (1969).

[4] Hunt, W. F. Jr., Pinkerton, C, McNulty, O. and Creason, J., A Study in Trace Ele- ment Pollution of Air in 77 MidwesteriTCities, Trace Substances in Environmental

Health-IV, Demphill, D. D., Editor, University of Missouri, Columbia, Missouri

(1971).

[5] Branscomb, L. M., Taming Technology, a Plea for National Regulation in a Social Context, Science 171, 972-977 (1970).
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 139

[6] Meinke, W. W., Analytical Chemistry - A Fading Discipline? No! Anal. Chem. 42, 26A-38A(June 1970).

[7] Shannon, J. A., Medical Research, /VeivEAZ^. 7. ofMed. 284,75 (1971).

[8] Research Grants: New Awards Bore Brunt of NIH Cutbacks (by R. Gillette, News

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Committee on Science and Public Policy, National Academy of Sciences, 525 pp. (1970). (Order from National Academy of Sciences ISBN 0-309-0 1770-X.)

[10] Cohn, S. H. and Dombrowski, C. S., Whole-body Counter for Neutron Activation

Analysis, IEEE Trans. Nucl. Sci. 18, 73 (1971). [11] Skeggs, L. T., An Automated Method for Colorimetric Analysis, /imer 7. Clin. Path. 28,311 (1957). [12] Henderson, L. J., Blood, A Study in General Physiology, Yale University Press, New Haven (1928). [13] Van Slyke, D. D., Acidosis and Alkalosis, Bull. N. Y. Acad. Med. 10, 103 (1934).

[14] Classon, P., Personnal communications of case histories of patients seen in Hartford

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Liquid Chromatography, /I Chem. 42, 1223 (1970). [18] Alpert, N. L., Automated Instruments for Clinical Chemistry: Review and Preview,

Clin. Chem. 15, 1198(1969).

[19] Kingsley, G. R., Analytical Reviews — Clinical Chemistry, Anal. Chem. 43, 15R

(April 1971). [20] Laboratory Notes in the yearly June issue of The Hartford Hospital Bulletin (copies

available on request). [21] Hopkins, F. G., Presidential address to the Royal Society, London, England, 1932, taken from the foreword to Enzymes, Dixon, M. and Webb, E. C, Editors, Academic Press, New York (1958). [22] Brauenstein, A. E. and Kritzmanon, M. G., Uber den Ab-und Aufbau von Aminosauren durch Umaminieung, Enzymologia 2, 1 29 ( 1 937). [23] Cohen, P. P. and Hekhuis, G. L., Rate of Transamination in Normal Tissues, 7. Biol. Chem. 140,711 (1941). [24] La Due, J. S., Wroblewski, F. and Karmen, A., Serum Glutamic Oxalacetic Transaminase Activity in Human Acute Transmural Myocardial Inidivcixon, Science 120,497(1954).

[25] Karmen, A., Wroblewski, F. and La Due, j. S., Transaminase Activity in Human

Blood, J. Clin. Invest. 34, 126 (1955). [26] Karmen, A., A Note on the Spectrophotometric Assay of Glutamic-oxalacetic Transaminase in Human Blood Serum, y. Clin Invest. 34, 131 (1955). [27] Warberg,0. and Christian, W.,fi/oc/iem.Z. 286,61 (1936). [28] Posen, S., Turnover of Circulating Enzymes, C/m. Chem. 16,71 (1970). [29] Bowers, G. N., Jr., Measurements of Isocitric Dehydrogenase Activity in Body

Fluids, C/m. C/iem. 5, 509 (1958). [30] Maxwell, J. C, Introductory Lecture delivered at Cambridge - 1871. [31] Report by the Sub-committee on Reference Materials, Committee on Analytical
140 ANALYTICAL PROBLEMS

Chemistry, Division of Chemistry and Clinical Technology, National Academy of Sciences - National Research Council: Standard Reference Materials as Viewed by

a Laboratory Supervisor - A Status Report, Anal. Chem. 40, 24A (March 1968). [32] Meinke, W. W., Standard Reference Materials for Clinical Measurements, Anal. Chem. 43, 28A (May 1971). [33] Peters, T. Jr., Proposals for Standardization of Total Protein Assays, Clin. Chem. 14,

1 147 (1968). [34] Bower, V. E., Paabo, M. and Bates, R. G., pH Standard for Blood and Other

Physiologic Media, Clin. Chem. 7, No. 3 , 292 ( 1 96 1 ). [35] Report of the Commission on Enzymes of the International Union of Biochemistry, 1961 and 1964, Macmillan, New York (1961 and 1964). [36] Atti del i Simposio Nazionale sui Methodi Enzimatici nella Diagnostica Clinica, Burlina, A., Editor, Puaderni Sclavo di Diagnostici Clinica e di Laboratorio 6, 1-288

(1970). [37] Standardization of Clinical Enzyme Assays: First Report of a Working Party of the Association of Clinical Bioanalysts with Special Reference to Alkaline Phosphotase

Estimations, D. W. Moss, Chairman (to be published in J. Clin. Path. 1971). [38] Standardization of Methods for the Estimation of Enzyme Activity in Biological

Fluids. A Report by the Commission for Enzyme Diagnostics and Standardizations,

German Society of Clinical Chemistry, Z. klin. Chem. u. klin. Biochem. 6, 658

(1970). [39] Bowers, G. N. Jr., Kelley, M. L. and McComb, R. B., Precision Estimates in Clinical Chemistry 1. Variability of Analytic Results in a Survey Reference Sample Related

to the Use of a Nonhuman Serum Alkaline Phosphatase, Clin. Chem. 13, 595 (1967). [40] Ohmori, Y.,E«zvmo/og/a4,217 (1937). [41] 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, 7. Biol. Chem. 164, 321 (1946). [42] Lowry, O. H., Roberts, N. R., Wu, M., Hixon, W. S. and Crawford, E. J., The Quantitative Histochemistry of Brain. li. Enzyme Measurements, 7. Biol. Chem. 207, 19

(1954). [43] Garen, A. and Levinthal, C, A Fine-structure Genetic and Chemical Study of the En- zyme Alkaline Phosphatase of E. coli. I. Purification and Characterization of Alkaline Phosphatase, Biochim. Biophys,Acta 38, 470 (1 960). [44] Amador, E., Zimmerman, T. S. and Wacker, W. E. C, Urinary Alkaline Phosphatase

Activity. IL An Analytical Validation of the Assay Method, 7. Am. Med. Assoc. 185, 953 (1963). [45] Morgenstern, S., Kessler, G., Auerbach, J., Flor, R. V. and Klein, B., An Automated

p-nitrophenyl Phosphate Serum Alkaline Phosphatase Procedure for the Autoanalyzer, Clin. Chem. 11,876(1965).

[46] Bowers, G. N. Jr. and McComb, R. B., A Continuous Spectrophotometric Method for Measuring the Activity of Serum Alkaline Phosphatase, CUn. Chem. 12, 70 (

1 966). [47] Hausamen, T. V., Helger, R., Rick, W. and Gross, W., Optimal Conditions for the Determination of Serum Alkaline Phosphatase by a New Kinetic Method, Clin. Chem. Acta 15,241 (1967). [48] Bowers, G. N. , Jr. and McComb, R. B. , A Comparison of Buffers for Serum Alkaline Phosphatase, Clin. Chem. 17, 560 (1971). (Paper to be presented at 23rd National Meeting AACC - Seattle Washington, August 9- 1 3 , 1 97 1 ).
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY 141

[49] Edisbury, J. k. , Editor, Practical Hints on Absorption Spectrometry, Hilger & Watts

Ltd., London 0966).

[50] Standard Methods of Clinical Chemistry, Vols. 1-6, Academic Press, New York

(1950s to 197bs). [51] Tonks, D. B., A Study of the Accuracy and Precision of Clinical Chemistry Deter- mination in 170 Canadian Laboratories, Clin. Chem. 9,217 (1 963 ). [52] Barnett, R. N., Medical Significance of Laboratory Results, /4m. 7. Clin. Path. 50,671

(1968). [53] Cotlove, E., Harris, E. K. and Williams, G. Z., Biological and Analytic Components

of Variation in Long-term Studies of Serum Constituents in Normal Subjects. III. Physiological and Medical Implications, C/zT?. Chem. 16, 1028(1970).

[54] Young, D. S., Harris, E. K. and Cotlove, E., Biological and Analytic Components of Variation in Long-term Studies of Serum Constituents in Normal Subjects: IV. Results of a Study Designed to Eliminate Long-term Analytic Deviations, Clin. Chem. 17,403 (1971). [55] Pybus, J., Feldman, F. J. and Bowers, G. N. Jr., Measurement of Total Calcium in Serum by Atomic Absorption Spectrophotometry, with Use of a Strontium Internal Reference, Clin. Chem. 16, 998 (1970). [56] Williams, J. H., Kuchmak, M. and Witter, R. F., Purity of Cholesterol to be Used as a Primary Standard, J. Lipid Res. 6,461 (1965). [57] Williams, J. H., Kuchmak, M. and Witter, R. E., Evaluation of the Purity of Cholesterol Primary Standards, CUn. Chem. 16,423 (1970). [58] Savory, J., Wiggins, J. W. and Heintges, M. G., Measurements of Calcium and Magnesium in Serum and Urine by Atomic Absorption Spectrometry, Amer. J. Clin. Pathol. 51,720(1969).

[59] Gimblet, E. G., Marney, A. F. and Bonsnes, R. W., Determination of Calcium and Magnesium in Serum, Urine, Diet, and Stool by Atomic Absorption Spectrophotometry, Clin. Chem. 13, 204 (1967). [60] Pybus. J., Determination of Calcium and Magnesium in Serum and Urine by Atomic

Absorption Spectrophotometry, Clin. Chim. Acta 23, 309 (

1 969).
142 ANALYTICAL PROBLEMS

APPENDAGE A

Volume and Precision^ on Individual Tests

(Hartford Hospital 1969-70)

Test Volume

Automation Section

Glucose 48,391 3.0

Urea 39,650 3.2

Osmolality 5,511 1.9 Creatinine 16,121 5.3

Sodium 36,176 1.2 Potassium 34,350 2.3

Chloride 27,540 2.1

Carbon dioxide 27,051 4.0

Uric acid 6,925 2.9

Phosphate 7,256 4.3

Bilirubins 11,345 20

Thymol 1,592 16

uepn. r loc. C. S. F. Protein 2,871 o.u Acetone 496

Xylose 1 1Q

Amylase \ 4:,D04 1 9

oweat 1 est

Electrolyte Section

pH 5,942 0.3

Micro pH 47

5,054

Micro bilirubin 704

Calcium 9,600 1.3 Magnesium 1,462 2.4

Lithium 2,382 1.2 Barbiturate 316

Salicylates 130

Bromide 21

Doriden 10 Calculi 214

Cu, Zn, Pb 90

Hemog. S. 110

Iron 1,187 9
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY

Appendage A (continued)

Test Volume % RSD

Protein--Enzyme Section

Alk. P-ase 12,965 8

Acid P—ase 2,030 3.5

Lipase 1,411 2.3 Choi inesterase 413(77) 6

SGOTransaminase 19,448 11 SGPTransaminase 18,193 17 Lactic dehydrogenase 17,687 8

Ceruloplasmin 31

Serum electrophoresis 4,544 4.5

Hgb electrophoresis 189

Urine electrophoresis 85

CSF electrophoresis 78

T. Protein 5,642 1.5 A/G by electrophoresis 4,448

Endocrine Section

5HIAA 128

Cortisol, plasma 1,178 5=2

Protein bound iodine 2,468 5.4 Chromatog. urine AA 135 — —

17—Ketosteroids 640 17 17—OH corticosteroids 585 30

Cholesterol 5,880 4.5 Triglycerides 4,461 s0

Metanephrine 89

VMA 10

Creatine kinase 4,051 8

Nights

Glucose 4,770

Urea 3,647

pH 4,827

Sodium 4,035

Potassium 4,419

CO2 2

Micro bilirubin 135

Acetone 321

Amylase 1,221
144 ANALYTICAL PROBLEMS

Appendage A (continued)

Nights (continued)

Test Volume

Barbiturates 152

Salicylates 117

Doriden

Osmolality 16

CSF Protein 15 Calcium ~^ 1 PO4 1 (Irons) ^ 889

Microchemistry

Glucose 15

Urea 42

Osmolality 1 Sodium 103

Potassium 105

Chloride 97

Chemical micro 5

Bilirubin

Alk-Phos. 8

SCOT 5

SGPT 5

LDH 5

Total protein 11

A/G by electrophoresis 10

Gulactosemia 3

Acetone 1 Based on 80 to 120 days of a 'blind' serum pool at multiple levels.
BIOMEDICAL RESEARCH AND CLINICAL CHEMISTRY

APPENDAGE B

Tests Referred to Outside Laboratories

(Hartford Hospital 1969-70)

Aldolase

Aldosterone

Bile acids

assays

Butanol extractable iodine

Catecholamines

Cortisol secretory rates Cortisone, urinarv free 17—OH Corticosteroids

(by Porter—Silber)

11% Hydroxysteroids

Estrogens, fractionated

Fecal fat Free fatty acids

Free thyroxin F.S.H. pituitary gonadotropins

Folic acid

Growth hormone

Haptoglobin

Iodine excretion

Luteinizing hormone

Epinephrine—norepinephrine

Mucopolysaccharides

Oxalate

Phospholipids

Pregnanediol

Pregnanetriol

Renin

Testosterone

Thyroid stimulating hormone

Total lipids

Triiodothyronine

PANEL DISCUSSION

Anal>"tical Problems in Biomedical Research

and Clinical Chemistry

CHAIR]VL\>

Robert S. Melville. Chief. Clinical and Applied Sciences Section. National

Institute of General Medical Sciences. National Institutes of Health

A

PANEL

George G. Guilbault. Associate Professor of Chemistry. Louisiana State

Lniversity. New Orleans. Louisiana

Bairv- L. Karger. Associate Professor of Chemistrs". Northeastern Lniversity. Boston. Massachusetts

Henry M. Fales. Chief. Laboratorv" of Chemistry. National Heart and

Lung Institute. National Institutes of Health. Bethesda. Marvdand

Howard V. Malmstadt. Professor of Chemistrv*. Lniversity of lUinois. Lrbana. lUinois

George N. Bowers. Jr.. M.D.. Director. Chnical Chemistry Laboratory'.

Hartford Hospital i Speaker

i

147
148 ANALYTICAL PROBLEMS

Fales — There are really two kinds of analytical chemistry connected

with this topic: biochemical and clinical. Biochemical analysis is essentially a precursor of clinical analysis so that measurements are usually

first made in the biochemical research laboratory, and then when the

techniques are worked out, the method goes to the clinical laboratory.

There are two misconceptions which may pop up when analytical

chemists consider the area of clinical chemistry. One is that if the analytical chemist dreams up some kind of instrument that works well, but is ex- pensive, that it is automatically out. In practice this does not really seem

to be the case. If the instrument gives the right answer, clinical chemists

and biochemists will accept it and make good use of it. A second idea is that there must necessarily be good theory relating the

measurement to the disease condition. Certainly, it's fine if one can do

this but often all one has is the correlation itself. Perhaps the SGOT

measurement falls in this category; much more needs to be known

about why it works — but it does work and it is being widely used. In clinical chemistry extraordinary specificity is required. A number of

years ago it appeared that gas chromatography and thin-layer chromatography would provide the answers to many of the clinical chemist's

problems, and it seemed that the methods would be used widely in the

clinical laboratory. Unfortunately, because of their lack of specificity, the

methods have never gotten off the ground in the average clinical environment. They definitely have utility today, but more to confirm results, and

for research purposes than in the routine clinical laboratory. So a technique which sometimes looks very, very good indeed often is discarded because of lack of specificity. What is really needed in the clini- cal field is a system which is routine and, hopefully, mechanizable.

In contrast to situations in the clinical field, scientists in the purely

biochemical areas are often confronted with the need for a one-shot analysis in order to solve a particular problem. In fact, several months might be

spent gearing up to run a single analysis if the question is important

enough. Furthermore, in biochemical analysis the scientist is trying to get

the widest range of information possible from a given technique. Therefore he uses broad range methods such as nuclear magnetic resonance and mass spectrometry because they give such a complete picture of the total system. There is also more of a tendency to deal with ultra-sensidve

methods in biochemical research as contrasted with clinical problems.

Sometimes in the clinical field there are special requirements; for example, in the field of drug analysis rather poor quantitation is satisfactory but

there should be no false identifications. If a laboratory reports that a per- son has been taking morphine, this report must be made with a very high
PANEL DISCUSSION 149

degree of reliability since inaccurate information could damage a person's

reputation or dictate improper treatment. For example, recently the District of Columbia contracted for drug analyses in the methadone program

and the requirement was that there would be no more than one false positive in two hundred analyses! It seemed to me that it would be very dif- ficult for any clinical laboratory to meet these specifications but in fact one laboratory has met the specification and been awarded the contract. Another area that has special requirements is the emergency identifica- tion of drugs in overdose cases. Here the requirement is that the analysis

should be very fast, i.e., less than an hour, and there must be an absolutely

positive identification of the drug, i.e., not just a "barbiturate" but what

bartiturate. Or: is it doriden? Quantitation is much less important.

One of the main requirements for such analyses, touched on by Dr. Bowers, is that there must be a very close association between the clinical chemist and the physician. Too many hospitals seem to have their clinical chemistry controlled by a very non-chemically-oriented pathologist. It must be realized that if a person is in the field of clinical chemistry, he is there primarily because he is interested in applying his chemical knowhow to help sick people. Thus, he ought to be given an opportunity on oc- casion to observe the patient at first hand along with the doctor. In this way the clinical chemist would feel much more a part of the overall clini- cal picture. The more such cooperation can be arranged, the more reliable the advice of the clinical chemist will become, since he will be able to see

at first hand how his measurements contribute to the overall clinical situation. Regarding more exotic methodology, particularly techniques which

might be in the near future, it seems to me that mass spectrometry may

well become a widely used method in the clinical laboratory. After all, one

of the fundamental properties of matter is mass; if one knows the mass of a molecule, one really knows quite a bit about it — including information as

to whether it has one or two nitrogens, etc. The mass limit of modern

mass spectrometers is only somewhere between 500-3000 mass units and

right now it is necessary to volatilize samples. Someday we should be able

to get around this requirement. It really is rather ludicrous to start with an

ionic substance, derivatize it in order to vaporize it, then return it to an

ionic form so that it can be analyzed in the mass spectrometer.

EPR will make an important contribution in the future. The "FRAT"

system of the SYVA Corporation has the combination of the specificity

found in enzyme systems combined with the sensitivity available from

measurements of electron paramagnetic resonance. Admittedly, the

technique is only in the early stages of development at the present time,

and confirmation by other methods is still desirable.
150 ANALYTICAL PROBLEMS

Fourier transform NMR methods are coming into use and may help us

to get rid of the slits and scanning systems in spectrometry. This is a way

to increase sensitivity by a factor of ten or decrease measurement time by

a factor of one hundred, but it requires the use of a computer. Another in- teresting area is radiative detection of NMR signals which, in special cases, could provide NMR with the sensitivity available in radioactivity.

X-ray crystallographic analyses will surely be more widely used — the sur- face has just been scratched. It is important to find ways to obtain good x- ray data from smaller and smaller crystals. Along with this, we need to find new ways to fish small amounts of pure crystalline material out of

biological systems without a lot of work-up.

Much more work must be done on separation methods in general. Present day methods must somehow be speeded up. Some sort of immunological-type detector is needed. If it is known what compound is to be

measured, there ought to be a way to "sensitize" a detector specifically to that compound. Nothing like this has been done to my knowledge. There

needs to be some way to get total elemental composition data on pure

materials at the picogram and nanogram level; microwave discharge has

been suggested as a way of doing this. But all of these things will not come to much unless we can find a way

to increase the interaction between the analytical chemist, the clinician

and the biochemist. It's a team effort today. This means we, as analytical

chemists, are going to have to publish with biochemists and physicians in the biochemical and clinical journals.

An additional thought comes to mind. In these days when many organic

and physical chemists are out of work there might be some possibility for

retraining interested individuals in the area of clinical chemistry. Perhaps some support from NSF or another organization is required. (Others

pointed out that there are committees working on these problems to develop a specific plan and thus detailed discussion at this time would

probably be premature.)

Guilbault — A few comments are called for on the status of teaching of

clinical analytical chemists in the colleges and universities today. Many

years ago almost all of the premedical students and the medical technicians went through a good, thorough quantitative analysis course. But

today, many of them do not. The reason for this is as much the fault of us

in analysis as anybody else. To a certain extent we have "turned off' the

M.D.'s and the people interested in the biomedical types of things by our

old-fashioned courses.
PANEL DISCUSSION 151

To reverse this trend, a number of places have revitalized their courses. For example, now at LSU there is a course strictly in clinical methods for

the medically-oriented people, the Med. Tech's., the M.D.'s, etc. In this course they do not do many of the old classical experiments but do a lot of new things. For example, they do experiments in atomic emission and

atomic absorption for sodium and potassium in blood and urine. They

study electrochemical methods, using sodium and potassium electrodes,

and even use, experimentally, the new urea electrode. These methods

have the advantage of selectivity and will be used a lot in the future to measure activity rather than just concentration, since activity is a more

important parameter in many clinical measurements.

Measurements using fluorescence, for example, are made for magnesium in blood. They do TLC separations for drug identification; they also use GLC in the same way. Also spectrophotometric methods are used for urea and glucose in blood. These are examples of methods commonly

used in many clinical laboratories. This gives the medically oriented people a much better idea of what types of analyses they will be running into

in the future. At least the M.D. that comes out of this course in the next

few years will have some kind of idea about what types of clinical tests are being performed for him.

Comments are appropriate on four specific problems in this area of

clinical measurements. The first problem, that of standards, was well

described by Dr. Bowers. There are a number of good standards available

today that can be used for ion-selective electrodes, blood pH measurements, cholesterol, etc.\ as well as actual biomedical samples, such as orchard leaves for calcium, potassium, iron, sodium, etc. Hopefully we

can expect many more from NBS; this is a terrific service.

A second area to be mentioned is that of accuracy. Just what is a normal

range? In many tests it has been shown that a so-called normal range is three-fold or more greater than what it should be. So how do we better

define these limits? Presumably one could do a better job of getting a more representative "normal" individual or a whole range of representative individuals. But just how do we do this to improve the accuracy of the

determinations?

Thirdly, better methods of analysis are needed, particularly for many of

the new enzyme tests. It is estimated that enzyme tests grow at the rate of

about 30 percent per year, and for many of these, better tests are still needed. Many of them lack analytical acceptance. New fluorescent

iViethods, ion-selective electrode methods, enzyme electrodes, elec- trochemical methods, and others should improve the enzyme procedures

of the future.

428-999 O - LT - 72 - 11
152 ANALYTICAL PROBLEMS

Fourthly, technician error is something that many of us do not recognize. When working with a large hospital in terms of trying to get some

idea as to the errors involved in a test, there are many small things that the

technicians might do which would invalidate or introduce greater errors

into a particular procedure. A definite advance can be made through the

development of technician-proof or "goof-proof methods which require a minimum of solution preparation and manipulation on the part of the

technician. This is the philosophy behind the development of enzyme

electrodes. These will be very simple to use just like a pH electrode with

essentially no special preparation. There are now electrodes for several

materials such as urea, D- and L-amino acids, asparagine, glutamine,

phosphorate ion, and glucose.

Another aspect that is being approached is the so-called solid surface

monitoring system which would completely eliminate all solution preparation. The technique here is to develop small pads upon which quantitative

measurements can be made. One has a series of bottles with these pads.

On the pads are all the reagents necessary to perform a particular assay.

All a technician would have to do is to take a pad out, put it on a strip, and

add the solution, whether it's blood or urine, in order to get the ap- propriate test. The pad would then be put in the instrument to take a reading, after having standardized the instrument. This would reduce greatly

the possibility for technician error. In summary there appears to be a great opportunity for the analytical

chemist in clinical chemistry. It is almost like the Gold Rush Days in California — today there is a wealth of important analytical problems in

clinical chemistry that are just waiting for the analytical chemist to rush in and offer his expertise.

Karger — A number of months ago Drs. Melville and Purdy described

the critical shortage that exists for trained people in clinical chemistry. It

is interesting to note that in a time when so many fields, including some of

the chemical fields, are having difficulties in placing students that the area

of clinical chemistry has such a diversity of opportunities.

To help remedy this situation the Department of Chemistry and the

College of Pharmacy at Northeastern University in Boston are proposing

a Masters Degree program in clinical chemistry for evening school parttime students. Students would be accepted with a BS degree in chemistry,

biology, pharmacy or medical technology. Clinical chemistry spans

several fields and it is unlikely that most students would have all the

requirements one would like them to have. Thus in addition to the BS

degree in one of the above fields, it is expected students will have two
PANEL DISCUSSION 153

quarters of physical chemistry, two quarters of organic, one quarter of

analytical chemistry, and two quarters of human physiology. If a student

does not have some of these courses, he would be able to take them in the

part-time evening BS program.

The program will include about 20 courses, 10 of which will be required

including courses from both analytical chemistry and from the pharmacy

school. Included are a course in separations dealing with chromatography

and other techniques, a course in electrochemistry, and a course in optical

methods; three courses in biochemistry, and two courses in clinical chemistry where analytical techniques as applied to clinical chemistry will be discussed. There will also be a course in radioisotope techniques and

one in concepts in toxicology. There will also be an elective core which

could include chemical instrumentation and computers in chemistry; also

special topics in analytical and clinical chemistry. Some students also

might be interested in taking business courses since they might hope eventually to manage clinical laboratories.

One of the problems is that of clinical practice (for example, in the case

of retraining persons whose primary background is physics, engineering,

etc.). It is hoped to establish a cooperative program with one of the local hospitals in which a person would actually get practical laboratory train- ing and experience over a period of three months.

Leaving the educational role analytical chemistry departments can play

in clinical analysis, let us next turn to the research side. It is felt that highspeed liquid chromatography will play an important role in clinical analysis in the future. One example of future potential is in the area of steroid

analysis. If one uses gas chromatography for steroid analysis, one must

first have a hydrolysis step to obtain the free steroids from the conjugates.

Then one may have to form derivatives before the sample is introduced

into the gas chromatograph. In high-speed ion-exchange liquid chromatography one can separate directly the conjugates leaving out the

hydrolysis and derivatization steps. The key here is that in liquid chromatography one can come close to the biological system with the sample

that is injected. Liquid chromatography could also be very important in drug analysis, for example for the separation and analysis of barbiturates. In forensic analysis also, liquid chromatography can be of great value.

If you ask the question "Why has gas chromatography not become a more powerful tool in clinical chemistry?" you can get several answers.

Dr. Fales has brought up the lack of specificity although one can point to many cases where very high specificity exists, e.g., in the pesticide field. The problem appears to be more that the people that work in the field find

gas chromatography a little too sophisticated to use routinely in the clini-
154 ANALYTICAL PROBLEMS

cal laboratory at the present time. Breakdown also appears to be excessive in relation to other methods such as simple colorimetric analysis.

Malmstadt — Analytical research chemists have a responsibility to make

a study of all of the basic processes that occur in scientific measurements.

George Bowers pointed out that 91 percent of the measurements made in his laboratory involved some kind of optical or spectrophotometric

measurement. As analytical chemists we should be interested in the

types of transducers and basic processes inherent in encoding chemical

information as electromagnetic radiation information. At the same time

we should be very much interested in the basic processes involved and the

methods used in instrumentation to decode this information. We should

not just be content to take some instrument as it has been given to us in the laboratory and use it for making scientific measurements. But we

should be aware of all the basic processes that are involved in encoding

and decoding and reading out of the information.

George Bowers mentioned the use of atomic absorption and flame

emission techniques as well as the introduction of atom fluorescence

methods which are today being used quite often in the clinical laboratory. In spite of the fact that there are many instruments available, there are many chemical processes involved in the encoding of the chemical sample

elemental composition in the form of electromagnetic radiation, which are poorly understood. For example, more must be known about the desolvation processes, the vaporization and atomization processes, etc., in order

to make atomic absorption and atomic fluorescent measurements much

more reliable, even though they are producing quite satisfactory results at the present time. In the area of atomic fluorescence methods there are some exciting possibilities for the future in the use of tunabie dye lasers, which should significantly improve sensitivities. There are also interesting recent experiments in atomic fluorescence and atomic absorption measurements in working directly with the solid sample without preliminary preparation.

And this, of course, is of great interest for the clinical laboratory so as to eliminate the elaborate treatment of solid samples.

Another area where there should be concern is in the mechanisms of

reactions. Too much of the early work was done on a very empirical basis,

and that is one of the reasons why the clinical laboratories were not as good in the early days as today when they are reaching for more basic ap- proaches.

Although this morning a lot was said about the problems in solid-state

research it should be pointed out that scientists in this field have done
PANEL DISCUSSION 155

such a tremendous job that their devices are completely revolutionizing

the instrumentation used for analytical measurements. As analytical

chemists we are just beginning to see the fruits of that new foundation of

electronics where entirely new design concepts have resulted from

microcircuits and other innovations of the solid-state scientists. What significance would a study of this new electronics have for the

analytical chemist? Whereas several years ago it would take considerable

time to try out certain instrumentation ideas, now an idea can be tested in a day or two. Thus it's a whole new ball game! If one has an idea of

something that would improve sensitivity or remove interference, instead

of taking six months or a year to develop it, it can often be tried out in a few days. Obviously, this can greatly expedite the development of new in- struments and will have a great impact in the area of clinical chemistry.

Much has been said this morning and this afternoon about interfacing

between individuals, the interface between the analytical chemist and the

physicist, the metallurgist, and the physician. These are major problems,

but there is also another type of interface that is of increasing importance

and that is the interface between the analytical chemist, the clinical chemist and the machine — /.^. the man-machine interface. In the past it has been important to interface with instruments to try to know how to get

the most out of them. But that kind of interface is going to become trivial in relation to problems of the very near future. In the past, time scales of

years were available to adjust to a new, essentially manually-controlled

instrument. But now as new automated instruments and automated

laboratories come in to being, a whole new set of problems evolve in the

man-machine interface.

Certainly this will be the decade of complete laboratory automation.

This is from the point of view not only of routine work but also of research

work. It is now possible to consider automating research approaches.

Thus one can not only automate an instrument but automate entire

research projects so that information which previously had taken many

years to obtain might be obtained in a month or so. In the medical laboratory there is the possibility of automating the interpretation of a complete

series of automatically obtained clinical analyses.

For our summer program in Digital and Analog Electronics for

Scientists at the University of Illinois there has been much interest from

people in the biological and medical sciences. Often they come in quite

poorly prepared in instrumentation in relation to the physicist and engineer, but after spending several weeks in the course they are able to see how some of the new concepts in electronics and instrumentation can help

them' solve some of their problems. Not that they can necessarily design
156 ANALYTICAL PROBLEMS

the circuits or instruments, but they do know how to communicate with

the people who would help them out. This year, of the 400-500 applications for our summer courses, only

about 10 were analytical chemists. Now this either means that -analytical

chemists are very well prepared in the new instrumentation and are ready

for the new era of automation in the laboratory, or else they don't think

it's important. Earlier in the day it was pointed out that analytical chemistry was a core discipline for analysis. It should be stated here that unless

analytical chemists really learn about the new approaches to instrumentation and automation in the next few years, analytical chemistry will not be

the core discipline for analysis.

Bowers — The rate of growth in terms of new tests for the clinical laboratory has essentially precluded the laboratory from going into some of the more sophisticated instrumental methods such as gas chromatography;

the clinical chemistry laboratory in most institutions has just been

overwhelmed! For example there is often a mandate to get a new test going almost immediately or the test will be sent to an outside laboratory.

These pressing demands have frequently been for tests which amounted

to thousands of samples a year such as blood gases at any hour— day or

night — in contrast to the few which would employ gas chromatography for one or two analyses/week, usually for steroids or the area of drug analysis. It takes a great deal more time from senior analytical people to develop gas chromatography methods than most persons realize. One just

doesn't walk up to the instrument and start turning out valid answers. As regards the "Gold Rush" mentioned earlier it should be pointed out

that in the past the clinical chemistry laboratory has often covered in its charges losses incurred from other hospital operations. There is now more

competition from private Taboratories, and the hospital laboratory

charges are today more nearly reflecting costs. When hospital chemistry activities just recover chemistry costs, then there will be little rea- son for non-chemists to control clinical chemistry laboratories and I would hope a good chemist would be placed in charge of these vital laboratories.

A person coming into a clinical laboratory with a Ph.D. in analytical

chemistry should plan on spending a minimum of two years in a laborato- ry as a post-doctoral fellow. The problem of interfacing with medicine is a major one. You just don't go into a clinical laboratory and suddenly

change something where there are already strongly entrenched attitudes

fixed by years of medical tradition. There needs to be an apprenticeship
PANEL DISCUSSION 157

approach to learn how to make these changes in a positive way. It can be

a very exciting time but one does need to become familiar with the clinical surroundings and the peculiarities of medicine. However, there are many

laboratories today where even undergraduate laboratory skill in analytical

chemistry can provide an important contribution. If a person is really

going to contribute to clinical chemistry in a major way, it is my contention he should equip himself with the knowledge and training of a Ph.D.

chemist or/ and M.D.

George W. Irving, Jr., Administrator, Agricultural Research Service, U.S.

Dept. of Agriculture, received a B.S. degree in chemistry and M.A. and

Ph.D. (1939) degrees in biochemistry from George Washington University. He completed part of his graduate studies at the University of Illinois,

Cornell University Medical College, and the U.S. Dept. of Agriculture

Graduate School. He was assistant in chemistry at Rockefeller Institute

of Medical Research after earning his degree and held this position until 1942 when he joined the Department of Agriculture at the Southern Regional Research Laboratory, New Orleans. He held many positions in the

Department until 1965 when he was appointed Administrator. Dr. Irving

is a member of the ACS, AAAS, the Society of Biological Chemists, and

the Institute of Food Technologists. He is the author of more than 50

scientific articles and books on pituitary hormones, amino acid and

protein chemistry and metabolism, plant and animal proteolytic enzymes,

antibiotics and plant growth regulators, and an equal number of articles on

various aspects of research administration.

159

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 351,

Analytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

Held June 16-18, 1971 , Gaithersburg, Md. (Issued August 1972). CHAPTER 4

ANALYTICAL PROBLEMS IN AGRICULTURAL

SCIENCE

G. W. Irving, Jr., and W. C. Schaefer

Agricultural Research Service U. S. Department ofAgriculture

Washington, D. C. 20505

This paper presents a review and analysis of the widespread applications of

analytical chemistry and analytical instrumentation in the research and regula- tory programs of the Agricultural Research Service of the Department of

Agriculture. Analytical chemistry is one of the essential sciences needed in ad- vancing the commodity and people-oriented programs which can achieve the

national goals of the Department. Chemical analysis will be shown to play a

vital role in assuring an adequate supply of farm and forest products and in as- suring the consumer a better product while minimizing costs for processing and distribution. In people-oriented programs, such as consumer health, safety and pollution abatement, analytical methodology is the key procedure for providing the basic data to get action programs into motion. Along with the far-ranging applications of analytical chemistry in agriculture come many unsolved analytical problems. In fact, few agricultural scientists

will admit complete satisfaction with any of their current methodologies. Yet some problems are more important than others. An assessment of these problems is made including an analysis of their relative importance in conjunction with the urgent needs of agriculture. Possible ways of classifying or

categorizing these problems will be suggested. For example, amino acid analysis touches almost every branch of agricultural science whereas pesticide analysis is more restricted but has a great many more complexities. For any given

pesticide, methodologies may be needed for controlling its application and for measuring its residues in free and bound form, its metabolites, trace contaminants, and the inert ingredients in its formulation. Other problems are discussed for which there appears no current solution. This will challenge the ingenuity and creativity of analytical chemists in the future.

Keywords: Agricultural pollution; agricultural products analysis: agricultural science; amino acid analysis; biomedical research; clinical chemistry; diagnostic chemistry; food analysis; pesticide residue analysis.
162 ANALYTICAL PROBLEMS

I. Introduction

Agricultural research is an extremely diverse field of effort. Agricultural commodities include fiber crops, oilseeds, animal products, fruits and vegetables, cereal grains, forages, sugar crops, tobacco, and new

crops. Research on these commodities is carried out for a variety of pur- poses such as protection from losses, efficient production and quality improvement, product development and processing, efficient marketing, improvement of human nutrition and consumer satisfaction, and elimination

of environmental pollution from the production and processing of foods.

This diversity of commodities and objectives presents us with an extremely wide range of analytical problems.

To facilitate the consideration of some of our current problems by experts in the various analytical competences, we have arranged the

problems topically rather than by commodity or type of activity. The

problems, therefore, are arranged under the following subjects: elements

and water, proteins and related nitrogen compounds, lipids, fibrous constituents of plants, nutrients and their availability, seed and plant constituents, animal products, sensory characteristics of foods, microorganisms in foods, animal health, biologically active substances, agricultural

pollutants, molecular properties of natural polymers, and automation and

instrumentation.

II. Elements and Water

Starting with the simplest problems, from the standpoint of chemical

composition, we would like to mention four problems that involve in- dividual chemical elements and two problems concerning determination

of water. Recent unprecedented FDA actions in removing from or keeping off the market swordfish found to contain more than 0.5 ppm of mercury has

made the public suddenly aware of widespread mercury contamination.

Fortunately methodology for determining mercury at trace levels had

been developed so that it was feasible to arrange to expand greatly and

quickly the monitoring of fish for mercury content. The method now in general use involves chemical oxidation and reduction steps before the

mercury is measured as vapor by atomic absorption. A determination

requires only a few minutes. Perhaps this procedure could be automated
AGRICULTURAL SCIENCE 163

at least for similar samples in which the level of mercury content does not vary more than 10-fold. Nonetheless, it would be desirable to have a method which could be used to continuously monitor the mercury content of a water supply or effluent stream, with the possibility of using the out- put as a feedback to automatically control, perhaps, the operation of a mercury decontamination device which requires periodic regeneration.

Another useful development would be portable equipment which could be

used in the field or on a boat. It would also be prudent to seek similar

rapid, automated methods for monitoring other toxic elements such as

lead, cadmium, and arsenic in streams and in foods.

Of these elements, lead especially concerns us because it is exhausted

into the atmosphere from automobile engines. Lead-contaminated crops

are certainly used for human and animal food and lead-contaminated air

is used in many food drying processes. How can this growing contamination be monitored?

The amount of lead now in foods must be measured in parts per billion. The classic dithizone procedures have this sensitivity, but are extremely

tedious and would be very expensive for routine analysis. The recently

developed atomic absorption methods are simple and fast, but are not sensitive enough for the low concentrations of lead found in foodstuffs. Fortunately the technological explosion in the electronics industry has

provided the means to solve most of these problems. In polarography, for

example, the recent development of techniques such as the pulse and

the fast scan oscillopolarographic methods now permit analysis for

metals such as lead near the part per billion level. The main problem areas now appear to be in the relative complexity of

the techniques and the sophistication required of the analyst. Instruments

designed for routine repetitive analysis with a minimum of operator atten- tion would be useful. Some method is needed for the easy acquisition of

permanent recording of rapid scan oscillographic analyses. This would

permit later careful study and could be of importance in regulatory

procedures.

Another element that concerns us is bromine. Methyl bromide gas is widely used as insecticidal fumigant on cereals and cereal products such

as flours and meals, on nuts and nutmeats, on dried fruits, and on some

fresh produce. Experimental treatment of grain is shown in Figure 1 . Annual U.S. production of methyl bromide in recent years ranged from 103

to 179 million pounds. Depending on the nature of the product treated,

the concentration and exposure period may need to be carefully controlled to avoid exceeding the allowable tolerance for bromide residue,

which varies from 50 ppm for cereals and pulses to 125 ppm for dried
164 ANALYTICAL PROBLEMS

Figure 1 . Fumigant gas being fed into a grain storage bin for insect control.

fruits to 200 ppm for nuts. In the case of wheat it has been found that all residual bromide is inorganic within a few days of fumigation. The method

generally used for determining residual bromide is that developed by

Shrader, Beshgetoor, and Stenger [1] or some modification of it. It in- volves oxidation of bromide to bromate and determining the bromate

iodimetrically. Its principal drawback is that it is time consuming and not

sufficiently sensitive for determining a few parts per million. Much faster and sensitive methods such as neutron activation and x-ray fluorescence

have been successfully used, but these require expensive equipment not
AGRICULTURAL SCIENCE 165

usually available in analytical control laboratories. A more sensitive and

preferably simpler procedure than the chemical one now in use is still needed for routine control analysis for bromide residues in foodstuffs.

Sulfur is of interest because the current emphasis on replacing natural

protein with nonprotein nitrogen in ruminant diets necessitates an evaluation of sulfur requirements. This has not been a problem in the past

because natural protein contains adequate sulfur. The current sulfur methods are not suitable, especially for feed and feces samples when

sublimed sulfur has been added to the diets of ruminants. The recovery

rates are low, the equipment needed is expensive, and the procedures are slow and tedious. An efficient, accurate, and inexpensive method of measurement is needed.

Water activity of foodstuffs is an important consideration because the

stability of many dehydrated, formulated, and other types of foods to vari- ous degradative processes depends on both the temperature and the

moisture content. The thermodynamic activity of water in such a product

is measured by the relative humidity of the atmosphere or gas phase in equilibrium with it. The relation between the water content and the

equilibrium relative humidity (ERH) at a constant temperature is called

the water sorption isotherm. Because sorption isotherms for different

types of food components (such as sugars, fats, proteins, and car- bohydrates) are quite different, those for different food products depend

upon the composition. The sorption isotherm of a food product also varies

with temperature.

For food formulation and new food product development work, stabili- ty characteristics are best observed and correlated in terms of ERH. On

the other hand, most process and product control of moisture is more conveniently done through measurement of water content. The sorption

isotherms needed to relate the two are now seldom determined because

there is no rapid convenient method of doing so. Present methods involve

either a point-by-point measurement of water content of material which

has equilibrated with an atmosphere of known relative humidity (RH) or an estimation of this value from the rate of change of water content as the

material equilibrates. Needed is automated instrumentation for determining sorption isotherm data on a suitably prepared sample over the RH

range of approximately 5 to 85 percent for a wide range of temperatures

and in a period of an hour or less. The need also exists for a rapid response method for control of product

moisture content in continuous fast drying of heat-sensitive materials.

The quality of a number of dried food or feed products is sensitive to the

temperature-moisture content history of the material during the drying
166 ANALYTICAL PROBLEMS

process. An important example is the rapid, continuous drying of alfalfa to produce dehydrated meal, which is of value as an ingredient in mixed

feeds for poultry because of its xanthophyll content (pigmenting power)

as well as its protein content. The xanthophyll pigments undergo serious

degradation under the conditions existing in a dehydrator and the degree

of loss (which varies from 30 to 70 percent) is related most closely to moisture content of the product at the discharge end even though rear end oven temperature is held constant. Because the leaf and stem portions dry at different rates, the moisture

content of the product as it leaves the drier is not uniform. Extremely

careful control of operating variables such as inlet temperature, speed of

rotation, air velocity, rear end temperature, and feed rate is essential for

minimizing damage to the leaf fraction while drying the stem fraction suf- ficiently for good stability and handling properties. Needed is a rapid

response method for measuring average moisture content of the moving

product stream at the drier outlet, which could be used for automatic control of operating variables such as air speed and temperature to result in continuous production of a product with optimum moisture content. For some commodities, possibly alfalfa, monitoring the surface temperature

of the product in the exit stream may be as effective as measuring

moisture.

III. Proteins and Related Nitrogen Compounds

The rapid measurement of essential amino acids and proteins is one of our primary needs in this area of analysis. We also need improved

methods for determining nutritional availability of proteins and amino

acids, for hydrolyzing proteins prior to their analysis, and for differentiat- ing between vegetable and animal proteins; and we need to eliminate en- vironmental pollution caused by mercury waste from the Kjeldahl method

for nitrogen determination.

A. Rapid Measurement of Essential Amino Acids and Proteins

Even the most fully automated systems presently available do not

satisfy requirements for use in plant breeding, crop management, and marketing of cereal grains. There is need for rapid, fairly reliable, simple, in-
AGRICULTURAL SCIENCE 167

expensive micromethods. These methods might involve measurements of

behavior of component electrons, protons, neutrons, or a combination of

them without destruction of the sample or individual grains which could

then be used by plant breeders. Other possibilities include componentspecific dyes which may then be detected by colorimetry. With nondestructive dyes the grain may be used for improvement purposes. Still another possibility is development of newer chemical procedures that

could be adapted to automation. Automated devices should be simple to operate and chemical procedures reliable and fairly accurate. A small

amount of accuracy could be sacrificed for speed of analysis. More

refined techniques, many of which are now available, could be used to confirm the discoveries from the more rapid methods.

Rapid methods for the determination of sulfur-containing essential

amino acids would be extremely useful because of the nutritive significance of these amino acids. A chemical method for methionine and

cystine would provide information on the relative nutritive value of foodstuffs without relying on long and tedious animal feeding studies. Present methods are complicated by the instability of these two amino

acids during acid hydrolysis, especially in the presence of carbohydrates.

So extensive is the destruction of these amino acids that hydrolyzates of

foods upon chromatography generally exhibit no measurable cystine peak

and methionine is partially oxidized to the sulfoxide.

The most accurate method to quantitatively determine the amount of

sulfur-containing amino acids in food products requires oxidation of the

products with performic acid to convert cystine to cysteic acid and

methionine to methionine sulfone. These reaction products are stable during acid hydrolysis of the food proteins to their amino acids, but this extra oxidation step adds an extra day or two to the analytical procedure.

A more rapid and accurate method to determine these amino acids or to

stabilize them to hydrolysis, especially in the presence of carbohydrates

would be extremely useful to the food industry in establishing nutritive

value or specific deficiencies in foodstuffs.

Similarly, more rapid and reliable methods are needed for tryptophan.

Tryptophan is an essential amino acid which is one of the limiting amino

acids in many proteins. Because of its acid lability, tryptophan cannot be

determined on the usual hydrolyzates used for determination of other

amino acids by ion exchange and gas — liquid chromatographic (GLC)

techniques. Alternative methods of hydrolysis of proteins such as alkali or enzymatic methods have been developed. Also, many colorimetric re- agents have been used. However, most of these procedures are time con- suming, and a review of the literature shows that the reported values vary

428-999 O - LT - 72 - 12
168 ANALYTICAL PROBLEMS

widely. For example, reported tryptophan values of defatted soybean

meal range from 1.09 to 1.6 grams per 16 grams nitrogen. As a result,

modifications of the tryptophan analysis procedures are continually ap- pearing in the literature without any assurance that the values obtained by

these procedures are accurate or that they can be duplicated in other

laboratories.

Problems with high-color blanks, poor recovery of added tryptophan,

and interfering substances in the reagents have been reported [2,3].

Procedures which involve enzyme pretreatment of the protein may

require as much as 96 hours of hydrolysis to release the peptide-bound

tryptophan from soy protein [3]

.

Accurate and rapid procedures for tryptophan are especially needed to evaluate the biological value of proteins by the system adopted by Food

and Agricultural Organization of the United Nations [4] where each essential amino acid is reported as a proportion of the total of essential

amino acids (milligram per gram total essential amino acids). These values

are then corrected to percentages of hen's egg essential amino acid pattern. Development of a method for determination of an acid-stable derivative of tryptophan along with the other amino acids in an acid hydrolyzate

of a protein would seem to be a worthwhile undertaking for the future. Rapid, accurate analytical methods are needed for determining the

protein content of dairy products. Payment for milk in commerce is made

on the basis of its fat content. This assigns a premium to a component

which is at an economic disadvantage with respect to vegetable fats. Milk

fat is also frequently implicated in health problems when consumed in ex- cessive amounts. If a rapid, accurate method for milk protein determinations was available, payment might also be made on the basis of protein

content. This would tend to publicize a component in milk which is qualitatively superior to many other proteins, and de-emphasize high-fat

milk. It might also lower the minimum fat contents specified for many

dairy products.

Rapid response methods for control of protein content in mixed feeds

would be important economically. Formulation of mixed feeds by the

larger feed suppliers is now done on a least-cost basis by a computer pro- gram utilizing as input data current market prices and nutritional values of

the various components of the ingredients and specified minimum nutri- tional values of the product. Because of variations in composition of vari- ous lots of ingredients purchased from different growers, it would be

economically advantageous to the feed mixer to be able to allow for such

variations during the mixing process rather than use proportions which

ensure minimum specified protein and energy contents based on spot
AGRICULTURAL SCIENCE 169

analyses of ingredients. Such a capability would also result in more

uniform nutritional quality of product with potential advantages to the

purchaser.

Moisture and protein contents are the key values to be measured continuously in streams of ingredients being mixed. This information would

be instantly introduced to a computer process control system which

would automatically adjust the proportions of the ingredient streams

being mixed. The methods currently used for indicating protein measure

nitrogen content, which is satisfactory for this purpose. However,

methods now in use are not fast enough to be used for essentially instantaneous control. Closest approach to date appears to be the automated

determination of nitrogen by high-energy neutron activation in which

there is a lag time of 5 minutes from introduction of sample to availability

of result [5].

B. Nutritional Availability of Proteins and Amino Acids

Chemical analysis for nutrients in foods and feeds often yields results W'hich exceed the actual nutrient value of the sample. This discrepancy

arises from the fact that the digestive system of the animal does not completely digest some nutrients due to the chemical bonds present.

Nutrient unavailability can be created during plant growth as exemplified

by bound niacin in corn, or it can result from processing as with lysine

losses due to heating. Present methods of determining the extent of

digestibility and availability involve feeding studies. These studies are generally carried out using rats. Other animals are sometimes employed.

Feeding studies are time consuming and can be expensive depending on

the animal used. Kohler et al. [6] have reported in vitro method for deter- mining protein digestibility in w^heat millfeeds using a proteolytic enzyme

digestion followed by treatment wdth chick pancreas amylase. This

method or a variation of it must be evaluated as to its general applicability.

A rapid, reproducible laboratory procedure that is correlated to each

animal type would be an outstanding contribution to the science of

nutrition. Lysine and methionine are the essential amino acids most often found

to be "limiting"' in grain and other vegetable food and feed proteins; i.e., these compounds are most likely to be present in smaller quantities than

other essential amino acids relative to the amounts required for good

nutrition. In particular, lysine has been added to bulgur and other wheat-
170 ANALYTICAL PROBLEMS

based foods supplied in nutritional aid programs to developing countries.

The total lysine content of proteins is apparently reliably measured by the

standard amino acid analytical methods, but not all of the lysine present

is nutritionally available to the consumers of the protein-containing foods

or feeds. Much evidence indicates that nutritional availability of lysine is correlated with the amount of lysine present with free €-amino groups.

A method has been developed for measuring the lysine-free e-amino

content of proteins through formation of the 1 -fluoro-2 ,4-dinitrobenzene

derivative. This method has been widely used as a measure of nutri- tionally available lysine, but it appears to be only partially satisfactory and

is less so for vegetable than animal proteins. Another method employs

2,4,6-trinitrobenzene sulfonic acid, a reagent which also reacts with free primary amino acid groups to form a colored derivative. Although more

rapid than the fluorodinitrobenzene procedure, this method is subject to interference from hexosamines leading to erroneous results. There is also much evidence that not all the methionine content of

proteins is nutritionally available, but so far there is no chemical method

to eliminate the need for bioassay measurement of available methionine.

Accordingly there is need for reliable chemical methods which can be

used in food and feed formulation and quality control work for determining nutritionally available lysine and methionine contents of proteins.

Such methods must show good correlation with results obtained by the

laborious animal feeding studies.

C. Other Problems

A critical problem in determining total amino acids in proteins of foods

and feeds is the lack of a hydrolysis procedure that yields amino acids

truly proportionate to their composition in the unhydrolyzed protein.

Hydrolysis is usually conducted with constant boiling hydrochloric acid

at elevated temperature and, even under the most carefully controlled

conditions, some destruction of labile amino acids occurs. At the same

time some peptide linkages are peculiarly resistant to hydrolysis, so that prolonged periods of heating are required to break them completely.

Thus, tryptophan is usually destroyed entirely, cystine is altered significantly, serine and threonine are degraded at more or less predictable

rates, and other amino acids — notably methionine and tyrosine — may be

somewhat labile. On the other hand, long periods of heating are needed to

liberate valine and isoleucine to the fullest extent.
AGRICULTURAL SCIENCE 171

In the analysis of purified proteins the problem has been solved

reasonably well but at the cost of additional time-consuming procedures.

Special methods for tryptophan, cystine, and methionine are available.

Analysis of acid hydrolyzates prepared by heating for different periods of

time permits extrapolation to zero time for good estimates of serine and

threonine content, while the results obtained from long-term hydrolyzates

represent maximal yields for valine and isoleucine.

The problem is more serious in the analysis of foods and feeds containing relatively little protein. The presence in foods and feeds of car- bohydrates, especially those like arabinose or pectin which yield furfural- dehyde when heated with acid, enhances the destruction of several amino

acids. Another consequence is diminished recovery of other amino acids

because of formation of insoluble humin under these conditions. Attempts

have been made to minimize these effects by hydrolyzing foods or feeds

in the absence of oxygen and with large excesses of acid, and by filtering off and washing insoluble humin before analysis of the hydrolyzate.

Another approach has been to prepare protein concentrates from the

foodstuff for analysis.

Numerous investigators have attempted to bypass the problems of acid

hydrolysis by the use of enzymatic hydrolyses. These efforts have not yet

provided a quantitatively satisfactory method. There is, however, within

this approach the potential which may make it possible to pour a solubil- ized protein solution onto the top of a column and collect the constituent

amino acids from the bottom. The use of a layered column of immobilized

enzymes could once more make science fiction a reality. In addition to the use of a number of different proteolytic enzymes, it would also be necessary to shift the optimum pH of activity through the chemical reaction

used in immobilization to a pH value mutually compatible with the dif- ferent enzymes and with the solubilized protein. This is not beyond the

realm of possibility.

Another need in protein analysis is a means of differentiating between

vegetable and animal proteins. This problem is gaining in importance

because of the substitution of vegetable for animal protein in products that formerly contained only animal protein. Rapid, accurate, economical

procedures are needed not only by regulatory agencies to test and verify

product compliance, but also by manufacturers for quality control to avoid economic losses. A recent method, not quite in vogue, utilizes immune absorption techniques to prepare species specific antisera for use in immunochemical detection of species adulterants formerly impossible due

to cross reactivity [7] . A simple, more rapid, and accurate immunochemical test would be of value.
172 ANALYTICAL PROBLEMS

We also need a method for distinguishing between endogenous and

plant origin of fecal nitrogen. The digestibility of feed protein is usually

estimated by determining the difference in the ration nitrogen and the

fecal output nitrogen. Since the nitrogen of feces arises from endogenous

excretions and some tissue material, as well as the residual nitrogen in the

undigested ration, the true digestibility of the ration or feed cannot be

determined directly. Furthermore, the nitrogen content of bacterial cells which contain nitrogen originating in the ration and extracted from it, but

residing in the excreted bacterial cell, represents another fraction of in- terest. The elucidation of the true digestibility and efficiency of absorbing

nitrogen from various feed components would be greatly enhanced if the

several nitrogen components could be distinguished in the fecal material

The final need in the area of protein analysis is for elimination of en- vironmental pollution caused by discharge of mercury in the waste of

Kjeldahl nitrogen determinations. The official method for determining

nitrogen in foods, feed, fertilizers, tobacco, and coloring materials is the

Kjeldahl test, which requires the use of mercuric oxide as catalyst.

Analyses for nitrogen are made in thousands of laboratories. Based on

estimates that 80 percent of the approximately 224,000 pounds of mercury consumed annually in the United States for catalytic purposes [8] is used for such tests, this source produces 180,000 pounds of waste

mercury per year. Either an alternate, noncontaminating catalyst or method must be developed, or mercury must be recovered from present

analytical wastes.

IV. Lipids

The chemical determination of lipids has lagged far behind that of other

chemical analyses. Unless scientists are specifically conducting studies in

this area, they tend to ignore the lipid content of biological materials

because of the cost and amount of equipment involved, time required, use

of solvents that require special handling procedures, and the limited

number of analyses that can be conducted per day. It is felt that this analytical area has suffered because of the lack of technology. A rapid and

simple method of analysis is needed and, if developed, should go far in en- couraging additional research in the lipid area. A. Rapid Measurement of Lipids and Fatty Acids

A specific need that we would like to mention is for a rapid determination of fat in corn grits. The market for brewers' grits is an important out-
AGRICULTURAL SCIENCE 173

let for the corn dry-milling industry. Brewers specify the maximum al- lowable content of extractable fat, usually about 1 percent in the grits they

purchase. During milling operations, corn is passed through a Beall degerminator mill to fracture the corn and separate the high-oil germ fraction

from the low-oil grit fraction. The brewers' grits fraction may be loaded

directly into a rail car for shipment. However, before the grits can be

shipped they must be analyzed for fat content. The current extraction

procedure for this analysis requires several hours. The millers need a

rapid procedure that can be performed in a few minutes. Prompt analytical information would allow changes to be made in milling operations

when fat content of product exceeds specification and would allow the

miller to produce more of the higher priced grit product. Wide-line nuclear

magnetic resonance (NMR) has been proposed as a rapid method for determining oil content, but this technique has not been shown to have the

required accuracy at low-fat levels. Our plant breeders need a nondestructive method for fatty acid deter- mination in connection with their efforts to breed oilseeds with specified

fatty acid composition. Problems of flavor stability, such as rancidity, are associated with oxidation of unsaturated fatty acids in vegetable oils. Striking progress has been made in changing the fatty acid composition

of rapeseed oil and safflower oil by plant breeding. In these cases, the

desired changes in fatty acids were simply inherited. Success was

achieved despite the necessity of destroying the seed in order to obtain a sample for analysis by GLC.

Soybean oil contains about 7 percent linolenic acid. Sharp reduction or

elimination of this acid would probably significantly increase stability of

soybean oil. Unfortunately, soybean germ plasm free of linolenic acid has

not been discovered. It is therefore necessary for the plant breeder to use

various techniques to apply "selection pressure" in favor of those genetic

types which have lower than normal linolenic acid. Thus he might ap- proach his goal of an acceptable level of linolenic acid in stepwise fashion.

To do so he needs a nondestructive method of fatty acid analysis. Such

a method would avoid destruction of the very seeds with the most desira- ble attributes. In GLC analysis the desirable seeds are identified only

after they have been destroyed.

The plant breeder ordinarily must examine thousands of plants in order

to find the rare one with desired qualities. The method must therefore be

simple, accurate, and suitable for rapid analysis of many samples.

NMR is a nearly perfect tool for oil analysis for the breeder of oilseed

crops. A method or instrument of similar capabilities and moderate cost

is needed for fatty acids.
174 ANALYTICAL PROBLEMS

We anticipate a possible need in the not too distant future for rapid, simple, economical methods to measure the degree of saturation of fatty acids

and the amount of cholesterol in meat, dairy, and egg products; baked

goods; and fats and oils. This need is arising from the national concern

about dietary cholesterol and saturated fats. One of the recommendations

in the "Report of Inter-Society Commission for Heart Disease

Resources" [9] was that efforts be made to "modernize regulations on

labeling and definition of foods so that consumers can identify nutrient contents such as the amount and type of fat and cholesterol." If more

detailed labeling practices should be initiated, there will be a need for massive routine analysis of food for type and amount of lipid. Present

techniques, which involve solvent extraction of lipids, ascertaining the nature of fatty acids by methylation and GLC and determination of

cholesterol spectrophotometrically, will not meet the future need for

rapid, simple, economical methods.

An incompletely answered question in this field is, "What are the

physiological effects of ingesting trans isomers of polyunsaturated lipids in humans and animals?" Some evidence of detrimental effects from trans polyunsaturated lipids has been obtained from laboratory animal feeding

experiments. Laboratory rats fed diets high in trans polyunsaturated

lipids showed increased blood clotting time and lowered prothrombin

levels in their blood. Because of the possibility of other damaging effects from trans polyunsaturated lipids, it is important to have a rapid, sensitive

analytical technique for estimating the level of each individual isomeric

polyunsaturated lipid present in processed foods and edible vegetable oils. Also, such a technique could be used to study trans polyunsaturated lipid levels as related to processing conditions in edible vegetable oils and lipidcontaining foods.

The current analytical technique used for determining cisltrans ratios

in edible vegetable oils depends on two independent measurements. The

iodine number gives an estimate of the total carbon-carbon double bond

concentration in a vegetable oil. In order to ascertain the trans isomer

content of an edible vegetable oil, it is first necessary to make a series of

infrared analyses of standard mixtures of methyl elaidate in carbon disulfide. A standard curve is then constructed by plotting the depth of the

970-cm-i band against concentration of the elaidate solutions. Then an in- frared analysis of the edible oil is made and the depth of the 970-cm-i

band is obtained from the recorded spectra. The concentration of the

trans carbon — carbon double bonds, expressed as concentration of

methyl elaidate in the oil, is obtained from the standard curve.
AGRICULTURAL SCIENCE 175

The current analytical procedure does not give detailed information on

the levels of each isomeric trans polyunsaturated lipid present. In order to

intelligently plan biological experiments and understand the food

processing conditions responsible for trans polyunsaturated lipid production, a new analytical technique is needed.

Another problem associated with vegetable oil processing is the need to know how much natural linolenate and linoleate remain in soybean oil after partial hydrogenation. Partially hydrogenated soybean oil contains

as much as several percent of linolenate and linoleate isomers different

from the naturally occurring forms. In such oils the content of the natural

isomers can now only be determined by the alkali isomerization method.

Because of the nutritional significance of natural linoleate and linolenate

and because most soybean oil is hydrogenated, the alkali isomerization

method of analysis is used extensively. GLC does not distinguish

between various nonconjugated isomers.

The Official Alkali Isomerization Method is laborious and requires

about 2 man-hours per sample. A recently developed new procedure combines isomerization and GLC for determining linoleic acid, other conjugatable dienes and nonconjugatable dienes in triglyceride esters, simple

esters, and fatty acid mixtures [10]. Compared to the conventional alkali isomerization-spectrometric method there are advantages of ease, microscale capability, speed, and versatility. However, this new

procedure is not suitable for samples containing large amounts of triene

and is not proposed as a replacement for the more accurate alkali isomerization-spectrometric method. It is suggested as a technique for the

analyst having a GLC and needing to determine linoleic acid in oil which

contains a small amount of triene. For process control uses and especially

for research studies, it would be very desirable to have available a rapid, accurate method for determining the content of natural linolenate and

linoleate in partially hydrogenated soybean oil.

B. Compositional Changes in Lipids

All oils and lipid-containing food products can suffer flavor and odor

deterioration due to oxidation of the lipids. Better methods are needed for

monitoring production and storage of such food products. This need will intensify if food distributors are required to include on the labels of their

products the date of manufacture and/or the date when the product should

be removed from the store shelf Urgently needed is a reliable, rapid predictive test which will indicate the approximate shelf life of a product.
176 ANALYTICAL PROBLEMS

Tests run at temperatures considerably higher than normal are generally not accurate. Simple Warburg techniques are useful, but until now, the

Warburg apparatus has not been automated in a commercially successful

instrument. There has been developed recently a new mini-gasimeter

which can continuously record gas uptake or evolution [11]. It is not yet

known if this type of measurement (using only mild temperature increase,

exposure to light, and exposure to 100 percent oxygen in place of air) will give a reasonably accurate automated predictive test for shelf life of lipidcontaining products.

In addition a method is needed to determine how far a food material has

progressed toward becoming unpalatable or objectionable because of oxidative rancidity. Use of such a test could allow the manufacturer or dis- tributor to extend the period during which a given lot of product could be

marketed. This problem is a very difficult one because the human nose and palate are more sensitive than most common instrumental techniques.

In the preservation of meat and meat products, oxidative deterioration

of the lipids is by far the most important source of quality loss. Autoxidation of the unsaturated fatty acids and decomposition of the resulting

hydroperoxides produce aldehydes which are principally responsible for

the rancid flavors that develop. The flavor effects of individual aldehydes

is cumulative so that traces of several individuals have a serious total effect. Thus, quantitative isolation and identification of these aldehydes

have been considered important to measurement of rancidity, and an un- derstanding of the chemistry involved.

Development and application of chromatographic procedures have

enabled the identification and measurement of the aldehydes as 2,4-

dinitrophenylhydrazones. The aldehydes which are characteristic of a given oxidized unsaturated fatty acid have been identified and measured.

Peroxide decomposition studies have shown that with respect to aldehyde

formation the scission of hydroperoxides is a selective process which is influenced by the proximity of a double bond. This fact explains the

predominance of certain aldehydes which is not supported by the amounts

of the precursor hydroperoxide isomers known to be present.

We still know very little of the nature and rates of reaction of the inter- mediate present. However, the complexity of hydroperoxide decomposition reactions is emphasized by the fact that scission decomposition to al- dehydes represents only a small proportion of the total hydroperoxide

decomposition. The nature of the other decomposition products is mostly

not known. A smaller volatile dicarbonyl fraction has been detected in oxidized pork fat from adipose tissue. These volatile dicarbonyl compounds

have not been identified. However, they are probably vicinal and of
AGRICULTURAL SCIENCE 177

moderate chain length. A nonvolatile dicarbonyl fraction is believed to exist, but this has not been explored in meat fats. Study of the dicarbonyl

compounds generated in oxidized fat is a useful line of investigation.

Meat contains two fat or lipid systems. Nonpolar lipids, which are represented by the triglycerides, compose most of the adipose tissue and

the fat marbling in the lean tissue. Autoxidation of the triglycerides is fairly well understood, and the methods referred to above are applicable.

The second fat system is the polar lipids, represented principally by the

phospholipids. Little is known about the oxidation of phospholipids,

although in some meat products they are a source of rapid deterioration. In uncooked meat cuts the polar lipids are stable. They are apparently

protected by meat enzymes in the lean tissue, of which the phospholipids

are an integral part. The enzyme systems maintain a reducing state which

prevents oxidation. Processing which inactivates the enzymes renders the

polar lipids very vulnerable to oxidative attack. These lipids contain much

more polyunsaturated fatty acids than the triglycerides. In the meat tissue

environment, hydroperoxides do not accumulate and apparently are immediately decomposed by the catalysts present. Identification and measurement of the carbonyls and other rancid flavor compounds formed and

their probable interaction products with amino acids and proteins are unexplored analytical problems in meat research.

When fats are heated in air, as in the case of deep fat frying with vegetable oils, they are partially transformed into volatile chain-scission

products, to nonvolatile oxidized derivatives, to cyclic substances, and to dimers and polymers. If the heating or oxidation is severe, the fats lose part of their nutritive value, probably because they contain substantial

levels of unabsorbable polymers. Further thermal or oxidative abuse may

cause the fat to become toxic. Certain cyclic derivatives and the oxidized

dimers are probably among the toxic compounds.

Since the cyclic compounds formed at low levels during the heating of

a fat are among the few components for which reasonable, convincing

evidence of actual toxicity has been obtained in studies with laboratory

animals, it would seem most desirable to have a dependable method for

their determination.

Many tests already exist for measuring either fat quality or the level of

deterioration products in fat. However, a dependable method for the

determination of cyclic esters is not available. The separation of the whole

complex of cyclic and branched compounds from noncyclized fatty acids

has always depended upon urea adduction which is not suitable for use in an analytical method. The development of an analytical method for quan-
178 ANALYTICAL PROBLEMS

titatively determining cyclic esters in heated oil would aid in resolving

their contribution to the toxicity of heated fats. We have a second problem related to cyclic substances, but this problem concerns naturally occurring cyclopropenoid fatty acids in contrast to cyclic acids that are formed in heated oils. Some 1 0 years ago a program was initiated to develop analytical methods for the quantitative

determination of the cyclopropenoid fatty acid content of cottonseed oil. This was an urgent problem at that time because of the potential biological

implications of these cyclopropenoid components on animal and human

well-being. As a result of this research program a number of analytical

methods were developed for determining the amount of cyclopropenoid

material present in oils and meals, on both a micro- and semimicroscale.

These included infrared spectrophotometric methods [12,13], a number

of titration methods utilizing a titrant of HBr in acetic acid [14-16] and

modified Halphen methods [17].

Analytical procedures now are needed for determining the composition

of the reaction products formed during processing and after ingestion of

cyclopropenoid material.

Another problem is related to the quantitative analysis of heat-sensitive

fatty derivatives that are surface active. These derivatives usually have

molecular weights between about 350 and 2,000. They usually are in mixtures which contain many isomers. They are easily degraded by heat,

acids, and alkalies. Examples of these fatty derivatives are esters of

sucrose, glucosides, polyglycerols, and other polyols. Sucrose and a given

fatty acid can form up to 255 different compounds (8 monoesters,

28 diesters, 56 triesters, 70 tetraesters, 56 pentaesters, 28 hexaesters,

8 heptaesters, and 1 octaester).

TLC of a sucrose ester product produces a series of spots. Qualitatively, the groups of mono- and diesters can be identified with certainty, but

the others cannot. Quantification of the different types of esters is even more difficult. Ester spots on a silicic acid plate can be made visible by

any one of a number of reagents, one of the better being a solution of urea and phosphoric acid. However, the determination of the amount of ester

in a given spot is far from accurate. For that matter, all quantitative determinations of spots on TLC plates are relatively inaccurate. Measurements of the amounts of pigments, either by transmission or reflection

densitometry, do not give the necessary accuracy even assuming that the

pigments formed in proportion to the amounts of compound present.
AGRICULTURAL SCIENCE 179

C. Other Problems

There is a great need for a procedure to distinguish various fats and oils from one another and when in admixture. This information is necessary

for use in normal trading and to detect adulteration of fats and oils, which

is a problem that has been of considerable concern to the U.S. Food and

Drug Administration.

Standards have been in use for many years as a basis for trade within

countries and between countries. These standards are based on classical

determinations of iodine values, refractive index, saponification value,

relative density, and others. Employing all these analyses, many oils could

not be distinguished one from another, let alone in admixture.

The Codex Committee on Fats and Oils, sponsored by the Joint

FAO/WHO Codex Alimentarious Commission, has recently proposed

identification of fats and oils from their fatty acid composition as determined by GLC. This is a considerable step forward which will reduce the

number of fats and oils that cannot be distinguished and affords a better means of detecting admixtures.

At the present time, improved procedures in GLC andTLC allow considerably more information to be determined about the biological source

of the samples of fats and oils. Determination of the amount and chain

length of the triglycerides present and the amount and type of sterols present in the sample apparently will greatly reduce the number of oils that cannot be distinguished.

From limited information, it appears that each vegetable oil has specific

sterols in a ratio to each other that exhibit little change, regardless of the

environment during growth and the maturity of the plant. Various animal

fats appear to have somewhat different triglyceride combinations but

similar sterols. Much more work needs to be undertaken along these lines,

but should these preliminary findings prove to be true, there would be

very few fats or oils that could not be identified; and indeed, it would be

possible to identify many in admixture.

What other types of analyses can be used to supplement the present

GLC-TLC approach to this problem?

The presence of dispersed plastics in rendered fats, particularly

rendered inedible tallow, presents an increasingly urgent problem. Plastic

materials, predominantly polyethylene, become evenly distributed

throughout the fat during the rendering process. The polymeric material

is usually present in about 50 ppm concentration as microcrystalline parti- cles in solid fat or as a fine haze in melted fat. Although the problem has
180 ANALYTICAL PROBLEMS

been intensified by the use of polyethylene drum liners that may find their

way into the finished animal fat, it is generally thought that the major

source of polymeric materials may well be packaging films that enter into

the rendering tank when off-condition packaged meat is sold to the

renderers. It is economically infeasible to remove packaging film from

large quantities of meat prepackaged in retail cuts. The haze which dispersed plastics impart on melted tallow is objectionable to producers of fat-enriched feeds, possibly mainly for aesthetic reasons. More serious are the problems caused to the fatty acid producer

by the presence of these polymers. More than 600 million pounds of inedi- ble animal fats are used for the production of fatty acids each year in this country. During the fat splitting process the polymeric materials accumulate in the pumps and splitting towers, causing frequent and serious equipment failure. The economic removal of dispersed plastics from animal fats represents an urgent problem. Even more urgent, however, is the development of a

rapid and dependable analytical method capable of detecting the presence

of plastic materials in concentrations ranging from < 5 to 100 ppm. Both

the Fatty Acid Producers Council and the National Renderers' Association are actively concerned with this problem and are supporting research

aimed at solving it. Several analytical methods have been proposed, but none has as yet been found which is rapid, accurate, and applicable to low

concentration ranges. One method which has been proposed is based on

extraction of the fat with petroleum ether, filtration of insoluble material,

and solution of the latter in hot benzene, reprecipitation of the polymeric

material with methanol, and filtration and weighing of the polymer. Confirmation of the polymer as polyethylene is carried out by infrared analysis. Other methods depending on infrared spectroscopy and on GLC have

also been proposed.

What is needed is a simple, direct, sensitive, and rapid method which

gives reproducible results. Such a method should be capable of being car- ried out by personnel having limited technical training.

Another problem to be solved is that of sequential analysis of lipids in membranes of various cells in nature. Cell membranes consist of

lipoprotein which can be isolated in fairly pure state. The protein moiety can be analyzed by various techniques available to protein chemists, but

the lipid moiety cannot be analyzed. The coordination of phospholipids,

cholesterol, small amounts of free fatty acid and triglyceride to form the

lipid portion of lipoprotein is poorly understood. All that is really known

is the fact that the lipids are not covalently bound to protein and that they

are extractable from lipoproteins. The various forms of the lipids can be
AGRICULTURAL SCIENCE 181

separated into the major classes and quantitatively analyzed. But the manner in which the lipids are organized in the cell membrane is unknown. No methodology is presently known for solving this problem.

An understanding of membrane structure is critical for comprehension of

transport mechanisms across cell membranes. Many of the aging and dis- ease processes of cells are intimately related to this problem. The analytical chemist has yet to devise a method to solve this problem.

V. Fibrous Constituents of Plants

Information on crude fiber content of feeds and cereals is used extensively in the milling and cereal industry. Crude fiber values are reported

along with such other values as total protein, crude fat, and moisture contents on most packaged dry cereals. This analysis is thus related to all pro- grams involving cereal products. The present methods involve a high

degree of empiricism, and the total crude fiber may vary considerably unless official procedures are rigorously followed. Even day-to-day checks

by the same operator on the same sample sometimes yield a high relative

standard deviation. According to the standard procedures of American

Oil Chemists' Society (AOCS), Association of Official Analytical

Chemists (AOAC), and American Association of Cereal Chemists

(AACC), one man can accomplish no more than three to four analyses per

day. All of these methods are basically quite similar. The sample is subjected to treatment in boiling sulfuric acid (1.25 grams/100 cm^ water),

followed by a slow and tedious filtration through a cloth of exacting

specifications. The remaining material is subsequently treated with boil- ing sodium hydroxide (1.25 grams/100 cm^ water), and filtered again, fol- lowed by extensive hot water washing. The remaining material is dried

and weighed. This material must be ashed so that the mineral content may

be subtracted, since it is not considered part of the crude fiber. Since crude fiber content is an indicator of the quality of feeds and

processed foods, a crude fiber analysis has been considered a necessity by

the milling and cereal industry. The requirements of a more desirable

method would be greater reproducibility, less empiricism, and certainly,

from the economic point of view, fewer man-hours per sample.

Determination of cellulose and/or lignin contents of plant materials and

their derived products is important because this information is essential in agronomic and genetic studies, process control, maintenance of product
182 ANALYTICAL PROBLEMS

quality, and appraisal of economic potential of fibrous materials, especially for the pulp and paper industry. The widely divergent backgrounds

and sources of lignocellulosics contributes to great dissimilarity in compositional characteristics and in presence and extent of extraneous sub- stances that interfere with the determination of either cellulose or lignin.

Accurate measure of the contents of both in original plants, materials in process, and in pulps and papers is essential to the Department's program

to develop processes for producing commercially acceptable pulps and

papers from kenaf and other nonwood plant materials. Present analytical techniques are empirical and generally depend upon

selective solution of the undesired constituents and contaminants so that either cellulose or lignin remains and can be measured gravimetrically.

Because of the types of materials examined and their histories, elaborate

time-consuming- schemes are necessary to eliminate undesired components. Frequently, purification procedures that must be employed contribute to degradation products, as exemplified by beta and gamma cellulose. A number of so-called direct techniques are also employed. Oxidation-reduction equivalents, viscosity of solutions from metallo-ammonium

or amine complexes, and optical densities determined under closely controlled conditions are some of the procedures employed. All suffer from

lack of suitable reference standards to correlate with lignin or cellulose contents in materials examined.

Rapidity, accuracy, and economy in analyses are essential if we expect

to decrease the gap between raw material supplies and demand for

lignocellulosic fibers in the years immediately ahead. Scanning electron

microscopy, differential scanning calorimetry, electron spin resonance, wide-band NMR, and other developments of the past decade offer excit- ing new possibilities for achieving these objectives in measuring cellulose

and lignin in plant materials at all stages of processing.

VI. Nutrients and Their Availability

Concern with nutritional status of various segments of our population

has called attention to the need for accurate data on quantity, form, and

availability of trace minerals and other nutrients, such as vitamins in soybean and cereal products. Literature values for these important

nutrients often vary widely which indicates that the analytical results are

suspect. In some instances analytical values are nonexistent [18].
AGRICULTURAL SCIENCE 183

In addition to total amounts of a given nutrient, it is necessary to know

the relative amounts of various forms of the nutrient that may be present,

because biological availability varies with different forms. For example,

minerals such as iron may occur free, bound to protein, and as ferric phytate. With some nutrients the forms in which they exist are still un- known; thus, there is no methodology available for their determination.

To determine the distribution of other nutrients such as phosphorus in inorganic, acid-soluble, phosphatide and phytate forms [19], tedious

extraction procedures followed by ashing and colorimetric methods are used. Because soybean and cereal products are used in a large number of

food products, there is a need to develop more rapid and more accurate methods that could be applied to the various food items now available.

There is also an imperative need for reliable data on the amounts in foods of three nutrients — folacin, vitamin Be, and vitamin B12, whose

dietary importance has only recently been recognized. This information

is essential for monitoring the nutritional adequacy of our present-day

foods, many of which are being used in highly processed forms. Since

1968, when the Food and Nutrition Board of the NAS-NRC first issued

recommendations for the amounts of these essential B-vitamins, the demand for data on the content of these vitamins in foods has greatly intensified. As vitamin Be occurs in foods in two and often three forms, which

future research may find to differ in biological value for man, determinations for each of the three forms are needed.

A major problem in the determination ot the vitamin content of foods is the complete release of the bound forms. Analysts tend to adopt a single

procedure for all types of foods regardless of the diversity in physical and

chemical composition. Collaborative studies are needed to determine the

best hydrolysis procedures for each of these vitamins and for each type of

food.

Currently the best data available for these three B-vitamins in foods, in- cluding the different forms of vitamin Be, are from microbiological assays. These procedures are much too time consuming and expensive to be practical. Alternative methods are needed for determining the content of these

B-vitamins in foods. Although some effort has been exerted in this direction, the challenge remains.

Several attempts have been made to devise chemical methods for these

vitamins. In the 1950's, Japanese workers reported on a procedure in- volving ion-exchange separation and a fluorometric assay of the vitamin

Be components in foods. To our knowledge, however, this work has not been duplicated. When the eluates used by our Human Nutrition Laboratory for the microbiological assay were tried fluorometrically, greatly in428-999 O - LT - 72 - 13
184 ANALYTICAL PROBLEMS

terfering fluorescent substances were found present. Further work would

be needed to obtain fractions suitable for fluorometric measurement of the

three components of vitamin Be in foods.

For folacin in foods, a fluorometric method has been described recently

in a Russian publication [20]. The authors reported that their folacin

values for the foods tested — cheese, carrots, and cabbage — compared

well with their results from microbiological assays with Streptococcus

fecalis. This fluorometric procedure may have potential for use. It would

need to be tried with a wider variety of foods and should be compared

with microbiological assays using Lactobacillus casei, the organism

presently preferred.

If satisfactory alternative methods cannot be developed, far more stan- dardization is essential in the microbiological assays as currently used.

The vitamin Be assay developed in our Human Nutrition Laboratory, and

now designated "Official, First Action" by the AOAC, represents a significant advancement. Total vitamin Be is obtained by an ion-exchange

separation of the three forms and microbiological assay of the three eluates. Thus the net effect of the destruction and conversion of one form to another can be followed during the processing and storage of foods. Few

processed foods have been assayed for the three components of vitamin

Be. Ways of speeding up this cumbersome procedure would be highly

desirable.

A recent report describes a semiautomated and a fully automated

method for estimating folacin activity in human serum [21]. The antibiotic chloramphenicol base and a chloramphenicol-resistant strain of

the preferred test organism eliminate the need for sterilizing the test material. Attempts might be made to adapt this method to the analysis of

foods.

Microbiological methods for vitamin B12 continue to present problems.

Agreement is lacking on many of the details in methodology including the

choice of the test organism. If no other means of determining vitamin B12 can be devised, the microbiological assay should be thoroughly rein- vestigated. Part of the problem with microbial determinations is caused by instability of test organisms. The reliability of the method can be improved by

use of the same batch of frozen inoculum over a period of time. The or- ganism for the vitamin B12 assay was found to remain stable for over 1 year when held frozen in liquid nitrogen. Another procedure for the

liquid-nitrogen preservation of the organism for the vitamin Be assay, when used with additional special techniques, resulted in reduction of the
AGRICULTURAL SCIENCE 185

incubation time from the usual 16-22 hours to 6 hours. Confirmation of

this technique is needed.

If organisms can be stabilized, some of the organisms discarded in the

past should be retested. The search should continue for organisms of

equal or greater sensitivity which require less time of the analyst.

The need also exists for a standardized method of determining the absolute assimilability of iron by humans and animals. Recent food con- sumption surveys have revealed widespread deficiencies of iron in the

diets of many Americans [22]. Such surveys normally assume a 10 per- cent absorption of iron from food. Biological assimilability data are frequently obtained by testing small animals. Investigators making such

tests have reported figures for the bioabsorption of dietary iron ranging

from 5 to 90 percent. Different iron compounds and food combinations

are known to affect the bioavailability of iron. Also such a wide variation

may reflect the lack of a standardized method. In view of the current ef- forts being made to enrich a variety of foods with iron, a reliable, stan- dardized method of measuring the absolute assimilability of iron by both

humans and animals is needed.

VII. Seed and Plant Constituents

The Department of Agriculture engages in extensive efforts, research

and otherwise, to promote industrial, nonfood uses of farm products; and

the problem of suitable analytical methods for constituents that are important in such uses is very relevant to these efforts. A desired constituent

may be extracted or otherwise removed from the commodity in question

for use or, alternatively, may impart essential properties for industrial applications of the agricultural product per se. In either event, accurate knowledge of the amount, availability, and functionality of the constituent

in the product is important to ensure necessary yield or performance for

the processor and/or user and a fair price for the producer. This information can also aid in defining practices to be recommended for maintaining

consistent and high contents of the valuable ingredients, levels of which

may vary significantly with genetic history of the crop, growth conditions

(geography, soil type, climate, agronomic treatments), maturity and

moisture content at harvest, and treatment after harvest.

Many examples could be cited, but three will serve to illustrate the

general nature of the problem: (1) the content of the key acid in certain

species of high-erucic acid oilseeds ranges from zero to approximately 60
186 ANALYTICAL PROBLEMS

percent depending primarily on the seed variety; (2) many oilseeds contain smaller quantities of highly unsaturated acids when grown in the

South than when grown in cooler northern areas; (3) drying of com at lemperatures above 60 °C can create problems m the subsequent manufacture of starch from it. In these examples, present methods vary in adequacy and complexity. Extraction of the seed oil, conversion of the

acids in it to methyl esters, and analysis of esters by GLC are the steps in- volved in determination of erucic acid content. A titration is used to determine iodine value, a measure of unsaturation. No better method than car- rying out of a wet-milling operation in miniature has been developed for

measuring the available starch in com that may have been heated excessively.

Recent work with wide-line NMR is one example of the kind of modern

techniques wherein the answer may lie. Single-step instrumental ap- proaches could provide the needed speed and procedural simplicity to

facilitate commerce and research progress involving the constituents and

commodities in question. These approaches developed for the examples

cited should be applicable with minor adaptation to related useful constituents; e.g., hydroxy acids and epoxy acids in the case of seed oils, and

amylose in the case of starch. In the area of marketing, rapid methods of compositional analysis are needed for grain and oilseed grading and quality determination to facilitate commercial trading. These rapid methods should also be helpful in determining nutritional value of grain products such as feeds and in distinguishing varieties of grain, particularly wheat. Present methods involve visual

inspection and laboratory testing for grading and certification. Carlots are held while this procedure is carried out. This practice is too slow for

today's market activities. On-the-spot testing of grain prior to shipping

would be beneficial to both farmer and tradesman. Delays while samples

are sent to the laboratory for testing can no longer be tolerated. Therefore,

the trend in analytical testing is toward nondestructive instrumental

methods, preferably automated, to give a number of analyses in a few

minutes. As soon as these methods can produce satisfactory accuracy

they will, no doubt, be adapted to inspection procedures.

A relaied problem exists in the small lot grading of oilseeds, where

there is need for rapid methods of compositional analysis. Since rapid

methods are not available, all oilseeds in small lots are graded on an "asis" basis, that is, on the average composition for the area concerned.

Present chemical methods of grading oilseeds are applicable only to the

trading of large lots, where the time and expense of analysis can be

tolerated. Small lot grading must be rapid, simple, and inexpensive. Such
AGRICULTURAL SCIENCE 187

grading must be based on instrumentation, preferably of nondestructive

nature. Our new infrared reflectance analysis of ground soybeans for

determination of oil, moisture, and protein is a step in the right direction.

This method must be further refined before it will meet all requirements.

For other oilseeds (cottonseed, safflower, sunflower, and flax) other ap- proaches may be required but in all probability should be based on an in- strumental method of analysis.

We have another marketing problem that is arising because of the possible future packaging and storage of processed grain and other commodities in polyethylene-film containers instead of the paper and cotton bags

that are now in use. The problem arises from the effect of the new

moisture-impermeable film containers on the storability indicators that are now used.

Percent moisture content and fat acidity value are two of the most used

chemical indicators of grain storability. After 1 month of unfavorable

storage conditions, the percent moisture content for commodities stored

in paper and cotton bags will normally rise severalfold and the fat acidity

value will drop to near zero, concurrent with a loss of oil. For

polyethylene-film containers stored under the same conditions, preliminary results indicate that percent moisture content does not increase

significantly and the fat acidity value does not drop to near zero. Thus,

there is a need for a rapid analytical indicator of grain storability for

commodities stored in the new moisture-impermeable containers.

We also anticipate a need for rapid, simple tests to identify high-oil,

high-lysine, and high-protein com kernels when these corns become

available on a commercial scale. These specialty corns will be sold, at least initially, on an identity preserved basis. Therefore, mixing with regular dent corn must be avoided during drying, storage, transport, and

milling. Methodology apparently does not exist which would provide the

elevator operator, shipper, or miller with tests for making on-the-spot

identification of the new corn types. In the area of plant physiology, we need nondestructive, highly portable, and self-contained methods and apparatus for analysis of plant constituents in studies on absorption, translocation, and metabolism of

nutrients in plants. The nutrient status of a plant can be estimated by analysis of various plant parts. However, the plant-to-plant variability may be

very great, partially or completely obscuring day-to-day variation. At

present, tissue samples selected by empirically determined criteria are taken on some sort of schedule.

A nondestructive method would permit successive analyses of the same

plant or tissue, thus removing errors due to plant-to-plant variability. Ap-
188 ANALYTICAL PROBLEMS

paratus should be portable and sturdy enough to move to field or greenhouse and should have modest power requirements.

Elements of interest are N, Ca, P, K, Mg, S, and the minor ele- ments— Fe, Mn, B, Cu, Zn, and Mo. The minor elements may be present

in amounts from a few parts per million to less than 1 ppm.

Methods are needed for identification and analysis of "active" fractions

in plant cell contents. For instance, many plants store substantial quantities of protein in seeds or other tissues. Is such protein metabolically ac- tive? The plant scientist needs a rapid and unsophisticated means to address this question.

VIII. Animal Products

Our analytical needs in this area can be conveniently grouped according

to food and nonfood products. For food products, we are concerned with

measurements of quality and of composition. Our needs in the area of

nonfood animal products center around leather manufacturing.

A. Meat Quality

The standards for quality grades of carcasses are based in part on the

physiological age of the slaughter animal as determined subjectively by a

trained meat grader. Degree of ossification of bones, darkness of

myoglobin pigmentation, and texture of Longissimus dorsi are some of the

factors used in ascertaining physiological age of the carcass. The meat in- dustry would feel more confident of decisions placing carcasses in the top

portion of the next lower grade if objective methods for quality grading were used. We developed a procedure in which the resonant frequency

and/or the sonic velocity of the right metacarpal was related to the

chronological age of Black Angus steers. This technique needs to be

adapted for use with bones of the wholesale carcass. A rapid objective

method is needed to determine the physiological changes which occur

during maturation and thus provide an index of physiological age. In order to certify the wholesomeness of meat from slaughter animals, we must be confident that the meat did not come from dead, dying, dis- eased, disabled, or drugged animals. We have found that determinations
AGRICULTURAL SCIENCE 189

of pH , myoglobin and hemoglobin-myoglobin ratio do not serve as criteria for unwholesomeness. Objective tests for detecting such meat are lacking.

Instrumentation is needed to measure metabolic changes in the live animal that are indicative of unwholesomeness in the carcass. Criteria

which can relate changes in the composition of meat to the health of the

slaughter animal from which it originated are also desirable.

Over the past several years, meat research workers and livestock

breeders have made great strides in producing pork carcasses which contain a higher percentage of muscle and a lower percentage of fat. Accompanying this selection for a meat-type hog, there has developed in pigs a

stress susceptibility which in some cases results in meat quality loss due

to a pale, soft, and watery condition. In some cases, animals shudder and

die before slaughter.

The occurrence of pale, soft, exudative (PSE) pork is usually reported

from subjective observations of the loin muscle and exposed ham muscle

or pH measurements on these muscles.

Improved antemortem methods, such as analysis of blood or biopsy

samples, are needed for determining whether or not an animal is PSE

prone. If these animals can be classified before slaughter, then they could

be segregated into areas of greatest utility.

The Department of Agriculture requires that canned hams and similar

nonsterile canned meats be heated during processing to temperatures high

enough to inactivate certain viruses. In the case of imported meats especially, there is need for an analytical method which will allow Department

inspectors to determine to what temperature a particular lot of canned

meats was heated during processing.

A method based on observing the temperatures at which soluble

proteins extracted from the ham coagulated was found to be an imprecise

means, and a better method was needed. The heat inactivation of acid

phosphatases has been found by workers both in Europe and in the U.S.

Department of Agriculture to give an estimate of the thermal history of

the canned meat which for 95 percent of the samples should be within

±2.4 °F. of the actual temperature. The actual analytical procedure

consists of extracting the acid phosphatases, allowing the extract to react with the substrate disodium phenyl phosphate, and measuring the phenol

released by its reaction with 2,6-dibromoquinonechlorimide. Although

the acid phosphatase method is a considerable improvement on earlier methods, an accurate means of determining the thermal history of canned

products over wide temperature ranges is still one of our unsolved

analytical problems.
190 ANALYTICAL PROBLEMS

B. Composition of Meat and Dairy Products

Consumer interest in the composition of meat is becoming increasingly more of a factor in the kind and amount purchased and consumed in the

home. Years of study have shown that a number of factors related to the

final quality of the product can be controlled by selective breeding and

nutrition. These results were obtained from research studies of the

dressed carcass which are not only time consuming but too late to be helpful to the meat animal breeder and feeder. It has been shown the antipyrine method for estimating composition, especially fatness in live beef

animals, is satisfactory for determining fatness in that species, but not in swine or sheep. It is a long and involved procedure that requires trained

technicians. A rapid, but accurate, analytical chemical method to determine the composition of live animals is urgently needed.

Compositional studies of slaughter animals have been carried out for

many years, but have proven to be of limited value because (1) samples do

not always relate well with total animal population, (2) sampling

procedure is destructive, and (3) large samples are required. Accepted

procedures are macro or semimicro in nature and only partly automated.

A need exists for semimicro- and micromethods that are automated and

applicable to specific systems such as the musculature, the circulatory,

and secretive systems.

Federal regulations presently limit the amount of fat that can be added

to meat products such as frankfurters. Buying and selling meat scraps and

ground meat on the basis of accurate estimates of fat content can result in more equitable distribution of the market dollar, and some assurance that

the consumer is not paying lean meat prices for fat. Connective tissue is less digestible than fibrillarsarcoplasmic protein and can be present in fresh ground meat in amounts approaching 20 percent. Knowledge about

amounts and distribution of connective tissue within a carcass and fresh

meat derived from it would aid in the determination of (1) meat quality

and (2) which muscles should be converted into further processed

products to increase collagen digestibility by heat treatment. Currently, fat content can be determined by ether extract, or methanol

chloroform methods, but these procedures are rather time consuming. A

commercially available Digital Fat Controller, which computes specific

gravity of the meat sample, can be used to estimate fat content within 0.1 percent in just a few minutes. This instrument is relatively expensive

(about $10,000) and cannot be used on meat samples below 32 °F. Another instrument employs x-rays for estimating fat content. A third in-
AGRICULTURAL SCIENCE 191

strument in the planning stage will use infrared for measurement of fat and

moisture in meat. Connective tissue can be determined using a

histometric point-counter method or can be characterized by chemical

methodology.

A spectrophotometric technique for determining moisture, oil, and

protein in soybeans is being developed in Department laboratories. Once

developed, its applicability to meat will be determined since such a method would be most welcome. Spectrophotometric and sonic methods

for determining connective tissue will be evaluated as an approach to nondestructive, rapid instrumentation.

Some processes used in the manufacture of dairy products such as evaporation, precipitation, coagulation, and crystallization involve rapid

changes in the concentrations of total solids, proteins, and lactose. Quick

methods for determination of these components are needed so that quality

control can be more accurately and efficiently maintained.

Reliable data on cholesterol in foods are urgently needed. Control of

dietary cholesterol has emerged as an essential part of treatment in certain

cardiovascular problems. In fact, some medical groups have been

pressing for curtailment or, at least, moderation in cholesterol content of

normal as well as therapeutic diets, particularly for such vulnerable

groups as the adult male.

Cholesterol is present only in foods of animal origin. The main sources

in the American diet are eggs, poultry, dairy products, fish and seafood,

and meat and meat products. For most of these sources only a few samples have been analyzed. Frequently the range reported by different in- vestigators for different samples of the same kind of food is so wide that an average value would have doubtful significance and if used would be

misleading. Information is fragmentary or lacking and is therefore ur- gently needed on the cholesterol content of such important items as cooked meats, luncheon meats, most varieties of cheeses, and for low-fat

dairy products such as skim milk and uncreamed cottage cheese. Such

values as are reported for some samples of skim milk and some cheeses

are more than 100 percent higher than the values reported by other in- vestigators. Content of cholesterol reported by various investigators for

salmon and oysters show even greater spreads, being in the order of 300

percent for salmon and 1 ,000 percent for oysters.

Reliable, reproducible methods are necessary before such problems as normal sample variation and species and seasonal differences can be stu- died and used in deriving guidance materials on cholesterol content of

foods and diet. The methodology must be tailored to be quantitative measurement of total cholesterol — free and ester— in a wide range of foods
192 ANALYTICAL PROBLEMS

and food mixtures. Methods satisfactory for use in clinical analyses of

blood and serum may require extensive modification to be suitable for

analyzing foods, with their far wider range of problems in extraction and

interferences.

Various combinations of solvents and different extracting times are in use for the extraction of total lipids from a food prior to determining

cholesterol. This lack of uniformity may account for some of the wide

range in values observed. Special attention is needed on procedures for

completely extracting cholesterol from products with little total lipid and

low cholesterol content such as skim milk, lean meats, and the many

mixed foods containing one or more animal products.

The principal method of cholesterol determination used to date on lipid extracts from foods relies on the Liebermann-Burchard color reaction in which cholesterol gives a color when combined with sulfuric acid,

chloroform, and acetic anhydride. Since this reaction is not totally specific

for cholesterol, a preliminary digitonin or tomatine precipitation is usually

made on the crude unsaponifiable extract to eliminate noncholesterol

sterols. The tomatine procedure is newer and more specific for

cholesterol than the digitonin, but both are subject to many interferences.

Other procedures of assay include the AOAC bromination, fluorometric,

and gravimetric digitonin methods.

A recently developed technique [23] for determining cholesterol by

GLC, while more specific than traditional colorimetric methods, has had

almost no application to analysis of foods. As presently published, the

GLC method requires about 2.5 grams of extracted fat for the analysis.

The weight of sample of low-fat foods required to provide this amount of

fat could be critical in some research. Therefore, specific standardized

methods for determining cholesterol in much smaller amounts of lipid extract are needed.

The widespread use of diethylstilbesterol in livestock production makes

knowledge of its metabolism imperative. Although there is vast literature on the use of diethylstilbesterol, a total metabolic scheme is not known in any animal species. The amount and nature of biological residues in edible

meats and the biological effects of these residues on the animals have not been well characterized. Conventional chromatographic separation and

colorimetric procedures are available for macroquantities of diethylstil- besterol. GC procedures are being applied to diethylstilbesterol deter- mination but adequate methodology does not as yet exist. A matter of concern related to the use of antibiotics in animal production is the possibility that residues of these compounds may be found in the edible tissues of livestock after they have been processed for human
AGRICULTURAL SCIENCE 193

consumption. This possibility exists, whether antibiotics have been routinely used for growth stimulation or for therapeutic purposes. Antibiotic

residues in animal tissues have been determined by means of microbiological assays. These microbiological methods are relatively slow and labori- ous, as well as being specific only for the compound in question. There is a need for simple and rapid analytical procedures which would identify not only the parent compound but also possible metabolic breakdown

products which might accumulate in various body tissues. Such

procedures would permit a more accurate determination of the fate of antibiotics in the animal after ingestion or injection and also allow for the

monitoring of larger numbers of meat samples in a consumer protection

program.

C. Hides and Leather

Analytical methods are needed to account quantitatively for all of the

sulfur-containing compounds in the lime-sulfide unhairing of hides. The

tanning industry is having considerable difficulty in reducing the quantity

of pollutants that it produces. One of the severe problems is the biological

oxygen demand (BOD) and sulfide content of the effluent from the unhairing system. The reactions involved are quite complex and have never been fully studied because of the lack of analytical methods suitable to fol- low the five inorganic (sulfide, polysulfide, hydrosulfide, sulfite, and

thiosulfate) and four organic (cystine, cystiene, lanthionine, and methyl

mercaptan) sulfur compounds suspected to be involved.

Sulfide is used up by the excess cystine in the hair, but may be

regenerated as sulfide or polysulfide by a second reaction which yields

lanthionine. Whether these reactions are directly involved in the hair- loosening or hair-dissolving step has never been established. Efficiency in the use of sulfide might be increased if more were known of the actual

hair-loosening reaction. The present analytical methods are not sufficient

to clearly establish all of the competing and serial reactions involved.

If the rate of the hair-loosening reaction could be increased at the ex- pense of the hair-dissolving reaction, a considerable decrease in the BOD

load and a reduction in the sulfide requirement might result. Since the same problem affects tanners throughout the world this is a problem of in- ternational significance.

The introduction of pickled cattlehides as a marketable commodity has

been hindered by a lack of adequate means of quality control and en- forceable specification parameters.
194 ANALYTICAL PROBLEMS

At present there is no standard procedure for determining the acid (pH

2) and salt (10 to 15 percent) contents of pickled hides. The acid is usually

sulfuric and the salt, sodium chloride, although formic, acetic, and

paratoluene sulfonic acids and sodium sulfate have also been proposed.

Ashing is unsatisfactory since some of the components will be volatil- ized or partially altered. Quantitative extraction from the hide involves

iengthly manipulation. The determination of pH values on a 1 0-fold water

extract after 1 2 or 24 hours has little reliability as a measure of acidity. Present attempts to assay hides utilize electrodialysis, surface specific

ion electrodes, and conductivity and capacitative measurements to determine total electrolyte contents and microelectrode probing to determine

stratigraphic distribution of electrolytes within hides.

Department research has led to the commercial use of glutaraldehyde

in the tanning of leather. Our research also disclosed that the aldehyde is bound irreversibly and not liberated upon hydrolysis of the tanned

protein. However, hydrolysis of glutaraldehyde-tanned hide substance

does produce ultraviolet absorbing species (maximum at 265 nm) which

correlate with bound glutaraldehyde as measured by loss of aldehyde

from the tanning solution. This method of analysis is adequate for

research purposes where the tanned hide substance may be analyzed before the finishing operations necessary to produce the finished leathers. Many materials used in finishing also absorb in this region so that this method for detecting and estimating bound glutaraldehyde is not applicable to finished leathers. This is a disadvantage from the standpoint of control of a new commercial tanning process and of specification of this new

leather. Fundamental research to establish the nature of the ultraviolet absorbing species can be expected to contribute to the solution of this analytical

problem. Because the tanning industry is lacking in research facilities and

technical staff, sophisticated and expensive techniques are to be avoided.

IX. Sensory Characteristics of Foods

Nearly all food research investigations require taste-panel evaluation of

food products or intermediates as a primary criterion of the efficacy of raw material or processing variations. Flavor and odor evaluations by

taste panel require a large enough group of tasters so that reliable statisti- cal evaluation of results can be made because of the variability of sensory

evaluations. For these and other reasons, food research involves con-
AGRICULTURAL SCIENCE 195

siderable expense. Instrumental methods should be developed to supplement and partially supplant taste-panel methods as an economic measure.

In certain of the beverage industries, for example, GLC analysis has been

used to advantage in this connection. However, it appears that flavor

compounds frequently are present in amounts far below the detection

limits of present-day instrumental sensors, since the most potent flavor

and off-flavor compounds are often detected by odor or taste, not instrumentally. Hence, further development of instrumental methods seems

warranted. Present knowledge of the essential components of cooked aromas, as well as the methods of estimation, are inadequate to provide processing

plant controls. Subjective odor panels are time consuming and expensive

and not adaptable to a continuous plant monitoring of the total production. Solution of this general problem will first require well coordinated

chemical, analytical, and sensory evaluation of the commercially important cooked food aromas. When the essential components and their criti- cal proportions are established, then relatively simple plant control

methods can be sought which will permit monitoring optimum flavor

development in the further processing of meat, poultry, and other heatprocessed foods.

Another important sensory characteristic of foods is "eye appeal,"

which is still measured largely by subjective techniques. Apparently, the

wealth of spectrophotometric equipment in the modern laboratory can tell the scientist little about the spectrum of light reaching the eye from the

surface of a peach, chocolate fudge, or a beef roast. Certainly the con- sumer easily notes color differences and reacts decisively to changes in the visible region that the photometers cannot detect or describe. In a typical instance, research on fortifying milk with iron to overcome

fairly widespread incipient anemias in certain population groups has been

hindered by the fact that the added iron causes color changes in some

foods in which milk is traditionally used, such as cocoa. The eye easily detects off-colored cocoa, but the finest reflective spectrophotometers are

blind to these changes. Obviously, much more effort could be spent in the

study of light reflected from complex surfaces of multiphasic systems

which have a measurable degree of translucence.

X. Microorganisms in Foods

There is a constant and ever-growing need to assess and control

microbial growth in our food products, in order to conserve our foods and
196 ANALYTICAL PROBLEMS

provide a wholesome, safe food supply with an adequate shelf life. Presently available and used methods of estimating total viable bacteria

almost always involve culturing and counting, which are laborious and

time-consuming operations. As a result, products are either unduly

delayed, or they move through channels of processing and distribution

without a current record of their bacterial load. Attempts so far to provide alternative chemical methods, such as dye

reduction methods, have fallen short of their goals. A workable method

should be general enough to react to and measure all viable microorganisms, and yet be specific enough to exclude all dead cells and foreign or- ganic matter. This is an extremely difficult goal for the analytical

biochemist, and probably would involve a basic search for suitable

transient intermediary metabolites to serve as the reactive chemical in- dicator of total viable bacteria present. Such compounds might be sought

in the general area of nucleotides and other biochemical organic

phosphates.

At the present time, standard cultural and serological techniques for the

isolation and identification of Salmonella require at least 4 days for positive identification. Since the methods are complex and time consuming,

there is general agreement that more rapid and reliable means of detection

are needed. Rapid methods are particularly needed for the assessment of

sanitation in food processing plants.

Department scientists have developed a new culture medium and test- ing procedure for Salmonella in dairy products which combines the advantages of a newly developed cultural procedure with the specificity of

absorbed antisera conjugated with fluorescein isothiocyanate. The

completed test for Salmonella requires about 24 hours.

Positive presumptive evidence of Salmonella is obtained in 18 to 24

hours by growth in a selective enrichment medium. Salmonellae are in- dicated by the medium changing color from red to black and/or yellow.

The medium contains a broad spectrum antibiotic for bacterial selectivity

and the enzyme trypsin, which digests milk casein and clarifies the medi- um. Confirmation of Salmonella in the 18 to 24 hours' growth medium is determined by the fluorescent antibody technique. Dairy products as- sayed by the new procedure have included raw and pasteurized milks,

milk concentrates, nonfat dry milk, whey, and cheeses. Although the new

procedure reduces the time for assay by one-fourth, a still more rapid

procedure is highly desirable for monitoring food processing operations.

Occasionally, canned whole tomatoes and some aseptically canned

products such as pea soup suffer flat sour spoilage in which acid is produced, off-flavor develops, and eventually some gas may be formed
AGRICULTURAL SCIENCE 197

due to bacterial action involving chiefly Bacillus stearothermophilus or

Bacillus coagulans. Fortunately, the incidence of such spoilage is in- frequent, the spoiled product is not toxic, and the likelihood of the spoiled

product being eaten is small. Nonetheless, the problem results in both

economic loss and consumer dissatisfaction. At present, there is no simple, objective, reliable method of detecting flat sour spoilage without

opening the can. One consequence of such spoilage is a marked decrease

in the viscosity of viscous products. This change may provide one possible basis for developing a nondestructive test for flat sour spoilage in canned products.

XI. Animal Health

Analytical problems in this area stem from the Department's surveillance efforts to detect foreign animal diseases and our regulatory and control program on veterinary biologicals.

A. Foreign Animal Diseases

There are numerous animal diseases exotic to the United States that would be devastating to our livestock and poultry industry should they

become established. The concept endorsed by U.S. officials is that any

outbreak of a foreign animal disease that would result in significant loss to the U.S. livestock industry must be quickly detected and eradicated

promptly. To accomplish this, a surveillance program has been

established, and each reported case suspicious of being a foreign animal

disease is investigated by a veterinarian with special training in the diagnosis of foreign animal diseases. Laboratory specimens such as tissues,

oesophageal-pharyngeal fluids, blood, and serum are collected and sent to appropriate laboratories for serological diagnosis. The time required for a tentative diagnosis can vary from hours up to several days and sometimes weeks. Since many of these diseases spread in an explosive manner,

this lengthy testing procedure may permit a disease to spread widely before eradication procedures can be initiated. More rapid tests, such as

analytical chemical methods, that could be developed to quickly and

economically determine the presence of a foreign animal disease would

allow control measures to be effected before the disease has a chance to spread widely.
198 ANALYTICAL PROBLEMS

Imported animal products have a disease-producing potential for

domestic livestock of the United States. Therefore, an accurate as possible determination must be made that these products do not contain animal

disease-causing microorganisms. Many animal products, including meat,

are visually inspected at ports of entry to determine whether the products

have been processed in conformance with Department regulations and

directives. At times, it is not possible to make a clear-cut decision on the

basis of visual examination. In many of these instances, samples are submitted to the laboratory for analysis for final determination. Development

of rapid, simple analytical methods which could be used at the point of

entry by laymen to determine the safety of a material would be beneficial. In addition to providing an immediate determination, test results would

provide backup support to the decisions of the inspectors.

Similarly, the chemical identification of foreign animal disease organisms would be beneficial in the Department's program of regulating the

importation and interstate movement of organisms capable of causing dis- ease in animals. Problems often arise when considering the importation of an organism because of the presence of an animal disease in the country

of origin which is not known to exist in the United States. Although the

organism which would be imported may be rather innocuous, fear of con- tamination of the culture, or the culture media, with a foreign animal dis- ease agent can result in the importation being refused or being allowed

only under stringent restrictions. These restrictions hamper some scientific exploration in the United States.

B. Veterinary Biologicals

One of the objectives of the Department is to conduct a regulatory and

control program for assuring the consumer that all veterinary biologicals are safe, pure, and potent. There are several important analytical chemistry problems that need to be solved in order to better meet this objective.

Three analytical problems that are of great interest to us are: (1) the

development of improved methods for the purification, isolation, and

identification of the active components of veterinary biologicals; (2) the

development of analytical methods that can be used for the evaluation of

fluorescent antibody reagents which are used as an aid in the identifica- tion and diagnosis of animal diseases; and (3) the development of analytical methods which permit correlation of chemical structure and biological

function and thus promote the development and use of synthetic antigens.
AGRICULTURAL SCIENCE 199

1. Active Components of Veterinary Biologicals

Many of our chemical problems in this area are bioanalytical in nature and require the correlation of chemical results with biological tests. Presently, we perform many product evaluations using in vitro biological

systems and others which can only be made by using host or laboratory

animals. These tests adequately evaluate the products but are often ex- pensive and time consuming to perform. Most of the biological products

are very heterogeneous and contain a large percentage of material other

than the active components. The bacterins and toxoids contain many

nonessential metabolic products along with the active components, and

the viral products contain large amounts of growth-supporting material

such as the residue from egg or tissue culture menstrum. The presence of

these residues have detrimental effects on any direct analytical approach

to quantitating the efficacy of these products.

One of the problems we face is to develop suitable purification

procedures that will permit more direct in vitro tests which can be used to verify the safety, purity, and potency of these products. Several purification procedures such as dialysis, ultrafiltration, and gel filtration are now

being used to help solve this problem.

2. Fluorescent Antibody Reagents

We are developing a program for the evaluation and control of

fluorescent antibody (FA) conjugates which are used for the identification

and diagnosis of animal diseases. FA, as the name implies, are specific antibodies which are conjugated to a fluorochrome. The fluorescent labeled

antibody permits the microscopist to readily identify the specific antigen

present in a bacterial smear or tissue culture preparation.

We presently produce and evaluate a number of specific FA reagents

both for our own use and for the use of biological producers. These re- agents are used solely as reagents for the evaluation of veterinary biologicals. A much larger number of FA reagents are commercially produced

for use in the diagnosis of animal diseases.

The procedures now being used for the evaluation of FA conjugates are very time consuming and require the microscopic examination of many

slides. Evaluation is further complicated and the work increased manyfold

by the requirement that conjugates must be specific and give a negative

reaction for a great number of similar antigens.

428-999 O - LT - 72 - 14
200 ANALYTICAL PROBLEMS

A major analytical problem is the development of a rapid and sensitive

method for the evaluation of FA conjugates used for the identification of

animal diseases. One method that is under consideration involves the use

of a flow-through laser photofluorometer. In this method, the antigenic

cells, either viral laden tissue cells or bacterial cells, are mixed with the

FA conjugate, incubated, and then passed through a microflow-cell in a continuous stream. The cells would be counted, sized, and their relative

fluorescence determined. An index could then be developed to correlate

relative fluorescence with the number of antigenic cells. The details of this method have not been worked out, but the general concept seems quite

feasible.

3. Synthetic Antigens

Most veterinary biologicals are very heterogeneous and contain many

substances other than the active components. Many biologicals contain

nonessential metabolic byproducts as well as large amounts of growthpromoting substances which have not been used by the organism. The active components have an extremely high level of biological activity and

yet only represent a very small amount of the total composition of the

product. Many toxins and allergins demonstrate biological activity in the

nanogram and picogram range of concentration. These two classes of

biological products probably will be among the first to be sufficiently well

characterized that they could be replaced by synthetic antigens.

A great deal of effort has been made to purify and characterize several

bacterial toxins. However, most of the work has been concentrated on

tetanus, diphtheria, and botulinum toxins with relatively little work on

toxins from other organisms. One of the main problems involved with

purification and characterization procedures is the lability of toxin during

processing. Most toxins are quite labile and readily degrade when they are separated from their native environment. Presently most commercial toxoids, chemically denatured toxins, are marketed as a mixture of detoxified

toxins, other metabolic products, and substances used to promote growth

of the organisms. The presence of such a large amount of nonspecific sub- stances puts an undue burden on the animal to be immunized as it causes

the immunological system to produce antibodies against all of these substances. Allergins, such as tuberculin, are another class of products that could

be sufficiently well characterized that a synthetic homologous antigen

could be produced. Tuberculin is presently used as a cell-free filtrate con-
AGRICULTURAL SCIENCE 201

taining all the metabolic products of the organism as well as the nutrients

required for its growth. An improved product is presently being evaluated

for use in animals, and it consists of only the "purified protein derivative"

(PPD) of the culture filtrate with most of the other metabolites and

unused media constituents removed during processing. Although it is called a "purified protein," PPD is actually a group of proteins. It has

been shown that the tuberculin PPD for use in human medicine contains a mixture of polymers. The basic component of the polymer has been

identified as a small molecule with an estimated molecular weight of 7,000

to 10,000.

The fact that many toxins and the basic component of tuberculin have

molecular weights in the 5 ,000 to 1 5 ,000 range enhances the feasibility that someday they may be produced synthetically. Technology is now

available that will permit synthesis of compounds of this size, as evidenced by the synthesis of insulin and ribonuclease. The main problem

facing us now is to purify the products and to perform the basic analytical

work which will identify their structural characteristics. Once the structural characteristics have been described, it will be necessary to correlate

the structure with biological function, and then we should be able to modify the structure of these components in order to obtain the necessary

immunizing effect. The advent of synthetic antigens is probably many

years in the future, but now we need to make our start on the analytical

work that is the necessary forerunner of such an event.

XII. Biologically Active Substances

Our interests in this area are varied, because Department programs

deal with some aspects of the whole spectrum of human, animal, and plant

life. We would like to discuss some problems related to the following substances: (1) indigenous plant constituents, (2) mycotoxins, (3) plant

growth regulators, (4) pesticides, and (5) nitrosamines.

A. Indigenous Plant Constituents

Compositional studies of plant products under our new crops program

have reminded us of the wide distribution of phytochemical substances

potentially harmful to animals or man. Familiar examples are solanine in
202 ANALYTICAL PROBLEMS

potatoes, tomatin in tomatoes, thioglucosides in crucifers, lathyrus factors

in certain legumes, indospicine and )3-nitropropionic acid in Indigofera,

oxalic acid in spinach, and cyanoglucosides in almonds, pecans, and

tapioca. There is little or no information to indicate that any of these compounds are so distributed and concentrated as to be harmful to animals or man when the plant materials containing them are used in ways that are conventional at present. However, there is always the danger that new

varieties, agricultural practices, or methods of processing may concentrate the undesirable principles beyond safe levels. Present analytical methods generally involve some form of extraction

and usually one or more fractionation steps applied to the extract to in- crease the concentration of the substance in question. Then, it is detected

and estimated in varying degrees of quantitativeness by colorimetry or some form of chromatography or spectroscopy. This multiplicity of steps

is time consuming, and each step contributes to the error range or magnitude of uncertainty in the final answer. Opportunities for marked improvement should exist in procedures analogous to the highly automated

ones currently used for amino acids and sugars.

Specifically, a need exists for simple and reliable methods to quantitate

the various steroidal alkaloids present in edible Solanaceous crops. There

has been a recent trend to use wild and uncultivated relatives of tomato,

potato, pepper, and eggplant in varietal development programs. These

wild relatives of the edible Solanaceous crops offer excellent sources of

pest resistance. However, the introduction of new genes from uncultivated species of Solanum presents a problem to plant breeders because some of these uncultivated species are not edible due to their alkaloid content. It is conceivable that the use of these species in breeding programs

could result in the release of new commerical varieties that contain alkaloid components not present in our older commercial varieties or varieties

that contain higher contents of the alkaloids which are normally found in our edible Solanaceous crops. For example, Solanum chacoense has been used in some potato breeding programs as a source of pest resistance. This wild relative of potatoes

contains a new class of steroidal alkaloids, the leptines, in addition to solanine and chaconine, the glycoalkaloids found in normal potatoes. The

leptines repel the Colorado potato beetle and its larvae, but because of the

taste and pharmacological properties of the leptines, they may be

undesirable in relation to food quality. At present, there is no simple

screening procedure to determine the quantity of leptines and related

water-soluble, steroidal alkaloids. A simple qualitative test for this class of compounds, as well as an improved quantitative method to determine
AGRICULTURAL SCIENCE 203

total alkaloids present in other edible Solanaceous crops, would greatly

benefit the plant breeder, the food technologist, and, ultimately, the

public.

This is but one example of the potential alkaloid problem involved in the breeding of Solanaceous crops and the need for improved analytical

techniques for alkaloids in these crops. Breeding of Solanaceous crops in- volving gene transfer from inedible wild relatives poses a problem not

generally encountered in breeding other agronomic crops. This problem

can be readily resolved once we have the methodology to analyze our edi- ble Solanaceous crops for these physiologically active alkaloids.

Another alkaloid is of interest in connection with fescue toxicity. Cattle

grazing on tall fescue grass, Festuca amndinaceae, sometimes develop a lameness termed "fescue foot." The condition is characterized by a dry

gangrene of the hind feet and tail. Cattle severely affected may die or recover with permanent defects of the feet. Animals mildly affected may

recover completely following temporary lameness. Much research has

been done to identify the toxic constituent or constituents with particular

reference to toxic alkaloids. One such alkaloid, perloline, has been found

to occur in the Kenwell variety of tall fescue and to peak at approximately

3,000 micrograms per gram dry weight for a period of 2 weeks. Up to 6,000 micrograms per gram dry weight has been found in some varieties

of tall fescue. Perloline is in itself apparently not toxic to animals, but it can reduce the digestibility of cellulose in the animal and thus influence its performance. Further work needs to be done with perloline and possibly

other alkaloids to determine their significance as toxic agents in forages.

Techniques need to be developed to readily identify such agents and to analyze plant materials not only for their presence but for the quantitative

levels produced.

A topic of increasing importance is the trypsin inhibitor content of

soybean products, because food uses of soybean proteins are expanding.

Present methods of determining trypsin inhibitor give varying results; thus, the absolute content of trypsin inhibitor in a given sample is still in doubt. Moreover, there are problems of reliability when the assay

methods are applied to processed soybean products. A recent report [24]

indicates that a significant level of residual trypsin inhibitor activity oc- curs in a commercial infant formula containing soy isolate. This infant for- mula gave a lower growth response when fed to rats than two other soy

isolate-based formulas containing very low residual trypsin inhibitor activity. This problem is a matter of concern to soybean processors.

Presently, four analytical methods are being used [25-28] . All of them are based on the principle of protein digestion and, consequently, are beset by
204 ANALYTICAL PROBLEMS

similar weaknesses. Soybean trypsin inhibitor (STI) is reported in relative

terms, such as tryptic units inhibited or STI destroyed when compared to an arbitrary standard. Thus, in order to report STI activity in terms of mil- ligrams per gram sample, absolute concentration of trypsin and protein

substrate must be determined.

Chemical constituents in plants are affected by all aspects of the en- vironment. Of particular interest is the recent knowledge that infection of

alfalfa {Medicago sativa L.) by foliar pathogens results in accumulation of

the estrogen, coumestrol, in plant tissues. Problems in animal per- formance have been attributed to feeding hay that is high in estrogen content. In some potent dehydrated alfalfa samples, coumestrol accounted

for 90 percent or more of observed estrogenic activity. Interestingly, dis- ease-free plants have about 2 ppm coumestrol while the coumestrol content of diseased plants often exceeds 500 ppm. No coumestrol is found in foliar pathogens that attack alfalfa. Information is needed to identify

biologically active constituents associated with quality factors in forages.

Also, we need to know the mechanism of biosynthesis of biologically active compounds like coumestrol and coumestans and the mode of interaction between plant and pathogen or other environmental variables. In other words, in a plant-disease relationship are other harmful constituents

synthesized? Why is coumestrol synthesized instead of some other substance?

B. Mycotoxins

Mycotoxins are a group of toxic metabolites — often car- cinogens—produced by the action of molds on agricultural commodities

and foods. The seriousness of the mycotoxin problem was not fully real- ized until the discovery of the aflatoxins, potent carcinogens, in 1960.

Since then, a vast amount of research has been conducted to determine

the extent of the aflatoxin problem. Figure 2 indicates the structural for- mulae of the aflatoxins with which we are primarily concerned. Presently,

other mycotoxins are being studied to discover their significance in agriculture.

As part of the evaluation of the aflatoxin problem, qualitative and quantitative methods were developed to determine the incidence and levels of

aflatoxins. Although procedures vary, basically each method consists of

sampling, defatting, extracting, purifying, and estimating by TLC.

Currently, two methods are officially recommended for the determination of aflatoxin in peanuts by the AOAC. One involves an extraction
AGRICULTURAL SCIENCE 205

M] M2

Figure 2. Structural formulae of aflatoxins.

with methanol-water and purification on Celite columns. The other

method, known as the "CB procedure," recommended for peanuts, in- cludes a chloroform extraction and a purification on silica gel columns. A

modification of this method was used to screen export cargoes of corn for

aflatoxin, ochratoxin, and zearalenone. In this study, ochratoxin was

discovered for the first time as a natural contaminant.

As part of a study of mycotoxins in grains under the AOAC, studies are being conducted to establish a method of analysis for aflatoxins in com

and soybeans that can be officially recommended. Studies will be ex- tended to include wheat, grain sorghum, oats, and barley in the future. These methods will probably be adopted by the AACC and the AOCS.

As other mycotoxins, such as sterigmatocystin, patulin, citrinin,

penicillic acid, and tremorgens, take on new significance as carcinogens,

suspected carcinogens, or toxins, the need for rapid, sensitive multitoxin

methods increases. A multitoxin screening method for five toxins has

been developed, but it lacks the sensitivity required for a quantitative

method. More effort in this field is necessary.

Accurate and sensitive analysis for aflatoxins became available when

automated fluorodensitometry was applied to evaluation of aflatoxin on TLC plates. This technique has been also applied to ochratoxin and
206 ANALYTICAL PROBLEMS

penicillic acid. GLC has been used to confirm the presence of

zearalenone in corn and studied as a confirmatory test for patulin. This

technique could be extended to other mycotoxins.

Aflatoxins Mi and Mo are carcinogens originally found in milk of cows

fed aflatoxin-contaminated meal. The development of recommended

procedures for determining Mi and M2 in agricultural commodities, particularly milk, has been delayed for two reasons. Aflatoxins Mi and M2

had not been separated on TLC plates, and supplies of toxins were not

available for standards. Both of these problems have been solved by Department scientists, and it should now be possible to develop reliable analytical methods for these toxins.

Although methodology can be improved and hastened by automation,

such as use of fluorodensitometers, mass spectrometers, and GLC, rapid

detection in the field to avoid delays in moving commodities will require

simplifications in methods at the sacrifice of accuracy. As new mycotoxins are discovered and evaluated, new methods will have to be developed.

In certain areas where cotton is grown under irrigation, invasion of

bolls hy Aspergillus flavus and the elaboration of high levels of aflatoxins

in the seed is a serious problem. Even within a narrow geographical area

the invasion is sporadic, with some fields heavily infested while others are

relatively free of attack. The result is that ginned seed lots entering a cot- tonseed mill may vary widely in aflatoxin contamination.

Presently, the only detection system depends on the presence of yellowish fluorescent "cat eye" spots adhering to the short lintered fibers when ginned seed are inspected under long wave ultraviolet illumination.

Unfortunately, industry experience shows a poor correlation between cat eye fluorescence and aflatoxin contamination.

The availability of a rapid detection system whereby a representative

sample of seed from an incoming truck or railway car could be analyzed

within 5 to 1 5 minutes would allow contaminated seed to be diverted to special storage areas for separate, special processing and would offer further assurance of absence of contamination.

Improved methods for mycotoxins would also be extremely beneficial

in the area of animal production. Most animal feeds are inhabited by a

vast array of microorganisms, some of which are capable of producing

materials which are highly to)^c to animals and/or humans. Conditions are usually unfavorable either for the growth of the organisms or the production of the toxins. Therefore, the development of clinical symptoms from

the ingestion of mycotoxins is not at all common. Because of the in- frequent occurrence of clinical symptoms, knowledge concerning the oc- currence of mycotoxin at subclinical levels is very incomplete. The possi-
AGRICULTURAL SCIENCE 207

bility that subclinical levels of mycotoxins are responsible for reduced

levels of animal production is frequently posed. Improved methods of

identifying and quantifying mycotoxins in feeds and animal products

would be of immense help in locating and eliminating the potential losses

from mycotoxins.

C. Plant Growth Regulators

Modern agricultural practice makes wide use of a variety of synthetic

plant growth hormones, stimulants, and inhibitors to produce desirable

modifications in the products we harvest. Our efforts are additionally in- fluenced, however, by an indeterminate number of native or natural

chemicals that powerfully influence plant growth.

Nowhere are these natural stimulants and inhibitors more important

than in the problems associated with control of weeds. Persistent, hard-tokill weeds depend, in many cases, upon efficient production of seeds,

buds, and rootstocks or rhizomes that become dormant during adverse

growing periods. The survival of these reproductive organs is dependent

upon their efficient dormancy mechanism as well as the fruitfulness of the

species. Subtle changes in biochemical intermediates, plant hormones,

and the plant pigment phytochrome help to regulate dormancy. The

problem of dormancy in plant tissues is an old one, and one that is criti- cally important in making progress in control of weeds.

Control or modification of dormancy could hold the key to efficient removal of many noxious weeds which pollute the environment. However, this control cannot be achieved until and unless our special analytical problems can be solved. The gibberellins, kinins, auxins, ethylene, and

other natural growth regulating compounds exert pronounced effects on

plants or plant parts that are entering or emerging from the dormant state. They occur in small amounts, frequently as labile esters, complexes, or

precursors. The many influential compounds involved vary greatly in chemical structure and reactivity, thus complicating analytical problems.

Detection in many cases depends on bioassay, making quantitation difficult. Modern chemical and physical methods of analysis may be

employed where identity is known and standards are available for

comparison. But in many cases related to dormancy, the identity of the

inhibitor or promoter is uncertain and standards are not available.

The most critical phase of analysis is the initial extraction of the

dormancy factors from the living tissue. Disruption of tissue during ex-
208 ANALYTICAL PROBLEMS

traction mixes the products being analyzed with a wide variety of natural

products which may chemically modify the material the analyst is seeking.

The extraction process may release or form substances that had no

physiological significance in the plant until the maceration occurred.

The burden of analysis is placed upon nondestructive methods of extraction that will not result in coincidental modifications during extraction

and separation.

One of the most difficult tasks we face in studies of tree physiology is the analysis of plant hormones. Plant hormones such as gibberellins,

cytokinins, abscisic acid, indoleacetic acid, and ethylene influence vir- tually all phases of plant metabolism. They usually act in combination in determining important tree characteristics such as height of the plant,

development of flowering, formation of seedless fruit, and resistance to stresses such as cold and drought.

Because the hormones act either in conjunction or in opposition,

usually more than one has to be analyzed in studying any given process. For example, cytokinins, gibberellins, and indoles are important in fruit growth; gibberellins, cytokinins, and abscisic acid in bud break and seed

dormancy; gibberellins and indoleacetic acid in stem growth.

There are presently available analytical techniques involving TLC,

GLC, fluorometry, and spectropolarimetry to determine each of the hor- mones. However, the extraction procedure is elaborate for each hormone,

and it is done separately for each compound. This makes the analysis of more than one hormone at any given time on a number of samples impossible. We need extraction and analytical techniques which allow us to extract more than one hormone at the same time. By developing such a method, we can expect to obtain much useful information about how to control plant metabolism.

Several chemicals are currently being evaluated for their effect on abscission as related to fruit harvest. Some materials hold the fruit on the

tree; others induce abscission to aid in harvesting of the fruit. With constantly increasing labor costs, the ability to manipulate crops to enhance

harvesting is essential. To properly evaluate any chemical for use on an agricultural crop, the

plant physiologist or biochemist needs to know the mode of action of the

chemical in question. In most instances, he does not know how a chemical

acts when he begins his initial evaluations but only the suspected

response. Thus, as his evaluation proceeds, he usually conducts experiments designed to answer this question. Often this involves precise analysis of the specific chemical in tissue extracts. However, this technique is only superficial since it will not tell the scientist where the chemical is in
AGRICULTURAL SCIENCE 209

the tissue. Knowledge of the location of the chemical in the tissue is criti- cal to understanding the mode of action. For instance, we know that cycloheximide induces abscission by influencing ethylene production

which comes from tissue wounding. We do not know exactly which

tissues are affected or the metaboHc pathways involved.

One of the most challenging problems for the analytical chemist is to develop techniques for the analysis of these chemicals in intact tissue at the cell level. Techniques of this type would enable the physiologist to pinpoint the location and concentration of the chemical in the plant and

relate this to the plant response obtained. Thus, the plants under test would not have to be sacrificed for analysis. In this regard, using an

isotopically labeled chemical does not solve this problem in most

instances.

D. Pesticides

Problems in this area can be conveniently grouped into the following

categories: (1) general problems, (2) pesticide complexes and metabolites,

(3) problems associated with manner of application, and (4) structural basis of herbicide action.

1. General Problems

We find that among our concerns of a general nature in the area of pesticides are analytical problems related to (1) confirmation of identity, (2) in- terfering chemicals, (3) new types of insecticides, (4) multicomponent

analysis, and (5) pesticide disposal.

a. Confirmation of Identity. — Valuable as GLC has been in determining

pesticide residues, the confirmation of identity, and the validation of

quantitative measurements by independent means are still needed. The

determination of GLC retention times on two or more columns or GLC

followed by TLC can provide strong supportive evidence, but not necessarily unequivocal proof of identity.

Areas where research is needed included: (1) More specific GLC detectors for halogen and nitrogen. — The flame photometric detector is highly specific for phosphorus or sulfur and very sensitive (0.01 to 0.001

ppm). Electron affinity detectors currently used for halogen compounds

are very sensitive but not selective. For nitrogen compounds, the best
210 ANALYTICAL PROBLEMS

methods now available are microcoulometric or electrolytic conductivity

detection as ammonia (5 to 10 ng). (See also carbamates.) (2) Cheaper

rapid scanning mass spectrometers and infrared spectrometers. — Present

instruments are either too expensive or not sufficiently sensitive. (3) New

methods of analysis. — One possibility is the plasma chromatograph in which an ionizing source charges molecules of a gaseous medium which

react with trace organics to generate trace ions which are then separated

according to their mobility (sensitivity reported at one part in lO^^). b. Interfering Chemicals. — The very extensive use of polychlorinated

biphenyls (PCB's) in industry has resulted in the appearance of these

compounds in wildlife and in agricultural products. PCB's are incorporated into asphalts, rubber tires, paints, plastics, and a variety of other

compounds. They have entered the air, water, and soil as a result of industrial and garbage smoke, automobile usage, and from industrial wastes. These compounds are similar in many respects to DDT, although analytical characterization is much more difficult. PCB's are an important source

of interference in the chemical detection of chlorinated hydrocarbon

pesticides. This is an example of an industrial pollutant, rather than a

pesticide, which exerts adverse biological effects and produces residue

contamination in agricultural products. This problem requires immediate

attention.

Methodology is not presently adequate for separation of the PCB's

from other chlorinated hydrocarbons and characterization. Pure samples

of PCB's and polychlorinated triphenyls are needed. The very complex

nature of the GLC patterns observed required better GLC systems to dif- ferentiate between compounds.

Another problem with interfering chemicals arises from trace contaminants in pesticides. Examination of technical pesticides often reveals

many impurities. Each of these impurities has a different toxicity to plant

and animal life. Identification and determination of trace contaminants in technical pesticide materials and formulations presents challenging

problems to the analyst. For example, dibenzo-p-dioxins with less than

five chlorine substituents have retention times on GLC columns similar

to the chlorinated hydrocarbon insecticides. This may present difficulties in analysis.

Thus, there is need for the development of methods for the confirmation of trace amounts of hazardous substances in environmental and

manufactured samples. Usage patterns, policy decisions, and even basic

changes in the current direction of American agriculture may depend on

the accuracy with which we can measure these impurities. Combined

GLC/mass spectrometry appears to hold much promise here. To enhance
AGRICULTURAL SCIENCE 211

the value of this method for the solution of some specific problems, an improvement in interfacing or enrichment techniques to permit the sample

to pass from GLC to mass spectrometer more efficiently is needed. The

full potential of the technique can best be realized by computer storage

and handling of acquired data to provide maximum information and rapid

recognition of trace contaminants.

c. New Types of Insecticides. — Because of persistence the chlorinated

hydrocarbon era is on its way out. Organophosphorus insecticides have

been intensively investigated and will be widely used. Methods of detection are available. On the other hand, carbamates are being developed and

used before good methods of analysis are available. Except for carbaryl,

the carbamates must be reduced or derivatized for analysis, which means

working with a variety of polar materials. A detector specific for nitrogen

is urgently needed. Undoubtedly, other types of compounds will be

developed for their degradability. Quantitative TLC with ultraviolet

fluorescence may be a useful approach.

d. Multicomponent Analysis. — Rapid, comprehensive, and reliable procedures for samples of unknown history are of importance to assure

safe food supplies. Some work has been done on GLC of phosphorus and

sulfur-containing pesticides and metabolites in representative foods in which interfering substances are not excessive. Certain crops such as onions, turnips, and radishes are not amenable to this method.

e. Pesticide Disposal. — There is no completely safe, economical, and

publicly accepted method of disposing of items such as used pesticide

containers and used, dirty, or otherwise unwanted pesticide mixtures.

Pesticide mixtures are currently supposed to be disposed of by dumping

on ground at sites which are inaccessible to livestock and where such

dumping will not contaminate surface or underground water supplies.

These conditions of disposal are often difficult to meet because of lack of

suitable disposal sites. Repeated disposal in the same area may result in pesticide buildup causing a spillover into the surrounding environment.

The qualitative and quantitative tests for pesticides are generally dif- ficult to conduct as well as being costly and time consuming. For that reason, the potential for producing pesticide residues and the actual

residue produced from dumping are usually estimated instead of being

carefully calculated by analytical means. Better, more rapid, and more

economical field tests for determining pesticide levels are needed. If such

tests were available for use by the field operators, they could more accurately determine whether unsafe residues would be deposited as a result of disposition of pesticide or pesticide-bearing materials. The collection

of a sample of well water for analysis is shown in Figure 3.
212 ANALYTICAL PROBLEMS

Herbicides seldom exist unaltered in the soil, in plants, or in water to which they have been applied. We are aware that herbicides do degrade in the environment, resulting primarily in harmless metabolites. We are less well aware, however, of the marked tendency of many herbicides to exist

in plants and soils as complexes, adducts, or conjugation products with

natural materials. The existence of these herbicide combinations presents a significant challenge to the analyst. Figure 4 shows how a sample of soil

is taken for measurement of pesticide residues.

A method of analysis that permits quantitative detection of the parent

herbicide in extracts of crops or soil may not include all of the non- degraded herbicide present in the original sample. Herbicides may exist

in biological systems as sugar esters, glycosides, amino acid adducts, and

complexes with proteins and lignins. In fact, a single herbicide may occur

in several of these forms simultaneously. For instance, the important herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) has been shown to complex with protein, to form an O-glucoside as well as a glucose ester, and to combine with amino acids. Chloramben (3-amino-2,5-dichlorobenzoic

acid) is converted almost quantitatively to its A^-glucoside in soybean
AGRICULTURAL SCIENCE 213

Figure 4. Soil sample being taken for measurement of pesticide residues.

roots and other plant tissues. Several carbanilate-base herbicides form

bonds with lignins that are resistant to hydrolysis.

It is not sufficient simply to degrade the complexes and measure the

released herbicide as is frequently done at present. The conditions of the

herbicide in the plant and in the soil determine its persistence and residual

phytotoxicity. Mammalian toxicity may also be significantly affected, and

usually reduced, by the existence of the herbicide as a complex. Thus, for

full understanding of the significance of the various states in which an herbicide can exist in nature, it is necessary to develop and use analytical

techniques that will be quantitatively accurate, but that will reflect clearly

the form in which the molecule exists at the time the sample is taken.

Another major problem of current interest and concern in the field of

pesticide residue analysis is the development of suitable methods for

isolation and quantitative determination of highly polar, water soluble,

pesticide metaboUtes. Until recently, the major emphasis in residue analy-
214 ANALYTICAL PROBLEMS

sis has been placed on the isolation and quantitative determination of the

nonpolar parent pesticide. The current extraction techniques and analytical procedures are often designed to examine only nonpolar parent pesticide residues to the exclusion of polar metabolites.

The justified and deep concern over what appears to be a substantial

void in our information on the ultimate fate of pesticide molecules in our environment makes this limited approach to pesticide residue analysis no

longer adequate or acceptable. The concentration, distribution, ultimate

fate, and significance of polar pesticide metabolism in plants, animals,

soil, and water have not been established. Indirect evidence now available

indicates that the increased environmental burden of polar, halogenated,

aromatic, and heterocyclic ring structures from pesticide applications may

be significant over a reasonably long period of time.

The major polar metabolites of many pesticides have been isolated and

identified, but rapid, reliable, and quantitative methods for the analysis of

most of these metabolites have not been developed. The development of

improved and selective extraction techniques for polar pesticide

metabolites needs to be investigated. New and improved methods for the

cleanup of extracts prior to quantitative analysis also need to be

developed. The rapidly developing technique of high pressure liquid chromatography seems to offer many possibilities for the rapid cleanup of

polar pesticide metabolites.

New and improved GLC techniques also need to be developed for the

quantitative analysis of polar metabolites. Investigations in this area should include the development of improved column supports as well as new and improved methods for the quantitative conversion of polar

metabolites to less polar or nonpolar derivatives before GLC analysis.

3. Problems Associated with Manner ofApplication

Problems in this area are to be expected in view of the variety of ways

in which pesticides can be used. We anticipate or have encountered

problems related to (1) admixture of surfactants with herbicides, (2) test- ing of liquid fumigants, (3) encapsulation of insecticides in plastic, (4) application of chemicals in foams, and (5) application in sprays. Most of

these problems are complex, and they probably will require a much more

sophisticated approach to analysis than now exists. However, these

problems cannot be ignored in view of the environmental implications of

pesticide usage.
AGRICULTURAL SCIENCE 215

a. Admixture of Surfactants with Herbicides. — Additives of various

types are becoming increasingly important in modern herbicide formulations. The surfactants added can completely change the activity of certain

herbicides from one type of selectivity to another. In addition to the mixtures and blends of a variety of surfactants included in commercial formulations, the farmer frequently adds additional surfactant to enhance or modify activity or to improve herbicide solubility characteristics.

As a result, a large tonnage of surfactants is applied with herbicides to plants and soils each year. Potential persistence of these compounds

varies, but the nonionics are most stable. Buildup of such materials in the

soil can influence the availability, movement, and persistence of herbicides in the soil profile. Continued usage of herbicide-surfactant mixtures

must be considered not only from the standpoint of alterations in herbicide persistence, but also from the standpoint of possible modifications in phytotoxicity patterns of herbicides applied later due to enhanced solubility in the soil. Attention must be given to the possibility that accumulation of the sur- factants themselves could result in harmful effects to plants, man, and

animals. Many of the common surfactants have been shown to have ad- verse effects upon biological systems.

The analytical problems associated with surfactants and their use in weed control are very difficult. A large number of nonionic, anionic, cationic, and amphoteric products are available for use in formulations or

for subsequent addition by the farmer. The identity of the surfactant in the

formulation is seldom available, and analytical procedures presently employed are complicated, expensive, and not sufficiently specific for clear- cut identification. Increasing use of surfactants with herbicides calls for a greater atten- tion by analysts to problems associated with detection and quantitation of

this important class of agricultural products.

b. Testing of Liquid Fumigants. — A liquid fumigant is applied to a stored

product and sufficient time is allowed for the liquid to vaporize and dis- tribute itself throughout the product. Samples of the vapor-air admixture

are collected and transported to the laboratory for analyses by means of

GLC.

If a researcher had a simple, rapid test by which to determine the concentrations of vapors in air, it would enable him to make immediate adI justments that could not be made otherwise. The equipment involved

should not be large, bulky, or expensive.

c. Encapsulation of Insecticides in Plastic. — Because of problems as428-999 O - LT - 72 - 15
216 ANALYTICAL PROBLEMS

sociated with the use of persistent chlorinated hydrocarbons, ways are being sought to prolong the effective life of organic phosphate insecti- cides. It appears reasonable to expect that phosphates encapsulated in plastic would be extremely effective because slow release would produce

high concentrations of insecticide over a long period of time. This possibility will be investigated in the Department for the control of insects during storage of dried fruit and tree nuts. Analytical methods are not now

available for measuring plastic-encapsulated insecticides without inter- ference from the plastic. In order to pursue this promising area of

research, it is essential that rapid, simple, economical procedures for en- capsulated insecticides be developed for analysis of laboratory formulations and residues on food commodities.

d. Application in Foams. — In recent months, much interest has been

shown in the possible use of foams as carriers for agricultural chemicals. In our research programs on the navel orange worm, preliminary experiments have indicated that foams have great possibilities as carriers for in- secticides. Owing to the foams' ability to hang on to the tree and nuts in al- mond orchards, insecticides would have a longer period of penetration. In

studies to date, methyl parathion, baygon, gardona, and fundal have been

infused into foams.

There is no methodology in existence that is applicable to insecticides

in foams. To date, in our studies on extraction and partitioning methods,

we have obtained inconsistent and incomplete recoveries of samples. In order to continue studies on foams as carriers, we need analytical

procedures for both foam formulations and residues on commodities after application.

e. Application in Sprays. — In this area, we are concerned about uniform

application or adsorption of insecticides or repellents onto fabrics,

packaging materials, and surfaces of storage facilities. It is extremely dif- ficult to correlate analytical residue data with time studies and effective- ness of pesticide treatments. Most likely, surface phenomenon are in- volved. The alkalinity of some spots on the surface may degrade the com- pound applied. Soluble dirts and stains on fabrics or cellulose may repel

the application. Active sites may attract the pesticide. In any case, the application is not as uniform as might be expected from the technique of

treatment. A study is needed to learn how and why these compounds are adsorbed or repelled by surfaces such as animal fibers, cellulose,

concrete, and paint. Such a study would benefit from a more rapid, simpler, more accurate, or more economical analytical method.
AGRICULTURAL SCIENCE 217

4. Structural Basis ofHerbicide Action

Intracellular target systems are being identified for at least some herbicides such as acylanilides, 5-triazines, and substituted uracils which might

be involved in the expression of phytotoxicity. For example, these herbicides interfere with electron transport and phosphorylation in isolated

chloroplasts and mitochondria. The end result of this interference is in- hibition of adenosine triphosphate (ATP) production. ATP energy is required to drive all cellular activity. Without energy, cellular functions

and growth are arrested. Hence, for the first time, the inhibitory action of

a large group of herbicides can be studied in vitro on processes that conceivably are related directly to their in vivo toxic action. Up to this time,

the studies have been primarily descriptive. Attempts are being made to elucidate the chemistry of the interaction, the bond relations between the

inhibitor and the affected site, and the conformation assumed by the in- hibitor at the target site. Inhibitors must possess properties that will enable penetration to the

target site and assumption of the precise spatial configuration required to complement the molecular architecture of the active center and, hence, to block the key reaction. Properties possessed by the inhibitors which could

conceivably be of importance include partitioning characteristics (hydrophilic/lipophilic balance), steric relations (the ultimate attainable configuration), resonance, keto-enol tautomerization, cis or trans relation of the

amide hydrogen and the carbonyl oxygen, and the possession of a critical charge by particular substituent groups which participate in intermolecular interactions at the active centers. Molecular orbital calculations should

indicate sites of probable reactivity on the herbicide molecule. However,

of the one attempt that has been published in which electron densities were related to inhibitory activity, for a series of acylanilides, informative

results were not obtained.

Very little information is available relative to the forces that are in- volved with the interaction of herbicides and active sites. Evidence obtained to date indicates that formation of covalent bonds is not involved;

instead the bonds are much weaker. The forces that might be involved

such as hydrogen bonds, nonpolar dipol-dipol interactions, and van der

Waals forces remain to be identified. To date, a few of the chemical and physical characteristics of the in- teraction outlined above have been elucidated. For the most part,

physiologists have attempted to obtain some of these answers with only

limited assistance from analytical chemists. Conceivably, answers could
218 ANALYTICAL PROBLEMS

be obtained more effectively with the assistance of highly skilled and in- terested chemists. While the implications of the elucidations at this time

are of fundamental interest, they could have real and practical importance

in designing and developing more specific and highly selective herbicides.

If the chemists charged with synthesizing new herbicides can be given

data relative to the molecular architecture of active sites, and the chemical

and physical properties that are required for inhibition of the target

system, it should be possible to tailor-make herbicides.

E. NiTROSAMINES

In 1956, Magee and Barnes [29] demonstrated that dimethylnitrosamine was carcinogenic, initiating a considerable amount of research on this class of compounds. Initially, the pharmacological activity was the important aspect but, since about 1962, the presence of nitro- samines, primarily dimethyl and diethyl, in a variety of food substances

has been reported. Since this becomes a matter of prime importance

to public health, the quantitative determination of nitrosamines in foods and other agricultural products assumes great current significance.

Two major techniques for determining microquantities of these compounds have been reported: (1) TLC and (2) GLC.

TLC methods developed by Preussman et al. [30] and by Sen et al. [31] involved separation on Silica Gel G by several solvent systems.

Both techniques require the use of duplicate spots and both methods depend on photolysis of the N-nitroso linkage for the identification reactions. Both techniques may be subject to interference due to artifacts extracted from foods {e.g., substances such as phenols, fatty acids, pigments, and amines). Sensitivity of this method is about 0.3 to 0.6 /Ag of

nitrosamine.

GLC has only recently been used for the quantitative determination of

nitrosamines. Two procedures, one developed by FDA [32] and the

other by the Department of Agriculture [33] , involve the use of the Alkali

Flame Ionization Detector (AFID) which is specific for nitrogen compounds containing two nitrogen atoms. The mechanism of action of the

AFID is unknown. Sensitivity to nitrogen compounds is about the same

as the normal Flame Ionization Detector, but sensitivity to hydrocarbons

is reduced by a factor of 10,000 which results in the selectivity. About 5 ng of dimethylnitrosamine can be detected by this method.
AGRICULTURAL SCIENCE 219

Regardless of whether TLC or GLC is used, the major problem in nitrosamine measurement at present is the isolation and cleanup

procedure. Nitrosamines vary widely in their characteristics and do not respond uniformly to recovery procedures. The cleanup factor is important because there are many food components that interfere with

measurement of nitrosamines.

With improved methods of obtaining relatively clean samples of

nitrosamines from food products, adequate procedures for determining

concentrations quantitatively will be no problem.

Xm. Measurement of Pollutants

Air pollutants that have been reported to affect agriculture [34] are of

two major categories: particulate and gaseous [35]. Particulates can be

described as being inorganic or organic in nature. Dusts derived from construction sites, feedlots, and cement facilities are referred to as inorganic,

while particles such as polynuclear aromatic hydrocarbons are organic

particles. Inorganic gases are carbon monoxide, carbon dioxide, sulfur oxides, hydrogen sulfide, nitrogen oxides, ozone, and hydrogen fluoride.

Organic gases and vapors are hydrocarbons, aldehydes, alkyl nitrates,

organic peroxides, tetra alkyl lead, pesticides, and methyl substituted

sulfides. Figure 5 shows damage to a tobacco leaf caused by photochemically generated oxidants.

Air pollutants are monitored by one or a combination of the following

methods: membranes, molecular sieves, atomic absorption, ultraviolet, or

infrared spectroscopy; or TLC, column chromatography, or GLC; or

titrimetric or conductivity measurements.

Inorganic particles are identified by light or electron microscopy,

characterized by x-ray diffraction, or by helium-neon laser, and the density in the atmosphere is determined by integrating nephelometers. Their

chemical composition is determined by atomic absorption spectroscopy.

Organic particles are collected on membranes, separated by chromatography, and characterized by ultraviolet, infrared, or fluorescence spectroscopy. Unless complete separation of the air components is achieved

by chromatography, the spectral analyses have been found to be incorrect. Carbon monoxide at 0.05 to 2 ppm is measured by GLC, but hydrogen,

olefins, and aldehydes interfere. Carbon dioxide can be measured by
220 ANALYTICAL PROBLEMS

Figure 5 . Tobacco leaf damaged by oxidant air pollution. GLC and mass spectroscopy with an accuracy of 1 percent in the

presence of nitrogen or air. Infrared CO2 gas analyzers are also used,

especially in laboratory studies. Sulfur dioxide is measured by conductivity or colorimetrically by West-Gaeke, but air contaminants (HCl, NH4,

and chlorine) interfere. A long path spectrophotometer with fine struc- tured bands (3 ,000 A region) measures low concentrations of SO2 without
AGRICULTURAL SCIENCE 221

interference. The loss of reflectance from a silver membrane surface is a

sensitive method for measuring hydrogen sulfide. Nitrogen oxides and ozone do not interfere. Nitrogen oxides can be identified by concentrating them on Molecular

Sieve 5A and releasing them at 250 to 300 °C for GLC. Sensitivity

is 0.19 ppm. Nitrogen dioxide is also measured coulometrically, but little is known about interfering substances. Ozone is generally measured by

Mast Ozone Meters which measure the current imposed against a 0.24 V

circuit from the oxidation of potassium iodide to iodine. Nitrogen oxides

and peroxy compounds interfere with the measurement. Long path in- frared can distinguish ozone from other materials, but the cost renders it useless for continuous analyses. Hydrogen fluoride is measured by GLC.

Hydrocarbons (C2-C12) are identified by GLC, using polyethylene

glycol or squalene columns. Aldehydes are found to be present by either

trapping them in bisulfite solutions or reacting them with dimedone for

fluorimetric determinations. They can be derivatized and separated by

GLC. Alkyl nitrates are characterized by GLC on polyethylene glycol at ambient temperatures. Organic peroxides are identified by either

colorimetric or ultraviolet procedures.

Tetra alkyl lead is trapped on activated charcoal and reacted with

dithizone in air samples of 100 to 300 m^, or by GLC with a range of 3 to 400 ng/m^ and 95 to 99 percent accuracy. Methylated sulfides are

identified by direct GLC.

Since the chemical diversity of constituents in the air is so great (3 to 4,000 identified), the challenge to the analytical chemist and the manufacturer is to develop instrumentation that is reasonable in cost, that is easily

operated and requires little attention in the field, that can employ analytical means for identifying several air components in a region of the

country, but with the added capabihty of screening out all but one component at a time. The instrumentation must be portable and have the sensitivity to be useful in field conditions.

We are concerned not only with pollutants that affect agriculture, but

also with pollutants that are caused by agriculture. For example, in connection with management of feedlots we need improved methods for

determining volatile nitrogen-containing compounds. Figure 6 shows a

feedlot on which management systems are being developed for limiting

pollution of streams and ground water. Results of ground water studies conducted below and near feedlots in Colorado and Nebraska suggest that minimum pollution occurs if anaerobic conditions prevail below the feedlot surface. However, air sampling
222 ANALYTICAL PROBLEMS

Figure 6. General view of a beef cattle feedlot. measurements show that appreciable amounts of ammonia and odors are generated under these conditions. In air sampling equipment placed at various locations in or near

feedlots, about 20 percent of the trapped nitrogen compounds were not ammonia. Qualitative tests indicate that these compounds are amines,

fatty acids, and indoles. However, standard laboratory techniques fail to recover sufficient quantities of these compounds to express them quantitatively. If feedlots are to be managed to avoid air and water pollution, a quantitative measurement is needed. Current methods are most in- adequate for developing management practices for minimum odor and

ammonia pollution.

Another area of concern is that of pollution caused by food industries.

Research is underway in many areas of agriculture to develop methods for

abating pollution from specific agricultural activities. One of the areas of

emphasis is the reduction of pollution in poultry processing plant ef- fluents. Researchers are charged with developing processes and

techniques that will reduce waste loading in poultry processing plant ef- fluents. One of the most important problems in this area of research is the
AGRICULTURAL SCIENCE 223

lack of a single, accurate, rapid method to measure the strength of organic

wastes in effluents. BOD is the test most commonly used. There is no measure of the accuracy of the BOD test on wastewater. It is possibly more of an art than a science. Even though it is a relatively simple

technique, its accuracy depends on choosing the right "seed." Samples

cannot be collected and stored before analysis; and single samples are al- most impossible to analyze since an error in seed choice would void the

sample analysis, but the error would not be known until 5 days later. Several other tests are in use today which in some way relate to or can

be related to BOD for specific operations, but they are not acceptable as a replacement for BOD. Chemical oxygen demand (i.e., reduction of oxidizing agent) is a 2-112 hour test and, in general, does not correlate well

with the BOD test. Total organic carbon can be determined in 2 or 3 minutes and has been strongly advocated by some because of its speed.

Suspended solids, volatile solids, and other factors are measured but, to

date, none of these measurements replaces BOD. Both in research and

plant scale evaluations, a test is needed that can be performed quickly and

which will give an accurate measure of the effect that a waste material will have on a natural stream. Preferably, the method should also be capable

of monitoring the available oxygen content of rivers, lakes, and streams

continuously.

XrV. Molecular Properties of Natural Polymers

In this area, there is need for conformational analysis of fibrous

proteins to identify the amino acid residues important to the general struc- ture of the protein. According to current views of molecular biology, the

primary structure of the protein (the sequence of individual amino acid

residues in the chain) determines its secondary and tertiary structure; namely, how the protein chain turns, twists, and folds upon itself The

physical and chemical properties of the protein depend almost entirely on

the topological identity of the amino acid residues; for example, the

groups that are exposed, the shape of the reacting site, and the residues

that form the reacting site. Some amino acids are more important to the

structure than others either because of their type, or their specific location

in the chain, or both. Substitution, addition, or deletion at these critical areas will profoundly affect the macromolecular protein structure and

function. If the intimate details of these composition-structure-functions

relationships were known, the chemical and physical properties of

proteins could be manipulated at will.
224 ANALYTICAL PROBLEMS

At present, there are no straightforward means for determining the configuration of individual atoms within the chain of fibrous proteins. Globular proteins, on the other hand, since they can be obtained in pure crystalline modifications, are amenable to precise structural determinations by means of x-ray diffraction techniques. Fibrous proteins, such as collagen,

the keratins, and silk, because of the absence of a submicroscopic identi- cally repeating structural unit, are not subject to detailed x-ray analysis.

The kinds of conformation presently obtainable relate to their macroscopic properties; molecular information is almost entirely inferential and incomplete.

Two routes offer promise for solving this problem. Scanning electron

microscopy of very high resolution may be able to identify the overall

chain conformations and the location of some specific amino acid

residues. Armed with sequence analysis data, one might be able to piece

together a fair picture of the protein structure. Another fruitful avenue of

research appears to be in certain branches of spectroscopy. With suffi- ciently high resolution and sensitivity, it would be possible to assign

specific energy transitions to given amino acid residues in the protein. By

observing the change in these transitions as the protein folds, unfolds, and

interacts with various solvents, it may be possible to establish the role played by many of the amino acids in the structure. NMR and Raman

spectroscopy, with the best instrumentation, appear to have developed to the point where studies of this nature are becoming feasible; as these

techniques improve, unusual opportunities will exist in these areas. Similar to this problem of conformational analysis of fibrous proteins is the problem of relating surface properties to polymer composition. In recent major oil spills, the relatively small portion of contaminated birds

that survived decontamination efforts is perhaps a dramatic indirect

reflection of how incomplete is our understanding of surface properties of

natural polymeric materials such as wool, mohair, and feather keratin.

Even though the overall composition of these materials is proteinaceous

and tends to be relatively hydrophilic, the nature of the surface layer is such that these materials display hydrophobicity to bulk water. As yet

there is only indirect evidence that these surfaces may be comprised lar- gely of methyl or methylene groups which mask the hydrophilic nature of a polyamide (protein) structure. The identification of organic groups at an

air/solid interface continues to be a problem of importance in characterizing surfaces more thoroughly in our search for understanding biological

systems and in defining adhesion and wetting phenomena. Some of the more important methods which have been used with some success include

Multiple Internal Reflection Spectroscopy (MIRS), Electron Spectrosco-
AGRICULTURAL SCIENCE 225

py for Chemical Analysis (ESCA), and surface wetting behavior {e.g., contact angle determinations).

MIRS is limited in usefulness in that the depth of penetration of the

reflected energy is more than the first molecular layer so that the spectra

obtained do not solely represent the constitution at the air/surface inter- face. ESCA is a promising, relatively new technique which is useful for

analyzing up to the first 100 A of a surface. The technique is sensitive to the oxidation state of the element and chemical environment of the atom.

At present, it has greater utility in the detection of noncarbon atoms, but

improved resolution may change this situation. Measurement of wetting

behavior of the surface is an indirect method, but one of the most sensitive

in detecting changes in surface composition. The difficulty, of course, lies in correlating wetting changes with actual changes in composition. Additional methods for determining the chemical nature of surfaces are clearly

needed.

The third problem in the area of molecular properties is related to the

evaluation of intramolecular heterogeneity of collagens. Much confusion

and uncertainty in collagen chemistry arises from the fact that while a given collagen preparation made from the tissue of a vertebrate animal

gives a consistent physical characterization, preparations from different

individuals of the same species display a wide variation in physical

characteristics. These differences seem to exist at all four levels of

molecular organization. Since it is very likely that function and viability of a given tissue is determined by all of its components, and collagen is in- variably one of them, a study of the structure of collagen in a given tissue

and how it correlates with the nutrition, development, and character of

that tissue seems important.

One application of such a study would be to correlate the structure of

collagen in meat with the various qualities sought by the consumer, such

as flavor and tenderness. Quality could in turn be related to the nutrition

and environment of the meat animal during its developmental phase.

At the present time, the only work of this nature being carried out is in the field of human geriatrics. Progress is slow because effort is limited to studies of amino acid distribution and sequence. A more fruitful approach seems to be the use of electric birefringence, which provides an extremely

sensitive technique for measuring subtle differences between molecules.

Our final problem in this area is that of determining molecular weight in the 1 ,000 to 15,000 range. The classical methods for determining molecular weights below 500 (namely, the ebuUioscopic and cryoscopic

methods) have largely been replaced by vapor phase osmometry. This

thermoelectric vapor-pressure method has been shown to be applicable
226 ANALYTICAL PROBLEMS

for molecular weights below 500 to 1 ,000 providing the correct solvent

and standard are used. In the case of larger molecules, light scattering for weight average

molecular weights and membrane osmometry for number average

molecular weights are applicable to materials as low as 10,000 to 20,000

if considerable care is exercised in the measurements. This leaves an area of roughly 1 ,000 to 1 5 ,000 for which no really reliable method is available. The problem is to find a suitable new method for this range or

possibly to extend the range of an existing method such as vapor phase

osmometry. The American Society for Testing and Materials and the

AOAC are both studying this problem with the hope of establishing an official method for determining molecular weights in this range. Any sug- gestions that would lead to a solution of the problem would be greatly ap- preciated by both groups.

XV. Automation and Instrumentation

Automation and instrumentation present the analyst with both opportunities and problems. We would like to mention some problems and opportunities related to (1) computer-aided analytical chemistry, (2) instrumentation suitable for increasingly smaller quantities of materials, (3) increasing the resolution of electron microscopy, (4) developing low-temperature

ashing procedures, and (5) computer control of vegetable oil refining.

Automated analysis is capable of producing more data at a faster rate than can be interpreted [36] . Mountains of recorder paper from our GLC

are forming in each of our laboratories. The answer lies in more assistance

from the computer, which can be programmed to present data in graphical

form ready for human interpretation.

Computer-aided analytical chemistry is not limited to the acquisition

and processing of experimental data, but it also opens the door to more ef- ficient analytical data management. Through computer control of sample

flow through the laboratory, the supervisor is assured that samples will not be lost, the analyst knows the backlog of work that faces him and how

much has been done, and the recipient of analytical data can receive an immediate update of all analytical information relating to the samples of

his interest. Computer-guided analysis enables us to make instant comparison

between theory and experimental results. This approach contrasts with

the conventional manner of waiting for days or weeks after an experiment
AGRICULTURAL SCIENCE 227

is completed to determine whether the range, number, and accuracy of

measurements are sufficient. Thus, parameters may be varied as needed

during the course of an experiment.

A major analytical problem facing the laboratory today is the need to accurately analyze increasingly smaller quantities of chemicals in animal

tissues and products. To meet this requirement, it is necessary that more

and more significant instruments and procedures be developed. This, in turn, requires that all chemicals used in the analytical procedure be of

highest purity — in many instances, of greater purity than is commercially

available. Instruments must have increasingly more specific and sensitive

detection systems which will increase the complexity while decreasing

flexibility. As a result, the "down time" of an instrument for repairs or to change or alter the detection systems can become a significant expense

item and a major cause of lost time in the laboratory. In many cases, it is necessary to have a technician come in from a regional office or the

headquarters of the company producing the instrument.

As procedures increase in sophistication and complexity, the chance of

error in manipulation increases. The scientist or technician at the laboratory bench must take ever- increasing precautions to prevent contamination of the sample being analyzed and to decontaminate the equipment

after use. Animal tissues and products must undergo increasingly more

rigorous and complex treatment to isolate the desired chemicals for detection, thus again increasing chances of loss or error. The localization of specific molecules within cells and subcellular components as an analytical adjunct to electron microscopy is just beginning.

The contrasting and imaging of organic molecules is limited by two factors. These molecules cannot be distinguished from the carbonaceous

matrix in or on which they are supported or from neighboring biological

molecules. In addition, the low inertia of the atomic components make

them susceptible to irradiation damage from the illuminating beam of electrons. Both of these problems could be solved by the addition of heavy ele- ments to specific organic molecules. The heavy elements would increase

contrasting ability and because of their mass, be more stable in the elec- tron beam.

Current methods have involved the use of metal-containing light microscopic stains or metal adducts of antibodies. Both of these techniques,

although they may be very specific in their mode of action, have resolution capabilities on the order of tens of angstroms instead of an order of

magnitude lower which would allow the imaging of specific molecules, not

aggregates of them.
228 ANALYTICAL PROBLEMS

If perfected, this technique would allow for localization of mycotoxins,

pesticides, antibiotics, and their metabolic derivatives. It would also

facilitate analysis of native components which play a role in spoiling, textural, and color changes. The current development of high resolution

analytical microprobes would, if coupled with this technique, allow the

simultaneous qualitative evaluation of several metal derivative compounds on a quantitative basis. Historically the analysis of the ash content of foods and fiber has al- ways held a role of importance in establishing quality and function.

Qualitative and quantitative analytical procedures in this area have advanced to the point where the rate controlling step of the analysis is now

the reduction of samples to ash. Thousands of burnt fingers and endless hours of waiting attest to the

deficiencies inherent in the muffle furnace or the wet digestion rack. In many instances recovery experiments using volatile metal complexes

have demonstrated the more serious shortcomings of classic procedures.

Advances in analytical procedures call for a concurrent advance in ashing

techniques.

Equipment has recently been made available which claims to reduce

ashing time and trouble by use of plasmas. Heralded as "cold" ashing

procedures, they remain to be more thoroughly studied and developed.

Certainly temperature of the plasma stream and sample temperature can- not be equated as similar.

It is most surprising that the development of the radically conceived

ashing devices employing reactive plasmas have been the subject of so lit- tle research and development. Theoretically they should already have a

routine place in modern laboratories devoted to food and fiber analysis.

An opportunity that we are now pursuing in the Department is computer control of vegetable oil refining and processing. Several advances

are needed to enable automatic control of the various steps involved in the

continuous refining and processing of vegetable oils to achieve most

economical production of consistently high quality products. The successive steps involved are alkali refining, bleaching, hydrogenation, and

deodorization. In the first of these operations, after oil has been treated with alkali solution, the resulting soapstock is separated from the oil by continuous

centrifugation. To minimize losses of refined oil, a small amount of soapstock is allowed to be carried over with the oil, which is subsequently

washed to remove this soapstock. When too much good oil is allowed to go with the soapstock, financial losses can exceed $100,000 per month in refineries which process 12 or more tank cars of oil per day. Needed is a
AGRICULTURAL SCIENCE 229

method for continuously monitoring the sodium content so that the oil loss in the soapstock and the sodium remaining in the oil are minimized.

Perhaps atomic absorption analysis could be adapted for this purpose.

The bleaching operation involves treatment with a bleaching clay at high temperature to remove objectionable color. With soybean oil, in particular, chlorophyll must be removed completely from the oil. Probably an

absorption spectrometric method could be adapted for continuous monitoring of color of the bleached oil. When oils are hydrogenated, the process is monitored relatively effec- tively by use of continuous refractometry, but this gives only the extent of

hydrogenation, not the amount of isomerization of cis to trans double

bonds. Too much isomerization raises the melting point of the fat and

causes excessive loss during subsequent winterization. A method for continuous monitoring of cisjtrans isomer composition is needed, based

possibly on infrared or Raman spectroscopy. This could be used to control hydrogenation conditions to minimize isomerization.

Deodorization is accomplished by steam stripping at high temperature

under vacuum. Currently there is no satisfactory way to determine when

this process is complete, so all oils are deodorized for a certain "safe"

period of time. Deodorizer throughput could easily be doubled if instru- mentation were developed to allow the oil to be carried through as soon as the process is complete.

XVI. Summary

Along with the far-ranging applications of analytical chemistry in agriculture come many unsolved analytical problems. In fact, few agricultural scientists will admit complete satisfaction with any of their current methodologies. For some of our problems, there appears to be no current

solution. These difficulties will challenge the ingenuity and creativity of

analytical chemists in the future.

XVII. Acknowledgment

The authors express their appreciation to the many scientists in the

Agricultural Research Service whose cooperation made preparation of

this paper possible.
230 ANALYTICAL PROBLEMS

XVIII. References

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428-999 O - LT - 72 - 16

PANEL DISCUSSION

Analytical Problems in Agricultural Science

CHAIRMAN

Philip D. LaFleur, Chief, Activation Analysis Section, Analytical Chemistry Division, National Bureau of Standards

PANEL

David Firestone, Head, Food Alterations Section, Biochemistry Branch,

U.S. Food and Drug Administration, Washington, D.C.

Herbert A. Laitinen, Professor of Chemistry, University of Illinois, Urbana,

Illinois Charles Merritt, Jr., Head, Analytical Chemistry Group, Pioneering

Research Laboratory, U.S. Army Natick Laboratories, Natick,

Massachusetts

Mario A. RoUier, Director, Center of Radiochemistry and Activation

Analysis, University of Pavia, Italy

George W. Irving, Jr., Administrator, Agricultural Research Service

(Speaker)

233
234 ANALYTICAL PROBLEMS

Firestone — It is most important to develop accurate and rapid methods

to analyze foods, plants and raw materials. It is particularly important that

methods be developed that can be used in regulatory laboratories at the

state and city level, that are not extremely sophisticated nor require highly

specialized equipment. The problem in these laboratories is not one of

total analysis but generally of analysis for a specific component, and

scientists developing procedures should keep that principle in mind.

Many of the methods that are available today are rooted in tradition and

therefore are slow to change.

In many areas of agricultural chemical analysis, extreme accuracy is not what is demanded. An example is the protein or fatty acid profile of

various foods. These profiles vary because we are dealing with natural

products. However, this should not imply lack of interest in, or neglect of,

accuracy. Much of the problem in the applicability of routine methods in the laboratory is really concerned with the availability of standards. The

instrumentation is and has been available, but it is only recently that

reference standards, supplied by commercial laboratories, have been

available for fatty acid profiles. These standards can be used either inter- nally or used without a sample to check on detector response. They are important for doing really accurate work in an absolute sense. An example of the problems in obtaining standards for these methods

is in the area of determining cyclopropenoids. These fatty acids, characteristic of cottonseed oils, are highly reactive and occur in fatty acid

profiles in trace amounts. The Halphen reaction, a highly sensitive and

specific colorimetric reaction, can be used to detect cyclopropenoids in biological tissue and can be used to determine that cottonseed oil is present in a mixture of other oils. These standards are difficult to prepare

and have to be handled so that they will not be oxidized. The individual

using this kind of methodology therefore has to prepare his own stan- dards, which is a fairly tedious procedure, or use secondary standardization methods.

Because of the demand of the medical profession to label foods as to

their fatty acid concentration, the analysis for fatty acid profile has

become much more important. This has prompted some foreign countries

{e.g. Canada) to develop an enzyme procedure for determining cis- polyunsaturates. This method has not yet been studied collaboratively but

is currently under study jointly by the United States and other countries.

The procedure has the advantage that polyunsaturates can be determined

rapidly. This is one example of the use of enzymes to provide sensitive

and specific methods.

It is also important that sensitive methods be available for the deter-
PANEL DISCUSSION 235

mination of mercury, lead, and cadmium and other toxic elements in food.

Problems still exist with respect to digestion and clean-up even though

sensitive methodology already exists for the determination of the cited

heavy metals, and more techniques are being developed. Complete

digestion and clean-up of fatty foods presents a special problem. It is, of

course, important when we are talking about mercury, lead and other

metals to take note of the form of the element: is it ionic or is it organometallic? For example, in the case of mercury the toxicities of the ions are much lower than those of some of its organo-metallic compounds, e.g. the

alkyl mercury halides.

The combination of gas chromatography and mass spectrometry (GLCMS) holds out promise for the analysis of many contaminants in foods. An

example is the chlorodibenzoparadioxins. These materials arise as con- taminants in chlorophenol and develop during the manufacture of

chlorophenols generally. One of the chlorodioxins, the symmetrical

2, 3, 7, 8-tetrachlorodioxin, is extremely toxic, thousands of times more

toxic than the usual chlorinated pesticides that we are familiar with. Thus

there is a great deal of interest in determining this compound, and its cousins, at very low levels. We find that chlorodioxins may occur in chlorophenols or in fats along with many other contaminants including

the polychlorinated biphenyls referred to by Dr. Irving.

There are many compounds that fit into this general class, the toxicities of which are not completely understood. At present there really isn't a

quantitative procedure, but one of the most useful analytical techniques

involved the use of a GLC-MS system where fairly low levels of

chlorodioxins can be determined; this system is particularly important

where several compounds may have the same GLC retention time. In many cases a peak from the GLC may represent four or five non-isomeric

compounds which, however, can be resolved by a mass spectrometry

system because of unique fragmentation patterns. However, there are problems in interfacing the gas chromatograph and the mass spectrometer. A new and better interface is required. The solution may be to have

the effluent from the GC flow directly into the ion source and to use a very large, efficient pump to eliminate carrier gas in the mass spectrometer.The work that has been done with insect attractants is an area which

should be fascinating to analytical chemists because in some instances

only a few micrograms of the material have been used to describe the

structure of some of these attractants. Perhaps they can be used to develop new approaches to controlling insect population and reducing the

widespread use of pesticides especially the chlorinated pesticides.
236 ANALYTICAL PROBLEMS

Laitinen — The approach taken in this symposium is intriguing. It is unusual and is particularly apt to be suggestive and productive in new

ideas because we are dealing not so much with analytical chemists doing

their "thing," as what we ought to be doing. One point about the agricul- ture system which makes it unique and particularly appropriate here is the

fact that we now have in universities, as well as in government and industry, two new concepts coming in terms of research. One is the systems ap- proach to research; that is, looking at a multivariant system (and now the one termed "ecosystem" is a very popular one). The other is "interdisciplinary"; that is, bringing to bear on a problem a large number of dif- ferent backgrounds.

These are extraordinarily important trends which are just in their in- fancy and will continue as long as we can foresee. If we look at agriculture

in its relationship to this approach, we see that it is unique in the sense

that it has components of all elements of the ecosystem, that is, the five

major components: the air, the water, the earth, the animal and the plant.

Agriculture is involved with all of these, and is of vital importance to society as well. Therefore, a real opportunity is presented to universities

and to government and industry, to use the systems approach and the in- terdisciplinary approach in agricultural research.

One definition of the systems approach would include the various levels of systems; i.e., the ecosystem first of all would consist of these five major

components and the transfer of materials and interconversions between

them, and the results of these transfers. We can speak of the systems ap- proach at lower organizational levels as well. Suppose we take one part of

the system, say the animal kingdom, and we consider one animal as a

system. He too has inputs and outputs and distributions within his in- dividual organs. We can then go down one more level on the organizational scale and consider one organ of this animal, say the liver, and consider its input, output, transfers, etc. We can then go on and on, down to the cell level, then to the membrane level, then to the liquid level. When

we get to the liquid level we are talking about the molecular system and

here is where chemistry really pertains.

The point is then, that at these highest organizational levels we are going to be performing research, the results of which will have the most

immediate impact upon society. At the lower levels we are going to per- form research that has the least immediate impact, but perhaps the

greatest fundamental and long range importance.

The systems approach to a problem as complex as agriculture, or ecology in general, ought to be attacked on at least five or six organizational

levels simultaneously, because you can't afford to wait to start on one
PANEL DISCUSSION 237

until you are ready for it. This really calls for concepts of interdisciplinary

research; it is no longer possible for an analytical chemist to have a complete background before having to solve these difficult and so- phisticated problems. It is necessary for him to learn to work with

others who bring him that knowledge; in turn he will bring to the solu- tion of the problem techniques that otherwise would be out of reach as

far as the practitioners of other disciplines are concerned.

One of the most important concepts that is necessary in analytical

chemistry is that of determining forms or species of the elements. Some of

you may have noticed the editorial in the June issue of Analytical Chemistry which was addressed to the importance of determining species; it is important to emphasize that point over and over again. Speciation is important not only in the more or less simple things, like the availability of

vitamins and proteins, but also in the complex interaction of ions,

molecules, oxidation states, etc., in nutrition and toxicology. The development of methods for species is an exceptionally difficult problem which

must be solved.

We will probably never be happy with the analytical methods available; not now, not 20 years from now, not 100 years from now — because there

is a type of evolution of an analytical method, or of science itself, which

makes it impossible for us to become satisfied. Consider a problem as dif- ficult as the aflatoxin problem that was mentioned by Dr. Irving. The first question is: Have we got any approach? Often the first approach utilizes the practical consequences of that particular material, but does not

directly identify it. If it is a poison, we can use a biological test. If it kills something, then there it is, and we can determine somewhat, the toxicity

level. This is unsatisfactory in the sense that we would like to have

methods that are simpler, more accurate, more rapid and more specific.

Add together all the desirable attributes of an analytical method: sensitivity, specificity, accuracy, speed, rehability, simpHcity, and economy.

Then, to top it all off, we would like a method that is nondestructive so

that we can test our little corn seed and still have it to plant. When are we

going to have an analytical method that does all of these? Then, if we start with a method that is so universally applicable and so sensitive and so

specific that you could put the sample in and immediately get an answer

out, you can be sure that the next complaint would be that it is too complicated: "I can't take it in my vest pocket and go out into the field and

make determinations." So you can't win this game, but that is what keeps

us busy and keeps us in business.

This brings us then to the point that where is this analytical work going

to come from? Certainly the work in agriculture is not going to come just
238 ANALYTICAL PROBLEMS

from agricultural scientists and it is the same in any other field you can name. We are going to have to call upon progress in all kinds of

disciplines, including physics, biology, etc. This is where communication comes in. We must have mediums of exchange, like this symposium and

like written publications, to transfer this information from the supplier to the user. What about the role of universities? In the past universities have suf- fered from one important drawback as far as application to problems of

society; e.g. they are disciplinarian in nature. This is, however, a very essential part of the university structure and this is the way that various

disciplines, not only scientific disciplines but social sciences, humanities,

etc., keep their validity. At the same time, there is need for something

beyond this, for something in addition to this; that is the interdisciplinary

approach.

Let us take a moment to define interdisciplinary in relation to another

term, multidisciplinary, which is similar and is sometimes used in- terchangeably. The Dean of the Graduate College at the University of Il- linois, Dan Alpert, has made a very useful distinction between these two

terms. The term multidisciplinary implies that a multiplicity of disciplines

such as physics, chemistry, biology, and so on, are brought to bear on a problem. The various disciplines, however, keep their separate identities and are brought to bear as needed.

On the other hand, the interdisciplinary approach is a problem-focused

approach in which various disciplines lose their identity to a certain extent. A biologist and a chemist and a physicist would team up together

daily to focus on a problem. That doesn't mean getting together once a week for a meeting, but actually working on a problem in detail together.

This is something very alien to the university campus as we have

discovered in the past years as we have been trying to set up such an or- ganization. The Agricultural Experiment Station doesn't even do it, and

the reason is that the agricultural colleges are essentially discipline

oriented. There are such things as departments of Soil Physics, Soil

Chemistry, and even Agricultural Economics. The reason for this is clear; people do not want to lose their identity within their own

disciplines.

It would be a terrible mistake for us to use the same pattern again in the

environmental field. We don't need to have, nor should we have in my

opinion, a Department of Environmental Chemistry, and environmental

this and that. Instead we should have this interdisciplinary mode of attack on the environmental system. This is something the universities will have

to do and yet not destroy the essential quality of their traditional disciplinary approach.
PANEL DISCUSSION 239

In concluding let me ask what would happen if 100 years from now, or, 1000 years from now, we were to have a summer symposium on Analytical Chemistry. What would it be like? What could it be like? One must

conclude that most, if not all, of the subjects that are in this symposium

would be appropriate 100 years from now. That is, we are definitely going

to be dealing with agriculture, with the environment, the atmosphere, the ocean, etc. Mankind is going to have to deal with these problems. What

would it be like as far as analytical chemistry is concerned? If you have a speaker 100 years from today talking about the human body, he might be

complaining about the fact that, although we have 1 100 different analyses

that can be run in 30 minutes as soon as the patient is admitted to the

clinic, look what is missing: we still don't know whether this particular en- zyme system is particularly active in this part of the liver or that part of

the liver and wouldn't it be nice to know—! And that we must have

methods which are more accurate, more sensitive, cheaper, and so on, and

so on, and so on.

Merritt — Because funds are usually limited, laboratories should be or- ganized to achieve a multi-objective mission with a minimum configuration. This means you must get the most work done with the fewest people.

This is not, of course, the ultimate solution. There is a need for more

trained analytical chemists and expanded educational programs, particularly those oriented toward problem solving and meeting specific needs in analytical chemistry. In the meantime it is necessary that analytical

chemists do an adequate job of selling their techniques and their relationship to relevant problems.

As an example, the U.S. Army Natick Laboratory has been involved in problems of coffee distribution to the Army in both field and garrison in- stallations. In the past, 95 percent of this coffee has been prepared from

ground beans. Because of low acceptability by the troops, instant coffee

of the spray or drum-dried types has not been used. However, freezedried coffee that meets the necessary acceptance standards is available.

This coffee can be prepared using analytical chemistry as an aid both in its development and in its quality control. When you present management

with the fact that annual savings of procurement costs of over $2,000,000

and, transportation cost savings in the world-wide distribution system of over $8,000,000 may be realized, it is obvious that it should not be dif- ficult to obtain $ 100,000 or so for sophisticated laboratory equipment.

Another major area that must be used more to advantage by analytical

chemists is that of serendipity, or spin-off. Serendipity in this context means that in developing methods of analysis, many other areas of science
240 ANALYTICAL PROBLEMS

may be studied other than analytical chemistry. In the area of analytical

chemistry the work of the Natick analytical chemistry laboratory is probably best known for some of the studies made in attempts to develop

methods of analysis for the trace amounts of volatile organic compounds

in complex mixtures. Twenty-five to thirty papers have been published in the last 10 years or so on gas chromatography, mass spectrometry, etc., and most of these papers have appeared in analytical journals. However,

what many analytical chemists may not know is that an equal number of

papers on flavor and odor studies, on mechanisms of irradiation damage

in liquids and proteins, on pyrolysis in proteins, etc., also have been

published. These papers have appeared in journals such as the Journal of

Agricultural and Food Chemistry, the Journal of Dairy Sciences, etc. In general the work at Natick is a two-fold activity: development of

methods on the one hand and the applications of those methods to specific

problems that require relevant solutions on the other. This may be a prag- matic approach but it is based on the real-life fact that very few agencies

will provide funds for laboratories to work only on the development of

analytical methods without a specific application in mind.

Another point of serendipity is that methods developed for one type of

analysis, for example food flavors, are often also applicable to a wide

variety of other studies with hardly any change except for the material in the sample flask. An example of a problem under study at Natick which

illustrates a large degree of serendipity is one that concerns one of the

larger Air Force Bases overseas that is using powdered milk in the mess

halls and the PX. The local water used to rehydrate the milk is polluted.

The problem was discovered because the milk tasted bad. The pollutants

from the water were isolated and identified. In this case they proved to be

from a chemical plant up stream from the air base. Developing the means

of isolating compounds from the dilute aqueous solution is primarily an

analytical problem, but now a method has been proposed for a large scale

purification of the water for use in large scale rehydration. This kind of

serendipity can produce a lot of support for analytical methods development.

Another example is the study of the changes that occur in foods upon

heating, e.g., the development of flavor in coffee beans brought on by

roasting. For this a combined pyrolysis-gas chromatographic-mass spec- trometry analysis system has been developed to study the thermolytic

decomposition of proteins using a combustion furnace. The same system

with a Curie point pyrolyzer becomes an analysis system for the identifi- cation of degradation-products for synthetic polymers. If the pyrolyzer is replaced with a high energy thermal source such as a carbon arc or solar
PANEL DISCUSSION 241

furnace, the degradation of clothing fabrics by a simulated nuclear thermal

pulse can be studied. The problems associated with the disposal of

packaging materials by incineration are being studied with a laser-gas

chromatograph-mass spectrometry system.

Although practical considerations may dictate that efforts be directed

to specific problem area needs, it must be recognized that all investigations of material composition move forward only as analytical methods

provide the means. In this context, funding agencies must take more cog- nizance of this fact and support the development of analytical methods

per se.

It requires very little imagination to realize the scope of applications

that a single analytical procedure might find. One of the most important

problems that must be overcome is that of communication between

analytical chemists and those whom they are to serve. Often other

scientists are not even aware of the fact that analytical chemists can contribute to the solution of their problems. Once this communication exists

analytical chemists can often solve the problems.

Rollier — Interdisciplinary research is becoming more and more important. The remarks of Professor Laitinen are certainly true. The solution to our problem is not to set up Departments of Ecological Chemistry or Ecological Physics or Ecological Biology, etc., but to have specialists in existing departments cooperate in interdiscipUnary ventures. This is certainly where the future of universities lies. One area that Dr. Irving did not have time to go into deeply is the

problem of the role of trace elements. This problem is an extremely important one for nutrition studies and for agriculture in general. The

University of Pavia had a contract with the International Atomic Energy

Agency to study the determination of the trace elements chromium and

selenium. Both of these elements are extremely important and their deter- mination in trace amounts is important not only in foodstuffs but also in human and animal tissues. Since that time a program has been undertaken

with the U.S. Navy Medical Unit stationed in Cairo, UAR, studying some diseases due to malnutrition, in which trace amounts of selenium

and chromium are being determined by activation analysis.

The problem of selenium is important because it appears possible that trace amounts of selenium, a fraction of a part per million in foods and in tissues, are persistently lower in the Mediterranean areas than they are in North America. Biologists know that the effect of selenium on growth is peculiar, especially at the 0.2-0.4 ppm level. It may be that a certain

amount of selenium is essential in the diet. Selenium deficiency may be
242 ANALYTICAL PROBLEMS

responsible for many different things, for instance for the fact that the

average height of the people in the Mediterranean area is less than that of

the people on the North American continent. This is, of course, at present

conjecture, but it is important to point out how essential accurate and

precise determination of trace elements in human tissues, in plants and

animals, and especially in foodstuffs may become in the near future. For

this reason chromium and selenium should be added to Dr. Irving's list of

minor elements such as iron, manganese, boron, copper, zinc, and

molybdenum. Neutron activation analysis is an excellent method for

determining many of these trace elements.

Determinations of trace elements in foodstuffs should be made routinely if we are to understand their role. The problem of detecting insecticide,

fungicide, herbicide, pesticide, etc., residues in foods and crops, is of the

greatest importance. Most of these chemicals contain phosphorus and/or

sulfur. Synthetic sexual lures, combined with chemical sterilizers usually

contain fluorine, phosphorus, sulfur, and bromine atoms in their

molecules, fluorine and phosphorus being predominant. Analysis by

neutron activation makes it possible to determine phosphorus and

bromine in tiny amounts of agro-chemical residues which may be in food

products. There are many nuclear reactors in nearly every country of the

world capable of doing these analyses.

It may be possible to tag agricultural chemicals with rarely occurring

but high nuclear cross-section materials so that pesticide residues could

be determined easily by activation analysis. It would, of course, be necessary to determine that correlations between pesticides and the added ele- ment were constant as a function of time. For this reason it would

probably be necessary to have the tagging material an integral part of the

molecule. This technique was suggested by scientists working in forensic

analysis some time ago, and has been applied in many cases. The amount

of tagging material being added would be very low and therefore probably

non-toxic although this would have to be proven.
A. P. Altshuller, is the Director, Division of Chemistry and Physics, Air

Pollution Control Office, Environmental Protection Agency. He received

his B.S. at the University of Chicago in 1948 and his M.S. and Ph.D.

degrees in 1950 and 1951 from the University of Cincinnati. From 1951

to 1955, he was an aeronautical research scientist engaged in fuels research with what is now the Lewis Research Center of NASA. Since

1955 he has held various research assignments in the air pollution pro- gram of the Public Health Service in Cincinnati. He assumed his present

position in January 1969. He has pubHshed about 100 papers related to spectrophotometric analysis, gas chromatography, coulometric analysis,

infrared spectrophotometry, photochemistry and kinetics, solution and

statistical thermodynamics, thermochemistry, and various aspects of at- mospheric chemistry. He was Chairman of the ACS Committee on Air

Pollution and of the ACS Division of Water, Air, and Waste Chemistry.

Dr. Altshuller received the Eighteenth Annual (1967) Cincinnati Chemist

Award of the Cincinnati Section, ACS, and the Frank A. Chambers

Award for outstanding achievement from the Air Pollution Control Association (1970).

243

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 351,

Analytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

Held June 16-18, 1971 , Gaithersburg, Md. (Issued August 1972). CHAPTER 5

ANALYTICAL PROBLEMS IN AIR POLLUTION

CONTROL

A. P. Altshuller

Director, Division of Chemistry and Physics

Research and-Monitoring Office

Environmental Protection Agency

Research Triangle Park, North Carolina 27711

The role of measuring techniques in activities ranging from research to regulatory activities of the Environmental Protection Agency will be discussed. Field and laboratory instruments and manual techniques wiir receive consideration. Both point-sampling and remote types of instrumentation develop- ments will be reviev/ed. Chemical and physical transformations contribute to the formation of new or modified pollutant species. Air quality measurements

must be capable of properly following such transformations. Especial emphasis needs to be given to the conversion of gaseous into various submicron particulate species. The mass, particle-size distribution and details of chemical composition of these particles is required. Examples will be given of new and improved types of air quality measurements, stationary source and mobile

source emission measurements which may be needed to meet air quality and

emission standards.

Keywords: Air pollution; air quality instrumentation; air quality measurements; anion analysis; Clean Air Act; elemental analysis; measurement of atmospheric gases and vapors; measurement of at- mospheric particles.

I. Introduction

A small program concerned with development of new or improved

techniques for measuring air pollution has existed for quite some time, but

particularly from the inception of Federal activities in 1955. Until 1967,

this program was primarily concerned with support of research and monitoring needs.

245
246 ANALYTICAL PROBLEMS

Between 1955 and 1967, a large number of laboratory analytical

techniques were developed for air pollutants. Considerable research was

done on development of colorimetric and chromatographic techniques.

These techniques were applied to air quality measurement and to motor

vehicle emissions research. During this period a small number of air quality instruments, particularly those for oxidants and hydrocarbons, were

evaluated and improved. Most of the colorimetric procedures used in air quality instruments were developed or improved during this period.

By the mid 1960's, it was apparent that new techniques and sensors were becoming available. Gas chromatographic techniques were well

enough developed to justify their use in monitoring instrumentation, but

the necessary resources were not available. The costs involved in incorporating a laboratory technique into an effective monitoring instrument

for routine general use are considerable.

The Clean Air Act, as amended in 1967, referred to instrumentation

requirements in two sections. Section 104 states: "The Administrator

may conduct and accelerate R/D of low cost instrumentation techniques

to facilitate determination of quantity and quality of air pollution emissions, including, but not limited to, automotive emissions." In Section

133, annual reports to Congress are required, including a report of

progress on "The development of quantitative and qualitative instrumentation to monitor emissions and air quality." These passages in the Act

stimulated for the first time the allocation of resources for instrumentation

R/D. The pace of these activities has accelerated slowly but significantly

since the passage of these 1967 Amendments.

It was clear by 1967 that most of the instruments available for measure- ment of air quality were deficient in sensitivity, specificity, or ease of

operation and maintenance. Emphasis was placed on the development of a new generation of instruments using physical principles rather than wet

chemistry. Many of these instruments can be used, with some modification, for monitoring emissions from motor vehicles and from stationary sources. The 1970 Clean Air Act also involves additional measurement capability for mobile emission sources. The development of new power sources

or propulsion systems requires that we have analytical procedures capable of qualitatively and quantitatively measuring pollutants "which cause

or contribute, or are likely to cause or contribute, to air pollution which

endangers the public health or welfare but for which standards have not been prescribed" in accordance with Section 202 of the 1970 Clean Air

Act. In general. Section 202 requires that measurement techniques be
AIR POLLUTION CONTROL 247

prescribed for any air pollutant, for which standards are promulgated, that

is emitted from motor vehicles.

Evaluation of instrumentation for measuring emissions from stationary sources has recently been initiated in accordance with provisions of the

1970 Clean Air Act. Section 110 (2F), on implementation plans, states that approval of such plans shall include, among other aspects, "requirements for installation of equipment by owner or operators of stationary sources to monitor emission." Section 114, on inspection, monitoring, and

entry, states: "The Administrator may require the owner or operator in carrying out provisions of Section 1 10, 1 1 1

, and 303 to (C) install, use and

maintain monitoring equipment or methods; (D) sample such emissions

(in accordance with such methods, of such locations, at such intervals,

and in such manner as the Administrator shall prescribe)." These provisions certainly will require the acceleration of both R/D and the evaluation and standardization of instrumentation and of sampling and analysis

procedures for emissions from stationary sources. A list of important areas of need for air pollution measurement capability are listed in Table 1. The listing is divided among air quality, motor

vehicle emission, and stationary source requirements. The order within

each area proceeds from research on pollutant composition and concentration, as related to atmospheric characteristics and biological effects, to regulatory needs. Most of the techniques presently available resulted

Table 1. Air pollution program elements requiring instrumentation and

measurements development, evaluation, and standardization.

• Air quality surveillance networks

• Atmospheric and plume research investigations

• Biological effects field investigations

• Air quality standards

• Motor vehicle emission composition investigations

• Motor vehicle certification and production line testing

• Surveillance of production vehicles with mileage accumulation

• Investigation of stationary source emissions composition

• Stationary source emission standards

• Determination of compliance with emission standards

428-999 O - LT - 72 - 17
248 ANALYTICAL PROBLEMS

from earlier R/D programs of air quality surveillance activities. However,

the Environmental Protection Agency will soon outrun this reservoir of

measurement capability when it begins impending regulatory programs.

It is essential that measuring requirements for R/D projects be supported

now, or the measuring techniques will not be available for regulatory

requirements later. The complexity of the process and the timing involved

will be considered in greater detail later in this discussion.

Important criteria for air quality instrumentation are listed in Table 2. Of course, these same criteria apply to many other instruments. Sensitivity requirements can vary greatly. For monitoring air pollution, the ability to measure 10 to 1000 /jLglm^ is required. It appears that at stationary source appHcations the concentration range will be 100 to 1000 times

higher. It follows that an instrument may have more than satisfactory sensitivity for monitoring stationary source emissions, but totally inadequate

sensitivity for measuring ambient air quality. Response times of 1 second

or less are needed for some motor vehicle applications, whereas periodic

analyses once every 5 or 10 minutes, or even less frequently, are required

for other air pollution measurements. Fortunately, it is possible to develop instruments with appropriate characteristics for the entire range

of applications.

Table 2. Criteria for air monitoring instrumentation.

• Compatibility with sampling system

• Sensitivity sufficient to meet monitoring application

• Specificity of response to pollutant of interest

• Adequacy of response time for application

• Simplicity of construction and operation

• Reliability and reproducibility of operation

• Compatibility with data handling equipment

A reasonable position is to develop the minimum set of different instru- ments. Such an approach will tend to minimize instrument development

costs, reduce problems of reliable commercialization of instrumentation,

and speed standardization and collaborative testing programs. This ap- proach must also produce instruments adequate for each of the applications listed in Table 1 at a reasonable cost per instrument.

A sequence of activities required to develop and commercialize an in- strument is given in Figure 1 . In many past and some present instrument
AIR POLLUTION CONTROL 249

Time Scale

12T- 18 months

28-42

months

6-8

months

6-8

months

4-8

months

1

Sensor research

investigations

i

Laboratory evaluation of

breadboard prototypes

r Development of prototype

instrumentation for field use ^

Field evaluation

f Component and circuit design simplification

f Preproduction prototype

c

i

Field evaluations

CO

r

>

Final design activities

o

f

Col

lab. Commercial production of

instrument

Applications

Second

generation

prototype

Figure 1 . Sequence of activities in developing, evaluating and standardizing air pollution

monitoring instrumentation.
250 ANALYTICAL PROBLEMS

projects, a pronounced tendency to bypass a number of steps in the given

process has prevailed. The net result has been either unusable instru- ments or expenditure of much work in rebuilding instruments. Aside from

the cost and inefficiency involved in them, such abbreviated approaches

actually prove more time consuming than an approach that systematically

follows through on the necessary steps as listed. As indicated in the time

scale, it is most unusual for this sequence to take less than 2 to 2-1/4

years; the sequence can require more than 3 years if research problems

occur early in the process or if prototypes must be redesigned.

The substantial period of time indicated here as essential for development of a satisfactory instrument reemphasizes the need to anticipate in- strument requirements very much in advance of regulatory deadlines. Unless they are developed for R/D projects, prototype instruments will not be available when required for regulatory standards. Cumbersome and

often inadequate manual analytical methods must be substituted, to the

discomfort of all, while the overdue instrumentation is being developed.

II. Air Quality Measurements

A. Instrumentation for Measuring Atmospheric Gases and

Vapors

This discussion will be concerned primarily with the present status of

and anticipated needs for instruments that can monitor ambient air. Point

sampling instrumentation, along with remote and long-path-type instrumentation, will be considered for the monitoring of gases. Ambient air monitoring needs will be considered to include not only determination of

urban pollution, but measurement of pollution on a regional and global

scale. Because a number of atmospheric substances of concern do not

result from primary emissions, but from chemical reactions, atmospheric

transformations must be considered.

Methods of analysis and instrumentation available up to about 1966

have received detailed consideration in a number of reviews [1-7]. Therefore, this presentation will emphasize advances in the last several years. Our first concern is with the justification for the development of new or improved instrumentation. It will be necessary to consider the deficiencies of instruments to analyze ambient air that have been available until recently.
AIR POLLUTION CONTROL 251

Almost all of the air-monitoring instruments installed up to 1970 were

based on analytical approaches developed 10 to 15 years earlier. Many of

the individual types of instruments are cumbersome, of low specificity, of

limited sensitivity, and difficult to maintain because of complexities of

design. Some of the instruments suffer from only one or two deficiencies,

while others fail in almost all aspects. As a result, the amount of valid

measurement data obtained has frequently been limited. Attempts were made to improve some of these instruments with respect

to specificity of response for ozone [8] and hydrocarbons [9,10]. Such

activities were viewed as temporary expedients necessary because of the

lack of funds to develop new instruments. The approach usually taken was to utilize a substrate in the inlet system of the instrument which

would remove or hold back interfering substances. Experience has shown

that such systems do not work very effectively in routine monitoring

operations, although they can be handled successfully by R/D personnel

in field studies. No R/D activity can anticipate all of the problems as- sociated with operations under a variety of routine conditions in the field. Variability in time to partial or complete breakthrough of the inter- ferences through the substrate under varying atmospheric conditions is one of the problems often experienced. In general, instruments that

require use of auxiliary clean-up systems to achieve specificity are more

prone to give incorrect results than instruments whose basic sensing principles confer the required specificity.

A number of instruments have been in common use in monitoring net- works during the past 10 to 15 years. When these instruments are considered in terms of sensitivity, specificity of response, simplicity of construction and operation, or reliability and reproducibility of operation,

they have proved inadequate, usually with respect to several of these

criteria. As a result, few concerned persons have expressed satisfaction

with instruments available during the last 10 or 15 years in monitoring

networks. Unfortunately, little incentive and fewer resources were availa- ble to remedy the situation until recently.

Although the resources available in the last several years have been

modest, considerable progress has been made, particularly with respect to new or improved instruments for measuring inorganic gases. Instruments

have been developed which are now receiving or have received field evaluation for sulfur oxides, ozone, carbon monoxide, nitric oxide, and

nitrogen dioxide. In addition, a better technique for determination of non- methane hydrocarbon has been developed. Most of these instrument

developments have utilized sensors or laboratory equipment produced by

research in recent years. However, a considerable range of activities has
252 ANALYTICAL PROBLEMS

been required to convert these into usable air pollution monitoring instru- ments. Sensors had to be evaluated with respect to sensitivity and

specificity for urban air pollution requirements. Long-term stability and

reproducibility or operation had to be evaluated. Breadboard laboratory

equipment had to be packaged as field instruments. Field studies have

been required to permit comparisons of instruments used for each of the

several pollutants under representative ambient air conditions. Such in- vestigations, from laboratory evaluation of the potential for applicability

of a sensor to air pollution needs through field evaluation, require several

years of continuing efforts. Field evaluations indicate that all of these in- struments have the sensitivity and specificity needed for urban air pollution applications. The instruments also should be capable for measuring

so-called near-urban pollution.

The gas chromatographic technique for carbon monoxide and methane

utilized in former years in laboratory photochemical studies [11-13] was

developed into a convenient monitoring instrument [14,15]. In combination with a capability for measuring total hydrocarbons, such a gas chromatographic analyzer provides a highly specific and sensitive means of

analyzing carbon monoxide and nonmethane hydrocarbons over a wide

range of atmospheric concentrations. As a carbon monoxide analyzer, the

gas chromatograph is much more sensitive and specific than current nondispersive carbon monoxide analyzers. This approach can also be ex- tended to include monitoring for other gaseous hydrocarbons. The direct

analysis for methane is much more desirable than the earlier attempt to use substrates to remove hydrocarbons other than methane [9,10]. This

type of technique has given erratic results in routine monitoring activities because of the care necessary to maintain the characteristics of the substrate utilized to provide the specificity required. Another technique

developed for methane and other hydrocarbons involves selective combustion, with subsequent detection by a water sorption sensor [16].

The present oxidant analyzers are unsatisfactory monitoring tools. The

"oxidant" has no exact meaning since the response obtained depends on

the presence of various interfering substances. The substrate used to eliminate sulfur dioxide interference oxidizes nitric oxide to nitrogen

dioxide and thus increases the signal caused by nitrogen dioxide [8].

Recently, it has been shown that hydrogen peroxide is an atmospheric oxidant, but the response of commercial analyzers to this oxidant is poor

[17]. It should be evident that each of the major oxidants should be measured separately. Several ozone analyzers have been developed and eval- uated under field conditions. All of these instruments are based on utiliza- tion of chemiluminescent reactions [18]. The three types of reactions in-
AIR POLLUTION CONTROL 253

volved are as follows: (1) reaction of ozone with Rhodamine B adsorbed on silica-gel disc (Regener Method), with emission at 0.59 micrometer; (2) mixing of ozone with excess nitric oxide, resulting in chemiluminescence

from excited nitrogen dioxide that extends from 0.6 to 3.0 micrometers

and (3) mixing of ozone with excess ethylene, resulting in a chemiluminescence peak near 0.43 micrometer (Nederbragt's Method). All of

these systems provide a specific and sensitive means of analyzing ozone

rapidly over an adequate linear range. However, both the Regener [ 1 9- 21] and the ozone-nitric oxide techniques [18,22] have several disadvantages, compared to the Nederbragt technique, for use in a routine air monitoring instrument [18,23,24].

The Regener approach requires preparation and calibration of the disc. Because of the activation and decay characteristics of the chemiluminescent surface of the disc, a 4-minute mode of operation is preferable;

the mode involves a calibration cycle, purge cycle, sample cycle, and

purge cycle [20,21]. This type of operation also complicates methods for

obtaining signal readout. The ozone-nitric oxide reaction requires use of

reduced pressure to avoid quenching the chemiluminescent reaction

[18,22]. Detection of emissions in the spectral region involved dictates

the use of photomultipliers near the edge of the chemiluminescent

response above 0.6 micrometers; thus cooling to —20 °C is required to obtain a sensitivity down to 0.01 ppm. The Nederbragt-type detector can

operate at atmospheric pressure without quenching and with a sensitivity

down to 0.003 ppm at 5 °C with a less expensive photomultiplier

[18,23,24]. The ozone-ethylene reaction can be used with a detector of

less complicated design, excellent operation, and good cost and size

characteristics.

The flame photometric detector has received considerable attention in our program for measurement of sulfur dioxide, hydrogen sulfide, and

other sulfur compounds [14,25,26]. This technique involves a response

to both inorganic and organic sulfur compounds because these substances

form S2 species in the flame zone which are responsible for the emission

observed. Several field evaluation studies already completed have demonstrated the effectiveness of this sensor, both as a sulfur analyzer and as a detector in a monitoring gas chromatograph [26]. This approach is more

attractive than most of the other techniques available for measuring sulfur compounds. The flame photometric sensor has the following characteristics: (1) high specificity for sulfur compounds if emission is measured

between 0.39 and 0.40 micrometers; (2) high sensitivity (0.005 ppm); (3) linear response in ambient air concentration range; (4) fast response

characteristics; (5) and a gas flow system (no liquid reagents). The flame
254 ANALYTICAL PROBLEMS

photometric sulfur detector is also the only detector sensitive to sulfur compounds that can be used practicably in a gas chromatographic

analyzer. When sulfur dioxide is the predominant sulfur species present,

the flame photometric analyzer has been used as a specific and sensitive

measuring instrument for sulfur dioxide, hydrogen sulfide, and methyl

mercaptan [26]. The gas chromatographic system has been readily

modified to measure a wider variety of organic sulfur compounds where

appropriate, such as in the vicinity of Kraft Paper Mills [27].

Several varieties of electrochemical or electroanalytical techniques

have been utilized in sulfur oxide monitors [28]. Research and development continues on these approaches. Electroanalytical techniques are

rarely specific. Liquid or solid substrates are often used to confer

specificity. One type of electrochemical transducer utilizes a selective

membrane along with a galvanic cell having an electrode potential

selected to reduce interferences. Work has been done on selection of elec- trode and electrolyte materials and membranes for development of sulfur dioxide and nitrogen oxide analyzers [29]. This sulfur dioxide analyzer is a compact, relatively low-cost instrument. Its sensitivity at present is adequate for short-term emission measurements but is marginal for continuous measurement of sulfur dioxide. The sensor utilizes l.OA^ H2SO4

electrolyte, a gold sensing electrode, and a lead dioxide counter-electrode

with a 1-mil polyethylene membrane. With potentiometric control, inter- ference from nitric oxide is low, but hydrogen sulfide constitutes a signifi- cant interference. Response time is less than a minute. This analyzer

requires improvement for use in ambient air analysis. Because of its mode

of operation, simpHcity of construction, compactness, and low cost, it of- fers possibilities for use in large monitoring networks for measurements

averaged over extended time intervals, such as 24-hour periods.

Measurement of nitric oxide in the ambient atmosphere has involved use of analyzers utilizing the colorimetric Griess-Ilosvay reaction for

nitrogen dioxide [29] , in which the nitric oxide is oxidized to nitrogen dioxide before analysis. A number of efforts have been made to overcome dif- ficulties in developing an oxidizing substrate capable of providing high

conversion efficiencies over a range of atmospheric conditions. The

stoichiometry of the colorimetric nitrogen dioxide reaction used has been

in dispute [30]. In addition, the nitric oxide and nitrogen dioxide

colorimetric analyzers have been difficult to keep in proper operation. No

electroanalytical or ultraviolet analyzer has yet been provided that is adequate for ambient air monitoring of nitrogen oxides. The need for improved instrumentation is particularly urgent for nitrogen dioxide.
AIR POLLUTION CONTROL 255

At present the most promising approach involves the use of chemiluminescent emissions from the electronic transition, NO* NO2-I- /zi^, produced in the reaction of nitric oxide with ozone [22]. The emission

spectrum extends from 0.6 to 3 micrometers, with maximum intensity near 1.2 micrometers. This method for nitric oxide determination is linear from 0.004 ppm up to 10,000 ppm. Photomultipliers responding

from the outset of emission at 0.6 micrometer to their cutoffs at 0.8 to 0.9

micrometer have been evaluated. To obtain a sensitivity down to 0.004

ppm, choice of photomultipliers must be optimized and the operating

temperature of the photomultiplier reduced to — 20 °C. Since such sensitivity is not needed in measurem.ent of source emissions, less sensitive

photomultipliers operating at room temperature can be used. For a wide

range of gases present in polluted air, quenching of the chemiluminescent

reaction has not been observed at reduced pressures. No interfering

species have been identified. This same reaction has been mentioned

eariier as a method for ozone measurement.

The reaction of nitric oxide with atomic oxygen produces excited states that emit weakly, with some line structure occurring between 0.35 and

0.45 micrometer, followed by a more intense unstructured band with a maximum near 0.65 micrometer [32]. Since atomic oxygen reacts with

nitrogen dioxide to form nitric oxide on a one-to-one basis, this reaction can be used to measure NO + NO2. This reaction can be used as the basis

of a rapid-response instrument for measuring nitrogen oxides. Use of the

emission in the 0.65 micrometer region only provides discrimination

against the chemiluminescent emissions produced by the corresponding

reactions of atomic oxygen with carbon monoxide and sulfur dioxide

[32].

Instruments have been fabricated for monitoring nitric oxide in ambient

air by reaction with O3, and NOx by reaction with atomic oxygen [33,34].

Field evaluation of these instruments will follow.

Optical techniques based on absorption of energy by pollutant

molecules in the ultraviolet, visible, infrared, or microwave regions have

found little application in ambient air monitoring. An exception is the nondispersive infrared carbon monoxide analyzer. An infrared fluorescent

analyzer has been developed for CO that has high sensitivity and

specificity [35]. Other pollutants existing at lower concentrations do not absorb sufficient energy to permit utilization of other present commercial

types of NDIR atmospheric analyzers. Improvements can be achieved by

multiplexing (the simultaneous observation of multiple spectral resolution

elements), which can be used to obtain higher signal-to-noise ratios [36].

Multiplexing can be obtained in a variety of optical devices, including
256 ANALYTICAL PROBLEMS

multiple-channel radiometers, interferometers, dispersive matched-filter

spectrometers, grating spectrometers, and cross-correlation nondispersive analyzers [36]. More use of this technique has been made in remotetuned closed-path instrumentation, but multiplexing can be used to advantage in both types of applications. In recent years, some of these optical techniques have been developed

with specific application for ambient air analysis. These approaches in- volve either differential spectrophotometry or correlation spectroscopy.

The technique of correlation spectrometry involves molecular absorption,

in which a portion of the ultraviolet or visible spectrum containing vibrational-rotational band spectra is compared with a replica of this spectral

region stored within the spectrometer. Point-sampling type correlation

spectrometers for SO2 and NO2 with an optical path length of 2.5 meters have been fabricated and evaluated in Toronto and Los Angeles

[37,38].

The second-derivative spectrometer electromechanically processes the

transmission versus wavelength function to produce a signal that is proportional to the second derivative of this function. This technique results

in enhancement of signal-to-noise ratio and improved specificity. A spec- trometer of this type has been fabricated to analyze nitric oxide, ozone,

sulfur dioxide, and nitrogen dioxide in the 190 to 400 nanometer region

[39]. Sensitivities ranging from 0.003 ppm for NO and SO2 to 0.02 ppm

for NO2 are reported for a 1 -meter white cell with 20 passes. Both

techniques make possible greater specificity by taking advantage of the

details of band structure. They also provide somewhat improved sensitivity. However, the electronic and optical systems utilized can be involved

and expensive. It is not clear whether these types of optical systems will be competitive in response characteristics, field performance, or cost-size

characteristics with the instruments discussed previously for ambient air monitoring.

Another approach is the use of a folded laser beam with white-cell optics. With a laser, a long narrow glass pipe could be used rather than the

usual large heavy steel tank [36,40]. To obtain the sensidvities needed

for ambient levels of pollutants, at least a 1 -kilometer path is needed,

requiring a 6-meter base length. Since both an analytical and a reference

wavelength are needed, tunable lasers are highly desirable. The tunable

semiconductor diode lasers, such as the lead-tin-telluride laser now under

evaluation, is attractive because of its small size and extremely high

resolution that results from continuous tuning over several tenths of a wavenumber around 10 micrometers [36,41]. Because of its small size, this type of laser plus a detector with excellent sensitivity, such as the
AIR POLLUTION CONTROL 257

mercury-cadmium-telluride detector in a common liquid nitrogen cooler can be used as the key elements in a closed-path optical instrument [36]

.

A spin-flip Raman laser providing tunable infrared radiation has been

used to measure nitric oxide in the ppm range [42]

.

All of the instruments or concepts discussed have involved point

sampling; that is, the atmospheric sample is pulled into the instrument

through a probe or inlet line of some type. Such types of instruments may

or may not integrate over time, depending on response characteristics or

deliberate provision for integration of the sample or the signal over time.

Integration over space is not involved, except as meteorological conditions influence the flow of pollutants past the sampling site. The measure- ments obtained are not necessarily representative of pollutant levels over extended areas of the city or over entire air quality regions.

A completely different approach to ambient air analysis involves the use of long-path or remote optical measurements. Such optical instruments would provide the ability to integrate over space in two or in three

dimensions.

The Research and Monitoring Office of the Environmental Protection

Agency has not only a program for development of point-sampling instrumentation, but also a program for development and evaluation of longpath and remote instrumentation. Correlation spectrometers have been

evaluated in several cities for long-path measurement of sulfur dioxide

and nitrogen dioxide, using near-ground-level paths with two-ended

systems utilizing active sources of energy [38]. Path lengths from 100

meters up to 1000 meters were used in these measurements. In the work

done in Los Angeles, two path lengths were compared, one a cross- freeway path and the other an off-freeway path. Long-path results were compared to point-sampling results, but the experiment also showed that point sampling could be unrepresentative of the average pollutant concentrations even in a 1000-meter path. These measurements can be limited

by aerosol scattering effects or excessive sunlight [38]. An open-path cir- cular-filter-type infrared analyzer, programmed for ozone measurement

but capable of measuring other pollutants in the 7 to 14 micrometer range, has been developed [43]. This instrument is designed to measure ozone between 0.01 and 1 ppm at path lengths from 400 to 1600 meters. Another investigation being supported is concerned with the potential of a CO2 laser system for long-path measurement of ambient air pollutant

concentrations [44]

.

The optical instruments discussed thus far involve either two-ended

systems, or use of reflectors over fixed path lengths. Ideally, the openpath system of choice might involve a pulsed signal with ranging capabili-
258 ANALYTICAL PROBLEMS

ty. If such a system could be developed with sufficiently extended range,

it could cover much of an urban area in three dimensions. Such a system

would probably require a highly developed pulsed laser system with complex data-handling capacity. A LIDAR-type system of this sort has

received some evaluation by several groups for estimating total particulate loadings in the atmosphere [45,47]. Similar systems do not presently

exist for measurement of gaseous pollutant molecules. The use of laser Raman spectroscopy offers possibilities, but sensitivities appear marginal

at present [48]. Considerable augmentation of the scale of the present ef- forts will be necessary to develop practical systems for routine monitoring

activities.

Still another area of interest involves the use of remote optical instruments mounted in aircraft. The instrumentation can be pointed at the

earth's surface to measure the albedo of the multiple path of sunlight

passing through the atmosphere to the surface and reflected upwards to the aircraft-mounted instrument. Correlation spectrometers for sulfur dioxide and nitrogen dioxide have been evaluated in flights over several

cities [38]. This technique also has applicability to monitoring of stack

emissions and plumes [38]. However, several problems exist in applying

the technique. In a clear atmosphere the equipment tends to operate

reasonably well, but scattering by aerosols limits use of the equipment and

also renders uncertain the appropriate path length to include in the calculation. In addition, such aircraft measurements are limited by any at- mospheric conditions that interfere with flight operations.

If aircraft operation of remote instrumentation is possible, various

earth-oriented satellite applications also are possible. Such satellites would not offer the flexibility, accuracy, and general utility of aircraft in taking measurements over urban areas of air quality regions. However,

satellites may have eventual utility for measuring long-lived pollutants

such as carbon monoxide and submicrometer particulates on a global

scale, although adequate measuring techniques are still lacking at the

present. NASA is supporting effort particularly for methods that analyze

carbon monoxide by utilizing both infrared correlation spectroscopy and

nondispersive infrared techniques. The latter approach was selected as applicable after a survey on numerous trace gas detection techniques

[49]. Applications of optical techniques in the upper atmosphere of the

earth or to other planetary atmospheres will not be considered in the

discussion.

The chemiluminescence ozone, and the gas chromatographic carbon

monoxide and methane instruments have already been used in low-concentration-level geophysical measurements [50,5 1 ] . The flame photomet-
AIR POLLUTION CONTROL 259

ric sulfur oxide and the chemiluminescence nitrogen oxide monitors may

be usable for nonurban or rural measurements, but do not presently have

the sensitivity for geophysical measurements. The initial emphasis has

been on development of equipment capable of continuously or periodically measuring pollutant concentrations in samples of ambient air col- lected in a very short time interval. However, for urban measurements as well as nonurban and geophysical studies, integration of concentrations over time periods of 1 to 24 hours is an alternative to the conversion of

continuous measurements to averaged values for these periods by data

reduction techniques. Work is just being initiated on the possibility of

utilizing several of the available types of sensors for integrated measurements. Monitoring capability is especially important for carbon monoxide, sul- fur dioxide, hydrocarbons, ozone, and nitrogen dioxide because air quality standards for these widespread pollutants will be established

throughout the United States [52]. In addition, a number of other gases

or vapors are of concern. These substances include hydrogen sulfide, or- ganic sulfur compounds, hydrogen fluoride, hydrochloric acid, chlorine,

and nitric acid. With the possible exception of hydrogen fluoride (and

water-soluble fluorides) the lack of data makes it difficult to estimate the

levels or wide-spread prevalence of these substances.

As already discussed, a sensitive and specific gas chromatographic

technique is now available for hydrogen sulfide and organic sulfur compounds [26,27]. Hydrogen fluoride is generally measured as fluoride

along with other water-soluble fluorides. However, a fully satisfactory

monitoring instrument does not appear to be available. The ion-selective

fluoride electrode may have the greatest potential because it has been re- ported to have acceptable selectivity and reproducibility [54]. Colorimetric methods utilized have required considerable processing, including

distillation, because of poor specificity. The ion-selective electrode has

not yet been incorporated into an operational field instrument.

No methods are available that have satisfactory specificity for

hydrochloric acid, chlorine, or nitric acid. Hydrogen chloride is emitted

from combustion of chlorine-containing coals and incineration of

chlorine-containing materials, particularly plastics, and this acid may also

be formed by atmospheric photooxidation of chlorinated solvents. Therefore, hydrochloric acid may be more prevalent than is often assumed.

Nitric acid is a direct emission product from nitric acid manufacturing,

but a much more important source may be photochemical reactions. Very

little of the nitric oxide emitted is ever accounted for in the form of particulate nitrate. Nitric acid has been shown to be an important product of
260 ANALYTICAL PROBLEMS

photooxidation of nitric oxide in the presence of hydrocarbons in laboratory experiments [55]. The kinetic mechanisms usually postulated for

conversion of nitric oxide and nitrogen dioxide to products also favor

nitric acid formation. Because of a lack of an acceptable technique for at- mospheric analysis, we cannot assess the importance of nitric acid as an

atmospheric pollutant. Optical techniques, including open-path instrumentation, should offer possibilities for measurement of nitric acid,

hydrochloric acid, chlorine, and hydrogen fluoride. Thus far, the discussion has greatly emphasized inorganic pollutants. In

part, this reflects the development of air quality criteria for only one group

of volatile organic substances — nonmethane hydrocarbons [52]. The lack

of additional differentiation among volatile organic substances does not

result so much from lack of laboratory analytical methods as from lack of

routine monitoring techniques.

Gas chromatographs with flame ionization detectors have been utilized

in mobile laboratories to analyze atmospheric samples for identification

and quantitation of 30 to 60 different aliphatic and aromatic hydrocarbons

[56,57]. The chromatographs have been used both for periodic monitoring and for grab sampling. The number of components analyzed has depended on the substrates used and the atmospheric concentration levels. Process-type gas chromatographs for nonmethane hydrocarbons are in development.

Peroxyacetyl nitrate also has been analyzed by gas chromatography

with electron-capture detectors [58]. Because peroxyacetyl nitrate is unstable, grab sampling is not satisfactory; however, on a given site monitoring can be conducted. Calibration problems and the lower stability of the

electron-capture detector considerably limit the utilization of this technique by monitoring networks. Formaldehyde, acrolein, and aliphatic

aldehydes also have been analyzed in atmospheric samples by colorimetric techniques [6]. Results reported have involved manual sampling and

analysis. Colorimetric analyzers have been fabricated and used in some

field applications [59,60]. Unfortunately, these colorimetric analyzers,

and other analyzers based on colorimetric techniques, would present considerable problems in large-scale routine monitoring use because of the

need for reagent preparation and use of liquid flow systems, and because

of varying response to individual aldehydes.

The monitoring that has been done for specific organics has been done

by a limited number of groups interested in improving understanding of at- mospheric chemistry or in identifying the contribution from various emission sources to hydrocarbon composition. Air quality standards now

being established are not likely to require differentiation among these or- ganic species.
AIR POLLUTION CONTROL 261

Analysis of nonmethane hydrocarbons has several fundamental limitations: (1) the measurements cannot be utilized to determine the effective- ness of control, over a given period, of any particular source of organics;

(2) response characteristics of the flame ionization detector differ for vari- ous hydrocarbons; and (3) the flame ionization detector does not respond

to formaldehyde and shows reduced response to other aldehydes.

Even a detailed gas chromatographic analysis for hydrocarbons will not make it possible to follow control of even the major sources of hydrocarbons. This difficulty results from a lack of specific hydrocarbons that can

be used as unique tracers of contributions from individual emission

sources. However, acetylene and ethylene can be used as indicators of

control of combustion sources. In downtown high-traffic-density areas, these hydrocarbons also should serve as indicators of the level of

hydrocarbons from vehicular exhaust. Ethylene measurements would be

of direct interest in some areas because of its plant-damage characteristics. Some steps must be taken to optimize flame ionization detector

response to minimize deficiencies in response to individual hydrocarbons

in atmospheric analysis. Calibration with a single saturated hydrocarbon

is the accepted procedure. Such a calibration limits the accuracy of comparative measurements of atmospheres in which the hydrocarbon composition varies. There is a definite need to include aldehydes in the measurement of

volatile organics. Aldehydes can constitute a significant fraction of the

reactive organic substances present in atmospheric samples. Better methods for monitoring atmospheric aldehydes are required, however.

Optical techniques warrant more consideration for their potential in providing convenient means for monitoring aldehydes in the atmosphere.

B. Instrumentation for Measuring Atmospheric Particles

Particulate matter analysis is more complex and in some ways less advanced than analysis of gaseous air pollutants. Certainly, there is a clear

lack of instruments for analyzing particles by mass, size, and chemical

composition. The great bulk of available results have been obtained by

collecting samples on filters in the field and then weighing and analyzing

the collected sample in the laboratory. Particles larger than 10 micrometers settle readily, and they are usually associated with settled dust and

dirt. Particles in this size range are deposited in the nasopharyngeal region

of the respiratory tract and do not tend to penetrate effectively into the

pulmonary and tracheobronchial regions [61]. Consequently, these large
262 ANALYTICAL PROBLEMS

particles have not been associated with toxicological action. Particles

above 10 micrometers also do not have significant effects on light scatter- ing and on visibility. Sampling for these large particles has been accomplished with dustfall jars, adhesive coatings, cyclonic collectors (for

higher-volume samples), long horizontal tunnels (as fractional elutriators),

and various impactors [62]. Dustfall jars appear as satisfactory as more

complex and expensive techniques although the dustfall jar has poor time

resolution. The justification for the use of dustfall jars has decreased,

however, since they have become increasingly poor measures of particulate matter pollution as the level of atmospheric particles smaller than 10

micrometers has increased. In view of these circumstances, little justification exists for directing effort toward development of instruments that can measure particles larger than 10 micrometers.

Particles between 0.1 and 10 micrometers probably are those of the

greatest concern in air pollution. The particles in this size range contribute

most of the particle mass. These particles penetrate into the pulmonary

and tracheobronchial regions. The proportion of the particles that are between 0. 1 and 1 micrometer is particularly important. Particles in this size range are largely responsible for reduction in visibility, and for haze

and turbidity. Studies of physiological response to particulate matter in- dicate that particles smaller than 1 micrometer can have greater irritant potency than larger particles [43 ]

.

Measuring techniques for particles in the 0.1 to 10 micrometer range

have included the Volz sun photometer, the integrating nephelometer,

various other forward-scattering and right-angle-scattering instruments,

cascade impactors, electron microscopes and tape samplers [62]. None

of these techniques is adequate for providing a quantitative measurement

of mass concentration of 0.1 to 10-micrometers particulates. Several of

these techniques have been research tools not primarily intended to give an overall measure of mass concentration; nonetheless, they have given

valuable specialized measurements on particular characteristics of particles in this size range. The tape sampler has received considerable use in the measurement of

suspended particles. Visual color of the spots on the tape has been compared with a standard gray scale. More frequently, reflectance or trans- mittance measurements have been used. Transmittance measurements,

however, have been shown to relate poorly to the mass of particles.

Reflectance also is not ordinarily related to total suspended particulate

matter but to "dark suspended matter," inasmuch as reflectance is also a

function of absorbance, and absorbance is a function of "color." Both

techniques suffer from a number of complications related to variability in the characteristics of the deposits. Neither technique can be considered
AIR POLLUTION CONTROL 263

satisfactory for measurement of absolute concentrations of particulates,

but tape samplers can be used to obtain relative values. Careful stan- dardization is critical. Changes in the characteristics of the particulates over periods of years at a site because of fuel changes or control efforts may limit the usefulness of these measurements even for obtaining rela- tive values.

The integrating nephelometer has received considerable evaluation in recent years in an attempt to relate its response to mass concentration of

suspended particulate matter [64-67]. This instrument was designed to measure meteorological range, which in turn correlates well with visual range. However, a close correspondence between nephelometer response

and mass concentration obtains only if the particle-size distribution and

other particle characteristics remain constant. Unfortunately, the experimental measurements themselves show this not to be the case [68] . A

visual range of 7.5 miles can be associated with mass concentrations ranging all the way from 50 to 200 )Ltg/m^. Such a range of mass concentrations

represents the entire range of annual geometric mean concentrations of

suspended matter in urban areas. Similarly, a visual range of 5 miles can

be associated with suspended particulate loadings ranging from 75 /utg/m^ to 300 /Ltg/m^. Such results clearly indicate that nephelometer measure- ments are completely inadequate as a means of determining whether particulate emissions comply or tend to comply with air quality standards for

particulates. This instrument certainly is useful, however, in its original

purpose, which is the measurement of meteorological or visual range. Since light scattering is associated primarily with particles in the 0. 1 to 1 micrometer range, it would seem more useful to attempt to relate nephelometer measurements to mass concentration in the 0.1 to 1 micrometer range than to total suspended particulate matter. The equipment that has received the greatest use in particulate matter

measurement has been the high-volume sampler [62]. This device necessitates the replacement of filters that must be transported to the laborato- ry for weighing and chemical analysis. Ordinarily, a 24-hour-average sample is obtained. When such integrated total weights of suspended particulate are required, the high-volume sampler is an adequate device. However, this sampler can collect particles well above 10 micrometers and

may not be too efficient in collecting particles approaching 0. 1 micrometer. In addition, the sampling rate is sensitive to the mass of material collected. It is in the utilization of high-volume samples for subsequent

chemical analysis that a multitude of problems occur. The glass fiber filter medium as ordinarily used gives high blank readings for a considerable

number of cations and anions. Furthermore, reactions can occur on the

filter medium between collected materials. Oxidation or volatization of

428-999 O - LT - 72 - 18
264 ANALYTICAL PROBLEMS

some collected substances can and does occur. Other filter materials can

be used, particularly for special analytical applications, but such filter materials usually have limitations associated with their use as well.

It should be apparent that these sampling problems cause greater obstacles to overall analytical accuracy than those caused by limitations in the

analytical techniques themselves. Conversely, it seems futile to expend

much effort on improving analytical techniques if the greater source of uncertainty results from the sampling technique itself. The Anderson sampler has received considerable use as a particlesizing device. Although materials collected on the various stages must be

returned to the laboratory for weighing or analysis, this sampler can serve

as a useful interim approach for both mass and composition measurements. A number of types of instruments ought to be developed for the measurement of particulate matter. A continuous or periodic monitoring in- strument capable of measuring the mass concentration of aerosols below

10 micrometers is needed. An instrument has been developed that uses an

electrostatic precipitator to deposit aerosol particles directly onto a

piezoelectric quartz crystal microbalance [72]. The balance is sensitive

enough to permit measurements of incremental mass for time periods of

less than 1 minute. Collection can be significantly influenced by sorption or desorption of water vapor, but this effect can be reduced by use of a

dual-crystal detector. Adhesion of larger particles depends on the composition of the particles and the relative humidity. Addition of a surface

active agent to the flow of sample can help minimize this difficulty.

Because of these possible limitations, such equipment requires careful

field evaluation and comparison with normal particle-sizing collection

techniques [69-71].

Equipment capable of periodic monitoring of particle-size distribution

by mass concentration also is required. An investigation is underway of a new cascade impactor concept for particle-size fractionation, in which

beta-ray attenuation is used to measure material collected on a filter tape. In view of the difficulties in filter collection and chemical analysis,

development of on-site chemical analyzers for various chemical species

in particulate matter may be justified. Aside from monitoring needs, improvement of our knowledge of gas aerosol transformations in the ambient atmosphere requires instruments capable of the diurnal monitoring

of such species as sulfuric acid, total particulate sulfate, particulate

nitrate, and organic aerosol. All of these substances contribute to the

loading of particles in the 0. 1 to 1 micrometer range. Research studies

using particle-sizing samplers also have identified lead, sulfate, nitrate,

carbon-containing species and ammonium and chloride ions as con-
AIR POLLUTION CONTROL 265

stituents in the 0.1 to 1 micrometer range [69-71 ,73,74]. To properly as- sociate visibility with air pollution control effectiveness, both the total mass concentration within the 0. 1 to 1 micrometer range and the contribution of these chemical species to the mass concentration must be known.

Of course, the direct association with visibiHty also involves computing

the on-site contributions of individual species based not only on mass and

size distribution but also on optical characteristics of the particles.

Despite emphasis on automated field methods, laboratory procedures

will continue to be useful in particle-size and morphological analysis of

airborne particles [75,76]. For example, optical and electron microscopy

can be used for particle sizing. These techniques, along with x-ray diffrac- tion and other measurements of physical properties, have been used to characterize a wide variety of types of particles. An atlas of photomicrographs of various airborne particles is available to aid in microscopic

identification [77].

The electron microprobe has been used for identification and for esti- mation of relative concentrations of a variety of elements including Al, Si,

P, S, CI, K, Ca, V, Fe, Ni, Cu, Zn, Br, La, Ce, and Pb in airborne particles

[78,79]. In addition, Millipore filters and beryllium disks have been

scanned by area for individual elements. Lead has been found associated

with bromine and chlorine, but samples also have been scanned in which

the lead is present with sulfate. Particles can be located in which no ele- ments can be identified in the x-ray distribution patterns, probably

because they contain carbon and other lower-atomic-weight elements.

The monitoring of a variety of hazardous or potentially hazardous ele- ments, including Be, Hg, As, Cd, V, and Mn may require the development

of on-site analyzers. For this purpose, x-ray fluorescence techniques

should have potential. Work is underway to estimate better the potential

of x-ray fluorescence for analysis of airborne particulate fractions.

Atomic absorption already has been shown to have potential for continu- ous analysis of certain elements in air, such as lead [80]. Such equipment

would allow sampling each 24 hours or more often, 7 days a week, instead

of the more frequent analysis ordinarily associated with filter collections

and laboratory analysis. A monitoring technique for various elements also

may be desired for analysis of some types of stationary emissions.

1. Elemental Analysis

Particulate matter samples are usually analyzed for the elements after collection on a filter. Contamination of the filter media with trace quantities of the elements is a common limitation on analysis. The glass fiber
266 ANALYTICAL PROBLEMS

filter most commonly used for collection is not satisfactory for trace analysis for a number of metals because of such contamination. Other substrates, such as polystyrene membrane or Millipore filters, have been

used but still are far from completely adequate. For a few elements such

as mercury, other collection techniques are used, such as amalgamation

or use of gas bubblers.

Methods of analysis utilized included colorimetry for a few elements.

Usually physical techniques including optical emission spectroscopy,

atomic absorption spectrophotometry, neutron activation analysis, x-ray fluorometry, spark-source mass spectroscopy, and stripping voltammetry

have been used for elemental analysis. The status of techniques available

through 1966 has been reviewed with emphasis on wet chemical

techniques, ring oven methods and atomic absorption spectrophotometry

[81].

Optical emission spectroscopy has been used for elemental analysis over the past 15 years in Federal monitoring activities. This spectrographic technique has been applied to analysis of 17 elements — As, Be,

Bi, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sb, Sn, Ti, V, and Zn [82] . This

method involves considerable analytical skill but it is still only semiquantitative, lacks sensitivity for a number of elements, and is limited by

blanks in the filter substrate.

Atomic absorption is a more quantitative and specific technique and is very sensitive for some elements. This technique can be used for As, Be,

Na, K, Ca, Mg, Ba, Cd, V, Co, Cu, Zn, Ag, Ni, and Pb [70, 83-85]. The

method is limited by the availability of hollow-cathode lamps. A number

of these elements have been measured after particle-sizing of airborne

particulates [70]. Atmospheric precipitation samples have been analyzed

for Pb, Zn, Cu, Fe, Mn, and Ni [86].

The thermal neutron activation analysis of particulate matter in air has

received increased use in recent years [87,88]. Scintillation counfing

using a thallium-doped Nal crystal for samples on cellulose fiber was util- ized to determine Al, V, Mn, Na, CI, and Br. In much more ambitious use

of neutron activation analysis, airborne particulate samples were col- lected on polystyrene filters and counted by means of a lithium-doped Ge

detector. The procedure permits determination of up to 33 elements pro- vided that 5 minute irradiations are utilized with 3 and 1 5 minute cooling

periods and a 2 to 5 hour irradiadon with a 20 to 30 hour and a 20 to 30

day cooling period [89]. Even with this scheme, most of the low-molecular-weight elements cannot be analyzed. The sensitivities for a number of

the elements are marginal or inadequate because of various limitations, in- cluding interference of other substances in the sample or a high blank
AIR POLLUTION CONTROL 267

value. Included in the group of elements analyzed by this method are Mg,

S, CI, Ti, Ni, and Ag. Determination of Li, Be, B, C, N, O, F, Si, and P

could not be made. Therefore, only a few elements lighter than K could be

analyzed. In addition, Pb and Cd, biologically important elements that occur in airborne particulate matter, are not included in the scheme. A

number of the elements that can be measured are not presently of concern

biologically nor do they contribute significantly to the mass of particulates. These elements include Ga, La, Sm, Eu, Au, Sc, Ce, Co, and Th.

Elements such as Cr, Fe, Ni, Zn, Se, Sb, and Hg are determinable only

after a 20 to 30 day decay period. Therefore, although this neutron activation analysis scheme in principle appears very attractive, in practice it can

only be applied with one or more other analytical techniques if determination of a range of biologically important elements or a mass balance is desired.

A particularly interesting study made recently of elemental analysis of

particulates included use of optical emission spectrography, spark-source mass spectrography, x-ray fluorescence, atomic absorption, CHN analysis, and chlorine analysis by a colorimetric technique [79] . By this means,

77 elements could be determined or estimated in particulates collected in Cincinnati, Denver, St. Louis, Washington, Chicago, and Philadelphia.

Bound oxygen could not be included in this analytical scheme. This study

is one of the few that permits any evaluation of analytical results from two

or more methods on the same samples. The agreement was good between

the analyses by atomic absorption and x-ray fluorescence for Ca, Fe, Cu,

and Zn, often being within 20 percent or less of each other. The agree- ment was not as close for K and Pb in some samples. When the analyses

for Cu and Zn by optical emission spectrographic technique were compared with those by atomic absorption or x-ray fluorescence, the values

obtained by emission spectrography averaged a factor of two to three

lower. The Fe analyses by the optical emission spectrographic technique

also averaged appreciably lower than those by atomic absorption or x-ray

fluorescence.

It is likely that even with additional improvements in these techniques,

at least two if not three different analytical techniques will be required to cover the range of elements of interest with adequate sensitivity. It also

would seem of considerable importance to conduct concurrently some additional comparisons of each of the several techniques for elemental

analyses of the same sets of airborne particulate matter samples. If confidence is to be placed in these analyses being correct, on an absolute basis, to within 20 to 30 percent, the reliability of each of the applicable

methods to each element of interest must be ascertained.
268 ANALYTICAL PROBLEMS

2. Anion Analysis

Analyses for sulfate' and nitrate are routinely made on bulk particulate

matter samples collected on the high-volume sampler at urban and nonur- ban sites [82,95,96]. Sulfate is determined by the methylthymol blue

method by means of an AutoAnalyzer [96]. Nitrate is assayed colorimetrically following reduction to nitrite by alkaline hydrazine [96]. In in- vestigations on the particle-size distribution of sulfate, several analytical

procedures have been used. In one of these investigations, high-temperature reduction of sulfate to hydrogen sulfide was utilized, followed by an

iodometric, microcoulometric titration for the hydrogen sulfide [74]. In

other work [69,97] a modified turbidimetric procedure was used to analyze the size-fractionated material from an Anderson sampler [98].

Nitrate was also determined colorimetrically after reduction following

particle sizing [71]. Phosphate and chloride also have been analyzed

colorimetrically after particle sizing [71].

3. Organic Particulates

A considerable amount of analytical effort has gone into the measure- ment of various types of organic substances in particulate matter. Polynuclear aromatic hydrocarbons, particularly benzo (a) pyrene, have

received the most attention. Certain of the procedures involving column

chromatography followed by spectrophotometric analysis have inter- ference problems associated with them. Paper chromatography, but more

usually thin layer chromatography, have received considerable attention

as separation techniques. Spectrofluorometric analysis and

quenchofluorometry have been applied to analysis of polynuclear aro- matic hydrocarbons, azo heterocyclics, and polynuclear ring carbonyls

after use of column and thin-layer chromatography. Gas chromatography

has been applied to A7-alkanes in airborne particulates as well as to polynuclear aromatic hydrocarbons. However, adequate separation of

benzo (a) pyrene from benzo (e) pyrene and perylene has been a problem

in applying gas chromatographic procedures to the analysis of airborne

particulate matter. The details of these developments have been reviewed

up to 1968 in several publications [3,4,90,91]. The status of work on

pesticides, nonvolatile fatty acids and phenols is also considered in one of

these reviews [91].

More recently, a simple and rapid procedure has been developed for the

determination of benzo (a) pyrene, benz (c) acridines and 7H-benz [de]
MR POLLUTION CONTROL 269

anthracen-7-one [92] . Benzene extracts were separated by one-dimensional thin-layer chromatography followed by analysis of benzo (a) pyrene

by spectrophotometry or spectrophotofluorometry and the benz (c) acridines and 7H-benz [de] anthracen-7-one, in trifluoroacetic acid, by

spectrofluorometry. The procedure was applied to 6-month composites

from 52 cities. Most analyses for polycyclic organic materials are applied

to 24-hour integrated samples or composites representing even longer in- tegration times. A sensitive procedure, requiring less than 20 minutes,

that involves thin-layer chromatography and direct fluorometric measure- ment was applied to 3-hour sequential air samples for analysis of 7H-benz

[de] anthracen-7-one and phenalen-l-one [93].

Little recent work has been done on pesticides in air. Samples collected

at nine urban and rural sites were analyzed for 19 pesticides and their

metabolites [94]. Chlorinated pesticides were determined on two dif- ferent gas chromatographic columns utilizing electron-capture detectors,

while organophosphate pesticides were determined on two different gas

chromatographic columns utilizing flame photometric detectors. Only

p,p'-DDT and o,p'-DDT were found at all locations.

C. Stationary Source Emission Measurements

Sources of pollutants among stationary sources vary from thermal

power plants and incinerators to cement plants and petroleum refineries. Pollutants associated with such sources include particulate matter, sulfur oxides, nitrogen oxides, fluorides, carbon monoxide, and organics.

Emissions of pollutants from stationary sources may involve flue gases

or other vented gases. For some types of extended stationary sources

analytical measurements would have to be made by a network of sampling

sites or by long-path techniques. Such problems have not received much

attention in practice.

For flue gases or vented gases in stacks, techniques of measurement do

exist. In such situations it is important not only to measure the pollutants,

but also the gas flow velocity, excess air, moisture, etc. The sampling

technique is of great importance and it must be suitable for the specific apphcation. If sampling problems are handled adequately, the analytical

measurement requirements can be simplified.
270 ANALYTICAL PROBLEMS

The analytical measurements of pollutants can be accomplished by at least four approaches: (1) intermittent sampling on site with manual analysis in the laboratory, (2) continual instrumental analysis of pollutants col- lected through probes, (3) in-stack instrumentation, (4) remote instrumental techniques. The second group of instrumental methods would include

instruments capable of operating at emission concentration levels and the use of a proper sample dilution system permitting use of ambient air type

instrumentation. The in-stack instruments presumably integrate so that

probing the stack configuration is not necessary.

Remote instrumentation cannot be utilized continually, but this class of

instrument can be used for rapid surveys and checking compliance. The

problem of positioning instruments or probes in stacks is eliminated.

Problems associated with the correct relationship between in-stack parti- cle concentration and particle size distribution to that emitted out of the

stack into dilution air also are avoided. However, the practical development and utilization of remote optical instruments with the appropriate

characteristics is difficult, scientifically and technologically.

7. Manual Procedures

Methods in common use for measurement of pollutants from stationary sources of emissions have been manual rather than instrumental. Filters,

impingers, evacuated flasks and condensation procedures are the collec- tion techniques commonly used. To express pollutant mass loadings and

relate results to process variables, additional measurements are required.

These measurements include stack gas velocity with a pitot tube,

moisture in-stack gases determined gravimetrically on condensate, excess

air by Orsat analysis and carbon dioxide by the nondispersive infrared

spectrometric method [99-101].

Sulfur dioxide usually is determined by collection of the flue gas in impingers containing hydrogen peroxide [102-105]. The sulfur dioxide is oxidized to sulfate and analyzed after reaction with barium chloroanilate

colorimetrically at 530 nm in terms of the chloroanilate ion. A second

procedure often used involves determination of the sulfate by the barium

perchlorate thorium titration method [102,103]. The sulfur trioxide or

sulfuric acid mist, after separation by filter collection, is determined by

the same procedures.

Cations such as Al+3, Cu+2, Fe^^, Pb+2, and Zn+2 produce negative in- terference in the colorimetric procedure by precipitating chloroanilate ion

from solution. These interferences can be minimized by filtering the flue
AIR POLLUTION CONTROL 271

gas with glass wool at the probe and pretreating the solution with a cation

resin. Anions causing interference are not likely to be present in flue gases. The titration with barium perchlorate is interfered with by cations

such as K+, Na+, NH+ by reducing the volume of titrant needed. Cationic interferences are minimized by use of a particulate filter in the

probe and by percolation of the collected solutions through a cation

exchange column. Anions which can co-exist in the collected solutions

such as nitrate, chloride and fluoride can interfere also. These sulfur dioxide analytical procedures have practical ranges from

10 to 3000 ppm by volume. The sensitivities are 10 ppm for 25 to 30 liter samples. The precisions are about ±3 percent at 1500 ppm. For sulfuric

acid aerosol the range is 10 to 300 ppm with a precision at 10 ppm of

±5 percent.

Nitrogen oxides often are collected from flue gases in an evacuated

flask containing dilute sulfuric acid-hydrogen peroxide absorbing solution.

The nitric oxide or nitrogen dioxide are oxidized to nitric acid which is measured colorimetrically at 420 nm as nitrophenol disulfonic acid [106]

.

This technique has a range of from 15 to 1 500 ppm by volume with a sensitivity of 1.5 ppm. Halogens interfere in this procedure.

In common practice, particulate matter is measured gravimetrically

after removal of uncombined water [107-109]. The particulate is removed from the flowing gas stream under isokinetic conditions by filtra- tion and condensation. Impinger collection is used as part of this procedure with extraction of organic particulate matter from the impinger

solution with chloroform and ethyl ether. Acetone washing of the probe

and filter holder also is included in the procedure. The total particulate

weight of aqueous and organic sample components is obtained by totaling

the weights of components. Total sample volume or stack velocity and

other parameters are utilized in calculating particulate mass in stack gas.

2. Continuous Instrumentsfor Sampling Through Probes

A number of instruments based on optical techniques have potential for use at stack gas pollutant concentration levels. Nondispersive infrared

analyzers or ultraviolet analyzers have been designed for sulfur dioxide,

nitrogen dioxide and carbon monoxide. Optical techniques have received

attention for visibility of stack gases in plumes. Portable electrochemical

transducers recently developed have potential for use in measurement of

sulfur dioxide and nitrogen oxides in stack gases [29]. The instruments
272 ANALYTICAL PROBLEMS

Utilizing the chemiluminescent reaction of ozone with nitric oxide could

be utilized for measurement of nitric oxide in stacks [22,3 1-33].

Gas chromatographic instruments have received considerable evaluation for sulfur dioxide, hydrogen sulfide and organic sulfur gases in Kraft

mill effluents [26,27,110-112]. Gas chromatographic analyzers also

could be utilized to measure carbon monoxide and sulfur dioxide in vari- ous stack gases. A source sampling technique for particulate and gaseous fluorides in- volves use of a heated glass probe to convert hydrogen fluoride to silicon tetrafluoride [113]. This type of sampling procedure has been used with

a fluoride-selective ion electrode to analyze water-soluble fluorides in stack gases. An attractive sampling approach would be to convert the stack gases by

dilution and cooling to conditions approaching those in ambient air analysis. The same instruments could be used as for air quality measurements.

This approach would considerably reduce the number of types of instru- ments in use, calibration requirements and maintenance problems. Studies are needed on the appropriate sampling interfaces to provide dilution

and cooling without changing pollutant composition. Work is in progress on the evaluation of sulfur oxide and nitrogen oxide analyzers for stack

gases.

3. In-Stack Instrumentation

Instruments capable of operating within the stack itself usually have

been based on optical principles. Such equipment must be built to withstand dust, heat, corrosion and vibration. Thermal gradients can

cause considerable problems in optical alignments. Calibration of such in- struments requires spectra under experimental conditions closely similat- ing stack conditions.

4. Remote Stack Instrumentation

Remote sensing techniques offer several advantages oyer the tradi- tional methods of sampling through a probe introduced into a source of

emissions. These advantages are as follows: (I) more representative sampling by virtue of spectral integration across the diameter of a stack

plume, (2) no need for interfacing between stack and analyzer with probes
AIR POLLUTION CONTROL 273

and sample conditions (3) capability of measuring across an extended

source such as an oil refinery.

Electrooptical techniques can be utilized remotely to characterize particulate and gaseous emissions. The use of LIDAR systems for determining the opacity of plumes from power plants has received considerable at- tention [114-117]. A study of the optical properties of such plumes con- cluded that the optical transmission of the plume best characterized the

aerosol loading in emissions. A LIDAR system has been fabricated as a research tool for field studies. More practical field equipment will probably utilize the signal backscattered from the plume aerosol rather

than the signal backscattered from the ambient air beyond the plume.

Such equipment would use a low powered laser. Mass loading measure- ments of aerosols in plumes also can be made by LIDAR but interpretation of the signal would require measurements on other characteristics in- cluding particle size distribution.

The measurement of gases such as sulfur dioxide can be approached by

use of ultraviolet transmission, infrared emission or Raman scattering.

Field evaluations have been made utilizing correlation spectroscopy in the ultraviolet [38] and will be made with high resolution infrared emission spectroscopy. Nitric oxide presents a particularly difficult problem

because of the overlap of the nitric oxide bands in the S/jum region by

water vapor bands.

Raman scattering provides a means of measuring nitric oxide without

serious interference by other stack gas constituents. In a preliminary

feasibility study, insufficient signal to noise ratio was available [118]. Improvements in signal to noise ratio can be achieved by use of resonance Raman scatter and by fluorescent scatter techniques.

Detailed analysis of particulate matter in stationary source emissions

for elements or compounds presents many of the same problems as those

already discussed for atmospheric analysis. Elemental analysis has been

limited uniil the recent increase in concern about trace metals. However,

available techniques already discussed for atmospheric particulate samples appear adequate for most stationary source applications. A

reasonably extensive sampling and analysis program was conducted some

years ago to analyze up to 10 polynuclear aromatic hydrocarbons in samples collected from a variety of combustion processes and industrial

processes [119]. Analyses were made by ultraviolet-visible spectrophotometry on the benzene soluble fraction of the samples following

separations by column chromatography.
274 ANALYTICAL PROBLEMS

D. Mobile Source Emission Measurements

Research measurements of the detailed composition of emissions of

motor vehicles have been made for at least 15 years although emission

standards for hydrocarbons in blowby gases were not promulgated until 1963 in California and for exhaust hydrocarbons and carbon monoxide in 1966 in California and in 1968 for the United States. Subsequently,

nitrogen oxide standards have been established. Much more restrictive standards for hydrocarbons, carbon monoxide and nitrogen oxides will apply to 1975 and 1976 model year vehicles. Evaporative loss controls

also have been established. The limiting factor on implementing standards

has not been analytical methods, but control technology applicable to mass production vehicles. The original techniques used for hydrocarbons,

carbon monoxide and nitrogen oxides involved the use of nondispersive

infrared analyzers. The infrared analyzers for nitrogen oxides have suf- fered from water vapor interference as well as limitation in sensitivity.

Many techniques have been applied for detailed analysis of hydrocarbon emissions. These have included mass spectroscopy, dispersive in- frared analysis, and by coulometric and colorimetric methods [6]. Gas

chromatography was applied to automobile emissions shortly after its first use in the United States. However, thermal conductivity detectors were

limited in sensitivity and suffered from water vapor and carbon dioxide in- terference. Chemical pretreatment to remove interferences and concentration techniques greatly complicated the practical application of gas chromatography to automobile exhaust emissions in the late 1950's. In

the early i960's, the application of flame ionization detectors eliminated

these earlier limitations and accelerated the use of gas chromatography to measure the detailed hydrocarbon composition of automotive emissions

[120,121]. With gas chromatographic capability well established other

analytical methods have not been utilized in recent years. The flame

ionization analyzer has replaced the nondispersive infrared analyzer as

the motor vehicle certification technique for hydrocarbons. The nondispersive infrared technique was of somewhat limited sensitivity for the more restrictive standards and its response depended on composition in an undesirable manner.

Subtractive columns have been used for analysis of paraffinic, olefinic,

and aromatic hydrocarbons in vehicle emissions using a flame ionization

analyzer [122]. This technique provides a more rapid approach to class analysis of emission than does gas chromatography.

The use of open-tubular columns combined with solid absorbant or packed columns combined with temperature programming makes it possi-
AIR POLLUTION CONTROL 275

ble to make about any analysis desired for individual hydrocarbons in emissions. Considerable work has been done applying gas chromatographic techniques to organic oxygenates in exhaust. Recently a chemical

ionization mass spectrometer has been demonstrated to be capable of

measuring a number of aldehydes and ketones in exhaust. Work also has

been done on an electrochemical approach to aldehyde analysis, but this technique is not far advanced.

Nitrogen oxide analysis primarily for nitric oxide has been done by

several other analytical techniques in addition to the nondispersive in- frared analyzer. These other approaches included use of an oxidizing step

prior to colorimetric analysis or use of an ultraviolet analyzer. A mass

spectrometric analyzer for nitric oxide also was developed. More recently

the need to measure the lower concentrations of nitric oxide required by

future standards with use of constant total volume samples has stimulated

development of more sensitive analyzers. Electrochemical and particularly chemiluminescent types of analyzers have received recent evaluation. The chemiluminescent analyzers involving gas titration of nitric oxide

with ozone are highly specific, very sensitive and have very rapid

response times. A thermal decomposition stage has been used to decompose any nitrogen dioxide to nitric oxide. A closely related technique in- volves gas titration with atomic oxygen which results in equal responses

for nitric oxide and nitrogen dioxide.

The nondispersive infrared analyzer for carbon monoxide has had

adequate sensitivity, specificity and speed of response for vehicle emission applications. Therefore, there has not been much incentive to develop other analyzers. Gas chromatographic analysis for carbon

monoxide and the recent fluorescent infrared techniques provide more

sensitivity if needed.

All of the instruments or methods discussed previously have been

developed either for research, certification or surveillance needs. Inspection of motor vehicles or production line testing requires simple and inexpensive instruments. Some of the instruments discussed are useable for

these purposes although they are more expensive than desired. Catalytic

techniques have been shown to have potential. A simple, rapid response

optical instrument of moderate cost also has much appeal.

Considerable effort has gone into analysis of oxygenated hydrocarbons,

particularly aldehydes and ketones in internal combustion engine exhaust

[6,120,123,124]. Gas chromatographic and colorimetric methods have

been emphasized. Phenols also have received attention as products in au-
276 ANALYTICAL PROBLEMS

tomobile exhaust [125,126] by chromatography and colorimetric

techniques.

Polynuclear aromatic substances, particularly the hydrocarbons, have

been the subject of several measurement projects on automobile exhaust

[91 ]. The work cited on polynuclear aromatic hydrocarbons for stationa- ry sources also included analyses for passenger cars and trucks [119].

Recently, additional work has been done associating polynuclear aro- matic substances and phenols with fuels and fuel additives and engine

variables [127]. The presence of azo heterocyclic hydrocarbons in au- tomobile exhaust has been demonstrated [128].

Earlier work on particulate matter in automobile exhaust was con- cerned with amounts of lead-containing particles [ 129]. During the same

period lead was determined polarographically on each of a series of parti- cle sized fractions from auto exhaust [30] from the Anderson sampler,

the Goetz spectrometer and other devices. Total and benzene-soluble

particulate matter have been measured in two investigations [131,132] on

exhaust from a number of passenger vehicles and trucks. Auto exhaust

particulate matter after size fractionation in an Anderson sampler has

been analyzed for lead, nitrate, sulfate and chloride by atomic absorption

and by nephelometric or colorimetric procedures for the anions [133].

The ratios of water-soluble to water-insoluble lead also were compared.

An Anderson sampler as well as a constant volume sampler were used to obtain total particulate matter and size-fractionation with analysis for

lead, iron and zinc by atomic absorption and bromine by neutron activation [134]. Both leaded and nonleaded fuels were utilized. A tunnel type

sampling system capable of sampling auto exhaust for particulate matter

under realistic operating conditions has been developed [135]. This

system was utilized with the Anderson sampler and the Monsanto impactor to do light particle size distributions as a function of vehicle operation

conditions and driving history [135]. A similar sampling system was used

in a more comprehensive study of composition of particulate and gases

with fuels with various tetraethyl lead contents [136]. Total weight, size distribution and metal and nonmetal analysis on exhaust particulate

matter and on fuel and engine oil by optical emission spectroscopy, and

neutron activation, as well as particle characterization by electron and

light microscopy, and organics by mass spectrometric and ultraviolet

fluorescent analysis were all included in the measuring techniques applied

in a recent study of auto exhaust [136].

Diesel exhaust emissions usually are considered of concern because of

smoke and odor problems in the vicinity of individual vehicles. Levels of

carbon monoxide are very low from dieseis [137]. Hydrocarbon concen-
AIR POLLUTION CONTROL 277

tration levels can vary widely [137]. Because of the higher molecular

weight of diesel fuels, the combined emissions of fuel components, low

molecular weight cracked hydrocarbons as well as partially oxygenated

organic products presents a substantial analytical problem [120]. A portion of these organic components are responsible for the odors associated

with diesel exhaust emissions [138-140]. Nitrogen oxides also can vary

considerably in diesel emissions overlapping the concentration levels produced by vehicles with spark ignition internal combustion engines

[137]. Polynuclear aromatic hydrocarbons also have been measured at substantial concentration levels in diesel exhaust emissions [141].

Measurement of the concentration levels of a number of components

such as carbon monoxide, nitrogen oxides, low molecular weight

hydrocarbons and polynuclear aromatic hydrocarbons does not present

substantially different analytical requirements from those well established

for spark ignition internal combustion engines. Sampling and analysis of

fuel hydrocarbons and their partial oxygenated products still present opportunities for analytical activity [120]. Although substantial progress

has been made in recent years on identification of the odorous components of diesel exhaust, much more analytical work is needed [140]

.

Considerable use was made in earlier work by Scott Research Laboratories of dispersive infrared measurements for carbon monoxide, carbon

dioxide, nitrogen oxides and some hydrocarbons [138]. Nondispersive

infrared instruments were used in Bureau of Mines investigations for car- bon monoxide and nitric oxide while nitrogen dioxide was measured with

a nondispersive ultraviolet analyzer [137]. Colorimetric methods have

been used for nitrogen oxides, formaldehyde, acrolein and total aliphatic

aldehydes [138]. Gas chromatography has been used for analysis of

hydrocarbons in combination with column chromatography with identifi- cation by mass spectrometry for odor components [140]. Considerable

progress on odor has been made by this combination of techniques when

applied to the oily-kerosene odor fraction and similar techniques are being

applied to the smoky-burnt fraction. Column chromatography combined

with fluorescence spectroscopy has been used to identify and measure a number of polynuclear aromatic hydrocarbons in diesel exhaust emissions

[141].

The use of high molecular weight fuels presents a special problem with

respect to the actual form of the higher molecular weight products upon

emission. These materials may be present in the atmosphere as vapors, as

finely divided organic aerosol, or as large droplets that settle to the ground

rapidly. Additional investigation is needed to properly define this situa- tion under realistic operating conditions.
278 ANALYTICAL PROBLEMS

While smoke and odor have been the main concern from the standpoint

of possible regulations, other emissions may well have to be included in emission regulations in the future. If emissions from the passenger vehicle

equipped with spark ignition internal combustion engines are very effectively controlled by the middle 1970's, the residual contributions from

other propulsions systems such as the diesel engine may well become of

greater concern.

IV. References

[1] Lodge, J. P., Air Pollution, .4 C/zem. 33, 3R (1961).

[2] Altshuller, A. P., Air Pollution, ^/la/. C/zem. 35, 3 R (1963).

[3] Altshuller, A. P., Air Pollution, C/iem. 37, 11R(1965).

[4] Altshuller, A. P., Air Pollution, .4 aia/. Chem.39, 1 OR (1967).

[5] Altshuller, A. P., Atmospheric Analysis by Gas Chromatography, Advances in Chromatography, Giddings, J. C, Keller, R. A., Editors, Vol. 5, Marcel Dekker,

New York (1968).

[6] Altshuller, A. P., Analysis of Organic Gaseous Pollutants, ^/r Pollution, Stem, A. C, Editor, Vol. II, Academic Press, New York (1968).

[7] Katz, M., Analysis of Inorganic Gaseous Pollutants, Air Pollution, Stem, A.

Editor, Vol. II, Academic Press, New York (1968). [8] Saltzman, B. E., and Wartburg, A. P., Jr., Absorption Tube for Removal of Interfer- ing Sulfur Dioxide in Analysis of Atmospheric Oxidant, Anal. Chem. 37, 779 (1965). [9] Ortman, G. C, Monitoring Methane in Atmosphere with a Flame, Continuous Monitoring of Methane and Other Hydrocarbons in Urban Atmospheres, Chem. 38, 644(1966).

[10] Altshuller, A. P., Ortman, G. C, Saltzman, B. E., and Neligan, R. E., Photooxidation

of Propionaldehydes at Low Partial Pressures of Aldehyde, J. Air Pollution Control

Assoc. 16,87(1966).

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AIR POLLUTION CONTROL 279

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280 ANALYTICAL PROBLEMS

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AIR POLLUTION CONTROL 281

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282 ANALYTICAL PROBLEMS

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the Concentration and Particle Size Distribution of Sulfate in Urban Air, Atm. Environ. 1, 479 {1967). [70] Lee, R. E., Patterson, R. K., and Wagman, J., Particle Size Distribution of Metal Components in Urban Air, Environ. Sci. Technol. 2, 288 (1968). [71] Lee, R. E., and Patterson, R. K., Size Determination of Atmospheric Phosphate,

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AIR POLLUTION CONTROL 283

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States Precipitation, Environ. Sci. Technol. 4, 55 (1970). [87] Brar, S. S., Nelson, D. M.. Kanabrocki, E. L., Moore, C. E., Bumham, C. D., and

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in the Lower Atmosphere Between California and Hawaii, Environ. Sci. Technol. 3, 1207(1969).

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Chromatographic Procedure to the Determination of Benzo (a) pyrene, Benz (c) Acridines and 7H-Benz (de) Anthracene-7-One in Airborne Particulates from Many

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284 ANALYTICAL PROBLEMS

[102] Corbett, P. F., The Determination of SO2 and SO3 in Flue Gases, J. Inst. Fuel 24, 237(1961).

[103] Matty, R. E., and Diehl, E. K., Measuring SO2 and SO3, Power 101, 94 (1957). [104] "Methods of Measuring and Monitoring Atmospheric Sulfur Dioxide," Publication No. 999-AP-6, U.S. Department of Health, Education and Welfare, Public Health

Service, National Center for Air Pollution Control, Cincinnati, Ohio (1964).

[ 1 05] Walden Research Corp. , "Determination of Sulfur Oxide Emissions from Stationary

Fossil Fuel Sources," Contract CPA-22-69-95, U.S. Department of Health, Education and Welfare, Public Health Service, National Air Pollution Control Administration, Durham, North Carolina (September 1970). [106] "Standard Method of Test for Oxides of Nitrogen in Gaseous Combustion Products

(Phenoldisulfonic Acid Method)," ASTM Standards 1968, Part 23, D- 1608-60, 1915 Race Street, Philadelphia, Pennsylvania 19103 (1968). [107] Smith, W. S., Shigehava, R. T., and Todd, W. F., "A Method of Interpreting Stack Sampling Data," 63rd Annual Meeting of the Air Pollution Control Association, St. Louis, Missouri, June 14-19, 1970. [108] Smith. W. S., Martin, R. M., Durst, D. E., Hyland, R. G., Logan, T. L., and Haper,

C. B., "Stack Gas Sampling Improved & Simplified with New Equipment," 60th Annual Meeting of Air Pollution Control Association, Cleveland, Ohio, June 11-16, 1967. [109] "Specifications for Incinerator Testing at Federal Facilities," National Center for Air

Pollution Control, Cincinnati, Ohio (1967). [110] Walther, J. E. , and Ambug, H. R. , Continuous Monitoring of Kraft Mill Stock Gases

with a Process Gas Chromatograph, TAPPI 50, 108A (1967). [Ill] Anderson, K., and Borgstrom, J. G. T., Determination of Hydrogen Sulfide and Sul- fur Dioxide in Low Concentrations, Svensk Papperstid (Stockholm) 70, 805 (1967). [112] Adams, D. F., Koppe, R. K., Direct GLC Coulometric Analysis of Kraft Mill Gases, J. Air Pollution Control Assoc. 17, 161 (1961). [113] Dorsey, J. A., and Kemnitz, D. A., A Source Sampling Technique for Particulate and Gaseous Fluorides, J. Air Pollution Control Assoc. 18, 12 (1968). [114] Hamilton, P. M., The Use of LIDAR in Air Pollution Studies, Intern J. Air and

Water Po//. 10, 427 (1966). [115] Johnson, W. B., Jr., LIDAR Applications in Air Pollution Research and Control,

J. Air Poll. Control Assoc. 19, 176 (1969).

[1 16] Johnson, W. B., Jr., and Uthe, E. E., "LIDAR Study of Stack Plumes," Final Report, Contract PH-22-68-33, U.S. Department of Health, Education and Welfare,

Public Health Service, National Air Pollution Control Administration, Durham,

North Carolina (June 1969). [117] Uthe, E. E. , "Study of Laser Back Scatter by Particulates in Stack Emissions," Contract CPA 70-173, U.S. Department of Health, Education and Welfare, Public Health Service, National Air Pollution Control Administration, Durham, North

Carolina (1971). [118] Leonard, D. A., "Feasibility Study of Remote Monitoring of Gas Pollutant Emissions by Raman Spectroscopy," Final Report, Contract CPA 22-69-62, U.S. Depart- ment of Health, Education and Welfare, Public Health Service, National Air Pollution Control Administration, Durham, North Carolina (August 1970). [119] Hangebrauck, R. P., Von Lehmden, D. J., and Meeker, J. E., "Sources of Polynuclear Hydrocarbons in the Atmosphere," Publication No. 999-AP-33, U.S. Department of Health, Education and Welfare, Public Health Service, National Air

Pollution Control Administration, Durham, North Carolina, (1967).
AIR POLLUTION CONTROL 285

[120] Dimitriades, B., Ellis, C. F., and Seizinger, D. E., Gas Chromatographic Analysis of Vehicular Exhaust Emission in Advances in Chromatography, Giddings, J. C, and

Keller, R. A., Editors, Vol. 8., Marcel Dekker Publisher, New York (1969). [121] Dimitriades, B., and Seizinger, D. E., A Procedure for Routine Use in Chromatographic Analysis of Automotive Hydrocarbon Emissions, Environ. Sci. Technol. 5, 222 (1971). [122] Klosterman, D. L., and Sigsby, J. E., Jr., Application of Subtractive Techniques to the Analysis of Automotive Exhaust, Environ. Sci. Technol. 1, 309 (1967). [123] Fracchia, M. F., Schutte, F. J., and Mueller, P. L., A Method for Sampling and

Determination of Organic Carbonyl Compounds in Automobile Exhaust, Environ.

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Molecular Weight Substituted Organic Compounds in Emissions, Environ. Sci. Technol. 4, 150(1970).

[125] Smith, R. G., MacEwen, J. D., and Barrow, R. E., The Occurrence of Phenols in Air and Combustion Products, 7. ^m^r. Ind. Hyg. Assoc. 20, 149 (1959). [126] Barbor, E. D., Sawicki, E., and McPherson, S. D., Separation and Identification of Phenols in Automobile Exhaust by Paper and Gas Liquid Chromatography, Aurl. Chem. 36,2442(1964).

[127] Esso Research and Engineering Co., "Gasoline Composition and Vehicle Exhaust Gas Polynuclear Aromatic Content," CRC-APRAC Project CAPE 6-68, Contract CPA-22-69-56, U.S. Department of Health, Education and Welfare, Public Health

Service, National Air Pollution Control Administration, Durham, North Carolina (February 1970). [128] Sawicki, E., Meeker, J. E., and Morgan, M. J., Polynuclear AZA Compounds in Automobile Exhaust, y4rc/z. Environmental Health 11, 773 (1965). [129] Hirschler, D. A., and Gilbert, L. F., Nature of Lead in Automobile Exhaust Gas,

Arch. Environmental Health 8, 109 (1964). [130] Mueller, P. K., Helwig, H. L., Alcocer, A. E., Cong, W. K., and Jones, E. E., "Concentration of Fine Particles and Lead in Car Exhaust," Symposium on Air Pollution Measurement Methods, Special Technical Publication No. 352, American Society

for Testing and Materials, 1915 Race Street, Philadelphia, Pennsylvania 19103

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Hydrocarbons from Automobiles and Trucks, J. Amer. Ind. Hyg. Assoc. 27, 47

(1966). [133] Lee, R. E., Jr., Patterson, R. K., Crider, W. L., and Wagman, J., Concentration and

Particle Size Distribution of Particulate Emissions in Automobile Exhaust, ^/m. Environ. S, 115-23,1 {\91\). [134] Nimomiji, J. S., Bergman, W., and Simpson, B. H., "Automobile Particulate Emissions," Second International Clean Air Congress, December 6-1 1, 1970, Washington, D. C. [135] Habibi, I., Characterization of Particulate Lead in Vehicle Exhaust- Experimental

Techniques, Environ. Sci. Technol. 4, 239 (1970). [136] Moran, J. B., and Manery, O. J., "Effect of Fuel Additives on the Chemical and

Physical Characteristics of Particulate Emissions in Automotive Exhaust," Contract CPA-22-69-145 (July 1970).
286 ANALYTICAL PROBLEMS

[137] Marshall, W. F., and Fleming, R. D., Diesel Emissions Reinventoried, Report of

Investigations, Bureau of Mines, U.S. Department of the Interior, Washington, D.C.

(1971). [138] Linnell, R. H., and Scott, R. H., Diesel Exhaust Composition and Odor Studies,

J. Air Pollution Control Assoc. 12, 5 10 (1962). [139] Vogh, J. W., Nature of Odor Components in Diesel Exhaust, J. Air Pollution Control

Assoc. 19,773 (1969). [140] Arthur D. Little. Inc.. "Chemical Identification of the Odor Components in Diesel Engine Exhaust," Final Report, CRC-APRAC Project 7-68 (1-69), Contract No. CPA 22-69-63, U.S. Department of Health, Education and Welfare, Public Health

Service, National Air Pollution Control Administration, Durham, North Carolina

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PANEL DISCUSSION

Analytical Problems in Air Pollution Control

CHAIRMAN

James R. McNesby, Manager, Measures for Air Quality Program, National

Bureau of Standards

PANEL

Walter C. McCrone, Director, McCrone Associates, Inc., Chicago,

Illinois Peter K. Mueller, Chief, Air and Industrial Hygiene Laboratory, Cahfornia Department of Pubhc Health, Berkeley, CaHfornia

Phihp W. West, Professor of Chemistry, Louisiana State University,

Baton Rouge, Louisiana

Paul Urone, Professor of Atmospheric Chemistry, University of Florida,

Gainesville, Florida

A. P. AltshuUer, Director Division of Chemistry and Physics, Environmental Protection Agency (Speaker)

287
288 ANALYTICAL PROBLEMS

McCrone — The optical microscope may be considered a specific sensor. It provides the means to recognize a specific pollutant or to get some

characterization data, using the eyes for identification purposes.

Three kinds of problems may be defined. The "needle in the haystack"

analysis involves looking at a large number of particles — perhaps millions

of them — to find any having a particular composition. They might be

asbestos particles, quartz particles, or lead particles. The problem, for ex- ample, may be to look for lead particles in an urban atmosphere.

Sometimes, the same type of sample may be given and the analyst is asked to search for "anything that could cause trouble." Here, trouble has

to be defined in terms of who is paying the bill. It might be concerned with

hard particles that would harm a polishing operation or it might be con- cerned with public health. Often, it involves several things. In any event,

the analyst would look at the whole sample for any compositions that would cause the specified kind of trouble.

Finally, there is the problem of looking at a settled or suspended dust

sample to identify "everything in it." This is the situation when someone

wants to know what dust is settling in his area, or what are the particles he

is breathing. Most of the components would be innocuous, but all need to be identified. Most particles can be identified by optical microscopy if one has seen them before (with a name-tag). For example, asbestos particles, unless

very small, have a characteristic morphology and optical properties so

there is no difficulty in identifying them. Extensive experience increases

the number of identifiable substances. Prepared slides of known materials

and reference sources such as the "Particle Atlas" ^ are invaluable aids. Particles as small as a few fjum can be identified by an experienced

microscopist.

Electron microscopy offers additional opportunities. Scanning electron

microscopy (SEM) is a relatively simple technique which is very popular

because the sample requires very little preparation. Many modern SEM's

have solid-state detectors so that they can make x-ray fluorescence

measurements, and become in a sense, then, electron micro-probes.

With such an instrument one can make a morphological selection of a

particle, measure the x-ray wave lengths generated by the electron beam

striking it, and thus determine its elemental composition.

The transmission electron microscope has better resolving power than

the scanning electron microscope and can identify even smaller particles.

> McCrone, W. C, Draftz, R. G., and Delly, J. D., "Particle Atlas," Ann Arbor Science Publishers, Ann Arbor, Michigan (1967).
PANEL DISCUSSION 289

However, the techniques involved are more difficult. Neither electron

microscope, of course, is a field instrument.

X-ray diffraction is a means of determining the crystal form of a compound. Even though a particle may be identified as Ti02, it could still be

rutile, anatase, or brookite. Optical crystallography or x-ray diffraction

would answer this question but the latter is probably easier for most

analysts. Fairly simple modification of x-ray cameras permits investigation of particles down to 10-^^ grams (2-5 /xm in diameter). Measurements at the nanogram level are routine. Particles of 2 /xm or larger

diameter, mounted on glass fibers and centered in the x-ray beam provide

measurable x-ray diffraction patterns.

The electron microprobe (EMA) can make an essentially complete

analysis of all particles in a sample. You could also use it for "needle in a haystack" analysis. EMA requires about a minute-per-particle to obtain

a quantitative analysis. The instrument may be programmed for automatic

scanning to look for a combination of elements such as found in a crystal

of specific interest. In scanning the sample, it will recognize and analyze

particles containing the desired combination of elements.

Particles may be mounted on a beryllium block, to which most

microprobes are insensitive. The entire sample or array of particles may

be photographed with selected x rays of one of the constituent elements.

Thus a picture may be taken first with the x rays of lead to show the leaa- containing particles and then with back-scattered electrons to show all particles in the field. The ion probe (IMA) may be the ultimate analytical instrument. It comes as close to ion-by-ion or atom-by-atom analysis as anything we

have and will detect as few as 100-1000 atoms of any element. Accordingly, it is capable of analyzing particles down to a few nanometers in diameter or about lO-^^-lO-^^g. The IMA determines elemental composition by measuring the relative numbers of isotopes of all elements in the

periodic table. In all of the above methods, a reliable means of collecting samples is obviously required. The sample must be representative to be worth analyzing.

Mueller — The technology of particle analysis is the one most in need of

further development. No instrumentation presently exists for continuous

analysis so that laboratory determination of field-collected samples is currently practiced. The collection of representative samples offers many

problems and the collection of liquid particles is a major problem.
290 ANALYTICAL PROBLEMS

The sampling procedure may discriminate on the basis of particle size. For instance, large particles may have too much momentum to change

direction to enter a sampling tube and hence may be excluded from a sample. A sampler, consisting of rotating rods or plates, normal to an air stream, is capable of collecting large particles. However, for these collec- tors the nature of the surface that would optimize efficient collection in the requirements of subsequent chemical analysis needs further investigation. The cascade impactor deliberately changes the air speed and

direction to achieve sample size fractionation, based on changes of momentum.

Studies have been made in California to determine elemental compositions of particles as related to the size distribution. Samples have been

collected with the cascade impactor and analyzed by fluorescent measure- ments of X rays generated by alpha particles from a cyclotron. This work was done in cooperation with Dr. Tom Cahill of the University of California at Davis. A sampling time resolution of two hours coupled with sizing

into five classes was possible.

This work was concerned with identifying the source of particles. Is a

particular haze man-made, natural, or is it a combination? If the latter, how much of each is involved?

Chemical analysis for sodium and chlorine has shown that certain size

fractions have a marine origin. Lead and bromine determinations have

shown that most of the lead in the submicrometer size arises from au- tomobile exhaust. Vanadium determinations identify particle sizes arising

from power plant emissions.

Measurements such as bromine-lead ratios provide insight to at- mospheric reactions. It has been found that this ratio is higher at night

than during the day, thus indicating a photochemically initiated mechanism for bromine disappearance, involving a change from the parti- cle to the gas phase.

Attempts have been made to investigate compounds occurring in the at- mosphere. Unfortunately, wet chemical techniques are too insensitive to determine ammonium, nitrates, and sulfate with sufficient reliability on

the small samples available of the fractionation the particles collected in 2 to 4 hours at 50 liters per minute in five size classes. However,

photoelectron spectroscopy is now arising as a valuable tool for such

determinations. Analyses for lead, nitrogen, and sulfur have been made by

this technique in cooperation with Dr. T. Novakov of the Shell Development Company. Normalization of sulfur and nitrogen with respect to lead,

and correlation of such data with particle size, provides insights to their

origins.
PANEL DISCUSSION 291

This study also led to the demonstration that four kinds of nitrogen

compounds exist in urban particulate matter— amino, ammonium, nitrate,

and pyridinium. Nitrate containing particles were found to peak late in the

day, indicating a non-automotive source. The other three kinds of

nitrogen were found to correlate with automotive traffic patterns. This in- formation, together with the knowledge that ammonium nitrogen is found

in exhaust gases and that amino and pyridinium fragments can arise from

gasoline additives, implicates automotive emissions as the source of these

particles.

Urone — There are two areas where the analytical community can help

tremendously. One of course is the research area, not only in responding

to the everlasting demand for better, quicker, and more specific methods,

but also for broadening the horizon in environmental areas by looking for new compounds, new mechanisms, and investigating cause-and-effect

relationships between environmental pollution and the receivers, be they

people, animals, plants, or materials.

The other important area is in the development of man power. There is a critical need in many areas — local agencies, universities, junior colleges,

high schools, ^rc.,— for people with a reasonable knowledge about air pollution. The lack of knowledge in these areas is amazing. The analytical

chemist should not only help instruct on methodology but emphasis

should be given to the importance of careful analysis. As more people

enter the field, the validity and meaning of their measurements becomes

increasingly important.

New analytical methods are rapidly emerging. Measurements of pollutants are being made and interpreted by an increasing number of persons,

other than analytical chemists. Unfortunately, many of these have vague

or incomplete knowledge of the basic analytical principles involved. Accordingly, interpretations made over a period of time may be incompatible. This was illustrated vividly in a recent lecture by Professor HaagenSmit. He stated that the carbon monoxide concentration in 1967 at seven Los Angeles stations was 1 1.0 ppm. In 1969, the carbon monoxide

level for the same stations was 5.3 ppm. Quite a reduction! This is not

true. Los Angeles is getting worse. The explanation is based on the

improper use of the non-dispersive infrared method for the measurement. The 1969 measurements had not been properly corrected for

interferences by a variable amount of water vapor. There is a very great need at present for analysts who can identify and

measure the organic substances which are produced as primary or secon- dary products of photochemical and thermal reactions. The processes of
292 ANALYTICAL PROBLEMS

haze formation, the causes of air pollution episodes, such as those of

Donora or London, are not well understood. Laboratory experiments

tend to repeat the primary photochemical products but do not adequately

identify and measure those substances which form aerosols or respiratory

irritants in real atmospheres.

West — The analytical chemist has been very remiss in letting other people take over his work. I refuse to admit that anyone who wants to call himself an analyst is necessarily qualified to put out numbers that truly

represent a situation. Air pollution analysis is tricky stuff and reams of

data taken by a black box are not necessarily meaningful just because some pollutant meter is used and masses of data fall out of the box.

An example of a pitfall in pollution analysis is the sampling problem.

The high-volume sampler, while adequate for the determination of total mass of airborne material, is not especially well suited for the chemical

analysis of particulates. The values for volatile materials such as selenium

dioxide are completely erroneous. Also, the filter media themselves are often too contaminated to provide meaningful measurement because of a

high or variable blank.

The determination of certain materials in particulate matter is of special

interest. Arsenic, chromium, nickel, and asbestos are known to be car- cinogenic. Elements which have been indicated as possible carcinogens

include beryllium, cadmium, cobalt, lead, and selenium. Manganese is not believed to be carcinogenic but is associated with Parkinson's disease.

Even helpful elements such as zinc and copper need to be determined. A

sequential tape sampler is excellent for collecting these materials and can

provide the 20-30 micrograms needed by modern analytical methods.

The ring oven provides a simple field method for identification and

determination of the approximate levels of most of the above-mentioned

elements. Essentially it is a solvent extraction separation technique in which the sample is dissolved at the center of a filter paper and carried by

a solvent front to the circumference of a heated circle where it collects as a thin but concentrated ring. Sectors of the filter may be cut out and

treated with suitable reagents to provide colored ring sections that are compared with standards for their quantitative estimation. Detailed

procedures using this technique have been developed in the LSU laboratories for determination of submicrogram quantities of most significant

metals, including cadmium, cobalt, beryUium, chromium, nickel, zinc, and

lead. Comparisons with other methods, especially atomic absorption

spectroscopy, have been very favorable.
PANEL DISCUSSION 293

General Discussion — The analytical requirements of particulate analysis were discussed. Too little effort has been applied to the use of several

techniques on the same sample to estimate the accuracy of methods. The

lack of suitable Standard Reference Materials makes it impossible to compare results obtained by various investigators.

The requirements of accuracy in air pollution are not severe, at present.

The consensus is that errors should be no greater than ±20 percent at tolerance levels and results accurate to within a factor of two should be

obtained at subliminal levels. The ultimate accuracy may well be related

to the sampling problem.

The subject of pollution indices involving a combination of the levels of

total particulates and specific pollutants was discussed. The interpretation

of such indices is uncertain not only because of the paucity of data but the

known fact that what is present is more important than the total amount of

suspended material in the atmosphere. Agencies concerned with the state- of-the-environment should be encouraged to support efforts to collect the

detailed data needed for more definitive studies of the effects of air pollution. Support of research to develop instrumentation and methodology for

the continuous monitoring of specific elements and specific constituents

should also be endorsed.

Khalil H. Mancy, is Professor of Environmental Chemistry, School of

Public Health, University of Michigan. He received his B.S. from Cairo

University, Egypt, in 1952 and his M.S. and Ph.D. degrees from the

University of North Carolina in 1959 and 1962. Dr. Mancy held positions

at the University of North Carolina, the University of Cincinnati, and

Harvard University before joining the University of Michigan as an as- sociate professor in 1966. He is President-Elect and Chairman of the Pro- gram Committee of the ACS Division of Water, Air, and Waste Chemistry. He was a UN and WHO consultant to Poland in 1968 and 1969 and

Czechoslovakia in 1970. Dr. Mancy is a member of the American Water

Works Association, and the Water Pollution Control Federation, and is a

consultant to the Environmental Protection Administration and NASA.

His research interests are in water quality measurement sensors and in- strumentation, and the aquatic chemical dynamics of gases and metals.

He is the author of about 30 publications and has two chapters and a book

in press. 295

428-999 O - LT - 72 - 20

NATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 351,

Analytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

Held June 16-18, 1971 , Gaithersburg, Md. (Issued August 1972). CHAPTER 6

ANALYTICAL PROBLEMS IN

WATER POLLUTION CONTROL

Khalil H. Mancy

School ofPublic Health

The University ofMichigan

Ann Arbor, Michigan 48104

The analytical chemistry of natural and waste waters requires subtle correlation between theory and experience, an insight into the nature and mode of ac- tion of interferences, and the ability to interpret analytical results in correlation with pertinent field observations. Because of the complexity of the system

under investigation, water pollution characterization is one of the most chal- lenging tasks to confront the analytical chemist. Design of measurement systems begins with defining (a) WHY the analysis

is needed, (b) WHAT are the parameters to look for, and (c) HOW to per- form the analysis. Water pollution measurement programs usually incorporate

a multitude of physical, chemical and biochemical procedures. The charac-

*terization of physicochemical and biochemical transformations in aquatic en- vironments takes into account interactions between atmosphere, hydrosphere and iithosphere and their influence on the aquatic habitat. Trace characterization of persistent organic residues or heavy metals is based on defining their distribution dynamics in the aquatic system, e.g., storage and release by bot- tom sediments and accumulation and tolerance by aquatic biota. Recent trends in water pollution measurement rely heavily on advanced in- strumental methods and automated analytical techniques. In situ sensor systems and remote noncontact optical measurement have been applied for monitoring water quality in rivers, lakes and waste effluents. The effects of en- vironmental factors and interferences on the reliability of the measurement

system have been under intensive research. As America's water pollution problems become more complex, more complex measurement techniques are needed to cope with them. Above all, the ef- fectiveness of pollution control programs are limited by their measurement reliability.

Keywords: Aquatic environment; environmental analysis; trace analysis; water analysis; water pollution; water pollution control. 297
298 ANALYTICAL PROBLEMS

I. Introduction

It seems clear to thoughtful individuals that efforts to protect the quality of our environment are highly dependent on our ability for measure- ment of pollution. Subjective responses to environmental quality deteri- oration in terms of perceived awareness (or annoyance) to such things as

taste, odor or noise are ultimately of great significance. Nevertheless, lit- tle headway can be made in setting standards, devising quality indices and

guarding against pollution of natural resources unless there is a basis in objective physical measurement. This implies the ability to measure re- liably and quantitatively pertinent water quality parameters, establish

baseline values, and monitor changes in these parameters both in location

and time. Present water pollution control activities are limited to a great extent by

a lack of adequate measurement capabilities. This is not because the

characterization of water pollution is a unique impossible task, but rather,

it was only recently when this field gained its present popularity and at- tracted the attention of analytical chemists. Unfortunately, this situation seems not to be improving fast enough to keep up with the rapidly increasing demands of water pollution control technology. This slow progress, or

the lack of it, is to a certain extent the result of the failure of administrators and managers of water pollution control programs to recognize the

need for improving present measurement capabilities. As a result, the

majority of research and development appropriations in government and

industry include insignificant proportions, or a complete absence of allo- cations, for the development of advanced pollution measurement

techniques. Ironically, large amounts of funds are being appropriated for

the removal of pollutants that cannot be measured adequately or cannot

be measured at all. Perhaps the analytical chemist, as a citizen and a professional, is to blame for not getting involved and assuming a leading role in environmental problem solving. A majority of analytical chemists, especially in government and educational institutions, are "technique oriented" and

simply ignore to devote their intellectual energies to urgent problems of our time. In spite of its obvious necessity, over-specialization in analytical

techniques insures the inability to follow a problem through from start to

finish and thereby cripples the individual. There is a pressing need for the

"problem oriented" analytical chemist who can effectively participate in pollution problem-solving teams. This is a specialist in the characterization of water or air pollution. He should have the background, through
WATER POLLUTION CONTROL 299

formal training or practical experience, to speak the language and to participate in the planning and implementation of pollution control programs.

The analytical mind that has guided the analytical chemist to an exciting

and productive career is particularly suited to undertake this challenge.

One may ask, "How adequate are present capabilities for measurement

of water pollution?" and "what are the areas which need improvement?"

An attempt to answer these questions is given in this presentation. This

includes a brief review of the ever broadening analytical needs for water

pollution control with a focus on "problem areas."

IL The Aquatic Environment

Water quality characteristics of aquatic environments are the net result of a multitude of physical, chemical and biological interactions. Rivers,

lakes, estuaries are continuously under a dynamic state of change with

respect to their geological age and geochemical characteristics. This is manifested by a continuous circulation, transformation, and accumulation

of energy and matter through the medium of living things and their activities. A multiplicity of natural regulatory mechanisms play the important

role of controlling the physicochemical properties of the aqueous phase

and number and type of its biological population.

Through man's exploitation of his water resources the dynamic balance

in aquatic ecosystems is frequently disturbed which may result in such

dramatic responses as fishkill, foaming, taste and odor, etc. These ecological responses are described in terms of "pollution" since they interfere

with man's use of the water resources. Consequently, measurements of water quality in aquatic environments

should take into account physicochemical and biochemical interactions

between the hydrosphere and biosphere as well as interactions with the

atmosphere and the lithosphere {e.g., bottom sediments). To this extent

the characterization of water quality in rivers, lakes, etc. may require the

measurement of the quantities and rates of mass and energy transfer across environmental boundaries, e.g., CO2 or O2 transfer at the air-water

interface and the accumulation or release of chemicals by bottom sedi- ments. Examples of such environmental interactions are given in Figure 1, which illustrates dynamics and the relative proportions of major elements

in an aquatic ecosystem [ 1 ] . The ratios given in Figure 1 are based on the

number of atoms per atom of phosphorus. The model suggests a basic
300 ANALYTICAL PROBLEMS

phenomenon that phosphorus, nitrogen and carbon levels are largely dependent on the synthesis or decomposition of organic matter by the

aquatic biota [ 1 ]

.

AIR

WATER

SEDIMENT \ s"""^ ^iTF^^

SOi

10

10,000 40,000 10,000 400,000

Figure 1 . Interaction of some major elements in aquatic ecosystems.

The physicochemical characteristics of the aqueous phase have a direct

effect on the types and distribution of the aquatic biota. The reverse is also true, the physicochemical characteristics of the aqueous phase are

greatly influenced by the activity of the aquatic biota. These interactions can be easily illustrated by considering a case of a thermally stratified lake

as shown in Figure 2. Certain lakes and marine environments undergo

seasonal thermal stratification into a warm surface layer (epilimnion), an

underlying layer where temperature drops rapidly with depth (thermocline) and a bottom deep layer of cool water of higher density

(hypolimnion).

The hypolimnitic zone represents a condition where the biological

decomposition of organic matter has completely removed the dissolved

oxygen which cannot be replenished since these layers are stagnant and

are cut off from the atmosphere. As a result of the disappearance of dis- solved oxygen, anaerobic biological populations take over and start to
WATER POLLUTION CONTROL 301

Temp. Depth

(°C) (feet) Surface

19 - - 0

Epilimnion: Aerobic conditions, phytosynthesis,

Phytoplankton, Zooplankton, higher forms of

Aquatic Biota

17-- 40

Thermocline: Temperature gradient, Physical

Barrier

7__ 70

Hypolimnion: Oxygen Depletion, Denitrification

(NO3 NO2 NH3 and N2), Sulfide production

(SOf" —> H2S), Precipitation of metal sulfides.

Anaerobic Biosynthesis (CH4), High Organic

Content, Anaerobic Microorganisms

4 __ 120

Sediment: High Organic Content, Metal Sulfides,

Anaerobic Microorganisms

Figure 2. Thermal stratification physical-chemical-biological interactions.

reduce nitrates to nitrites and ammonia. After the nitrates and nitrites supply is exhausted, sulfate reduction takes place which results in the for- mation of free sulfides, e.g. H2S. Besides being toxic, the sulfides will form

highly insoluble metal sulfides which tend to strip the water of its heavy

metal content and enrich the bottom sediments with the formed

precipitates. This dramatic display of biological and chemical interactions

is typical of all aquatic environments.

In view of the above discussion, full characterization of water quality

for pollution control should account for (a) the distribution dynamics of

chemicals in the aqueous phase (soluble, colloidal or adsorbed on particulate matter); (b) accumulation and release by the aquatic biota; (c) accu- mulation and release by bottom deposits; and (d) inputs from land and at- mosphere e.g., airborne contaminants and land run offs.
302 ANALYTICAL PROBLEMS

One of the more important aspects of water quality measurement is the

ability to characterize the chemical species. For example, in the case of

metal analysis, it is highly significant to provide analytical data in terms of

the oxidation state and type of species i.e., free or complexed instead of a

total metal concentration. This is apparent in view of the fact that the

chemical reactivity of a given metal is dependent on its form and not

necessarily on its total concentration. Accordingly, the availability of

metal micronutrients to the biological system or their toxicity effects are dependent on the metal species rather than the total metal concentration.

It may occur to the reader at this time that water quality characterization for pollution control is an involved expensive task which requires a

lot of manpower. The fact of the matter is that in many cases only a few

measurements were found to be sufficient. Like a physician dealing with

a patient, the water analyst may make his diagnosis of water pollution

based on few measurements. The analyst experienced in water pollution

characterization can often make appropriate decisions based on practiced

intuition.

III. Design of Measurement Systems

Design of measurement systems begins with defining (a) WHY the

analysis is needed (Objectives), (b) WHAT are the parameters to look for (Parameters), and (c) HOW to perform the analysis (Methods).

A. Objectives of Analysis

Definition of the purpose and objectives of analysis is the first step in the design of any measurement system; this includes the definition of particular problems to which solutions are sought. Some of the more common

objectives of natural and wastewater analysis are as follows:

(a) determine the suitability of a water for its intended use and

to establish the degree of treatment necessary prior to its use,

(b) estimate the possible detrimental effects of a waste effluent on the quality of the receiving water for subsequent

downstream use,

(c) evaluate necessary treatment requirements in view of water

reuse,
WATER POLLUTION CONTROL 303

(d) determine the quantities of valuable by-products which

could be recovered from a waste effluent,

(e) evaluate and optimize industrial processes on a continuous

or batch basis, and

(f) provide background information on the present quality of

streams and lakes which can be used to demonstrate future

changes in their quality.

Natural bodies of fresh water can be classified according to intended

use, including: public water supply, fish or shellfish propagation, recreation, agricultural use, industrial water supply, hydroelectric power,

navigation, and disposal of sewage and industrial wastes. Water quality

criteria may be based on the quality to be maintained in a receiving water,

or may relate to the quality of the effluent itself. Criteria relating to the

receiving water are termed "stream standards" while those relating to the

waste discharge are termed "effluent standards." Both types of standards

have their strengths and weaknesses.

Stream standards, which have been developed to comply with the

provisions of the Water Quality Act, permit the full capacity of a stream

to be utilized in the assimilation of wastes. In establishing stream stan- dards the quality of the receiving stream is maintained at a level above

that required for the desired usage. This permits full utilization of the stream's capacity to assimilate wastes and forces materials discharged

into the different streams to be subjected to different degrees of treatment

prior to discharge. In contrast, if effluent standards were used, all discharges of the same type to streams designated for the same use would

be required to receive identical levels of treatment. The level of treatment would be consistent with the capability of advanced technology. The ef- fect of the effluent on the receiving waters would differ and there would

be no assurance that the concentration of pollutant in the stream would

not exceed that critical to the desired use.

It is readily apparent that either system of standards has its flaws. It is likely that future standards will incorporate features of both stream and

effluent standards.

The analysis of wastewaters which are to be discharged to municipal sewers is done principally for the purpose of evaluating compliance with

certain effluent criteria set by the municipality. Effluent standards in this case are established for the purpose of protecting municipal waste treat- ment plants from operational interference which might be caused by in- dustrial waste discharged and for protection of the sewer structure from

damage. Both the municipality and the industry may carry out periodic
304 ANALYTICAL PROBLEMS

analysis of the waste effluent for purposes of control and assessment of

charges, which are usually related to the strength and volume of a particular waste. It is important to point out that wastewater which is discharged

to municipal sewers usually becomes the responsibility of the municipality.

An important objective of many surveys of lakes and streams is to pro- vide a base of information to be used to evaluate future changes in water

quality. Such data have been used by Beeton [2] and others in demonstrating the eutrophication of the Great Lakes. These data are important

sources for other investigators. The U.S. Geological Survey has compiled

data on the quality and quantity of water of surface waters of the United

States and has publications on the quality of surface water for irrigation

and of municipal water supply for industrial use [3,4]. Other water quality data has been published by the Public Health Service and Federal

Water Pollution Control Administration [5].

B. Parameters for Analysis

After defining the objectives of analysis, the next step in the design of

measurement systems is to decide on particular constituents for which

analyses are to be made and what methods are to be employed. The

analyst experienced in water quality characterization can often make the

proper decision based on practiced intuition. In most cases, however, certain rather well defined guidelines should be followed.

Depending on the intended use of a receiving water, the parameters

listed in Table 1 are of significance for water quality characterization, and

this should serve as guidelines for analyses of water quality for purposes

of treatment and control [6]

.

The choice of parameters for analysis depends primarily on the type of

information sought. Certain tests are frequently used for the identification

of various types of pollution associated with industrial wastewaters. For

example, Table 2 lists a number of tests and their significance.

Some of the important and most frequently used tests in the analysis of

water are the nonspecific tests listed in Table 3. These tests often measure

a property of a group of substances as in alkalinity where the capacity of

the water to neutralize hydrogen ions is measured or a physical parameter

such as density or a physiological property such as odor. Many of these

tests are used to determine the suitability of natural waters for industrial or municipal use and to determine the type and degree of treatment needed to render them acceptable.
WATER POLLUTION CONTROL 305

Table 1. Parameters for water quality characterization

A. Domestic Water Supplies

Quality parameter Permissible criteria Desirable criteria

1. Color (Co-Pt scale)

Odor

Taste

2. Turbidity

3. Inorganic chemicals

pH

Alkalinity (CaCOs units)

Ammonia

Arsenic

Barium Boron Cadmium

Chlorides

Chromium (hexavalent)

Copper

Dissolved oxygen

Fluorides

Iron (filtrable) Lead

Manganese (filtrable)

Nitrates plus nitrites (as mg/lN)

Phosphorus

Selenium

Silver Sulfates Total dissolved solids

Uranyl ion

Zinc

4. Organic chemicals

Carbon chloroform extract (CCE)

Methylene blue active

substances

5. Pesticides:

Aldrin

Chlordane

DDT

Dieldrin

75 units Virtually absent

Virtually absent

6.0-8.5

30-500 mg/1

0.5 0.05

1.0

1.0 0.01

250

0.05

1.0 >4.0

0.8 to 1.7 mg/1

<0.3

<0.05

<0.05

<10

10-50 /xg/1

0.01

0.05

250

500

5

55

0.15

0.5

0.017

0.003

0.042

0.017

< 10 units

Virtually absent

Virtually absent

Virtually absent

6.0-8.5

30-500 mg/1

<0.01

absent

<25

absent

Virtually absent

Air saturation

1.0 mg/1

Virtually absent

Absent

Absent

Virtually absent

10/ig/l

Absent

<50

<200

Absent

Virtually absent

<0.04

Virtually absent
306 ANALYTICAL PROBLEMS

Table 1. (continued)

Quality parameters Permissible criteria Desirable criteria

5. Pesticides: (continued)*

Dieldrin 0.017

Endrin 0.001

Heptachlor 0.018

Heptachlor expoxide 0.018

Lindane 0.056

M^thoxyc'hlor 0.035

Organic phosphates plus 0.1

carbamates

Taxophane 0.005

Herbicides 2,4,D plus 0.1 2,4,5-T, plus 2,4,5-TP

6. Radioactivity

Gross beta 1,000 pc/1 < 100 pc/1

Radium 226 3 pc/1 <1 pc/1

Strontium-90 10 pc/1 <2 pc/1

B. Recreation and Aesthetics

The general requirements are that surface waters should be capable

of supporting life forms of aesthetic and recreational values. Hence,

surface waters should be free from

a. materials that may settle to form objectionable deposits or

float on the surface as debris, oil and scum,

b. substances that may impart taste, odor, color or turbidity,

c. toxic substances including radionuclides physiologically

harmful to man, fish or other aquatic plants or animals and

d. substances which may result in promoting the growth of undesirable aquatic life. Presently, there are no well defined water quality criteria for recreation or aesthetic purposes.

C. Aquatic Life, Fish and Wildlife

1. Turbidity:

Discharge of waste in receiving waters should not cause change in turbidity in the order of 50 Jackson units in warm—water streams, 25

Jackson units in warm—lakes, and 10 Jackson units in cold—water streams and lakes.

2. Color and Transparency:

Optimum light requirements for photosynthesis should be at least 10 percent of incident light on the surface.
WATER POLLUTION CONTROL 307

Table 1. (continued)

C. Aquatic Life, Fish and Wildlife (continued)

3. Settleable Matter: Minor deposits of settleable matter may inhibit growth of flora and

biota of water body. Such materials should not be discharged in surface waters.

4. Floating Matter:

All foreign floating matter should not be discharged in surface

waters. A typical pollution problem is that of oil waste discharges

which may result in the formation of

a. visible objectionable color film on the surface,

b. alter taste and odor of water,

c. coat banks and bottoms of water course,

d. taint aquatic biota and

e. cause toxicity to fish and man.

5. Dissolved Matter: The effect of dissolved matter on aquatic biota can be due to toxicity at relatively low concentrations or due to osmotic effects at relatively high concentrations. In general, total dissolved matter should not exceed 50 millimoles (the equivalent of 1500 mg/1 NaCl).

6. pH, Alkalinity and Acidity

The pH range of 6.0 to 9.0 is considered desirable. Discharge of waste effluents should not lower the receiving water alkalinity to less than 20 mg/1.

7. Temperature:

Heat should not be added to a receiving water in excess of the amount that will raise the temperature by 3-5 °F. In general,

normal daily and seasonable temperature variations should be

maintained.

8. Dissolved Oxygen:

It is generally required to maintain a dissolved oxygen level above

4 to 5 mg/1. In cold water bodies it is recommended to maintain the dissolved oxygen above 7 mg/1.

9. Plant Nutrients:

Organic waste effluents such as sewage, food processing, canning

and industrial wastes containing nutrients, vitamins, trace ele- ments, and growth stimulants should be carefully controlled. It is important not to disturb the naturally occuring ratio of nitrogen

(nitrates and ammonia) to total phosphorus in the receiving water.

10. Toxic Matter: Waste effluents containing chemicals with unknown toxicity characteristics should be tested and proven to be harmless in the concentration to be found in the receiving waters. Discharging

pesticides in natural waters should be avoided if possible or kept

below 1/100 of the 48-hrs. TL^ values. Levels of ABS and LAS
308 ANALYTICAL PROBLEMS

Table 1. (continued)

C. Aquatic Life, Fish and Wildlife (continued)

10. Toxic Matter: (continued)

should not exceed 1.0 mg/1 and 0.2 mg/1, respectively, for periods

of exposures exceeding 24 hours.

It should be noticed that the presence of two or more toxic agents

in the receiving water may exert an additive effect.

11. Radionuclides:

No radionuclides should be discharged in natural waters to produce

concentrations greater than those specified by the USPHS Drinking Water Standards.

D. Agricultural Water Use

1. Total Dissolved Solids or "Salinity"

This is the most important water quality consideration since it controls the availability of water to the plant through osmotic pressure

regulating mechanisms. The effect of salinity on plant growth

varies from one type to another and is dependent on environmental

conditions.

2. Trace Elements Tolerance for Irrigation Waters may be Summarized as Follows:

Short-term water use. Element Continuous water use fine texture soil mg/1 mg/1

Aluminum 1.0 20*0 Arsenic 1.0 10.0 Berillium 0.5 LO

Boron 0.75 2.0 Cadmium 0.005 0.05

Chromium 5.0 20.0

Cobalt 0.2 10.0 Copper 0.2 5.0 Lead 5.0 20.0 Lithium 5.0 5.0 Manganese 2.0 20.0 Molybdenum 0.005 0.05

Nickel 0.5 2.0 Sellenium 0.05 0.05

Vanadium 10.0 10.0 Zinc 5.0 10.0
WATER POLLUTION CONTROL 309

Table 1. ^continued)

D. Agricultural Water Use (continued)

3. pH. Acidity and Alkalinity

pH is not greatly significant and waters with pH values from 4.5 to 9.0 should not present problems. Highly acidic or alkaline waters can induce adverse effects on plant growth.

4. Chlorides

Depending upon environmental conditions, crops and iiTigation management practices, approximately 700 mg 1 chlorides is permissible

in irrigation waters.

5. Temperature

\'ery high as well as very low temperatures of irrigation waters can

interfere with plant growth. Temperature tolerence is highly dependent on the t\pe of plant and other environmental conditions.

6. Pesticide

A variety of herbicides, insecticides, fungicides and rodenticides can be present in irrigation waters at concentrations which may be

detrimental to crops, livestock, wildlife and man. As far as effect on plant growth and permissible levels are concerned, these are variable and highly dependent on the type of chemical, type of

plant, environmental factors and exposure time.

7. Suspended Solids

Suspended solids in irrigation waters may deposit on soil surface and produce a crust which inhibit water infiltration and seedling

emergence. In waters used for sprinkler irrigation colloids and

suspended matter may form a film on leaf surface which impair

photosynthesis and defer growth.

8. Radionuclides

USPH Drinking Water Standards are usually applied to irrigation waters.

Some of the more frequemly measured parameters in pollution studies

are listed in Table 4. The specific chemical analyses to be performed in a water pollution study will ultimately depend on the types of materials

discharged and on the desired uses of the receiving water.

C. Methods of Analysis

Following the establishment of the objectives of the measurement pro- gram, and the selection of parameters for analyses, suitable analytical
310 ANALYTICAL PROBLEMS

Table 2. Significance of parametric measurements.

Test or determination

Dissolved solids

Ammonia, nitrites, nitrates,

and total organic nitrogen

Metals

Cyanide

Phenols

Sulfides

Sulfates

Calcium and magnesium

Synthetic detergents

Significance

Soluble salts may affect aquatic life or future use of water for domestic

or agricultural purposes.

Degree of stabilization (oxidation) or organic nitrogenous matter. Toxic pollution.

Toxic pollution.

Toxic pollution, odor, and taste. Toxic pollution, odor. May affect corrosion of concrete,

possible biochemical reduction to sulfides.

Hardness.

Froth, toxic pollution.

Table 3. Non-specific water quality parameters.

Physical parameters

Filterable residues

Salinity

Density

Electrical

conductance

Chemical parameters

Hardness

Alkalinity and acidity

Biochemical oxygen

demand (BOD)

Chemical oxygen

demand (COD)

Total carbon

Chlorine demand

Physiological parameters

Taste

Odor

Color

Suspended matter

Turbidity
WATER POLLUTION CONTROL 311

Table 4. Tests used for the measurement of pollution of natural waters.

Nutrient demand

Dissolved oxygen

Biochemical

oxygen demand

Chemical oxygen

demand

Total carbon

Specific nutrients

Nitrogen:

Ammonia

Nitrate

Nitrite Organic nitrogen

Phosphorus:

Orthophosphate

Polyphosphate

Organic phosphorus

Nuisances

Sulfide

Sulfite

Grease and

oil Detergents

Phenols

Toxicity

Cyanide

Heavy metals

Pesticides

methods are then selected. There are no prescribed procedures which are

applicable to all situations but the best method for any given situation

must be based upon consideration of many factors. Some of the more important factors are: (a) required sensitivity, (b) accuracy of method,

(c) presence of interferences, (d) number of samples to be analyzed,

(e) necessity of field or in situ analyses, (f) speed required for results, (g)

availability of required instruments, (h) number and skill of laboratory

personnel, and (i) required use of standard or referee methods.

Another point for consideration in selecting analytical methods concerns the collection, transportation and storage of samples. Screening

tests should be conducted for purposes of approximating required sample

volumes, establishing desirable sites for and frequency of sampling, and

providing a rough estimate of the waste composition and strength.

Listings of "standard" and "recommended" methods for analysis of

natural waters and wastewaters are to be found in a variety of publications

sponsored by several water works, pollution control, and public health

agencies and organizations in this country and abroad. In addition, in several instances certain private industries have found it desirable to for- malize listings of more specific methods for analysis of particular types of

industrial wastewater.

While general procedures of analysis for specific waste constituents are

highly useful, the analyst must be careful to guard against overreliance

428-999 O - LT - 72 - 21
312 ANALYTICAL PROBLEMS

upon such procedures, and against the possibiHty of being lulled into a

false sense of security by results obtained from application of such

procedures in instances where they may not be applicable. Indiscriminate

application of general-purpose methods for analysis without due consideration of specific interferences and other problems must be avoided.

D. Intensive Versus Extensive Measurement

Physical and chemical characterization of water quality can be

categorized conveniently as intensive versus extensive measurements.

This categorization should not be considered in terms of rigorous ther- modynamic entities but rather in terms of conceptual quantitative properties of the system under investigation. It is for reasons of convenience,

which will become apparent later, that we should differentiate between in- tensive and extensive water quality parameters.

In textbook terminology extensive properties are additive in the sense

that the total value of a system is the sum of the individual values for each

of its constituent parts. Conversely, intensive properties are not additive,

and can be specified for any system without reference to the size of that system. This is illustrated in Table 5. The chemical potential or the molar free energy change (dGldn) t,p is further defined: = In a (1)

Table 5. Extensive and intensive parameters.

System

designation

Gravitational

Thermal

Electrical

Chemical

Intensive

parameter

Height ih)

Temperature (T)

Voltage (E)

Extensive

parameter

Mass im)

Heat capacity (C^)

Charge iq)

Work done by

the system

= mg(h2—hj)

Chemical potential ifi) Number of moles in) =n(yi2~y^\^

where h = height, cm m - mass, g

T = temperature, °C

E = voltage, V

q = charge, C

= acceleration, cm/s^

= heat capacity, cal/deg mol = the chemical potential, cal/mol = number of moles
WATER POLLUTION CONTROL 313

where a is the activity given in a molar scale and /x° is the chemical potential at a reference state. The activity can be related empirically to concentration C by the equation:

where y is the activity coefficient. Accordingly, the activity, a, is an intensive parameter and is a direct measure of the difference between the

chemical potential in the actual and in the reference state. In defining a chemical system it is important to distinguish between in- tensive properties based on chemical potential measurements and extensive properties based on counting the number of moles of a given substance. This can easily be illustrated in comparing data from potentiometric measurements of pH, pX or pM where X and M refer to anions and cations respectively, with those from titrimetric determinations of acidity,

anions or cations. In the former case measurement is based on potential

determinations which are essentially intensive parameters while in the

latter case measurement is based on stoichiometric calculation. Results of

analysis of either type may not agree particularly if interferences are present which may cause the activity coefficient to deviate from unity,

e.g., salting-in or salting-out agents.

Similarly, in the case of dissolved oxygen voltammetric membrane elec- trode systems the measured parameter is essentially an intensive factor,

since the diffusion current is solely dependent on the difference in the

chemical potentials of molecular oxygen across the membrane. The diffu- sion current equation at steady state conditions is as follows:

where id is the diffusion current, z is the number of electrons transferred,

F is the Faraday, A is the cathode surface area, P,„ is the membrane

permeability coefficient, b is the membrane thickness and ao2 is the activity of molecular oxygen.

Dissolved oxygen measurement by the membrane electrode (ME) does

not have to be equal to results obtained by titration methods, such as the

Winkler test. In the former case, the measured parameter is the activity of

molecular oxygen, while in the latter case, the total number of oxygen

molecules in the titrated sample is estimated by stoichiometric calculations. This difference can be easily realized on applying both the membrane electrode and the Winkler test to water samples containing saltingin and salting-out agents, as shown in Figure 3.

(2)

ia=\zFAP,

n

(3)
314 ANALYTICAL PROBLEMS

70.0

60.0

50.0

40.0

30.0

20.0 -

g 10.0,

o

>-X

o

3

o

5

CONCENTRATION

(WINKLER)

ACTIVITY (ME) • •

0.2 0.4 0.6 0.8

XCgHgOH

9.0

8.

7.0

6.0

5.0

4.0

• . •

I I ACTIVITY (ME)

— \

CONCENTRATION

(WINKLER)

ot

25.0

H 20.0

- 15.0

- 10.0

H5.0 2

(a)

1.0

3,0

- 2.5 Q

2.0

1.5

1.0 2.0 3,0 4.0 5.0

KCI mol/l

Figure 3. Effects of (a) salting-in and (b) salting-out. Po2=0.2J atmosphere, temperature = 25±0.5°C
WATER POLLUTION CONTROL 315

Results shown in Figure 3 were obtained with water samples containing

varying concentrations of potassium chloride and ethyl alcohol and were

equilibrated with one atmosphere of air (Po^^O.ll atm) at constant temperature. Being a sparingly soluble gas, oxygen solubility in water follows

Henry's Law and the following expression applies:

where H is Henry's Law constant. Consequently, the test solutions had

the same activity and varying concentrations of dissolved oxygen depending on the type and concentration of the salting-in or salting-out agents.

The results in Figure 3 show that the membrane electrode gave identical

diffusion current values indicating that the test solutions had the same ox- ygen activity. In the meantime in the presence of KCl, the salting-out ef- fect caused a decrease in oxygen solubility with increasing salt concentrations as shown by the results from the Winkler titration. In this case the

activity coefficient, yo^, increased with increasing ionic strength according

to the following relationship:

where Ks is the salting-out coefficient and / is the ionic strength.

Conversely, in the presence of ethyl alcohol, the salting-in effect caused

an increase in oxygen solubility with increasing alcohol concentration as shown by results from the Winkler titration. Under these conditions the

activity coefficient, yoz^ decreased with increasing alcohol concentration.

The above example may serve to explain the controversy in measure- ment of dissolved oxygen in natural and wastewaters as well as in biological fluids. This is exemplified by the present confusion in expressing dis- solved measurement in terms of oxygen tension, percent saturation, partial pressure, etc. Activity measurement, per se, is considered most significant in characterizing biochemical and physicochemical transformations in aquatic environments. For example, oxygen transfer across the

air-water interface or within the bulk of the aqueous phase under constant temperature and hydrodynamic conditions is solely dependent on the

gradient in activity, and not always in the direction of diminishing concentration. Thus, under certain conditions, "uphill" diffusion, counteracting

equalization of concentration may occur. Similarly, in biological systems

the availability of molecular oxygen for biochemical reactions depends

primarily on the activity level and hence respiration rates are more

precisely described in terms of oxygen activity than in terms of concentra-

(4)

(5)
316 ANALYTICAL PROBLEMS

tions. This distinction is significant in the characterization of waste ef- fluents, rivers, lakes and estuaries, although in many applications it may

be possible to assume that the activity coefficient is close to unity and the

difference between activity and concentration is negligible for all practical

purposes.

E. Sampling

The significance of a chemical analysis is no greater than that of the

sampling program. Ideally speaking, a representative sample does not exist. Attempts are made however to come as close as possible to sample

aquatic environment without disturbing its physicochemical and

biochemical characteristics. There is no universal procedure for sampling

applicable to all kinds of natural and wastewaters. The analyst has to design his sampling program based on a complete understanding of the

purpose of analysis and the type of parameters to look for. The most important requirements for a satisfactory sample are that it be

both valid and representative. For a sample to be valid, it has to be one which has been collected by a process of random selection. Random

selection is one of the most basic, yet most frequently violated, principles

in development of a sampling program. Any method of sampling that

sacrifices random selection will impair statistical evaluation of the analytical data. If nonrandom sampling procedures are contemplated — perhaps

for significant reasons of convenience — it is highly desirable to first demonstrate that the results of the analysis check those which would be

obtained by random sampling. This check would be essential prior to any

statistical evaluation of the data. A satisfactory sample is not only randomly drawn, but also is representative. This means that the composition of the sample should be identical

to that of the water from which it was collected; the collected sample

should have the same physicochemical characteristics as the sampled

water at the time and site of sampling.

Planning for a sampling program should be guided by the overall objectives of analysis. Major factors of concern for any sampling program are:

(a) frequency of sample collection, (b) total number of samples, (c) size of

each sample, (d) sites of sample collection, (e) method of sample collection, (f) data to be collected with each sample, and (g) transportation and

care of samples prior to analysis.

Frequency of sampling will depend to a large extent upon the frequency

of variations in composition of the water to be sampled. There are two
WATER POLLUTION CONTROL 317

principal types of sampling procedures commonly used for analysis of

natural and wastewaters. The first type is that which yields instantaneous

spot or grab samples, while the second type yields integrated continuous

or composite samples. A grab sample is a discrete portion of a wastewater

taken at a given time; a series of grab samples reflects variations in constituents over a period of time. The size of such individual samples will depend on the objectives and methods of analysis, and on the required ac- curacy. The total number of grab samples should satisfy the statistical requirements of the sampling program.

Composite samples are useful for determining average conditions,

which when correlated with flow can be used for computing the material

balance of a stream of wastewater over a period of time. A composite

sample is essentially a weighted series of grab samples, the volume of

each being proportional to the rate of flow of the waste stream at the time

and site of sample collection. Samples may be composited over any time

period such as 4, 8, or 24 hours, depending on the purposes of analysis.

Selection of sampling sites should be made with great care. A field sur- vey is often useful in planning for site selection. In the case of sampling of

a stream, special consideration should be given to sources of waste

discharge, dilution by tributaries, and changes in surrounding topography

[7.8].

Sampling of wastewater from pipes or conduits is more complicated

than stream sampling, especially when the water to be sampled is under

pressure. For example, in the case of a chemical treatment plant, selection

of sampling sites may require extensive investigations and preliminary

checking of samples from a number of effluent outlets. Proper positioning

of the sampling outlet within the cross section of a conduit is essential for

obtaining a representative sample, particularly for conduits of large

diameter. The choice of a sampling site within the cross section of a conduit is best done by examining and comparing samples drawn from several

points along the vertical and horizontal diameters of the conduit. The

cross-sectional area of the opening or inlet of the sampling line should be

such that the flow of water in this line is proportional to the flow of the

water in the conduit. An elaborate discussion on sampling of water from

pipes and conduits can be found in the ASTM Manual on Industrial

Water and Industrial Waste Water [9]

.

Wastes discharged by industry are of great variety, and sampling must

be tailored to suit the particular characteristics of a given wastewater.

Sampling procedures can be expected to vary widely from one waste- water to another. Special procedures have been reported for use with

water sampled under reduced or elevated pressure and/or temperature
318 ANALYTICAL PROBLEMS

[10]. Procedures and equipment used for the sampling of waters containing dissolved gases and volatile constituents susceptible to loss upon aeration have been described [10].

A certain amount of precaution is sometimes required in sampling

processes for reasons of safety. For example, strict precautionary measures [11] should be followed in taking samples from deep manholes to guard against accumulation of toxic and explosive gases and insufficiency

of oxygen.

Sampling can be accomplished by either manual or automatic means,

again depending on the purpose of analysis and method of sampling. A

grab sample is usually collected manually. When it is necessary to extend

sampling over a considerable period of time, or when a continuous (repetitive) record of analysis at a given sampling point is required, automatic

sampling equipment is commonly used.

Continuous sampling equipment, correctly designed and installed, will provide more frequent samples, tend to eliminate human errors, and in many cases be economically more feasible. A variety of automatic sampling equipment suitable for water sampling under variable conditions and

for different purposes, is presently available [ 10]

.

The maintenance of complete records regarding the source of the sample and the conditions under which it has been collected is an inherent

part of a good sampling program. This is of particular importance in field,

river, or in-plant surveys, where a great number of samples are collected

from different sources and under variable conditions. For illustrative pur- poses, the U.S. Geological Survey has defined the minimum data required

for samples of surface and groundwaters [11] (see p. 3 19). One of the most important aspects of the sampling process is the care and preservation of the sample prior to analysis. This point cannot be

overemphasized. A water analysis is of limited value if the sample has un- dergone physicochemical or biochemical changes during transportation or

storage. These changes are time dependent, but they usually proceed

slowly. In general, the shorter the time that elapses between collection of a sample and its analysis, the more reliable will be the analytical results. Certain constituents may, however, require immediate analysis at the

sample site. Certain determinations are more sensitive than others to the method of

handling of water samples before analysis. Changes in temperature and

pressure may result in the escape of certain gaseous constituents (e.g., O2,

CO2, H2S, CI2, CH4), or the dissolution of some atmospheric gases (e.g.,

O2). It is recommended, therefore, that determinations for gases be done

in the field, or, to "fix" such materials as O2, CI2 and H2S, the sample ^
WATER POLLUTION CONTROL 319

Minimum data requirements for water samples.

Surface Waters Name of water body

Location of station or site Point of collection

Data of collection

Time of collection

Gage height or water discharge

Temperature of the water Name of collector, and

Weather and other natural or other man-made factors

that may assist in interpreting

the chemical quality

Ground Waters Geographical and legal locations

Depth of well

Diameter of well

Length of casing and positions of screens Method of collection

Point of collection Water bearing formation(s)

Water level Yield of well in normal operations Water temperature

Principal use of water Name of collector

Date of collection

Appearance at time of collection, and

Weather or other natural of man-made

factors that may assist in interpreting chemical quality

should be treated upon collection with stable oxidizing or reducing agents.

It is also recommended that the temperature and pH of the water be deter- mined at the site of sampling. Changes in temperature and pH may cause changes in the solubility of dissolved gases and certain nonvolatile constituents, resulting in their separation from aqueous phase. Carbonic acidbicarbonate-carbonate equilibria may be shifted to release gaseous CO2,

or to precipitate certain metal carbonates. Similarly, shifts in hydrogen

sulfide-sulfide equilibria due to changes in pH and/or temperature may

result in the escape of H2S or the precipitation of metal sulfides. Heavy metals ions may undergo a variety of physicochemical transfor- mations during sample handling. It has been recommended that for analyses for Al, Cr, Cu, Fe, Mn and Zn, samples should be filtered at the

site of collection and acidified to about pH 3.5 with glacial acetic acid

[11]. Acidification tends to minimize precipitation, as well as sorption on

the wall of the sample container. Since acetic acid may stimulate growth
320 ANALYTICAL PROBLEMS

of molds, it may be necessary to add a small quantity of formaldehyde to the sample solution as a preservative.

Another major point of interest for handling water samples is the effect of biological activity on the sample characteristics. Microbiological activi- ty may be effective in changing the nitrate-nitrite-ammonia balance, in reducing sulfate to sulfide, in decreasing the dissolved oxygen content,

biochemical oxygen demand (BOD), organophosphorous compounds and

any readily degraded organic compound. Freezing of water samples is helpful in minimizing changes due to biological activity. Certain chemical

preservatives, such as chloroform or formaldehyde, are sometimes added

to water samples for this purpose.

That it is practically impossible to handle and process a water sample

without changing its characteristics should be recognized. The best

chance for error-free procedure lies in the use of in situ analyses. In the

end, the dependability of even a well planned sampling program rests upon the experience and good judgment of the analyst.

IV. Water Quality Surveillance Programs

Pollution control activities rely heavily on the ability to monitor and

survey the quality of natural and wastewaters. This includes rivers, lakes,

estuaries, as well as industrial and domestic waste effluents. Presenfly,

there are several state and federal water surveillance programs designed

to (a) acquire, evaluate and disseminate information on the quality of

waters for and from the varied local, state, interstate and federal agencies

together with educational, commercial, industrial and individual entities,

(b) determine the long term trends and variations of water quality, and (c) provide a rapid intelligence system for the preservation of the waters and

the protection of the water users, including compliance with water quality

standards.

Water quality surveillance activities may be based on manual surveillance, aerial surveillance, public surveillance and automatic surveillance

techniques.

A. Manual Surveillance

Manual surveillance is based on the periodic collection of water samples, their transportation and storage and subsequent laboratory analysis.
WATER POLLUTION CONTROL 321

Sampling sites and frequency are selected to give an overall evaluation of

water quality in drainage basins and effects of waste discharge.

B. Public Surveillance

In an attempt to increase the scope of the overall surveillance program

certain states [12] initiated public surveillance programs, sometimes

referred to as the "water watchers." Similar to traffic safety patrols for school children, citizen groups are encouraged to watch for the safety of

water resources and to telephone and report abnormal conditions of streams or lakes, e.g., oil or visually apparent chemical spills and fishkills.

C. Aerial Surveillance

Photography and remote sensing from aeroplanes are being used to gather information on (a) materials on the water surface, e.g., oil spills, (b)

suspended matter in the water, and (c) certain soluble compounds. Measurement is based on the interactions of electromagnetic energy with

matter floating, suspended or in solution. The electromagnetic energy

source may be the sun, in which case sensors depending on solar reflec- tions are referred to as "passive" systems, or the energy source may be an

"active" source, e.g., laser [13]. Applications of "passive" airborne

systems, the measurements in the ultraviolet, visible and infrared regions

of the electromagnetic spectrum have been recently reported in the litera- ture [13-15].

A number of chemical pollutants can be detected by this technique

based on their fluorescence properties. This includes a variety of compounds such as chlorophyll, phenol, ligninin-sulfonates, Rodamine WT

and oils. Fluorometric techniques seem to be more applicable to airborne

remote sensing than absorption techniques. Beside being more sensitive

than absorption techniques, fluorometric procedures were found to be

less affected by turbidity and quite useful in the analysis of mixtures [ 15]

.

As low as 5 ppb of Rodamine WT were detected using airborne

fluorometric measurement [15]. In this case, the sun was used as the excitation source; hence, the procedure was limited to daylight hours.

Remote multispectral scanning techniques have been used for the measurement of various pertinent water quality parameters. Temperature

measurement are usually made in the 8 - 13.5 nm region throughout the
522 ANALYTICAL PROBLEMS

day and night to an accuracy of 0.5 °C [13]. This was used advantageously in thermal pollution measurement which provided the abili- ty of a complete instantaneous mapping of large water areas. Detection of oil spills can be easily done with airborne remote sensing

using IR and UV multispectral scanning [14]. This is considered to be a very powerful technique because of its ability to follow the movement of

the oil slicks over large areas of water surface.

The University of Michigan Willow Run Research Laboratory

developed a unique multispectral system [13,14] with the ability to ex- amine a scene in 17 bands distributed between 0.3 and 1.35 nm (i.e., a 17

channel spectrometer with electronic processing capabilities). This

system was used for aerial surveying of oil spills and industrial waste

discharge in lakes, rivers, etc. The system is capable of automatically

determining the optimum wavelength, and in addition, the interactions of

radiation with the effluent at other positions in the electromagnetic spec- trum are also determined. Each effluent type and receiving body of water

has its unique spectral characteristics which are used to differentiate it from other sources. Aerial surveillance by multispectral remote sensing techniques seems

to have a great potential in pollution monitoring. Such monitoring systems

may not be able to provide information on the chemical composition of

the system under investigation. Nevertheless, it is considered to be a valuable tool in the study of the dynamic conditions in aquatic environments

and large scale water resources investigations.

D. Automated Surveillance Systems

Automated surveillance systems rely on the use of automatic, unat- tended measurement procedures operated on a continuous basis or inter- mittently at a determined frequency. Automatic water quality monitoring

systems are being widely used in this country and abroad in a variety of

water quality management programs [12,16-18]. The main advantages in using automated systems lie in the ability of maintaining a continuous, in- stantaneous record on water quality in rivers, lakes and estuaries, industrial effluents, etc. Changes in water quality may occur suddenly as a

result of intense storms, industrial spills, etc. In certain cases changes in water quality undergo rapid variation such as dissolved oxygen and salini- ! ty changes from natural tidal fluctuations. ' Manual surveillance techniques seem to be inadequate and certainly not practical to monitor sudden changes in water quality.
WATER POLLUTION CONTROL 323

The main objectives of automatic water quality monitoring activity are

to (a) detect violations of standards or other undesirable quality conditions so that remedial action can be taken quickly, (b) the establishment of

water quality baseline and trends, (c) verify and provide data for predictive calculations on water quality including computerized mathematical

models for the purpose of establishing design and operational criteria for

water pollution control facilities, and (d) determine short-range water

quality trends for program management and public information purposes.

Selection of monitoring sites is usually made at places where (a) rapid

fluctuations in water quality or quantity, (b) high potential for accidental

spills of wastes, and (c) hydrologic or other conditions which permit continuous evaluation of the quality response of the stream system to waste

discharges and other pollutional impacts.

1 . System Characteristics

There are two main approaches for water quality measurement in auto- matic monitoring systems. The first approach is based on in situ measurement by means of electrochemical transducers without altering

the physicochemical characteristics of the test solution. The second ap- proach is based on the use of automated repetitive wet analytical

procedures in which chemicals are added to the test solution and measure- ments are made after suitable chemical reactions have taken place, e.g., the Technicon AutoAnalyzer.

Monitoring systems in which the measurement is done by transducer

systems (without the addition of reagents) are being used widely for monitoring water quality of rivers, streams, estuaries and industrial and

domestic waste effluents. These systems are usually capable of measuring

temperature, electrical conductivity, dissolved oxygen, turbidity, pH,

sunlight intensity, chlorides, oxidation-reduction potential (ORP) and

alpha and beta radioactivity. Certain parameters are measured for specific

applications, e.g., the monitoring of fluoride ions, residual chlorine,

nitrates and hardness in drinking water supplies [19] and monitoring cyanides, sulfides, copper ions in certain waste effluents. Electrochemical sensors used for water quality monitoring systems can

be categorized based on the type of measurement, i.e., (a) conductometric, (b) potentiometric, and (c) voltammetric sensors. Table 6 lists examples of sensor systems used in water quality monitors and the underlying

basic relationship.
324 ANALYTICAL PROBLEMS

Table 6. Electrochemical sensors. Type

n

a. Conductometric L

b. Potentiometric Em

constant +

1. Glass electrode pH log %+

2. Inert metal electrode pE = -log o^jA 2. 3 RTF" 1) (redox potential)

membrane electrodes

c. Voltammetric membrane

electrodes (dissolved

oxygen)

specific conductance

K = cell constant c. = ionic concentration

A. = ionic equivalent conductance

z. = ion valency

measured electrode potential

F = the Faraday constant

selectivity coefficient = diffusion current A = electrode surface area P = m

membrane permeability coefficient

b = membrane thickness

Cationic = pM+ = -log

3. Potentiometric Anionic = pA = -log a^-
WATER POLLUTION CONTROL 325

Electrical conductivity is a gross estimate of the ionic strength of the

test solution. It is dependent on the ionic concentration a (number),

equivalent ionic conductance X,, ionic charge Zi and water viscosity. Conductance measurement cannot be used as an indication of the concentration unless all other variables in the conductance equation are held constant. Consequently, equality in electrical conductance of two different

waters may not mean equality in the total dissolved solids. Attempts are being made to incorporate the newly developed potentiometric membrane electrodes (selective ion electrodes) in water quality

monitoring systems [ 19] . Table 7 gives a listing of commercially available

electrodes with some of their properties [20] . It should be noted that the

measured potential is a function of the activity of free ions and these elec- trodes are insensitive to complexed ionic species. This is one of the main

advantages of these systems since this property can be used to dif- ferentiate between free and complexed metallic species.

Table 7. Potentiometric membrane electrode.

Bctrode system pH range Principal interference

Bromide 0 - 14 CN", r, s^-

Cadmium 1 - 14 Ag+, Hg2+, Cu2+, Fe2+, Pb^^

Calcium 5.5- 11 Zn2^ Fe2^, Ph^\ Cu^\ Ni^^

Chloride 0 - 14 Br", r, S^", CN", SCN", NHg

Cyanide 0 - 14 s^", r

Cupric 0 - 14 Ag-^, Hg2+, Fe^-^ Fluoride 0 - 8.5 OH"

Iodide 0 - 14 S^" , CN"

Lead 2 - 14 Ag+, Hg^+, Cu^-^, Cd^+, Fe^+

Nitrate 2 - 12 1" , Br" , S^" , NO2 , CN" , HCO3

cr, OAc", C0|", S0|", S O3"

Sulfide 0-14

Hardness 5.5 - 11 Zn^^, Fe^^, Cu^^, Ni^^, Ba^^, Sr^^
326 ANALYTICAL PROBLEMS

In cases where kinetics of interactions between free and combined ions

are fast, equilibrium relationships can be used in conjunction with Nemst

equation to measure free, combined and total species concentration [21]

.

For example, free sulfides [S^"] alone may be measured by the membrane

electrode. But if the pH is known, the following equilibrium relationships can be used to calculate [HS"] , [H2S] and total sulfides [St] , log [Sr] = log [S2-] + log

log [H2S] = log [S^-] + log^p^ (7)

log[HS-] = log[S2-] + log^^ (8)

Voltammetric membrane electrodes for dissolved oxygen measurement

of the galvanic or polarographic types are widely used in water quality

monitoring systems [22,23]. The electrode diffusion current is linearily

proportional to the activity of dissolved oxygen in the test solution. This

relationship is dependent on the membrane thickness "Z?" and permeability coefficient "F

In monitoring systems the most critical part is the sensor and the relia- bility of measurement is usually dependent on the reliability of the sensor

system. This is true whether the sensor is an electrode, a thermistor or a

photoelectric cell. A clear understanding of the operation characteristics

of the sensor and its dynamic response is essential. This is based on

proper calibration, servicing, maintenance and alertness for small clues

that may indicate malfunction.

Primary sensor characteristics are defined in terms of (a) sensitivity, (b)

response time, (c) selectivity, (d) long term stability, (e) accuracy, and (f) precision. Secondary sensor characteristics are those which define the en- vironmental effects, e.g., (a) temperature, (b) flow, (c) ionic strength, (d)

pH, (e) sunlight, etc. Primary Sensor Characteristics:

Sensitivity is usually defined in terms of the smallest change in the measured variable that causes a detectable change in the indication of the in- strument. It specifies the lower limit of detection of the sensor. Sensitivity

is directly proportional to the slope of the curve relating the signal magnitude to the amount of detectable material present. This will reflect directly on the ability to ascertain a difference between the signal and
WATER POLLUTION CONTROL 327

background noise at the detection limit, i.e., given adequate precision, the

greater the sensitivity, the better the detectability.

The limit of detection of analytical method is the lowest concentration

whose signal can be distinguished from the blank signal. This value depends on the sensitivity of the method, as well as the signal-to-noise ratio required to discern the response due to a sample. Advances in electronics

have brought about the design of instruments with greater inherent stabili- ty and, therefore, lower limits of detection. Use of an on-line digital computer in fast-sweep derivative polarography has permitted the resolution

of closely spaced peaks and extended the analytical sensitivity of the

technique by more than an order of magnitude.

The speed of the sensor response to changes in the test solution is referred to as the "response time." It is an indication of the time needed

for the sensor signal to follow 90, 95 or 99 percent of instantaneous full scale change in the measured variable. The response time should be

specified for each sensor indicating whether it is dynamic or static sensor

response.

Selectivity of the sensor refers to the effect of interferences resulting

from detectable ions or molecules other than the species of interest. Since

all sensor systems cannot achieve absolute or 100 percent selectivity,

then it is important to specify the selectivity limitations in a given test solution. If the type and amount of the interfering species are known, then

it is possible to incorporate the term "selectivity coefficient" in the sensors sensitivity expression. Also, in certain cases it is possible to incorporate interferences effects in the sensor calibration curve. This can be

done by means of the standard addition technique where known amounts

of the measured ions are added to the test solution and the proportional

signal values are recorded.

Long term stability usually refers to the change in the sensor's per- formance characteristics with time. This is used to decide on the frequency of checking the calibration or servicing the sensor. Long term stability

is a property of the particular system and is dependent on the presence of

interferences and the physicochemical characteristics of the test solution.

Deviations of results by a given sensor from the "true" value define the

accuracy of the system. If the source of error is found, and it is possible to correct for, this is called "determinate error." If the deviation from the

true value is compounded indiscriminately by many small errors, it is simply a "random error." Random errors are subject to statistical treat- ment of the data. Precision is defined in terms of the reproducibility of the sensor measurement. The more scatter in successive readings, the less precise are the

428-999 O - LT - 72 - 22
328 ANALYTICAL PROBLEMS

measurements. Usually, precision is closely identified with random errors and statistical theories.

An important distinction between precision and accuracy is that accurate measurements are always precise, but the converse is not necessarily

true. The precision of a series of measurements may be good, but every

result may be higher than the true value because of an unsuspected inter- ference. Only when determinate errors are minimized will precision also

imply accuracy.

Secondary Sensor Characteristics:

Secondary sensor characteristics refer to the effect of environmental

variables. This can be a result of changes in the sensors primary characteristics or changes in the physiochemical characteristics of the test solution. For example, temperature effects on conductance measurement are

quite complex since the temperature coefficient is dependent on both

ionic strength and temperature. The conductivity of sea water was found

to increase by 3 percent per degree increase in temperature at 0 °C, 2 per- cent increase at 25 °C and about 5 percent increase at 30 °C. It is therefore advisable to measure relative conductance rather than absolute

values [24]. This is done by measuring the ratio of the conductance of the

test solution to that of a reference solution at the same temperature.

Thermistors or resistances can be used instead of the reference solution.

It is always advisable to establish the primary and secondary sensor

characteristics for each sensor independently before using it for field applications. Not only these characteristics will vary from one sensor type

to another, but also differences between two sensors of the same type and

from the same manufacturer may occur. The second main type of monitoring systems is based on automating

wet analytical techniques, e.g., the Technicon AutoAnalyzer. Such

systems are essentially capable of automatic sampling, filtering, diluting,

reagent addition, mixing, heating, digesting, and after appropriate delay

for color development, colorimetric measurement are done. All these

steps, which are usually done by an analyst, are automated and performed on a stream of samples moved by a fixed speed peristallic pump.

This technique finds its widest application in laboratory operations

where large samples of waters are handled daily. There have been certain

attempts however to use this technique for water quality monitoring

where the AutoAnalyzer is kept in a trailer on a river bank [25] . In other

applications autoanalyzers were used for monitoring silicates and nitrates

in sea water off the coast of California [26] , and for monitoring nutrients

concentrations in Lake Erie [27]

.
WATER POLLUTION CONTROL 329

Table 8. Parameters measured by the Technicon CSM 6. Parameter Nominal range^ Detection

(ppm) (ppm)

Phosphate 0-8 0.08

Chromium (hexavalent) 0-5 0.05

Copper 0-10 0.10

Iron 0-10 0.10

Ammonia 0-10 0.10

Methyl orange alkalinity^ 0-500 5.0

Thymol blue alkalinity*^ 0-100 1.0 Hardness*^ 0-300 3.0 Sulfate^ 0-500 5.0

Phenol 0-5 0.05

Cyanide^ 0-3 0.03

Chemical oxygen demand 0-500 5.0

Chloride^ 0-10 0.10

Nitrite 0-1.5 0.015

Nitrite + nitrate 0-2.0 0.02

Fluoride^ 0-2.5 0.025

Orthophosphate 0-10 0.10

Silicate 0-15 0.15

^ From reference 25

^ Alternate ranges optional

^ Deviates from linearity

Sulfate is only available on continuous monitoring applications

One of the main advantages of applying the "AutoAnalyzer" for water

quality monitoring purposes lies in its abihty for measurement of a large

number of parameters. Table 8 shows a listing of water quahty parameters

that can be measured by such systems [25]

.

The application of the "AutoAnalyzer" for water quality monitoring

purposes has been faced with a number of Hmitations; (a) the effect of tur- bidity on colorimetric measurements, (b) these systems need more
330 ANALYTICAL PROBLEMS

frequent attendance for servicing, and (c) AutoAnalyzer systems seem to have a higher capital cost and running expenses in comparison to the

transducer-type monitoring systems.

2. Data Acquisition

Automatic surveillance systems generate large volumes of data. Careful

planning in data management is essential to the successful operation of

these systems.

The planning for data management should be based on the need for the

data, the frequency with which the data should be collected, the optimum

locations of collection, the provisions for data storage and the resources

for analysis of data. Depending on the size of the monitoring systems, a variety of computer

programs have been developed to control the operation of the system and

for data handling [28-3 1] . Computer programs to control the operation of

the system are designed to: interrogate each monitor, collect the data,

check for abnormal operating conditions in the monitor, compute for any

unusual rates of change of the incoming data, transmit functional com- mands to the monitors, display alert and alarm conditions on the remote

terminal, and allow human intervention when desired. In view of the large

volume of data generated by these systems, it is essential to use computers for data reduction, storage and retrieval. Computer programs permit data to be retrieved in the form of individual

values by date and time of collection or summarized in the form of central

tendency dispersion (maximum, minimum, arithmetic mean and standard

deviation) and percentiles for each location or a group of locations. Computerized annual summaries are printed routinely for widespread distribution to water users and other interested persons [28,3 1]

.

3 . System Appraisal

Effective implementation of water pollution control programs rely

heavily on adequate automatic water quality monitoring. Measurement by

manual systems is limited by the frequency with which samples can be

collected for analysis. The time delay between sampling and analysis can

result in certain changes in the sample characteristics causing it to be less representative of the body of water from which it was collected. The time

involved in making and reporting the analysis is time lost before any water

pollution corrective action can take place. In addition, based on the

availability and cost of labor, automatic monitoring systems are considered to be the least costly system.
WATER POLLUTION CONTROL 331

As stated earlier, the limiting step in automatic water quality monitoring

systems is the sensor system. There are only few sensor systems available

at the present time which are used for measurement of a limited number

of parameters. There is a pressing need for the development of sensor systems for parameters such as phosphates, trace metals (Pb, Cu,

Cd, As, Zn, Hg, etc.) and trace organic matter (pesticides, carcinogens,

phenols, etc.). The reliability of a sensor system is determined by its performance

characteristics under field conditions and the ability to compensate for

environmental variables such as temperature, hydrostatic pressure and

hydrodynamic effects. These sensor systems should also exhibit a certain

amount of long term stability, in order to minimize the frequency of servicing and checking the monitor.

It is also recommended to initiate research programs for adapting

recently developed sensor systems for water quality monitoring purposes.

A typical example is the incorporation of "selective ion electrodes" in present water quality monitors which would significantly increase their

measurement capabilities. Attempts should be made to utilize in situ separation and concentration techniques to increase the sensitivity and

selectivity of certain sensor systems, e.g., voltammetric membrane elec- trodes and in situ anodic stripping voltammetry.

V. Analytical Methodology

Analytical procedures for measurement of water quality characteristics

have been arbitrarily classified according to (a) the purpose of analysis

{e.g., biodegradability and toxicity tests), (b) the nature of the constituent

under test (e.g., gases, alkali metals, and heavy metals), and (c) the nature

of the analytical procedure itself (e.g., titrimetric, gravimetric, and elec- trometric). In this presentation, methods of analyses are classified according to the analytical information sought and whether it pertains primarily

to the physical, chemical or biological characteristics of the test solution.

The discussion includes a brief assessment of present measurement capabilities and future needs.

A. Physical Characteristics

Physical water quality characteristics of major significance are (a) density and viscosity, (b) surface tension, (c) temperature, (d) electrical conductance, (e) particulate, volatile and dissolved matter, and (f) radioactivity.
332 ANALYTICAL PROBLEMS

Adequate analytical procedures are presently available for measure- ment of the above physical characteristics. One of the physical parameters which is frequently neglected in water quality measurement programs

is surface tension. Measurement of surface tension can provide a very

sensitive indication of the presence of invisible surface films of oil or sur- face active material. The main reason for not using surface tension measurement is perhaps due to the absence of applicable monitoring

procedures for surface tension.

B. Physiological Characteristics

Physiological characteristics of water quality include (a) color, (b) odor,

(c) taste, and (d) turbidity. These parameters represent human physiological responses to physicochemical characteristics of water quality. Consequently they are considered to be most difficult to measure and in certain cases, cannot be expressed quantitatively. This is particularly true whenever human optical or olfactory senses are used to measure color or odor of water samples.

1 . Color

Color is a physiological response to physical optics. Until recently

color measurement has been based on visual comparison of the color of

water samples with standard colored solutions {e.g., platinum-cobalt color

solutions) or colored glass discs. Color determinations by visual comparison are subject to a number of

interferences and variables. The main drawback to this method is the subjectivity and variation in response of different individuals to color. It is obvious also that certain waters may have colors which cannot be

matched well by the standard platinum-cobalt scale. A more accurate determination of color can be accomplished by application of the tristimulus colorimetry technique in which color can be ex- pressed in terms which approximately describe the visual response of an

individual. One of these terms relates to the brightness and the degree of

saturation (pastel, pale, etc.) by purity. Luminosity and purity are usually

reported in units of percent, and the dominant wavelength in nanometers.

Tristimulus parameters are commonly determined from measurements

of the light transmission characteristics of a filtered water sample. Trans-
WATER POLLUTION CONTROL 333

mission data are converted to color classification terms by using stan- dards adopted by the International Commission on Illumination [32].

Chromaticity diagrams are used to describe the color numerically in terms

of the tristimulus parameters [33] .

2. Odor

Odor is a human physiological response to odorant volatile matter that stimulates smell in man.

Determination of odor is based solely on the olfactory senses of the

analyst, or on those of a group of individuals, and on the ability of the

analyst (or group) to distinguish between different levels and kinds of

odors. The testing is based entirely on arbitrary comparison since no absolute units or base for odor exist [33,3*4].

Several authors have attempted to characterize and classify the origin

of odor in natural and wastewaters [34-39]. Most of these studies treat

taste and odor as closely connected human responses. Taste determinations using human subjects are generally not recommended especially in cases of wastewater or untreated industrial effluents, and thus are ex- cluded from the present discussion.

Odor can always be related to the presence of volatile organic and/or in- organic species present in water. Odor intensity is a function of the

volatility and the concentration of the odor-causing species, as well as of

certain environmental factors such as temperature, ionic strength and

pressure. It has been claimed that there are only four basic types of odor:

(a) sweet, (b) sour, (c) burnt, and (d) goaty, realizing that the many odors

are in fact combinations of two or more of these groups.

Odors often can be related to the presence of certain biological forms in the wastewater, such as algae and actinomycetes. Such odor-causing or- ganisms are believed to secrete characteristic volatile oils during growth,

and upon decomposition and decay. Such poetic terms as musty, earthy,

woody, moldy, swampy, grassy, fishy, and wet-leaves have been used to describe odors [4 1 ,42]

.

Recent studies of odor characteristics and human response have led to a proposal of a steriochemical theory of odor [35,41]. This theory relates the response to odor to the geometry of molecules. It has been postulated

that the olfactory system is composed of receptor cells of certain different

types, each representing a distinct "primary" odor, and that odorous

molecules produce their effects by fitting closely into "receptor sites" on
334 ANALYTICAL PROBLEMS

these cells. This concept is similar to the "lock and key" theory used to explain certain biochemical reactions; e.g., enzyme with substrate, an- tibody with antigen, and desoxyribonucleic acid with ribonucleic acid in protein synthesis.

Seven primary odors are distinguished [44], each of them by an ap- propriately shaped receptor at the olfactory nerve endings. The primary

odors, together with reasonably familiar examples are (a) camphoraceous,

e.g., camphor or moth repellent, (b) musky, e.g., pentadecanolactone as

in angelica root oil, (c) floral, e.g., phenylethyl methyl ethyl carbinol as in roses, (d) pepperminty, e.g., methone as in mint candy, (e) pungent, e.g., acetic acid as in vinegar, and (g) putrid, e.g., butyl mercaptan as in rotten

eggs. A classification of odor by chemical type is shown in Table 9 [36].

It has been claimed that every known odor can be made by mixing the seven primary odors in certain combinations and proportions [36].

Odors resulting from mixtures of two or more odoriferous substances

are extremely complex. The mixture may produce an odor of greater or

lesser intensity than might be expected from summing the individual

odors, or a completely different kind of odor may be produced [35-37].

Accordingly, it is frequently necessary to characterize the odor of the

wastewater and that of the receiving stream both separately and in combination if the actual relationship and effect are to be determined.

Odor intensity is expressed in terms of the threshold odor number

[34,35]. By definition, the threshold odor number is the greatest dilution

of the sample that still leaves a perceptible residual odor. The test procedure is based on successive dilution of a sample with odor-free

water, disregarding any suspended matter or immiscible substances, until a dilution is obtained which has a barely perceptible odor. It has been

recommended that odor tests be run at 25 °C and 60 °C [33] or 40 °C and

60 °C [34]. In all cases the sampling and test temperature should be re- ported, since the threshold odor is a function of temperature. A given

sample, under fixed conditions, will emit a characteristic odor stimulus,

but the response to this stimulus and the judgment based upon this response are purely subjective matters, and their interpretation may vary

considerably from individual to individual [35,38,39]. Consequently, it is desirable to use a panel or group of judges, rather than a single analyst for

both qualitative and semiquantitative evaluation of odors in water or wastewater samples [33].

Quantitative measurement of odor causing matter seems to be the only

exact approach for odor measurement. Gas chromatographic procedures

have been used to identify and measure the quantities of odors causing

substances in water samples [41]. A positive identification is accom-
WATER POLLUTION CONTROL 335

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336 ANALYTICAL PROBLEMS

plished once a correlation is established between the isolated substance

and its human odor sensation. Additional research work is needed to reduce these techniques to a practical level for day to day operations and

automatic measurement procedure applicable for monitoring purposes.

3. Turbidity

The term turbidity is commonly used to signify a visual response to the

absorption and scatter of light by suspended matter in a given water. Accordingly, turbidity represents a physiological response to physical optics

similar to color. Turbidity is measured in terms of the amount of light scattered and/or

the light absorbed by matter suspended in the water. The classic

procedure for turbidity measurement is based on the preparation of a series of standard suspensions and comparison with the test solution using a Duboscq type comparator.

Measurements are usually done in reference to a standard suspension

of fine silica [33]. The standard procedure for the measurement of tur- bidity [34] is based on the use of "Jackson Candle Turbidimeter." The

test is based on measuring that length of light path through the solution at which the outline of the flame of a standard candle becomes indistinct. Results are arbitrarily reported in "Jackson Turbidity Units." This is done in reference to a standard silica suspension, or a formazin polymer

suspension which is easier to prepare than the silica suspension.

The turbidity of a given water is a function of both the amount of light

absorbed and light scattered by the sample. Light scattered by suspended

solids depends on the number, size, shape and refractive index of the particles as well as on the wavelength of the exciting light. If the number of

particles in suspension is small, particles act as independent scatterers and

the measured scatter is proportional to the particle concentration. At

higher particle concentrations the light scattered is rescattered and inter- particle interference occurs. Turbidity measurement is usually made by light absorption and light scatter techniques. Light absorption measurements are usually applied to water with high turbidity.

The term "optical turbidity, J" is frequently used [43] as a measure of

turbidity in terms of light scattered by a test solution in a given cell light

path. This is defined as follows:

I = he-''^ (9)
WATER POLLUTION CONTROL 337

or

\og {hlI)^ = A^=T (10)

where h and / are intensities of incident and transmitted light and b is length of light path. Light scatter measurements are usually made at 90 ° to the incident light beam with a precision in the order of 1 percent.

Waters of turbidity in excess of 1000 Jackson units are diluted prior to measurement. For waters of low turbidity (less than 25 Jackson turbidity

units) light scattering techniques are most commonly used [33,34]. Black

and Hannah [43] have discussed the theoretical and procedural aspects

of turbidity measurements with the Jackson Candle method and more so- phisticated methods. A simple low-angle photometer that may be

calibrated with clay suspensions in terms of Jackson turbidity units is described and recommended for use with low turbidity waters. Several

commercial turbidity monitoring systems are available, and have found

wide use for monitoring water quality [44]

.

New developments in turbidity measurement should stress the following areas (a) the effect of color of the test solution, (b) instruments capable

of measurement over a wide range of turbidity, and (c) a correlation

between turbidity and human visual responses.

C. Analysis for Metals

1 . Significance

Metal analysis for water pollution control is usually concerned with (a) alkaline earths (hardness metals, e.g., Ca and Mg), (b) iron and manganese, and (c) trace metals {e.g., Cu, Cd, Pb, Zn, Cr, Ni, Mo, Hg, and

As). The most challenging aspect in metal analysis is considered to be

species characterization at the concentration levels present in the aquatic

environment. This is particularly true with trace metals.

Metals such as Cu, Cd, Pb, Zn, Cr, Ni, Hg, and Mo are frequently

found in the aquatic environment in the part per billion range (lO-^M to lO-^^M). Some of these metals play a significant role in biologically

mediated reactions in natural waters. In trace quantities, many of these

metal ions serve as essential micronutrients for enzymatically mediated

transformations, but in high concentrations they may exert inhibitory or
338 ANALYTICAL PROBLEMS

toxic effects on biological systems. Lead and mercury, in particular, are

toxic to biological systems and are considered to be direct signs of man- made pollution.

It has been generally assumed that the availability of metal

micronutrients and the toxicity of metals to biological systems are solely

dependent on metal concentration. This has been the basis for establishing tolerance limits and water quality standards.

This assumption is not quite valid, since the availability of metal

micronutrients to biological systems and metal toxicity effects are highly

dependent on the degree and type of metal complex formation. The

biological response to trace metals in aquatic environments is greatly in- fluenced by the presence of metal binding ligands of natural or pollutional

origin and whether the metal species are in solution or in the form of colloidal particulates.

Trace metals in the aquatic environment also may serve as "markers"

to provide a finger-print identification of water masses. The distribution

and relative abundance of metals may offer valuable information on the

geochemical history and pollutional characteristics of natural waters. Transport dynamics of metals between the hydrosphere and biosphere,

lithosphere and atmosphere provide pertinent information on the

geochemical cycle and the effect of man's activity.

Trace metals in public water supplies have great public health significance. It has been known that trace metals are necessary consitutents

in many metabolic processes in the human body. Such metals as Cu, Pb,

Cd, Co, Zn and Mo have been found in the human body in trace quantities, although their function and effect are not yet known. However, the

presence of excessive quantities of such metals in human water intake causes toxic effects. In spite that the normal daily metal intake from food

and drinking water may not seem harmful, the human body has the capacity to concentrate some of these metals to a point where it may cause a "long-term" toxic effect. Insofar as the water supply industry is concerned, trace metal concentration can be used as a diagnostic criterion to determine the adequacy of

the water source and the safety of the distribution system. This has been

particularly useful in detecting signs of certain industrial pollution of the

water source and corrosion problems in the water distribution system.

In recent years there has been a strong interest in the analysis of trace metals in drinking water. This has been particularly significant in certain

epidemiological studies which infer a relationship between trace metal

content in drinking water and cardiovascular diseases, dental caries in school children and certain diseases of the bone.
WATER POLLUTION CONTROL 339

2. Metals in Aquatic Environments

Metals in aquatic environments may be found in one or more of the fol- lowing forms:

a. Soluble species, (i) free hydrated metal ions and (ii) metal

complexes with inorganic or organic ligands.

b. Insoluble particulates, (i) colloidal particulates of metal complex or aggregates of hydrous metal oxides and (ii) metal complexes adsorbed or suspended particles.

A full characterization of metals in natural waters should take into ac- count the above mentioned forms. The existence of the metal in one form

or another is highly dependent on the temperature, pH, pE, and the ionic

strength. Transformation from one form to another may be chemically or biochemically (enzymatic) mediated.

A schematic diagram showing metal dynamics in an environmental seg- ment is shown in Figure 4. Inputs from the atmosphere may be in the form

of aerosol or metal particulates. Metal accumulation and release from bottom sediments may occur mass transport of interstitial waters or reversible adsorption or ion exchange reactions with bottom sediments. In the

aqueous phase, metals may exist as free metal, complexed with inorganic

ligands, e.g., OH~, CO^- or complexed with organic ligands, e.g., amines,

proteins, vitamins, humic acid, etc. These forms of metals may be in solution, insoluble particulates or within the aquatic biota, e.g., algae, plankton, etc. The effect of biological activity on metal transformations in aquatic en- vironments is shown in Figure 5. Mercury discharges in the form of metallic mercury Hg*', inorganic divalent mercury Hg2+, phenyl mercury

CeHsHg^ or alkoxy-alkyl mercury CH.sO-CH2-Hg", can all be converted

to the highly toxic methylmercury CH3Hg+ or dimethyl mercury

(CH3)2Hg in aquatic environments. The biological methylation of mercury occurs primarily in bottom sediments through the activity of certain

microorganisms.

3. Measurement Procedures

Metal analyses have undergone significant changes in the last three

decades. Prior to about 1940, most analytical techniques for metals were

either gravimetric or volumetric. Since the post-war years of the late
340 ANALYTICAL PROBLEMS

ATMOSPHERE

Soluble Organic

Complexes Biological

(Proteins, Vitamins,..) (Algae, Plankton)

-H

Interstitial Waters, Adsorbed, Ion Exchange

LITHOSPHERE

Figure 4. Metal dynamics in environmental segment.

1940's there has been a considerable increase in the use of the spec- trophotometric techniques, largely as a result of the development of vari- ous organic reagents such as dithizone, o-phenanthroline, sodium diethyldithiocarbamate and diphenylcarbazide which form color-producing compounds with metal ions in solution. Many of these reagents are highly

specific for particular metals and find wide application in water analysis.
WATER POLLUTION CONTROL 341

(C6H5)2Hg

C6H5Hg+ (CHjjgHg

i t i

> Hg CH3Hg+

CH3O-CH2- CHg-Hg"*"-

Figure 5. Biochemical transformations of mercury compounds in aquatic environments.

Compleximetric titrations with ethylene diamine tetra acetate (EDTA)

and a variety of specific metal ion indicators also are used extensively for

analysis of metal ions in waters and waste-waters. Perhaps the most

familiar example is the analysis for calcium, magnesium and water hardness by titration with EDTA.

a. Concentration and separation techniques. — Various separation and

concentration techniques are available for removing interferences, for extraction of colored organometallic complex compounds, or for concentration prior to titrimetric, spectrometric, electrometric, or radiometric analysis. In certain cases, the separation itself is sufficiently specific that it may be followed by a nonselective analytical procedure. In other cases, a

mixture of two or more metals, ions may be separated from solution and

then subjected to analysis by more selective analytical procedures.

Ion exchange techniques have been used extensively for separation of

metal cations in natural and wastewaters. The total free metal ion content

of a water sample can be determined by ion exchange [45-48]. The

technique involves passing a sample of water through a hydrogen-form cation exchange resin and titrating the equivalent quality of H+ released

with a standard base. Another aliquot may be titrated with EDTA for the

hardness metals. Analyses for NH4+, K+, Rb+ and Cs+ ions in water have

been carried out by precipitation with tetraphenylboron. The precipitate

is dissolved in acetone and the solution passed through a cation-exchange

resin in the hydrogen form; the resulting free tetraphenylboron is then

titrated [48].

Ion exchange chromatography offers an effective method for concentration and separation of ions from wastewaters. The ions are first concentrated on a suitable ion exchange column and then selectively eluted

to be determined polarographically, radiometrically, spectrophometrically
342 ANALYTICAL PROBLEMS

or spectrographically [49-51]. Iron, commonly found in industrial waste

effluents, may be collected on cation-exchange columns as ferric iron,

reduced to the ferrous state by a dilute ascorbic acid solution, and then

eluted with a strong acid [47]. The selective separation of metals by ion

exchange chromatography often can be markedly improved by using complexing agents.

Attempts have been made recently to use ion exchange membranes for

the separation and concentration of metal ions prior to analysis [52,53].

The technique involves mounting a cation-exchange membrane, of two to

five mils thickness, on the surface of an indicator electrode (usually car- bon, platinum or gold) in a voltammetric system. The membrane serves as an ion exchange preconcentration matrix as well as a rigorously defined

diffusion barrier for surface active or electroactive interferences present

in the test solution. The exchange rate of metal ions between the test solu- tion and membrane is accelerated by applying an appropriate e.m.f. across

the membrane.

Perhaps the most interesting aspect of ion exchange membranes in this application is their suitability for in situ separations. Additionally, the ion

exchange membrane itself can be used as a preconcentration matrix for

subsequent determinations by activation analysis, emission, or absorption

spectrophometry.

The main problem associated with using cation-exchange membranes

for transition and heavy metals in natural waters and wastewaters is their

lack of specificity. This is particularly significant since the alkalies and al- kaline earths are usually present in great excess. Another limitation to the use of cation-exchange membranes for natural

waters and wastewaters is the fact that most metals are found in the form

of inorganic or organic complexes. The separation of such complexed

metal ions by ion exchange is sometimes not possible unless the complex

is first disrupted.

Solvent extraction is frequently used for metal separation. Valuable

schemes for the separation of a large number of metal ions by successive

extractions employ different complexing agents and organic solvents, e.g., dithizone (diphenylthiocarbazone), oxine, cupferron, diethyldithiocarbamate,itetraphenyl arsonium salts, quaternary ammonium compounds

and various chelating agents.

Selection of an appropriate solvent-extraction system depends on its specificity and its suitability for subsequent analytical procedures.

Partial freezing has been used for the concentration of Fe, Cu, Zn, Mn,

Pb, Ni, Ca, Mg, and K in water samples in concentrations ranging from

0. 1 to 10.0 mg/1 [54] . Increasing the mixing rate up to some limiting value
WATER POLLUTION CONTROL 343

increases cationic recovery. The effect of initial pH on recovery efficien- cy depends on the nature of the cation. Alkali metals (K, Ca and Mg) concentrate best at low pH while heavy metal cations (Pb, Ni and Cu) concentrate best under alkaline conditions [54]

.

b. Analytical techniques. — Ideally speaking, a suitable analytical

procedure should have (a) high sensitivity and a low detection limit applicable to water analysis without sample pretreatment, (b) high selectivi- ty and minimum effect of interferences commonly present in natural

waters, (c) ability to characterize free and complexed metallic species, (d) in situ measurement, (e) portability and ruggedness for field operations,

and (f) suitability for continuous monitoring purposes. Unfortunately,

such an ideal analytical procedure is not presently available. Nevertheless, the analyst can rely on a number of techniques which offer some of

the above advantages. A listing of some of the applicable analytical

procedures and their sensitivity limits is given in Table 10. Atomic Absorption and Atomic Fluorescence Spectrophotometry.—

Among the optical methods, atomic absorption and atomic fluorescence

spectrophotometry are frequently used for metal analysis in natural and

wastewaters. These optical procedures have the unique advantage of vir- tual specificity. Exceptions are those few cases in which unfavorable

matrix components are present in the sample solution. This is largely a

result of the presence of certain compounds which combine with the metal

under analysis form relatively nonvolatile compounds which do not break down in the flame. Calcium in the presence of phosphate exhibits

this effect [55]. This may be remedied by sequestering the calcium ion

with EDTA. Matrix effects may be minimized by separation or by adding

approximately the same amount of matrix component to the standard

solutions. In contrast to flame photometry, there is very little interelement inter- ference in atomic absorption spectrophotometry. Also, while sensitivity

in flame photometry is critically dependent on flame temperature, this is not the case for atomic absorption spectrophotometry.

Over 60 elements can be determined readily by atomic absorption in the part per million range without sample pretreatment and with an accuracy of ± 1-2 percent. This sensitivity can be vastly increased to the part

per billion range by scale expansion or by extracting the metals in a non- aqueous solvent and spraying it into the flame. Microgram per liter quantities of cobalt, copper, iron, lead, nickel, and zinc have been determined in saline waters by extraction of metal complexes with ammonium pyrrolidine dithiocarbamate into methyl isobutyl ketone [56]. The use of or- ganic solvents may alter the flame temperature, which, in contrast to

428-999 O - LT - 72 - 23
344 ANALYTICAL PROBLEMS

Table 10. Sensitivity limits of common analytical procedures.

Method of analysis

Molecular absorption spectrophotometry

Molecular fluorescence spectrophotometry

Atomic absorption spectrophotometry

Atomic fluorescence spectrophotometry

Optical and emission spectroscopy

Neutron activation analysis

Potentiometry with metal, specific glass or membrane electrodes

Classical polarography

Derivative polarography

Square—wave and linear- sweep voltammetry

Anodic stripping voltammetry with hanging

mercury drop electrodes

Anodic stripping voltammetry with thin film

or solid electrodes

flame photometry, will generally have no significant effect. An increase of

about 60 percent in the atomizer efficiency can be achieved with the use

of certain organic solvents.

Although atomic absorption spectrophotometry is a relatively new

technique, it is being appUed widely for analysis of metal ions in natural

waters and wastewaters. In addition to its selectivity and sensitivity,

atomic absorption spectrophotometers are easy to operate and maintain.

Atomic fluorescence spectrophotometry offers no increase in selectivi- ty over atomic absorption spectrophotometry since the latter is virtually

specific for each element. It is possible, however, to increase the sensitivi- ty of measurements with atomic fluorescence spectrophotometry by in- creasing the intensity of irradiation, or by increasing the amplification

until the system becomes noise limited [57]. The increase in sensitivity

Sensitivity

mole/1

10-° - 10-^

10-7 - 10~^

10-*^ -10-7

10-7 - IQ-^

10-5 - 10-®

10-9 -10-10

10-4 - 10-5

10-5 - 10"'^

lo-*' -10-7

10-7 - 10-®

10-7 -10-9

10-9 - 10-10
WATER POLLUTION CONTROL 345

can be seen on comparing the light absorption and fluorescence equations.

For absorption spectrophotometry, the absorbance, /4, is directly related

to the concentration of the absorbing species, C, the length of the light

path through the absorbing solution, / and the molar absorptivity of the

absorbing species, e; i.e..

where h and / are the intensities of the incident and emitted light,

respectively.

The fluorescence equation may be expressed as follows:

where F is the amount of fluorescence generated, is a constant related

to the efficiency of fluorescence, /© is the intensity of incident radiation, e

is the molar absorptivity at a given wavelength, / is the pathlength in cm,

p is a fractional constant, and C is the concentration. Hence, F measured

in terms of the signal response of a photomultiplier tube sensitive to fluorescence radiation is proportional to the analytical concentration C,

while the parameters /©, / and p are instrumental factors, and the parameters (j) and e are functions of the efficiency of the fluorescent reagent

system.

It is evident from equation (11) that for absorption spectrophotometry

that any increase in h will be accompanied by a matching increase in /, with no net gain in the absorbance, A. However, for fluorescence

spectrophotometry, any increase in /<, will be matched by a corresponding

increase in the analytical signal F, as indicated in equation (12). Also, any

increase in the amplifier gain in absorption spectrophotometry will amplify lo and / correspondingly, whereas in fluorescence spectrophotometry,

this will result in an increase in F. For these reasons the sensitivity of

fluorescence spectrophotometry is inherently greater than that of absorption spectrophotometry.

The main limitations of atomic absorption and atomic fluorescence

spectrophotometry techniques are (a) inability of species

characterization, (b) lack of sensitivity for direct measurement without

concentrating the sample, (c) these procedures are primarily laboratory

procedures unsuitable for field use, and (d) inability for in situ measurement.

Activation Analysis. — Very high sensitivity can be attained by activation analysis, which finds several applications in water pollution control

activities. Sensidvities of the order of IQ-i- are obtainable with neutron

^ = log(/o//) = e/C (11)

F= [2.303 </)/oe//?]C (12)
346 ANALYTICAL PROBLEMS

fluxes of 10^^ sec"i cm-- [58, 59]. Application of activation analysis to natural waters and wastewaters generally requires radiochemical separation of the sample and comparison of the activities of the unknown sample

and of a known mass of standard treated under identical conditions.

Characterization of elements in a given water sample may be done by

identifying type, energy, and half life of emission. The concentration of a

given element in a sample is determined by quantizing its characteristic

emission, /.e., Mass of element X in sample _ Total activity from element X in sample

Mass of element X in standard Total activity from element X in standard

Some of the factors governing quantitative measurements are: the concentration of stable elements, activation probability, neutron flux,

relative abundance of stable isotopes, and time of irradiation. A wide

range of separation techniques have been associated with activation analysis procedures, some of which have been automated effectively [60].

Considerable improvement in the resolution of individual peaks in spectra

has been accomplished by replacement of sodium iodide detectors with

germanium detectors for gamma-ray scintillation spectrometry.

Detailed procedures for activation analysis of natural waters and waste- waters have been reported [61-63]. Generally, the accuracy and precision of these procedures are about ± 10 percent. Principal errors in analysis are due primarily to self shielding, unequal flux at the sample and stan- dard positions, inaccurate counting procedures and counting statistics. The problem of identifying the components of a complex spectrum has led

to the use of computers to facilitate interpretation of available data [64].

Computers may be used also to determine optimum conditions for irradia- tion of particular samples. Some of the main advantages of activation

analysis are: (a) its very high sensitivity, (b) the rapidity of analysis, and

(c) the nondestructive nature of the test. Although activation analysis offers the high sensitivity required for trace metal analysis, its use is limited by the availability of reactor facili- ties; furthermore, it is an elemental analysis procedure that offers no in- formation about oxidation state or degree and type of metal complexation.

Electroanalytical Methods. — Comparatively speaking electroanalytical

techniques find limited applications in water quality analysis for pollution

control. This is surprising since electroanalytical techniques, which are most suitable for metal analysis, offer a number of unique advantages not shared by other methods of analysis. Generally speaking, the main advantages offered by electroanalytical procedures are (a) ability of species

characterization, (b) in situ analysis, (c) suitability for field operation
WATER POLLUTION CONTROL 347

(portability) and ruggedness), (d) high sensitivity {e.g., anodic stripping

voltammetry), and (e) suitability for automation and continuous monitoring purposes.

For the purpose of this discussion, electroanalytical methods are conveniently classified as being either based on the passage of faradaic current, e.g., voltammetry— or based on electrode equilibrium and nonfaradaic current measurement, e.g., potentiometry [65]. A summary of some of the more important basic relationships underlying electroanalytical procedures is given in Table 1 1

.

Polarographic Techniques. — As a result of the capacitance current used

in charging the double layer, the sensitivity of classical polarography with

the dropping mercury electrode is limited to approximately lO'^M. However, by means of preconcentration techniques it may be possible to ex- tend the sensitivity range significantly. Copper, bismuth, lead, cadmium,

and zinc have been measured in the range of 0.01 mg/liter after extraction

with dithizone and carbon tetrachloride [66]. Preconcentration by ion

exchange, freeze drying, evaporation, or electrodialysis may be used.

A significant problem in the application of classical polarography to natural wastewaters is the effect of interferences by electroactive and sur- face active impurities. Such impurities, frequently present in wastewaters,

may interfere with electrode reaction processes and cause a suppresssion

and/or a shift of the polarographic wave [67]

.

Modifications of polarographic techniques, such as "differential

polarography" and "derivative polarography," may be used to increase

the sensitivity and minimize the effect of interferences [68]. Pulse

polarography has the advantage of extending the sensitivity of determination to approximately 10-^M. The technique is based on the application of

short potential pulses of 50 ms on either a constant or gradually increasing

background voltage. Following application of the pulse, current measure- ments are usually done after the spike of charging current has decayed.

The limiting current in pulse polarography is larger than in classical

polarography. Derivative pulse polarography, which is based on superimposing the voltage pulse upon a slowly changing potential (about ImV/s)

and recording the difference in current between successive drops versus

the potential, is even more sensitive than pulse polarography [68]

.

Cathode-ray polarography or oscillographic polarography has been

used for analysis of natural waters and wastewaters, with a sensitivity of

10-W being reported [69-71]. This technique involves the use of a

cathode-ray oscilloscope to measure the current-potential curves of applied (saw-tooth) potential rapid sweeps during the life-time of a single

mercury drop. Multiple sweep techniques are also applicable. The peak
348 ANALYTICAL PROBLEMS

Table 11. Basic electroanalytical relationships applicable to metal analysis.

I. Methods based on faradaic current measurement

A. Transient techniques

Chronoamperometry: E = constant, / = f(c,t) Cottrell equation: = zFA

^j^J ^

Chronopotentiometry: i = constant, E = f(cJ)

Sands equation: 5 = pFAD^^j c

Applications include pulse, square—wave, and sign—wave

voltammetric techniques.

B. Steady state techniques

Ilkovic equation (polarography): / Voltammetric membrane electrodes: /

"/e3Dl/2^2/3^1/6j^

'zFAP ^Ic mb\

C. Modifications

Anodic stripping voltammetry (ASV)

Plating step: = Qq d-e'^O

Stripping step:

Randies—Sevcik equation (HDME):

[/^^3/2^Dl/2,,l/2]c

Ep = ^1/2 - ^-^T/zF

Roe and Toni equation (Thin film ASV):

= \zFSl^v\ C

E = + M log

^
WATER POLLUTION CONTROL 349

Table 1 1 . (continued)

II. Methods based on non—faradaic current measurement

Potentiometry (membrane electrodes)

Nernst equation: E ^ SJL U \a + K.a.^'^^J

\

current (ip) in the resulting polarogram is related to the concentration of

the electroactive species for a reversible reaction.

Oscillographic polarography has the advantages of: (a) relatively high

sensitivity, (b) high resolution, and (c) rapidity of analysis. Traces of Cu,

Pb, Zn and Mn can be determined at 0.05 mg/ml level in natural waters by

this technique [71].

Anodic Stripping Voltammetry.—One of the most useful electrochemical approaches to metal analysis in trace quantities is anodic stripping

voltammetry. This technique involves two consecutive steps: (a) the

electrolytic separation and concentration of the electroactive species

to form a deposit or an amalgam on the working electrode, and (b) the

dissolution (stripping) of the deposit. The separation step, best known

as the pre-electrolysis step, may be done quantitatively or arranged to separate a reproducible fraction of the electroactive species. This can

be done by performing the pre-electrolysis step under carefully controlled conditions of potential, time of electrolysis, and hydrodynamics

of the solution.

The stripping step is usually done in an unstirred solution by applying

a potential— either constant or varying linearly with time — of a magnitude

sufficient to drive the reverse electrolysis reactions. Quantitative deter- minations are done by integrating the current-time curves (coulometry

at controlled potential) or by measurement of the peak current (chronoamperometry with potential sweep). Several modifications of the

separation and stripping steps have been reported [72]

.

Hanging-drop mercury electrodes of the Gerischer's type [73] or of

Kemula's type [74] have been widely used for anodic (or cathodic)

stripping analysis. Greater sensitivity has been achieved by use of elec- trodes which consist of thin films of mercury on a substrate of either

platinum, silver, nickel, or carbon [75]. Errors due to non-faradaic

capacitance current components can be minimized by proper choice of

stripping technique.
350 ANALYTICAL PROBLEMS

The main advantage of stripping voltammetry is its applicability to trace

analysis. The technique has been applied for metal analyses in sea water

[76], natural waters [77,78] and wastewaters [78], and drinking water

supplies [79].

Anodic stripping voltammetry can be used to measure metal complex

formation in aquatic environments. Metal ions in natural waters may be

complexed with simple inorganic ligands such as water, halides, car- bonate, and sulfate, or they may be tied up with complex organic ligands

such as amino acids, organic acids, vitamins, porphyrins, humic acids, and

tannins.

Qualitative and quantitative information on free and combined metal

ions and metal-binding ligands and their distribution can be gained by

varying the sample pretreatment and the anodic stripping procedures. A

flow chart showing various analytical steps is given in Figure 6. Total

metal content can be determined by digestion of the sample before anodic

stripping analysis, and filtration separates dissolved from particulate frac- tions on the basis of variations in particle size. In addition, it is possible to exchange the metal in many complexes by

additions of acid (H+) or metal cations; the freed metal ions subsequently

may be determined by anodic stripping voltammetry (Table 12).

SAMPLE

digestion filtration

total digestion

particulate dissolved

addition of material variation of ASV parameters

electroactive metals non—eiectroactive metals

sweep rate plating time

stripping medium

plating potential

mixing

Figure 6. Analytical scheme for anodic stripping voltammetric determination of metals in natural waters.
WATER POLLUTION CONTROL 351

Table 12. Trace metal concentrations in various natural and waste

waters determined by anodic stripping voltammetry.

Sample Location

Concentration (/xg/1)

Acid

Free exchangeable

Cd Pb Cu Pb Cu

1 Rouge River 0.5 mile (0.8 km)

above Ford Motor Co. turning

basin

<0.1 1.1 2 15 27

2 Rouge River turning basin 7.4 2.2 14 37 108

3 Rouge River mouth 1.5 0.4 8 11 19 4 Detroit River, Trenton Channel 0.8 0.4 18 5 28

5 Detroit River, Livingston

Channel

L9 0.4 11 6 29

6 Lake Erie, central basin,

surface water

1.8 6.8

7 Lake Erie, central basin,

bottom water

0.3 0.07 0.5 L6 L8

8 Lake Michigan, Waukegan,

Illinois LO 0.2 0.8 3.3 18

9 Lake Michigan, Ludington, 0.6 0.4

Michigan

The metal-complexing ability of natural waters, which is a measurable

characteristic, depends on the type and concentration of simple or complex ligands. Quantitative and qualitative information on metal-binding

ligands can be obtained by a complexometric titration procedure in which

an electroactive metal ion is used as the titrant. In this technique the stepwise metal-ligand complex formation can be followed and the

stoichiometric end points can be detected by anodic stripping. Similarly,

the rates of metal-Ugand dissociation on titration with a strong acid can be

determined.

Some of the more pertinent diagnostic criteria that can be drawn on

using anodic stripping voltammetry [80] are (a) ip measurement can dif- ferentiate between free metal ions and labile metal complexes on one hand

and nonlabile metal complexes on the other hand, (b) Ep measurement can
352 ANALYTICAL PROBLEMS

differentiate between free metal ions and complexed metal compounds,

(c) ip measurement, together with Ep, can differentiate between free metal

ions and labile metal complexes, and (d) metal-organic titration techniques in which electroactive metals are the titrant can differentiate

between labile and nonlabile and complexes as well as the competitive in- teractions between metal-organics complex formation.

The analytical feasibility of anodic stripping voltammetry can be ex- tended greatly by varying the deposition time, the potential sweep rate, the stripping medium, and the mixing regime. These variations can be

used to advantage to characterize free and complexed metal ions in the

aqueous environment.

Potentiometric Techniques. -Recently developed potentiometric membrane electrodes, known as ion selective electrodes, are extremely useful

for measurement of a variety of metals in natural and wastewaters. Electrode systems are available for the measurement of Ca2+, Mg-+,

Cu2+, Cd^+ and Pb-+ [70]. The detection limits for these electrode

systems are in the range of 10"^ to IQ-^M. Since Ca2+ and Mg^+ are usually found in the concentration range of 10-"* to 10~^M in natural

waters, they are easily detected by ion selective electrodes [81]. Hardness

measurement (divalent metal ions) is a typical example where these

selective ion electrodes prove to be useful. Selective ion electrodes cannot be applied directly for measurement of

Cu2+, Pb^+ and Cd^^ in natural waters, since these metals are usually

found in trace quantities IQ-^M to lO-^W. They may be applied however,

for measurement of heavy metals in certain industrial waste effluents such

as plating industry, pickling waste and steel effluents. Selective ion electrodes are sensitive to the activity of the electroactive

species. In order to use such electrode systems for determination of concentrations rather than activities, it is important to consider the effects of

ionic strength on the activity coefficient of the electroactive species and

the liquid junction potential between the test solution and the reference

electrode. To avoid the uncertainty of estimating an activity coefficient,

it is useful to adjust the ionic strength of the sample to that of a standard

solution. Since the total ionic strength of the solution determines the activity coefficient for a specific ion, the activity coefficient of the ion being

analyzed in the test sample will be identical to that in the standard solution. A constant ionic strength can be obtained by using a "swamping

electrolyte." This technique is frequently referred to as the 'ionic medium" method. In cases where the ionic strength of the test solution does

not change significantly, direct potentiometric measurement can be made.

This is the case with most water supplies.
WATER POLLUTION CONTROL 353

Generally speaking, the analyst is advised to determine the selectivity,

sensitivity range and long term stability of the selective ion electrode before its application. This is particularly significant in applications to natural and wastewater systems where interferences may occur. Perhaps the most significant feature of selective ion electrodes is their

selectivity to free ions. This feature can be used to an advantage for the

characterization of metal species. Through a sequence of potentiometric

titration techniques, it is possible to construct distribution diagrams of

metal species as a function of certain ligand concentrations. Such information is most valuable in the understanding of the aquatic chemistry of

metals and should give added insight in the applicability of equilibrium

diagrams calculated from thermodynamic constants. Furthermore, selective ion electrodes can be used in the study of the

kinetics of metal complex formation. The rates of interactions between

certain metals, e.g., Ca, Mg, Cu, Cd, etc., and certain organic or inorganic

ligands can be used to elucidate the distribution dynamics of metals in aquatic environments. Moreover, this information will offer a better un- derstanding of the effect of discharge of certain waste effluents in natural

waters. A schematic diagram of an analytical scheme for selective ion electrode

application is shown in Figure 7.

SAMPLE

digestion

total digestion

filtration

particulate dissolved

addition of material

potentiometric titration kinetics of complex formation

Figure 7. Analytical scheme for selective ion electrode determination of metals in aquatic environments.
354 ANALYTICAL PROBLEMS

D. Analysis for Organic Matter

7. Significance

Organic material in water may be naturally produced within the water

body, such as plankton waste products, or derived from outside the water

body, such as leaves detritus or by man-made waste products. Organic

matter may be present in particulate, colloidal, or dissolved form, depending upon physicochemical characteristics of the water, i.e., temperature,

pressure, pH, ionic strength.

Organic constituents of certain industrial wastewaters are of general concern for one or more of a number of effects they may have upon

receiving waters. The most readily apparent effect is that exerted directly on aesthetic quality by certain organic pollutants as a result of foaming,

formation of slicks and films, offensive odors and tastes, discoloration,

etc. Of equal significance are the effects of biologically oxidizable organic

wastes on the depletion of dissolved oxygen levels in receiving waters,

with concomitant disruption of the natural ecology of these waters. Other

organic wastes may not degrade biochemically, but may rather persist and

accumulate, thus constituting insidious long-term potential health hazards.

The damaging effects of organic wastes on the quality of water for particular uses are summarized in Table 13. The ability of organic matter of natural or pollution origin to complex

with metals in aquatic environments is of utmost significance. For example, the complexation of ferric iron by certain organic compounds may

prevent it from precipitating to the bottom and renders it more available

to the aquatic biota. Organic complex formation with trace metals, e.g., Cu, Pb, Cd, Zn, Co, Cr, etc., may result in solubilizing these metals and in effect raises their concentration in the aqueous phase. The ability of

aquatic biota to utilize organometallic complexes vis-a-vis free metal

ions is under investigation and is considered to have significant ecological implications.

Several approaches are commonly used for characterization of organic

matter in industrial waste effluents. These can be classified into two main

categories. In the first category the damaging effect (poUutional effect) of

the organic waste matter on the receiving water is estimated. This may be

accomplished by diluting the waste effluent with the receiving water to a

level corresponding to the dilution which will result in the receiving

stream, and then characterizing appropriate pollution parameters, such as odor, color, carbon demand, chlorine demand, fresh-fish tainting, per- sistence (resistance to biodegradation) and treatability.
WATER POLLUTION CONTROL 355

Table 13. Characteristics of organic pollution.

Water use

Domestic^ or industrial

process supply

Production of fish

Recreation

Agricultural irrigation

Watering of livestock

Damage to water quality for particular use Taste and odor

Carbon demand

Chlorine demand

Interference with coagulation

Color

Corrosion promotion

Carbinogenic properties

Toxicity to humans

Toxicity to fish or fish food

Taint fish flesh

Deoxygenation of water Promotion of filamentous organisms

Sludge deposits

Odor

Color

Floating matter Suspended matter Sludge deposits

Toxicity to plants

Toxicity to animals

This damage £^plies in cases where industrial waste is discharged to municipal sewers for combined treatment with domestic waste.

The second approach is based on both qualitative and quantitative

analyses for the organic compounds. Analyses for organic compounds

may involve either nonspecific or specific analytical methods. In the

former case, analysis is done by measurement of oxidizable organic

matter, such as the biochemical oxygen demand or the chemical oxygen

demand, or by determining the total organic carbon or total organic

nitrogen present in a given sample. Specific analysis, on the other hand,

includes identification and quantitative determination of specific species

present in the test solution.
356 ANALYTICAL PROBLEMS

2. Nonspecific Analysis

Nonspecific analytical methods are often based upon measurement of

quantities of oxidizable organic material, either by biologically-mediated

oxidation or by strictly chemical oxidation (by wet or dry combustion

procedures). The procedure most commonly employed for measurement

of the susceptibility of a waste to biological oxidation is the biochemical

oxygen demand (BOD) test, while the chemical oxygen demand (COD)

test is widely used for measurement of concentrations of organic matter

oxidizable by a dichromate reflux method. Detailed descriptions of stan- dard procedures employed for BOD and COD measurements are readily

available in the literature [32,33].

a. Biochemical Oxygen Demand.- Basically the biochemical oxygen

demand test is a bioassay procedure for determining the amount of

biologically oxidized organic matter in terms of the amount of oxygen

consumed. The test is done at controlled conditions. Measurements

are based on the assumption that since the amount of oxygen required

to convert a given quantity of biologically-oxidizable organic compounds

to CO2 and H2O is fixed, it is possible to interpret BOD data semiquantitatively in terms of gross concentrations of organic matter as well

as oxygen consuming tendency. This is illustrated as follows:

where [CHO] refers to biodegradable organic compounds. Measurement

is given in terms of the amount of oxygen consumed within five days

"BOD5. " Similar to any bioassay technique the variables in the BOD test are (a) types, numbers and ages of microorganisms, (b) types and concentration

of the organic substrates, (c) effects of temperature, mixing, etc., (d)

presence of antibiotic agents, (e) illumination which may promote

photosynthetic oxygen production, and (f) presence of predator

organisms.

One may certainly question — and many have — the use of such a simple

expression for description of a complex biochemical process. The formulation of such a complicated reaction has been widely discussed, and nu- merous modifications have been proposed. It is beyond the scope of this presentation to cover this aspect of the BOD test, and the reader is referred to other sources for detailed discussions of this matter [82,83]

.
WATER POLLUTION CONTROL 357

It is important to note that under most favorable conditions, the precision of the BOD test is in the order of ± 20 percent.

Because the dissolved oxygen content in streams, rivers, or lakes is of

major concern from the standpoint of water pollution control, the BOD

test has been extensively used for providing an estimation of the amount

of oxygen likely to be consumed by a given amount of waste upon

discharge to a receiving water. By utilizing a "seed" taken directly from

the receiving water in question, actual conditions can be closely paralleled

in that the measured consumption of oxygen will be that corresponding to the biological activity of the particular organisms indigenous to that receiving water. Recent studies [84] have pointed out the advantage of determining the

rate of biochemical oxygen demand, rather than the BOD5 per se. In an

attempt to predict the effect of disposal of organic matter on the oxygen

balance in the stream, the authors described a laboratory procedure to evaluate the assimilative capacity of receiving waters. The technique is based on the simultaneous determination of the rate of biochemical ox- ygen utilization and the rate of atmospheric oxygen uptake, using stream

and waste waters, in appropriate rations.

b. Chemical Oxygen Demand.-The chemical oxygen demand test (COD) measures concentrations of organic materials which may be

oxidized by a strong oxidizing agent. Depending upon the nature of the

organic compounds present in water, the COD test may represent a

fractional measure of total organic content. A number of oxidants have been tested'for possible use in the COD

method, including: dichromate, permanganate, persulfate, eerie sulfate,

perchloric acid, periodic acid, nitric acid, and numerous combinations of

these and other reagents. Dichromate has been selected for the standard

method on the basis of comparative testing with other reagents such as those listed above [33]

.

The COD test was originally intended to serve the combined functions

of providing a more complete measure of the organic content of waste- waters than is afforded by the BOD test, while at the same time giving a

rapid approximation to the ultimate oxygen requirements of the waste. Unfortunately, the test as presently described accomplishes neither of

these objectives completely, in that oxidation of many organic compounds

is not complete under the conditions of the test and. because certain

biologically-oxidizable substances are not measured as COD while

others, which are not available for biological oxidation are oxidized by the dichromate-sulfuric acid mixture.
358 ANALYTICAL PROBLEMS

3. Specific Analysis

a. Elemental Analysis.-A gross estimate of organic matter in water can be achieved by determining the total organic carbon (TOC) and in certain cases total organic nitrogen (TON) or total organic phosphorus

(TOP).

The total carbon analysis test determines total soluble carbon within a matter of minutes and the method requires less than 0.5 ml of sample

[85] . If particulate matter is excluded the test represents total soluble car- bon and if inorganic carbon is excluded or corrected for, the results represent dissolved organic carbon.

The aqueous sample is injected directly into a combustion tube, heated

to 970 °C in a constant flow of oxygen gas. Any organic matter is oxidized

completely to CO2 and water vapor on an asbestos packing impregnated

with catalyst and these are carried from the combustion tube by the ox- ygen stream. Dry combustion of the organic matter is usually done in the presence of

a catalyst {e.g., cupric oxide, cobaltic oxide, or asbestos-supported silver permanganate at elevated temperatures of 900 to 1000 °C).

Organic nitrogen determinations are commonly done by the Kjeldahl

method. This method is applicable to many types of organic compounds

although it is sometimes referred to as a method for aminoid or albuminoid nitrogen.

The procedure is based on destroying organic material with sulfuric acid in the presence of various catalysts, and the nitrogen is converted to ammonium acid sulfate. The ammonia is liberated and either titrated or determined colorimetrically [32,33]. One of the principal problems of this method is that certain organic-bound nitrogen structures cannot be easily

transformed to ammonia [86]. Mercury is considered an adequate

catalyst for the digestion of this type of compounds.

Several methods utilizing various combustion techniques separately or

in conjunction with the Kjeldahl procedure have been proposed. The

reader is referred to the recent review articles [86,87]

.

For determination of organic phosphorus, organic material is first broken down by wet combustion with mixtures of sulfuric and nitric acids.

The phosphoric acid produced then can be determined either gravimetrically as ammonium phosphomolybdate or colorimetrically [32,33]

.

b. Constituent Analysis. — Specific analysis of the organic constituents

in natural and wastewaters is not an easy task. Direct application of conventional analytical techniques is frequently not possible because the

quantities present may be well below detectable limits. Additionally, the
WATER POLLUTION CONTROL 359

presence of interferences may create difficulties for the application of certain separation techniques. One of the major problems associated with the

isolation and separation of organic compounds from water samples is the

biochemical degradation or chemical transformation of these compounds

resulting from bacterial action, temperature effects, surface catalysis,

solvent-solute and solute-solute interactions, etc.

It is frequently necessary to concentrate sample solutions to bring them

within the detectable limits of certain analytical procedures. Concentration by removal of water can be achieved by either evaporation or freezing. For evaporation, vacuum distillation at low temperatures is generally

preferred. Partial distillation and steam distillation techniques have also

been employed [88], and freeze concentration techniques have been

used very effectively [89]. One of the main advantages of freeze concentration is that the materials to be concentrated are kept at low temperature

during the entire process; thus the chances for loss of volatile constituents

and/or alteration of the nature of organic substances of interest are minimized.

Reverse osmosis with cellulose acetate membranes can be used effectively to concentrate both low and high molecular weight organic compounds [90]. In this technique, pressure greater than the osmotic pres- sure is applied to the sample solution, resulting in the flow of water

through the membrane, with a high degree of retention of the organic

solute. The advantage of the membrane-osmotic method for concentrating

dilute solutions of organics is that it is an in situ separation technique

which does not involve changes in phase or changes in temperature.

Carrying the same concept further, all the water can be removed from

the sample by evaporating till dryness or freeze-drying. These techniques,

however, are used primarily to get a rather rough estimate of the nonvolatile organic content.

c. Separation Techniques.-A variety of separation techniques may be

used, singularly or in combination, for isolation and concentration of or- ganic matter in natural and waste waters. Among these are distillation [88], solvent extraction [91], precipitation and crystallization [92], adsorption [93], gas chromatography [94], paper chromatography [95],

and thin layer chromatography [96]

.

Partial distillation, in which the sample flows through the distillation

cell continuously and only a small portion is distilled (10 percent), has

been applied to oil refinery waste effluents [88]

.

Adsorption chromatography is one of the best methods available for

rapidly concentrating or extracting solutes from dilute aqueous solution.

Compared with the volume of the solution used, a relatively small volume

428-999 O - LT - 72 - 24
360 ANALYTICAL PROBLEMS

of absorbent is required for the separation. The solute normally can be

recovered from the absorbent in a small volume eluting agent.

Adsorption chromatography with activated carbon has been used for more than a decade for separating organic compounds from surface

waters and certain industrial effluents [93,96]. Much of the work leading

to the development of this procedure was done at the Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio, in the early 1950's [96]. For

this procedure, a carbon column, often referred to as the "carbon filter"

is used in conjunction with a sand prefilter and/or a presettling tank. The

efficiency of the method is a function of the rate of flow of the sample

solution, particle size of the absorbent, type and characteristics of the adsorbate(s), and physicochemical properties of the sample solution such as temperature, pH, ionic strength and turbidity.

The organic matter collected on the adsorption column is recovered by

solvent extraction using chloroform and ethyl alcohol. These two extracts are further fractionated by means of various solvent extraction schemes

as shown in Figures 8 and 9. The carbon filter technique, as it is used now [32,33,98] does not quantitatively separate the total organic content of a water or waste solution.

Recoveries may range from approximately 50 to 90 percent, and replicate

samples may agree within only about ± 10 percent. In spite of these drawbacks, the carbon filter technique is useful for qualitative collecting of or- ganic matter from dilute waste effluents. It can be used to advantage for

screening purposes, as well as for monitoring industrial waste effluents. Gas chromatography has proven to be a useful tool for analysis of natural waters and wastewaters, being used in the majority of cases to separate

and identify components of extracts from carbon filters or from other

separation processes [91-94]. In a few cases, however, water samples

have been directly injected for analysis of organic content [94,100,101].

Paper chromatography and thin-layer chromatography also find application for separation and identification of organic constituents in wastewaters. Phenols, cresols, xylenol, and other industrial and biological

phenols have been determined in the microgram per liter concentrations

in polluted waters by paper chromatography [102]. Chlorinated

hydrocarbon pesticides have been determined at concentrations of two

micrograms per liter or less in carbon-chloroform extracts [103]

.

Thin-layer chromatography has been used effectively for removing in- terfering substances from pesticides prior to gas chromatographic analysis

[96]. Christman has discussed the advantages of thin-layer chromatography over paper chromatography for separation and identification of or- ganic chromagenic agents in natural waters [89]. Similarly the separation
WATER POLLUTION CONTROL 361

WEIGHED SAMPLE

add ether, filter

Ether Solution

extract with H2O —

I

Residue

evaporate, weigh

ETHER INSOLUBLES

I Ether Layer

extract with HCl

Water Layer

evaporate, weigh

WATER SOLUBLES

Ether Layer

extract with NaHCOs

Water Layer

make basic, extract with ether

I

Water Layer

make acid

extract with

ether

I

Ether Layer

dry, evaporate

and weigh

BASES

Water Layer

discard

Ether Layer

dry, evaporate

and weigh

STRONG ACIDS

Ether Layer

extract with NaOH

1 Water Layer

discard

Water Layer

make acid

extract with ether

I

Ether Layer

dry, evaporate

NEUTRALS

Ether Layer

dry, evaporate

and weigh

WEAK ACIDS

! Water Layer

discard

Figure 8. Liquid extraction scheme for organic compounds.
362 ANALYTICAL PROBLEMS

NEUTRALS

adsorb on silica gel column

2 3

elute with

i so—octane

elute with

benzene

elute with

chloroform/methanol

(1:1)

AUPHATICS OXYGENATED COMPOUNDS

of various herbicides can be effectively done by thin-layer chromatographic techniques [96]

.

d. Identification Techniques.-Spectrometric techniques can be used for

the identification of organic compounds in natural and wastewaters, e.g., infrared, ultraviolet, mass spectroscopy and nuclear magnetic resonance

techniques. For analysis of natural and wastewaters, analytical spec- trometry must generally be preceded by separation of the organic material

from the wastewater sample by one or more of the techniques discussed

previously.

Near-infrared spectrometry offers some advantages when interferences occur in the "fingerprint" region from 7 to 12 /x,m. Measurement in the

near-infrared region (1 ixm to 3 /xm) are only useful for detecting and subsequently identifying functional groups which contain unique hydrogen

atoms, and are not characteristic of the organic molecule as a whole.

A modification of infrared spectrometry which offers unique possibilities for analysis of organic matter in water involves attenuated total reflectance [104,105]. This method is based on the passage of a monochromatic or monochromatically scanned light into a crystal of

suitable material, in contact with the test solution, and detection of the

transmitted light intensity. Changes in light intensity are related to changes in the type and concentration of "light active" substances in contact with the crystalline material. The principle is based on energy reflec- tion at the interface between media of different refractive indices [106]

.

UV-spectrometry finds wide appHcation in industrial processes for

monitoring the composition of reaction mixtures. Monitoring of trace or-
WATER POLLUTION CONTROL 363

ganic material in industrial effluents by UV-spectrometry has been re- ported [107] . The method is based on measuring UV absorption spectra

for compounds having aromatic or conjugated unsaturated molecular configurations. The instrument employed for these analyses used a mercury

discharge lamp with a principal UV-radiation at 2537 A. The author has

reported a sensitivity of 10 mg/1 of phenol in water. Mass spectrometry has not as yet been widely used for analysis of wastewaters. Melpolder [108] has described the distillation of volatile or- ganic compounds from oil retlnery wastewater and subsequent identifica- tion by mass spectrometry. Because of the high training level required for operating personnel, the data handHng problems, and the cost of instru- mentation, this technique has been restricted primarily to research applications, and then to only a few major water pollution control laboratories.

The technique does, however, offer considerable possibilities; for example, mass spectrometry has been used to detect phenols m nanogram

quantities in the effluent from a gas chromatographic column [ 109]

.

Fluorescence spectrophotometry has been applied successfully for

analysis of lignin sulfonates in the waste effluents from Kraft sulfite pulping operations and for analysis of organic color-forming compounds in natural waters [110]. Traces of carbohydrate in the nanogram per liter range have been determined in water samples using spectrofluorometry

[111], The sensitivity of the method and the effects of environmental fac- tors have been discussed [111].

4. Biodegradability of Organic Compounds

Biodegradabiliiy tests are basically designed to estimate the extent

to which organic compounds may be oxidized biochemically and an

industry must be concerned with the question of whether its organic

products or waste materials can be degraded or assimilated efficiently

by existing biological waste treatment processes. Biodegradability may

be considered as a measure of the susceptibility of organic material to microbial metabolism. This property is not well defined and there is no single standard test for its measurement. It constitutes, however,

a dominant mechanism for the removal of organic pollutants from water,

both in self-purification processes in natural waters and in the ac- celerated biological processes of waste treatment. Biodegradation

can occur aerobically or anaerobically. depending on the availability

of atmospheric oxygen. The process is affected by a variety of environ-
364 ANALYTICAL PROBLEMS

mental conditions. As discussed previously, organic compounds are not equally susceptible to biodegradation; some are readily metabolized, others are more resistant (refractory compounds). Principal

factors involved in any biodegradation process are as follows: (a) type and

number of microorganisms; a mixed culture of organisms, as in sanitary

sewage, possesses a remarkable capacity to adapt to strange or different

organic materials while single cultures may not be effective, (b) structure and concentration of organic materials; certain organic compounds, e.g., certain pesticides and surfactants, are relatively resistant to biodegradation in comparison to simple carbohydrates. The concentration of the or- ganic material is also significant. High concentrations of "sugars" in certain wastewaters may inhibit biodegradation, yet upon dilution in a receiving water the sugars will be easily degraded, resulting in a water pollution

problem due to oxygen depletion, (c) environmental factors; factors such

as temperature mixing, viscosity, etc. of the wastewater are quite signifi- cant in dictating the extent and rate of biodegradation and its effect on the

ecology of the receiving environment (air, water, or soil). Much of the work which has been carried out on biodegradation has

been concerned with surfactants, and in particular with the "hard"

branched-type ABS and the "soft" straight chain LAS [ 11 2, 11 3].

A number of techniques have been used to test for biodegradability,

such as the Warburg respiration technique [114], activated sludge tests [115], shake-flask experiments [115], and river die-away measurements

[116]. A comprehensive critique of various biodegradability tests has

been reported [117]. Sewage microorganisms are commonly used for

biodegradability measurements because of availability, and to avoid the

need for maintaining standard cultures [117]. Biological oxidation has

been followed by measurement of one or more of the following parameters: (a) the rate of disappearance of the organic compound under test, (b) the rate of appearance of biodegradation by-products, (c) growth rate of

microorganisms, and (d) dissolved oxygen consumption.

E. Analysis for Inorganic Anions

1 . Significance

Inorganic anions of significance in water pollution characterization may

be categorized into (a) halides, e.g., F", Ch, Br" and I", (b) nutrient

anions, e.g., PO^-, NO3 and NO2 , (c) sulfur anions and (d) cyanides
WATER POLLUTION CONTROL 365

and thiocyanate. The significance of each group, insofar as water quality

is concerned, varies widely and generalities cannot be drawn. Of particular interest to the public nowadays is the problem of disposal of

phosphates in natural waters. It is claimed that increasing the phosphorus

level will result in the acceleration of the rate of eutrophication of receiving surface waters. This is considered a serious problem since current sewage treatment processes do not remove phosphorus from sewage ef- fluents.

2. Separation and Concentration Techniques

Separation and concentration techniques for metal ions are generally

apphcable also for nonmetal inorganic species. Evaporation, precipitation, ion exchange, solvent extraction, and partial freezing are frequently

used ion exchange being particularly well suited for anion separations.

Anion exchange chromatography has been used rather extensively for

separations of species found in waters and wastewaters [117].

3. Measurement Procedures

Phosphorus may be present in industrial waste effluents either as inorganic phosphates (ortho-, meta-, or poly-phosphates) or in organic combination. The most common analytical method for inorganic phosphorus

is based on the colorimetric determination of the phosphomolybdenum

blue complex [32,33]. The test is sensitive to orthophosphates and not condensed phosphates. Polyphosphates and metaphosphates are then

estimated as the difference between total phosphates (hydrolyzed samples) and orthophosphates (nonhydrolyzed samples).

Orthophosphates react with ammonium molybdate in acid medium to form the phosphomolybdic acid complex, which when reduced yields the

molybdenum blue color which may be determined colorimetrically. The

sensitivity of the test is largely dependent on the method of extraction and

reduction of the phosphomolybdic acid. Aminonaphthol-sulfonic acid

[33], stannous chloride, (Deniges Method) [118]. metal sulfites (Tschopp reagent) [119] , and ascorbic acid [120] have been used in the

reduction step. The stannous chloride method is considered most sensitive and best suited for lower ranges of phosphate concentration.
366 ANALYTICAL PROBLEMS

A number of substances have been reported to interfere with the

phosphate determination [33]. Arsenic, germanium, sulfides, and soluble

iron above 0.1 mg cause direct interferences. Tannins, lignins and hexavalent chromium will cause errors only for analysis of phosphate concentrations below mg/1.

Indirect UV spectrophotometry and atomic absorption methods have

been developed for phosphates and silicates [ 121] . These techniques are based on the selective extraction of molybdophosphoric acid and molybdosilicic acid, followed by ultraviolet molecular absorption spec- trophotometry and/or atomic absorption spectrophotometry. The

molybdophosphoric and molybdosilicic acids are formed in acidic solu- tion by addition of excess molybdate reagent. Molybdophosphoric acid is extracted with diethyl ether from an aqueous solution which is approximately 1 M in hydrochloric acid. After adjusting the hydrochloric acid

concentration of the aqueous phase to approximately 2 M, the molybdosilicic acid is extracted with 5:1 diethyl ether-pentanol solution. The

extracts of molybdophosphoric and molybdosilicic acids are subjected to acidic washings to remove excess molybdate. Each extract is then contacted with a basic buffer solution to strip the heteropoly acid from the or- ganic phase. The molybdate resulting from the decomposition of the

heteropoly acid in the basic solution is then determined either by measurement of the absorbance at 230 nm using ultraviolet spec- trophotometry or by measurement of absorbance at the 313.3 nm

resonance line of molybdenum by atomic absorption spectrophotometry.

The optimum concentration ranges are approximately 0. 1 - 0.4 mg/1 of

phosphorus or silicon for indirect ultraviolet spectrophotometry and 0.4 - 1.2 mg/1 for indirect atomic absorption spectrophotometry.

4. Potentiometric Membrane Electrodes

The recently developed "selective ion electrodes" offer unique analyti- cal capabilities for the determination of F", CI", Br", I", and CN", SCN",

NO3, and S^~. The principle and performance characteristics of these

electrode systems were discussed earlier. The fluoride ion membrane electrode finds its widest application in the

analysis of fluoridated water supplies [80]. Free fluoride ions are deter- mined in water samples buffered to pH 5.2 by an acetate buffer. Total

fluorides are determined after the addition of a masking agent, e.g., citrate, which forms stable, nonvolatile complexes with aluminum and
WATER POLLUTION CONTROL 367

measuring fluorides at pH 5.2. The addition of a masking agent is necessary to release the bound fluorides, e.g., (AlFe)^". In this procedure the

fluoride ion-potentiometric membrane electrode is used to provide quantitative information on free and complexed fluorides. This should be

highly significant in assessing the effect of free vis-a-vis complex fluorides on the prevention and control of dental caries. Similarly, the sulfide ion membrane electrode can be used to characterize free sulfides in natural and waste waters [21]. Potentiometric measurements of S^" can be related to the concentrations of H2S, HS~, and St

(analytical concentration of free sulfides).

Cyanide, nitrate and chloride ions, in natural and wastewaters, may be

also determined by selective ion electrodes [80]. These applications are

subject to the same general limitations on sensitivity and selectivity as those for previously mentioned membrane electrodes. It is important,

however, to establish the electrode performance characteristics in any

given water prior to its application. For example, the nitrate electrode is quite accurate and precise (1-5 percent error) in pure nitrate solutions,

and the lower limit of sensitivity is about 0.6 ppm NO" which is sufficient to give meaningful electrode response at any nitrate level in surface and

underground waters in the USA. Nevertheless, the electrode's liquid ionexchanger deteriorates sufficiently to cause significant errors after three

to four weeks of operation, after which the electrode must be rejuvenated

and recalibrated.

One of the main problems with the nitrate electrode is the effect of in- terferences by Cl~ and HCO", commonly found in all types of waters.

F. Analysis for Dissolved Gases

1. Significance

The gases of significance in water pollution characterization are

primarily those associated with biological activities in the aquatic environment e.g., O2, CO2, H2S NH3, CH4, etc. Carbon dioxide and oxygen are

particularly of significance in assessing biological productivity. Hydrogen

sulfide, methane and gaseous ammonia, if present in natural and wastewaters, are considered indicators of anaerobic conditions.

The analysis for CO2, H2S, and NH3 is frequently based on acid-base

equilibrium calculations. For example in the case of CO2 the following
368 ANALYTICAL PROBLEMS

reactions occur: CO2 (gas) ^ CO2 (solution) (13)

CO2 (solution) + H2O ^ H2CO3 (14)

H2CO3 ^ HCO3- + H+ (15)

HC03-^C02- + H+ (16)

In spite that the hydration of dissolved carbon dioxide and the dehydration of carbonic acid are relatively slow, it is possible to derive equilibrium expressions for CO2 concentrations [123] . By combining the expressions for carbonate alkalinity, [ Alk] , the solubility of carbon dioxide and

carbon dioxide ionization constants (A^i and K2), it is possible to derive

the following expression:

[CO2] = [Alk] —-T-^TFl (17) aol .

, 2K2 ]

where

[Alk]=[HC03-] + 2[COi-]+[OH-]-[H+], (18)

__ aH[HC03-] aH[COr]

aH.oaeo. [HCO3-] ('^^

and ao and as are the solubility coefficients of carbon dioxide in pure

water and sample, respectively.

2. Separation and Concentration Techniques

Dissolved gases in natural and waste waters may be readily separated

by vacuum degasification or by one or more of various stripping

techniques. Stripping is essentially a gas-liquid extraction procedure in which an inert carrier gas is bubbled through a sample to carry off the dis- solved gases for further separation, concentration, or detection. Gas

transfer efficiency in such systems is primarily dependent on the gasliquid interfacial area and on the degree of mixing.
WATER POLLUTION CONTROL 369

Gas-exchange separation can be carried out as either a batch or a continuous flow process. In one design, a continuous mixed stream of sample

and carrier gas (nitrogen or hydrogen) is forced through an aspirator nozzle under fifty pounds of pressure [124]. In another design, the dissolved

gases are stripped from the test solution by means of multiple spinning

discs rotating at high speed [125,126]. Detection of the stripped gases in the stream of carrier gas may be done by measurement of paramagnetic

susceptibility, thermal conductance, etc. [124].

The gas stripped from a wastewater sample may be separated into its various components by gas chromatography [126]. Several modifications

of this technique have been reported [124-128]. By choosing appropriate

detectors, it is usually possible to analyze simultaneously for almost all gases of interest in a water or wastewater sample.

2. Sampling

In situ analysis is probably the most effective way to analyze for dis- solved gases e.g., dissolved oxygen. Certain precautions should be taken

in cases where water samples are collected and stored for subsequent

analysis. The sample must not remain in contact with air nor be agitated;

either condition will cause a change in dissolved gas levels. Samples from

any depth or from waters under pressure require special procedures to eliminate the effects of changes in pressure and temperature on sampling

and storage. Detailed description of procedures and equipment for proper

sampling of waters under pressure as well as waters at atmospheric pres- sure are available in the literature [34]

.

4. Measurement Procedures

For illustrative purposes, the analysis for dissolved oxygen is briefly

discussed in this presentation. A detailed discussion on this subject can be

found elsewhere [129].

The oldest and one of the most popular methods for the analysis of dis- solved oxygen is the Winkler Test [130]. Originally reported about 75

years ago, the Winkler procedure is still the basis for the majority of

titrimetric procedures for dissolved oxygen. The test is based on the quantitative oxidation of manganese (II) to manganese (IV) under alkaline

conditions. This is followed by the oxidation of iodide by the manganese
370 ANALYTICAL PROBLEMS

(IV) under acidic conditions. The iodine so released is then titrated with

thiosulfate in the presence of a starch indicator. In applying the Winkler Test for oxygen determinations in natural and

wastewaters, full consideration must be made of the interfering effects of

oxidizing or reducing materials in the sample. The presence of certain oxidizing agents liberates iodine from iodide (positive interference), and the

presence of certain reducing agents reduces iodine to iodide (negative in- terference). Reducing compounds may also inhibit the oxidation of the

manganous ion. Certain organic compounds have been found to interfere

with the Winkler Test in a different way. Surface-active agents for example, have been reported to hinder the settling of manganic oxide floe, thus

partially obscuring the end point of the final titration with thiosulfate

[129].

Several modifications of the Winkler Test have been devised to minimize the effect of interferences found in different waters [129]

.

The precision and accuracy of titrimetric procedures for dissolved ox- ygen may be considerably improved by using better end-point detection

techniques. Potentiometric detection of the iodometric end-point improves sensitivity to about ±0.001 mg/1 of iodine [129]. "Dead stop"

end-point detection (an amperometric technique) offers an extremely sensitive as well as accurate measurement [129] . The procedure is quite simple and utilizes two smooth platinum electrodes with a small potential dif- ference (from 15 mV to 400 mV, depending on the sensitivity required).

Diffusion current is measured during the course of the titration. No at- tempt is made to control the potential of either electrode; only the potential difference is controlled. The end-point is indicated by discontinuation

of current flow in the cell. As long as free iodine remains in the solution,

the chief electrode reaction under the influence of the applied voltage is the oxidation of iodide to iodine at the anode and the reverse process at the cathode. At the end-point when all free iodine has been removed, the

iodine to iodide reaction can no longer occur and the cell current comes to a "dead stop." Since the thiosulfate/tetrathionate reaction is highly ir- reversible and proceeds at only a minute rate under the influence of the applied voltage, no detectable current is observed at and beyond the endpoint. Ordinarily, the end-point is so easily detected that there is no need

for a graphical estimation of its position. By using sensitive current- measuring devices the end-point can be established to an accuracy of ±

0.01 microgram iodine in a 1 00-milliliter sample, or 1 part in 10 billion. Coulometric titration of dissolved oxygen by in situ electrochemical

generation of iodine has been used with considerable success [131]. The

procedure consists of the successive additions of standard solutions of
WATER POLLUTION CONTROL 371

MnS04, KOH -f KI, H2SO4, and an excess of Na2S203 to the test solution. The iodine formed electrolytically reacts with the residual thiosulfate

in solution. The electrolytic current is held constant by varying the potential, and the equivalence point is conveniently detected by the dead-stop

end-point method.

The procedure is very accurate, to within|0.02 /xg/1. It has a distinct advantage in that, because the titrant is generated in solution, errors caused

in conventional titrations by contact with air are eliminated.

Direct colorimetric methods of analysis for dissolved oxygen are based on the interaction of molecular oxygen with an oxidation-reduction in- dicator to give a color change. One of the most commonly used indicators

for the detection of oxygen in solution is methylene blue; others are indigo

carmine and safranin-T. In the presence of dissolved oxygen a reduced methylene blue solution

exhibits a blue color, and in the absence of dissolved oxygen it is colorless. This indicator has been used in the relative stability test for sewage

effluents [33]. Quantitative colorimetric determinations of dissolved ox- ygen can be made with indigo carmine dyes. Indigo carmine in the

reduced state reacts with oxygen to give a color change through orange,

red, purple, blue, and finally a blue-green in the completely oxidized form.

Colorimetric procedures are subject in general to a variety of inter- ferences which limit their applicability to industrial wastewaters.

A radiometric procedure for monitoring dissolved oxygen is based on

the quantitative oxidation of radioactive thallium-204 by oxygen in the

test solution [132]. Thallium-204 is primarily a beta emitter with a halflife of 3.6 years; therefore, decay over several months does not greatly

reduce the sensitivity of the technique. The apparatus consists of a column of radioactive thallium electrodeposited on copper turnings, and

two flow-type Geiger-Mueller counters. The technique involves passing the test solution by one of the GeigerMueller counters to detect background beta-activity, then through the

column where the following reaction occurs: 4 Tl + O2 + 2 H2O 4 T1+ + 4 OH- (20)

The radioactive thallium in the effluent from the column is detected by the

second Geiger-Mueller counter. One milligram of oxygen liberates

25.6 X 10-3 g Qf 204 ji Yhe counting rate is directly proportional to the

oxygen concentration in the test solution.

The sensitivity of the test using a column with a specific activity of 2.04

millicurie per gram of thallium is about 0.2 mg/1. That is to say, a test solu-
372 ANALYTICAL PROBLEMS

tion containing 0.2 mg/1 produces a 20471 counting rate equal to the

background counting rate of the detector. As a rule, because of the ran- domness of radioactive disintegrations, the precision of this method is± 2 percent. It is important to note that oxidizing agents and changes in the

pH of the test solution may interfere with the test. A few coulometric methods of analysis for dissolved oxygen have been

reported. For purposes of orientation, it is helpful to note that air-satu- rated water at 25 °C and under 750 mm Hg air pressure contains 8.18 mg

of dissolved oxygen per liter. This in terms of coulometric response is 0.1083 A-s/g, a rather large quantity. Two coulometric procedures are of

interest [129,133]. In one method chromous ions are generated electrolytically and are then reoxidized by the ox^/gen in solution. Detection

of chromous ions marks the end of the titration. Another coulometric

method is based on the interaction between oxygen and an ammoniacopper complex Cu(NH3)2. This is followed by the reduction of the oxidized ammonia-copper complex on a platinum cathode.

Use of a constant-potential derivative coulometric system was reported

recently [134]. The working electrode was composed of tiny metal

spheres packed in a ceramic tube. No field experience has been reported

with this system, however.

Voltammetric analyses for dissolved oxygen have been carried out with

various degrees of success using rotating platinum electrodes and

dropping mercury electrodes. The main difficulty in using such electrode

systems in natural and wastewaters is the presence of surface active and

electroactive interferences which frequently cause "electrode poisoning."

A detailed discussion of the effects of surface active agents on the polarographic oxygen determination is available in the literature [135].

Various modifications of the dropping mercury electrode system have

been developed for continuous monitoring of dissolved oxygen

[125,135]. In the absence of interferences, the sensitivity of this technique ranges from 0.05 to 0. 10 mg of dissolved oxygen per liter. Oxygen-sensitive galvanic cells have been used for some time for analyses of water effluents [135]. These are made of galvanic couples of an inert metal cathodes (e.g., lead, zinc or antimony) [136]. The cathodic

reduction of molecule oxygen results in a galvanic current proportional to the concentration of dissolved oxygen in the test solution. Change in the

pH and the conductivity of the test solution influences the oxygen measurement.

The usefulness of this type of cell is limited because the electrode

system may be easily poisoned. The electrode is in direct contact with the

test solution and surface-active compounds as well as other suspended
WATER POLLUTION CONTROL 373

material frequently adsorb on its surface, particularly in wastewaters. To

prevent incrustation, a small amount of HCl may be added to the water by

a dosing device ahead of the galvanic cell [135].

At the present time it appears that voltammetric membrane electrodes

offer one of the most useful techniques for analysis of dissolved oxygen in wastewaters. The unique feature of such electrode systems is that of the

membrane separating the electrode from the test solution. Two main

types are presently available, the voltammetric type [136] and the galvanic cell type [137]. The two types are similar to operating characteristics; in the voltammetric type an appropriate e.m.f. source is needed,

however, while the galvanic type is basically an oxygen energized cell. Oxygen membrane electrodes have three main components, the membrane, the oxygen-sensing element, and the electrolyte solution. The

membrane, the unique feature in such electrode systems, serves in three

different capacities. First, the membrane acts as a protective diffusion

barrier separating the sensing element from the test solution. Since plastic

membranes are permeable to gases only, oxygen molecules pass through,

but electroactive and surface-active contaminants present do not. The

possibility of poisoning the sensing element is thus minimized.

Second, the membrane serves to hold the supporting electrolyte in contact with the electrode system and thus makes it possible to determine ox- ygen in gaseous samples as well as in nonaqueous solutions such as industrial wastes [138].

The third advantage, discussed previously, is that the membrane constitutes a finite diffusion layer, the thickness of which is independent of

the hydrodynamic properties of the test solution.

A detailed discussion of the principle, operating characteristics, applicability, and limitations in the use of oxygen membrane electrodes may

be found elsewhere [139,140].

Polymeric membranes used with oxygen membrane electrodes show

selective permeability to various gases and vapors. Gases reduced at the

potential of the sensing electrode {e.g., SO2 and halogens) cause errone- ous readings, but these gases rarely exist in a free state in aqueous

systems. Other gases capable of permeating plastic membranes may contaminate the sensing electrode or react with the supporting electrolyte,

e.g.,C02 and H2S.

Oxygen membrane electrode systems have been used extensively in laboratory and field analysis as well as for continuous monitoring purposes. Some of the main operational problems associated with the use of

these electrode systems have been the effect of mixing in the test solution on the electrode sensitivity and short term stability which necessitates
374 ANALYTICAL PROBLEMS

frequent calibration. In monitoring operations, the accumulation of inert or biological material on the membrane surface has caused a lot of

nuisance. In addition, it has been practically impossible to reproducibly

change and mount the membranes without a change in the membrane

thickness and permeability characteristics.

These problems can be minimized by the use of a "three electrode" vol- tammetric membrane electrode system and pulse voltammetry instead of

steady state measurements [141-143]. Using an optimum pulse time and

frequency, the new electrode system exhibits 100 times higher sensitivity

and a remarkable long term stability. One of the main advantages of the

pulse voltammetric technique is that measurement is independent of the

membrane permeability and thickness. This is icalized by comparing the

steady state and transient diffusion current equations,

Equation 22 indicates that the peak current ip is independent of the membrane permeability coefficient "Pm" and thickness Under these conditions deposition of material on the membrane and changes in the mixing

characteristics of the test solution will have no effect on dissolved oxygen

measurement.

Enforcement of water pollution control programs is primarily based on

water quality analyses. Too frequently, the analytical chemist is asked to provide data on which certain legal actions have to be made. Under these

conditions the analytical chemist may have to provide evidence on the

authenticity of the sample and the reliability of the measurement.

Enforcement procedures and its legal basis are beyond the scope of this

article. However, it may be useful to illustrate the demands on analysis by

taking for an example the federal enforcement procedures.

ioc = zFAPm-r2Lo^ (21)

(22)

G. Legal and Administrative Implications

1. Enforcement Procedures
WATER POLLUTION CONTROL 375

Water quality standards for interstate streams and their classifications are being adopted. The first responsibility for enforcement lies within the

state and the final responsiblity is that of the federal government.

In the case of a violation, a notice is issued to the violator and to the

state for taking corrective actions within 180 days. Enforcement in court

is primarily based on providing evidence of violation and to prove that the

violation endangers human health and welfare. Meanwhile, it may be

necessary to prove that compliance to the water quality standards is technologically and economically feasible. Another enforcement mechanism available to the federal government

is that of the "Federal Conference Procedure." If pollution from one state

is causing harm to human health and welfare in another state or causing

economic damages to interstate fish or shellfish marketing, a federal conference is held and the states are invited to participate. The conference

does not constitute judiciary proceedings. A federal report is presented

and the states are asked to execute corrective measures within six months.

Usually federal conference procedures result in extended programs of

further fact gathering. Only after several other steps the case may be

finally submitted to the Attorney General for court action.

More recently, the "Oil Pollution Act" gives enforcement

responsibilities to the Coast Guard and the Environmental Protection

Agency. Enforcement requires the proof that pollution is caused by oil, a petroleum product or traces of oils in products. Penalties for discharge

and cost of cleanup are decided in court. Pollution of surface waters by discharges from vessels, e.g., pleasure

boats, barges, cargo ships, etc., falls under the "Oil Pollution Act." The

Environmental Protection Agency is preparing standards for effluent discharges from such vessels. Enforcement will be the responsibility of

the Coast Guard.

The Rivers and Harbors Act of 1899 (Refuse Act), which has been in- active for several decades, was recently applied to water pollution control. This is based on requiring industry to apply for permits for discharge

of effluents into navigable waters. AppHcations for permits should be ac- companied by information on the waste composition. It is the applicant's

responsibility to supply these data. The U.S. Corps of Engineers is the agency authorized to issue these

permits. Evidence of violation is provided by the Environmental Protection Agency and enforcement is under the jurisdiction of the Justice Department. Violators may be brought to court under criminal proceedings

428-999 O - LT - 72 - 25
376 ANALYTICAL PROBLEMS

in cases of nonpermitted discharge or under civil proceedings (injunction)

where irreparable damage of the receiving water is proven.

2. Problems of Collecting Evidence

One of the main problems facing water analysts is how to collect evidence in case of violation of water quality regulations. This may in- volve the collecting of "valid" samples and performing certain analyses

which may be challenged in court. Sampling and analytical procedures, commonly used by regulatory

agencies and industry, are listed in the "Standard Methods of Analysis for Water and Wastewater" [33]. The validity of these methods of analysis

is generally accepted for purposes of legal proceedings. If the analyst

chooses to use different methods of analysis, he has to provide the

evidence that they are at least equivalent and are not less reliable than the

standard methods.

One look at the latest Standard Methods [42] will give the impression

that these procedures are generally out of date and in certain cases they

lack the required sensitivity and accuracy. This is particularly true in the case of trace metals or trace organic analyses.

Another point of concern is related to the availability of adequate

analytical capabilities to certain industries and government in order to en- force water quality standards and pollution control regulation. Presently,

there is a scarcity of qualified analytical chemists, with appropriate

backgrounds who are interested in water quality characterization on a day-to-day basis. In most cases, the responsibility of water quality analysis has been left to technicians or plant operators.

The seeming lack of opportunity or the nonattractiveness of the water

pollution control field to analytical chemists should be blamed, in part, on

the managers and program directors. There is an apparent apathy, especially in government agencies, to give the analytical chemist more

responsibilities on the managerial level. This stems from a belief that chemical analysis per se is a supporting function to the main effort and

responsible decision-making positions should be filled by engineers,

lawyers or physicians. This is reflected in salary and promotional opportunities which are in contrast to the recognition earned by analytical

chemists in other governmental and industrial agencies.

More exasperating is the fact that little emphasis is given in our educational institutions to the analytical chemistry of environmental quality.
WATER POLLUTION CONTROL 377

Formal teaching in analytical chemistry is exclusively technique oriented

with little or no emphasis on applications. A majority of educators believe

that if the student is taught the fundamentals, he is capable to deduce the

applications himself. This may be true, but it is indeed a very slow, expensive process and for a nonexperienced student, it may take him years to "deduce" these applications on his own. If we believe in the merit of specialization, then it is appropriate to offer problem oriented analytical

chemistry courses dealing with health and pollution control. On the national level, the National Bureau of Standards should participate and take a leading role in formulating and adopting standard

methods for the Examination of Water and Wastewater. This could be in the form of analytical reference services for the purpose of establishing

the apphcability and limitations of classic and advanced water pollution

measurement procedures.

VI. References

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[2] Beeton, A. M.,Limnol. Oceangr. 10,240(1965).

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378 ANALYTICAL PROBLEMS

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WATER POLLUTION CON! ROL 379

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[46] Collins, A. G., U.S. Bureau Mines, Rept. Invest., 6641, 18 (1965).

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[53] Mancy, K. H., Unpublished work. School of Public Health, University of Michigan

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lege Station, Texas 77843, 123, (1965). [61] Allen, H. E., 155th National Meeting American Chemical Society San Francisco,

California (April 1968). [62] Blanchard, R. L., Leddicotte, G. W., and Moeller, D. V/.,J.Amer. Water Works Assoc. 51,61 (1959). [63] Turekian, K. K., U.S. Atomic Energy Commission Rept. 29 1 2- 1 2, 60 ( 1 966).
380 ANALYTICAL PROBLEMS

[64] O'Kelley, G. D., "Application of Computers to Nuclear and Radio Chemistry," U.S. Atomic Energy Commission Rept., NAS-NS 3017, p. 156 (1962). [65] Laitinen, H. A., Trace Characterization by Electrochemical Methods in Trace

Characterization — Chemical and Physical, Meinke, W. W., and Scribner, B. B., Editors, NBS Monograph 100, U.S. Government Printing Office, Washington, D.C.

20402(1966).

[66] Heller, K., Kuhla, G., and Machek, P., Mikrochemie 18, 193 (1935). [67] Mancy, K. H., and Okun, D. A., Anal. Chem. 32, 108 (1960). [68] Schmidt, H., and von Stackelbert, M., Modern Polarographic Methods, Academic

Press, New York (1963). [69] Hetman,J.,J.^/?/?/. C/zem. 10, 16(1960).

[70] Pohl, F. /K.,Z.Anal. Chem. 197, 193 (1963).

[71] Whitnack, G. C, Electroanal. Chem. 2, 1 10 (1961); see also Maienthal, E. J. and

Taylor, J. K., Advances in Chemistry Series, No. 73, 172-182 (1968).

[72] Farendicht, E., Electroanal. Chem., Bard, A. J., Editor, 2, 53, Marcel Dekker, Inc. (1967). [73] Gerischer, H., Phys. Chem. (Leipzig), 202, 302 (1953).

[74] Kemula, W., and Kubik, Z. ,Anal. Chim. A eta 18, 1 04 ( 1 958).

[75] Shain, I., and Lewinson, J., /Jnfl/. C/j^m. 33, 187(1961).

[76] Matson, W. R., Ph.D. Dissertation, Massachusetts Institute of Technology, Cambridge, Massachusetts (1968). [77] Matson, W. R.,Roe,D. K., and Carritt, D. E., Anal. Chem. 37, 1594(1967).

[78] Allen, H. E., Matson, W. R., and Mancy, K. H., J. Water Pollution Control Federation 42,573(1970).

[79] Peterson, T. L., Brant, D. O., and Mancy, K. H., 160th National Meeting, American

Chemical Society, Chicago, Illinois (September 1970). [80] O'Shea, T. A., and Mancy, K. H., "The Aquatic Chemistry of Copper," Research

Progress Report, The Environmental Chemistry Laboratory, School of Public Health, The University of Michigan, Ann Arbor, Michigan 48 1 04 ( 1 97 1

). [81] Mancy, K. H., and McClelland, N. I., "Water Quality Monitoring in Distribution Systems," Research Progress Report, National Sanitation Foundation, Ann Arbor,

Michigan (1971). [82] Fair, G. M., Geyer, J. C, Water Supply and Waste Water Disposal, New York

(1954). [83] Moore, E. Q., Thomas, H. A., Jr., and Snow, W. B., Sewage Ind. Wastes 22, 1343

(1950). [84] Gates, W. E., Mancy, K. H., Shafie, F., and Pohland, F., Proceedings of the 21st Purdue Industrial Waste Conference, Purdue University, LaFayette, Indiana, p. 238

(1966). [85] Montgomery, H. A. C, and Thom, N. S., Analyst 87, 689 (1962). [86] Schoniger, W., in Advances in Analytical Chemistry and Instrumentation, Reilley, C. N., Editor, Vol. I, p. 199, Interscience, New York (1965). [87] Feigl, F., Belcher, R., and Setephen, W. I., in Advances in Analytical Chemistry and

Instrumentation, Reilley, C. N., Editor, Vol. 2, p. 1, Interscience, New York (1963). [88] Melpolder, F. W., Warfield, C. W., and Headington, C. E., Anal. Chem. 25, 1453

(1953). [89] Christman, R. F., and Ghassemi, M., "Progress Report to Public Heahh Services on

Research Grant WP-00558," The University of Washington Press, Seattle, Washington (1966).
WATER POLLUTION CONTROL 381

[90] Andelman, J. B., and Suess, M. J., 151st National Meeting, American Chemical

Society, Pittsburgh, Pennsylvania (March 1966).

[91 ] Rosen, A. A., and Middleton, F. M.,Anal. Chem. 27, 790 (1955). [92] Otsuki, A., and Hanya, T., Nippon Kagaku Zasshi 84, 798 (1963); Chem Abstr. 60, 2640e(1964).

[93] Baker, R. A.,y.^mer. Water Works Assoc. 56,92 {\96A). [94] Weiss, C. M., Johnson, J. D., and Kwan, B.,J.Amer. Water Works Assoc. 55, 1367

(1963) . [95] Lure, Yu Yu, and Nikolaeva, Z. V., Zavodsk. Lab. 30, 937 (1964); Chem. Abstr. 61, 13035e(1964).

[96] Breidenbach, A. W., U.S. Public Health Service Publication, 1241 (1964). [97] Bartow, H., Chem. Eng. 72, 177 (1965). [98] Rosen, A. A., personal communication, Cincinnati Water Pollution Control Laboratory, FWPCA, USDI (1966). [99] Nemtsova, L. L, Semenov, A. D., and Datsko, V. G., Gidrokhim. Materialy 3S, 150

( 1 964) ; Chem. Abstr. 62, 1 5902x ( 1 965). [100] Baker, R. A.,J.^m£'r. Water Works Assoc. 5H,75 \ (1966). [101] Faust, S. D.,and Aly,0. U.J.Amer. Water Works Assoc. 54,235 (1962). [102] Goodenkamf, A., and Erddei, J.,y.^mer. Water Works Assoc. 56, 600 (1964). [103] Abbott, D. C, Egan, H., Hammond, E. W., and Thompson, J., Analyst 89, 480

(1964).

[ 1 04] Fahrenfort, J., Spectrochem. A eta 17, 698 (1961). [106] Wilks Scientific Corp. Publication, South Norwalk, Connecticut. [107] Bramer, H. C, Walsh, M. J., and Caruso, S. C, 151st National Meeting, American

Chemical Society, Pittsburgh, Pennsylvania (March 1966). [108] Melpolder, F. W., Warfield, C. W., and Headington, C. E., Anal. Chem. 25, 1453

(1953). [109] Gohlke, R. S.,Anal. Chem. 34, 1332 (1962). [110] Christman, R. F., Proceedings Water Pollution Control Federation Annual Meeting, 40th Symposium on Water Quality Analysis, p. 8, New York (1967). [Ill] Rogers, C. J., Chambers, C. W., and Clarke, N. A., Anal. Chem. 38, 1853 (1967). [112] Soap and Detergent Association, Subcommittee on Biodegradation Test Methods,

J. Amer. Oil Chemists' Soc. 42,9S7 (1965). [113] Anon, Ind. Eng. Chem. 57,45 (1965).

[1 14] Umbreit, W. W., Burris, R. H., and Stanffer, J. F., Manometric Techniques, Burgess

Publishing Co., Minneapolis, Minnesota (1957). [115] Sweeney, W. A., and Foote, J. K., J. Water Pollution Control Federation 36, 14

(1964). [116] Allred, R. C, Stezkom, E. A., and Huddleston, R. l..,J.Amer. Oil Chemists' Soc. 41, 13(1964).

[117] Bunch, R. L., and Chambers, C. W.,y. Water Pollution Control Federation 39, 181

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Joint Committee, y4Ai6(/3'5f 83, 50 (1958).

[

1 20] Tschopp, E., and Tschopp, E., Helv. Chim.Acta 15, 793 ( 1 932). [121] Fogg, D. N. , and Wilkinson, N. T. , nalyst 83, 406 ( 1 958). [122] Hurford, T. R., and Boltz, D. F., 154th National Meeting, American Chemical

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382 ANALYTICAL PROBLEMS

[123] Skirrow, G., "The Dissolved Gases-Carbon Dioxide" in Chemical Oceanography,

Riley, J. P., and Skirrow, G., Editors, Vol. 1, p. 264, Academic Press, New York

(1965) . [124] Thayer, L. C, and Robinson, D. A., Proceedings First International Congress of In strument Society of America, Philadelphia, Pennsylvania, Vol. 9, p. 2 (1954). [125] Swinnerton, J. W. Linnenbom, V. J., and Cheek, C. H.,Anal. C/zem. 34, 483 (1962). [126] Williams, D. D., and Miller, R. R., Anal. Chem. 34,657 (1962). [127] Szekely, G., Raoz, G., and Traply, G., Period Polytech., Chem. Engr. 10, 231

(1966) ; Chem Abstracts 67, 500 (1967). [128] Sullivan, J. P., "Determination of Dissolved Oxygen and Nitrogen in Sea Water by

Gas Chromatography," Marine Science Department, Report No. 0-17-63, U.S. Naval Oceanographic Office. Washington, D.C. (1963). [129] Mancy, K. H., and Jaffe, T., Environmental Health Series, Public Health Series Publication, 999-WP-37 (1966). [130] Winkler, L. 'H.,Ber. Deat. Chem. Ges. 21, 2843 (1888). [131] Hissel, J., and Prie, J., Bull. Centre Beige Etude et Document Eaux 44, 76 (1959). [132] Richter, H. G., and Gillespie, A. S., Sr., Anal. Chem. 34, 1 1 16 (1962). [133] Barbi, G., and Sandroni, S., Com. Naz. Energia Nucleare CNI-55, 3 (1960). [134] Eckfeldt, E. L., and Shaffer, E. V/.,Anal. Chem. 36, 2008 (1964). [135] Mancy,K. H., and Okun,D. A., ^/ifl/.C/zf-m. 32, 108(1960).

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(1970).
PANEL DISCUSSION

Analytical Problems in Water Pollution Control

CHAIRMAN

John K. Taylor, Chief, Microchemical Analysis Section, NBS National

Bureau of Standards

PANEL

James N. Butler, Head, Physical Chemistry Department, Tyco Laboratories, Inc., Waltham, Massachusetts

David M. Hercules, Associate Professor of Chemistry, University of Georgia, Athens, Georgia

Robert E. Sievers, Senior Scientist, Analytical and Inorganic Chemistry,

Aerospace Research Laboratories, Wright-Patterson Air Force Base,

Ohio

Vincent P. Guinn, Professor of Chemistry, University of CaHfornia, Irvine,

California

Khalil H. Mancy, Professor of Environmental Chemistry, University of Michigan (Speaker)

383
384 ANALYTICAL PROBLEMS

Butler — The understanding of water pollution in all of its ramifications

depends upon the existence of models, while the sophistication of such

models depends on the availability of suitable techniques of observation.

Thus a simple model might require knowledge of the presence of a single

element in a system, which would be a relatively easy task, while a highly

sophisticated model, beyond the capability of present technology, would

require a detailed knowledge of every chemical species present in a given

system.

Any concept with predictive value is a model. Thus a theory, an

analogy — ^.g. a mechanical model, and water quality criteria are examples

of models commonly used. Their complexity may range from intuitive analogies to intricate computer programs.

The simplest model, involving the distribution of elemental species, is the most capable of implementation by presently available techniques.

Trace element analysis is highly advanced, yet there are serious disagreements in the results of elemental analyses by several methods of determination, due to measurement problems, but also because the model may

have been oversimplified. In the first case, the use of Standard Reference

Materials (SRM's) offers a means to intercompare and improve meas- urement capabilities. In the latter case, a better understanding of the

fundamental nature of a given water system will lead to better meas- urement techniques which will in turn provide increased quantitative

knowledge.

The simple atomic distribution model can provide limited understanding, at best. The distribution of groups of atoms, the nature of distinct compounds that may be present, and the equilibria of mutual interactions

must ultimately be known. The equilibria between metals and ligands are known for only a relatively few cases in clean, aqueous systems. The

number of possibilities is staggering and the problem of mixed ligands has

been barely treated, even in simple systems.

Kinetic models, especially those involving transport and interfacial phenomena, are at the frontier of knowledge, at present. Analytical

techniques providing sound quantitative data are indispensable for un- derstanding these problems. One of the areas of special concern is the

water-sediment interface. Until such equilibria are better understood, ef- forts to improve many ecological problems may be meaningless. For ex- ample, phosphate release by sediments may be rate regulating rather than

the rate of its introduction into certain bodies of water. Another area of knowledge which needs further investigation is that of

fundamental physico-chemical relationships in the water system.

Knowledge of the thermodynamic activity of important chemical species
PANEL DISCUSSION 385

is very limited, yet is basic to a better understanding of many important

problems. This is true both for fresh water and for the ocean, although the

task in the latter case is somewhat simpler because the essentially constant salt concentration provides a medium of constant ionic strength.

Models have been explored in which biological phenomena are a part.

Thus, the biological removal of phosphate no doubt involves the steps,

sediment-water-algae-fish. In the last step phosphorous may return as sediment or be removed by man for use as food or for other purposes.

More complicated biological processes no doubt exist and their un- derstanding and measurement are intimately related. Microbial catalysis is well recognized, especially in redox reactions, but

the understanding is far from complete. Chemotaxis, the phenomenon of

orientation of organisms by chemical species, is also of great interest. It is now recognized that bacteria have chemical senses and that the excretion

of chemical substances is a means of communication between organisms,

as recent work on sex attractants of insects has demonstrated.

The ways in which pollution may interfere with natural processes to provide both beneficial as well as detrimental effects have been barely explored. Pesticides are probably the best known organic substances that cause adverse biological effects, but many of the compounds that occur in

oil and other organic wastes interfere with the chemical senses which

keep organisms interacting in the way which is most beneficial to them.

There may also be effects on the developmental biology of organisms but

this has barely begun to be studied.

There are also long-term low-level effects and it is difficult to identify

what is causing such effects. Pesticides are known to produce stresses at low concentrations. They decrease the insect population, thus producing

a lack of food for fish. So if you use pesticides too heavily you may lose

your fish, not because the fish are poisoned, but because they are starved.

We now find that heavy metals are removed and stored in animals.

Thus, mercury is stored in swordfish, so we can't buy swordfish in New

England any more. The removal of the phosphorus by fish has already

been mentioned. These are just a few examples.

The important point I want to make is that, whether we like it or not, we

make use of models in our thinking. If we are to be successful in un- derstanding and controlling nature, we should be aware of what models we use, and of their limitations. Although a simple model like elemental

composition may be satisfactory for some purposes, in other cases it may

be hard to understand anything without a sophisticated biochemicalphysical-ecological model.

The basic idea to remember is that everything we think about implies some model. The more aware we are of these models, and the more accu-
386 ANALYTICAL PROBLEMS

rately we make these models, the better equipped we will be to deal with

problems that arise.

Guinn — Neutron activation analysis (NAA) offers a number of potential advantages for the determination of elemental pollutants in water. This technique only determines elemental composition, but it is capable

of quantitatively determining some 75 elements with sensitivities equal to or better than many other conventional analytical techniques. In many

cases, interferences are not a serious problem, and many samples can be

analyzed using instrumental procedures in which no sample destruction or dissolution, and no chemical separations, are required.

A good practical example of the utility of the instrumental NAA

method is its use as a rapid screening procedure to identify the presence

of certain elemental pollutants. Even quite low levels of many elements

(but often in the concentration range of concern) can be detected and measured rapidly, accurately, precisely, and nondestructively by the instrumental NAA method— in many kinds of matrices of practical importance.

The determination of mercury levels in seafoods is an example of this type

of application.

Instrumental NAA is found to be capable of determining mercury at the

0.5 ppm level to a precision and accuracy of ±0.02 ppm in many kinds of

biological matrices — including, for example, grains and fish. In such

matrices, the instrumental form of the NAA method can usually detect

mercury levels as low as 0.02 ppm. If one employs the destructive

radiochemical separation form of the method, mercury levels as low as 0.0001 ppm can still be accurately measured.

Another type of application of the instrumental NAA method is in multi-element characterization analyses, in forensic studies. One example

is its use to characterize the source of crude oils and fuel oils, as in the

identification of the source (polluter) of an oil slick found on the surface of a body of water. We have found that a fairly simple instrumental NAA ex- amination of a specimen of crude oil or fuel oil can detect and measure the

levels of as many as 20 trace elements in the specimen. Extensive measurements have shown that the patterns of the levels of these various

trace elements are characteristic of crude oils from each of the world's

producing zones and fields (and also of the fuel oils produced from them).

Thus, comparison of an oil slick trace-element pattern with those of oil samples taken from ships in the area can identify which one of the ships was the source of the pollution.
PANEL DISCUSSION 387

Hercules — Instrumental techniques offer many approaches to the

identification of trace organic materials in water. Fluorimetry is especially

attractive for the detection of traces of aromatic compounds. The recent

availability of frequency-doubled laser sources should permit determinations at dilutions as low as IQ-^^ molar.

The utility of electrochemical techniques should be emphasized, since

they permit the determination of concentrations of ionic species and especially the thermodynamic activity, which information is often the desired

parameter. Kinetic methods based on differences in reaction rate cata- lyzed by free and complexed metal ions, respectively, are also promising.

Catalysis of chemiluminescence represents a particularly attractive possibility. Differences in oxidation state of ions can also be determined by

chemiluminescence.

The role of sediments cannot be over stressed. Bottom materials, such

as clays, are recognized ion exchangers and can adsorb or otherwise remove significant amounts of specific pollutants from a body of water. Long after a pollution incident may have occurred, the adsorbed material

may still be present for redistribution in the water. There are many difficult analytical problems concerned with water

recycling. Present water usage is based on the concept of linear throughput from source-to-use-to-waste. Recycling, consisting of purification fol- lowed by re-introduction to the water supply, may become a common

practice, and this will require highly sophisticated quality control measures. Not only must one determine the levels of substances known to be

present, but constituents conceivably present must be sought. Suitable

analytical methods must be developed in advance of the technology of

water purification to make recycling feasible. This symposium has been very successful in its purpose to reveal the

real problems in areas of national concern, but how can the academic

community respond to them? It appears that analytical chemists may be

basically oriented in two ways. They may be oriented along problem-solving Unes, or toward specific techniques. Both kinds of interests can coexist within the same departmental framework. There is no fundamental

conflict, and both kinds of chemists can make significant contributions.

The approach of the problem-oriented chemist is to pick out a problem

and solve it by whatever techniques are necessary. The techniqueoriented person would look at a list of problems to select one applicable to

his particular specialty.

Perhaps the most important service an academic chemist might perform

is to make his students aware of the real-life problems in a very positive

way. Approaches to such problem solving may be incorporated into the
388 ANALYTICAL PROBLEMS

training of graduate students without any great difficulty. These could be

included in special topics courses, seminars, and doctoral propositions.

It is also important that the graduate analytical chemist receive a broader background than was given in past years. This may be done by

encouraging minors in biochemistry, geology, oceanography, ecology, and

pharmacy, for example. Such minor studies would serve well to create an awareness and appreciation of problems needing analytical solutions.

It is unfortunate that recently created organizations such as the Environmental Protection Agency are so strongly crisis-oriented. It is granted that they are faced with many crises to which they must respond.

However, this response in many respects, overshadows any response in a

positive way to long-term problems.

The philosophy of long-range research needs to be cultivated. The solu- tion of the problems that may occur tomorrow but cannot be envisioned

today are perhaps more important than the solution of the problems of the

present.

Sievers —The important area of species identification in water pollution

has already been pointed out. It may not be enough to know total chromium, for example, but rather whether it is present as chromium(III) or chromium(VI), because of their differing toxicities. Likewise the precise

form and distribution of mercury compounds may be more important than

the total mercury concentration present. Clearly, the species problem is even more complex as one moves into organic chemistry. Better methods

appear to be needed for separating organic compounds, for differentiating

them, or both.

Analytical chemists have hesitated to make extensive use of nuclear

magnetic resonance methods for several reasons. One of these is expense,

but the more significant one is that the spectra are often extremely complicated. This is true even for single compounds. When mixtures are in- volved, the situation has been almost hopeless.

Recent advances in nuclear magnetic resonance methodology have

greatly improved its potential. Fourier transform procedures have pro- vided increased sensitivity for NMR so that the spectra of microgram

quantities or microliter samples can now be measured. Complexing

techniques recently developed may be used to greatly simplify NMR

spectra. For example, compounds with functional groups may be complexed with europium, which spreads out the spectra, greatly aiding their

interpretation. Such procedures have been used in pesticide residue deter- minations and no doubt have wide applicability. The fact that hydrocarbon spectra are not shifted provides the opportunity to detect small
PANEL DISCUSSION 389

amounts of compounds containing functional groups in the presence of

large amounts of related hydrocarbons. Probably the most effective shift reagent is Eu(fod)3, which was recently described in the literature {J.Am.

Chem.Soc. 93, 1522(1971)).

Gas chromatography combined with mass spectrometry has great

potential for the analysis of pollutants in water. This procedure utilizes the mass spectrometer to substitute selectivity for the highly sensitive but

relatively unselective electron capture detector. Simplification of the mass spectrometer is needed to fully utilize such a procedure.

One of the problems up to the present time has been that only expensive general-purpose mass spectrometers have been produced. A simplified inexpensive instrument is needed to provide the capability of monitoring a given mass during the course of a chromatographic separation

with high sensitivity. It should also be possible to change the mass detected during a separation, depending upon the retention times of the compounds of interest and the motivations of the experimenter. It would seem

that such an instrument could be produced for about $ 10,000 as compared

with the $50,000 to $70,000 cost of the more versatile equipment on the

market today.

The instrument suggested would sacrifice resolution for sensitivity (unit mass resolution at m/e 100-200 would be adequate). The differentiation,

now provided by resolution, would be obtained by using a gas chromatograph at the inlet. An example of application is the determination of chromium or beryllium after conversion to the trifluoroacetylacetonate. The electron capture

gas chromatographic procedure is relatively simple for air filter samples

which are customarily ashed before analysis and quantitations at the ppb

(10-^) level are achieved. No difficulties are encountered because the

ashed samples are relatively free of electron-capturing interference On

the other hand, in looking for chromium at the 5- 10 ppb level in biological

matrices such as blood or serum, electron-capturing impurities are often

encountered. These interferences can be avoided by the use of a mass

selective detector.

General Discussion — The general discussion dealt largely with the

problem of the interpretation of analytical data. Too often, the ideas as to research needs come from within pollution control agencies. The scientific community needs to inject more in-put into the development of pro- gram plans.
390 ANALYTICAL PROBLEMS

Analytical chemists need to present their data in terms in which it can be understood. Moreover, they cannot equivocate. Decisions must be

made using the best judgement, based often on incomplete data. While

this is contrary to best scientific practice, the chemist must interpret his own measurements, lest they be interpreted by others with less scientific perspective.

With respect to the reliability of specific analytical measurements, a

plea was made for the more general use of Standard Reference Materials

to validate procedures. There are presently only a few SRM's that are applicable and the list needs to be greatly expanded. The cooperation of

water analysts and pollution technologists is required to identify the most

urgent needs.
James H. Carpenter, is Professor of Earth and Planetary Sciences at Johns

Hopkins University. Dr. Carpenter received his B.A. in chemistry and

biology in 1949 from the University of Virginia. He earned an M.A. and

Ph.D. in 1951 and 1957, respectively, from Johns Hopkins University

with a major in oceanography. He was active in the Chesapeake Biological Laboratory (1949-50) and has been connected with the Chesapeake

Bay Institute as a research assistant (1950-54); research staff assistant

(1954-57); and research associate (1957-60). He became Assistant

Professor of Oceanography at Johns Hopkins in 1958 and a research

scientist at Chesapeake Bay Institute in 1960 (a position he still holds). In 1967, he was made an associate professor at Johns Hopkins. Dr. Carpenter is on the Board of Editors of the Journal of Marine Research and

is a member of the American Society of Limnology and Oceanography,

the American Geophysical Union, Sigma Xi, and AAAS. He is Past President of the Atlantic Estuarine Research Society and is Chairman of the

Panel on Chemical Methods, National Academy of Sciences Committee

of Oceanography. He has more than 20 publications connected with measurement techniques in natural waters. 391

428-999 O - LT - 72 - 26

xNATIONAL BUREAU OF STANDARDS SPECIAL PUBLICATION 351,

\nalytical Chemistry: Key to Progress on National Problems,

Proceedings of the 24th Annual Summer Symposium,

Held June 16-18, 1971, Gaithersburg, Md. (Issued August 1972). CHAPTER 7

PROBLEMS IN APPLICATIONS OF ANALYTICAL

CHEMISTRY TO OCEANOGRAPHY

James H. Carpenter

The Johns Hopkins University

Baltimore, Maryland 21218

The general character of oceanic and estuarine systems poses special

problems in applying analytical chemistry to our description of these environ- ments. The chemistry of sea water is described in general terms and the result- ing analytical problems are identified by type examples. For the more abundant constituents, analytical precision and accuracy approaching the classical atomic weight measurements is required if improved data are to be obtained. For the moderately abundant constituents and, in particular, for those ele- ments that participate in biological processes, large time and space variations occur and the requirement of large amounts of data favors automated analyses,

if precision and accuracy can be retained. For the trace constituents including

many of the heavy metals, inaccuracy produced by current techniques and

analysts has produced a nearly useless body of data.

Keywords: Chemical analysis; oceanography; pollution; water analysis.

I. Introduction

Oceanography is the study of a very large, naturally occurring object,

rather than a system that is arbitrarily defined for intellectual purposes.

This large mass of water has been repeatedly evaporated and condensed

to produce a massive leaching of the solids at the earth's surface. This

process may be observed today and we see that the rates of addition and

loss of compounds of the mobile elements (high aqueous solubility — little other reactivity) are millionths of 1 percent per year of the existing quantities in the oceans and even for relatively immobile (low aqueous solubili- ty—high chemical reactivity) elements the fractional change in inventory

is of the order of 0.01 percent per year. Mixing rates based on flow mea393
394 ANALYTICAL PROBLEMS

surements or on the distribution of cosmic ray produced radiocarbon are roughly 500 to 1000 years. The solution is well stirred and, therefore,

rather uniform.

If the rates of the energetically favored reactions were rapid, with half

times of decades to perhaps a century, it would be possible to write a concise thermodynamic description of the oceans and that would be that. However, many of the reactions involve complex mechanisms. Activation energies to pass through intermediates appear crucial. Some reactions appear to occur only at solid surfaces and even then at rather slow

rates. Goldberg [1] estimates the rate of growth of manganese nodules,

which are widely distributed in the surface sediments, at 1 to 100 atomic

layers per day (0. 1 mm per thousand years).

The other complicating aspect of the oceanic system is the presence of

living organisms. Constituents are assimilated, the biological structure moves in contrast to the water motion and an altered distribution is produced. Swimming is important but sinking from gravitational attrac- tion is the major process. The effects of biological activity vary from ele- ment to element depending on the degree of participation in the biological process. Productivity of the photosynthetic organisms in the oceans is limited by light and the availability of nutrients. As pointed out by Ryther

[2] , the situation in the ocean is rather different than on land, which he il- lustrates by considering the availability of fixed nitrogen. For a terrestrial environment with fertile soil one meter thick, the fixed nitrogen content of

approximately 0.5 percent could support a crop or yield of approximately

50 kg of dry organic matter per meter square. The structure of the ter- restrial photosynthetic organisms leads to conversion of solar energy to an organic yield of 10 g per day or an annual production of several kilo- grams per meter square and the local supply of nitrogeo is adequate for maximum growth for many years. In contrast, the oceanic environment

contains 0.00005 percent fixed nitrogen and the surface layers that have

sufficient light intensity to permit plant growth (the euphotic zone)

average 0.00001 percent fixed nitrogen. This productive layer is roughly

100 m thick and the reservoir of fixed nitrogen is 10 g. Complete conversion to organic matter would yield roughly 100 g of dry plant tissue. Self

shading as the crop develops reduces the rate of production and the total possible crop is not achieved. The factor of 500 between the possible ter- restrial yield and oceanic yield suggests that feeding the world's human

population will be based on better management of the terrestrial system.

Limited areas of the oceanic system have high yield because the producer

extracts nutrients from a large volume of water, i.e., attached plants such

as kelps and turtle grass or fish that maintain themselves in areas where
OCEANOGRAPHY 395

upwelling continuously brings nutrient rich water into the euphotic zone. Many of the processes and phenomena studied by soil chemists and

agronomists are important in the oceans but the temporal and spatial in- tensities of the processes are quite different.

II. Some Historical Aspects of Oceanography

The scientific study of the oceans developed as a part of the disciplines

of physics, chemistry and biology, with valid observations beginning in the seventeenth century. As noted by J. P. Riley [3] in his historical

review, Robert Boyle might be considered the founder of scientific chemical oceanography in about 1670. Boyle's work included efforts to determine the salinity of sea water by evaporating samples, with results that were so variable that the method was considered unsatisfactory. Three

hundred years later, this wretched characteristic of sea water salts plagues

the modern analyst and we are unable to determine the simple property,

total dissolved solids, by the obvious operation of quantitatively removing

the water. Boyle and Hooke resorted to indirect estimates from measure- ments of specific gravity with a hydrometer or crude pycnometer as a more precise estimate. The use of indirect methods persists today and

conductivity or refractive index measurements are employed to estimate

the salinity of sea water samples.

During the eighteenth century, the first analyses of the composition of

sea water were undertaken by Antoine Lavoisier, using selective

precipitation during evaporation and selective extraction of the remaining

salts. The fractions isolated with Lavoisier's method were shown by John

Murray in the early nineteenth century to be quite impure and with the ad- vent of the ionization theory of Arrhenius, Murray proposed that the in- dividual "acids and bases" be determined as a sounder procedure for establishing the composition of sea water. The difficulties with Lavoisier's method that result from the multicomponent aspect of sea water are a recurring point of distress in the Hterature of the analytical chemistry of sea water. Analytical properties have been ascribed to one constituent

and subsequently shown to be influenced by unsuspected constituents.

The development of oceanography as the integrated application of the

scientific disciplines to the study of the oceans received major impetus

from the planning and work undertaken on the world-wide expedition of

H.M.S. CHALLENGER from 1873 through 1876. The analyses of samples from this expedition by W. Dittmar [4] were reported in a de-
396 ANALYTICAL PROBLEMS

lightfully comprehensive report. The care and logic with which Dittmar

devised and tested analytical procedures and his realistic appraisal of the

probable accuracy and precision of the sample results are a model of

quality that modern analysts have difficulty in emulating. Considering the

limitations of chemical theory and analytical techniques available to Ditt- mar, his results are a remarkable achievement. The accuracy of his results as judged by comparison with modern results is better than 1 percent ex- cept for the magnesium results that appear to be 2.5 percent too high.

The methods for each constituent were tested and then adhered to

rigidly, so the systematic errors might be nearly constant and Dittmar

hoped that if subsequent investigations demonstrated systematic errors, a simple correction might be appHed to his resulte for the samples from all the oceans. In addition, the precision of the measurements was estimated

to be of the order of 0. 1 percent. Dittmar's data show variations that are

larger than would be expected to arise by chance, but he could not correlate them with depth or geographical position except for calcium and the

calcium/chloride ratios for deep waters were found to be larger than the

ratios for surface waters. The calcium variation has been "rediscovered"

every few decades since Dittmar's publication.

The results for the 77 samples studied by Dittmar established that the

major constituents of sea water are present throughout the oceans in nearly constant proportions, but that small variations might be present.

This principle of constant proportions has been used as a major feature of

oceanographic work. Each of the major constituents may be estimated

from any one of the others when the proportionality constants have been

determined. Many of the papers on chemical oceanography during the

twentieth century have been studies of the proportionalities, usually in the

form of the ratio of a constituent to chlorinity (the sum of the halides ex- pressed as chlorine equivalent using 1900 atomic weights — see below).

The importance of these results is that all the physical properties, and, in particular, density, may be expected to correlate to chlorinity with high

precision.

Salinity was estimated by Dittmar as the sum of the individually deter- mined constituents, a laborious procedure. The need for precise relationships between an easily determined quantity and both salinity and density

(more accurately, specific gravity) led to the formation of an international commission in 1 899 to investigate and advise on the definition and

determination of salinity. The work of the commission was reported by

Forch, Knudsen and S0rensen in 1902 [5]. S0rensen devised a method

for evaporating sea water samples that yielded a reproducible mass of

residue, a tedious procedure but one that the commission deemed ac-
OCEANOGRAPHY 397

ceptable. The analytical results from S0rensen's method were taken as being identical with a definition of "salinity as the weight in grams of the

dissolved inorganic matter in 1 kg of sea water, after all the bromide and

iodide have been replaced by the equivalent amount of chloride, and all carbonate converted to oxide." The results with S0rensen's procedure are 0.46 percent smaller than the mass of total dissolved salts in open ocean

waters and the precision can be 0.05 percent, i.e., the accuracy is an order

of magnitude poorer than the precision. The method was used on nine

samples, of which two came from the open ocean and the others came

from the North Sea, Baltic Sea and Red Sea. A correlation between measurements of chlorinity and salinity by the S0rensen procedure was found

and the best fit equation was

8 = 0.03 + 1.805 [CI] ing/kgor«/oo (1)

The commission felt that "salinity" could be estimated reliably from

either specific gravity or chlorinity measurements, but their report clearly

distinguishes between directly measured values of salinity and estimates

from another quantity. As Knudsen points out in the first sentence of the

section of the report dealing with salinity, "By salinity one means when no

other stipulations are made, the weight of dissolved salt per unit weight of sea water." Knudsen used the symbol Sci to designate values derived

from chlorinity measurements, but others have felt that this was too cumbersome and nearly all the twentieth century oceanographic literature conforms to the use of the symbol S and the word salinity to mean the

S0rensen-salinity estimated from chlorinity. It is necessary to guess the

meaning of salinity in various publications and it appears probable that authors using ordinary dictionaries and without special knowledge of

oceanographic nuances would mean total dissolved salts and that authors

publishing in oceanographic journals would mean the above peculiar

quantity.

During the past two decades, instruments for measuring relative conductivity of sea water samples with a precision of 0.005 percent have been

devised and are commercially available. The high precision results from

the use of inductively coupled cells rather than metallic electrodes with

their instability. The superficial simplicity of the manipulations required

in the use of such instruments has lead to great popularity. There is a seri- ous deficiency in the conductivity technique, which is that reproducible

and defined standards are not available. An adequate standard should be

defined to 0.001 percent and should be reproducible to the same extent. Current standards for electrolytic conductance based on solutions of
398 ANALYTICAL PROBLEMS

potassium chloride are reproducible only to approximately 0.04 percent. In lieu of an adequate standard, water samples from the ocean are being

used as a reference substance. An effort at building an instrument to determine the absolute conductivity of aqueous solutions has been in- itiated at the National Physical Laboratory, but, to my knowledge, has not been successful.

This deficiency associated with conductivity measurements has led to some unfortunate developments and misconceptions in the recent litera- ture. The problem has been considered by the UNESCO Joint Panel on

Oceanographic Tables and Standards and tables [6] of recommended

values of relative conductivity have been distributed. In addition, a note

that encourages the use of these tables has been published in several journals, [7] as an example. However, the note is titled "redefinition of salini- ty" and includes the statement "Expression (3) constitutes the recom- mended definition of salinity" and the referenced expression is S%o = - 0.08996 + 28.29720 Ri5

+ 12.80832 Rfs- 10.67869 Rf- + 5.98624 R^5- 1.323 1 1 Rp5

Ri5 is the ratio of the conductivity of a water sample to that of water

having a salinity of exactly 35<^/oo both liquids being at a temperature of

1 5 °C and under a pressure of one standard atmosphere.

Examination of this proposed "definition" shows some remarkable fea- tures, not the least of which is the fact tha^ it is illogical. Water with little conductivity would have a negative salinity, a difficult concept to comprehend. The definition seems mysterious unless there is some place some

water with a "salinity of exactly 35^/oo" that is known to the authors but

not generally known. The reasons that the measurements of Cox et al [8]

produce this strange empirical relationship seem to be the following.

1. Salinity was not measured, but rather estimated from

chlorinity measurements. The salinity estimates were made

using the expression S = 1.80655 [CI] rather than the relationship of Knudsen S = 0.03 + 1.805 [CI]. The Knudsen results had been criticized by Cox [8] as implying that salinity was

nonconservative in the ocean, i.e., if waters of different salinity

and chlorinity were mixed, the salinity and chlorinity of the

mixture could not be computed by simple proportionality.
OCEANOGRAPHY 399

2. Neither expression for estimating salinity from chlorinity has

universal validity. In the open ocean, chlorinity and presumably

salinity vary because of evaporation and precipitation of nearly

pure water and there should be direct proportionality between

chlorinity and salinity. The functional form of Knudsen's empirical relationship is due to the distribution of the samples, which came in a large part from areas where substantial mixing of sea water with land runoff had occurred. Since river waters draining

into the Baltic Sea contain relatively large concentrations of

salts as compared to chloride, the empirical expression predicts

appreciable salinity when the chlorinity is zero. Each expression seems to have a legitimate application but the correct one must be identified in each situation.

3. The samples on which the new tables and definitions are based included low salinity waters from the Baltic Sea. The

equation used to estimate salinities for these samples will produce an underestimate of approximately OA^Uo and the

negative intercept arises entirely from the manipulation of the

data. Most chemists find the mathematical transformation of the chlorinity to estimates of salinity not very useful. For example. Carter et al. [9] in 1933 stated "The salinity is ascertained from the determined chlorinity by

reference to the Hydrographic Tables, or in other words, the chlorinity is multiplied by a constant and 0.03 added to the resulting value. The term

salinity thus seems to be superfluous and its determination is a useless

gesture. The direct reporting of the nature of sea water as the chlorinity

appears to be the easier and more logical method." For several decades,

workers on the West Coast of the United States and Japan reported only

chlorinity. Now everything is converted to S0rensen-Salinity and the

several hundred thousand values recorded in the National Oceanographic

Data Center are a mixture of estimates from chlorinity titrations or conductivity comparisons.

There is a deep-seated desire by many physicists and biologists who

study the ocean to know the "saltiness" of various parts of the system.

The fact that there is no adequate method of measurement does not abate

that desire, and it does not appear to be possible to abandon the uncertain

estimating process described above. This is an area where analytical

chemistry is the paramount tool and a new technique with the required

precision and simplicity would find wide acceptance.
400 ANALYTICAL PROBLEMS

III. The Determination of Halides

The most numerous analyses of sea water have been the titrimetric esti- mation of the halides, using precipitation with silver nitrate solutions. The

work of the international commission [5] reported in 1902 put the measurement of hahdes on a sound basis. The commission recognized the ex- treme importance of an adequate standard and initiated the distribution of

ampoules of "standard sea water," which are samples of sea water that have been compared with the original standard water using the Volhard

method by weight to obtain a precision of 0.003 percent. This practice has

continued to today and we are in the very pleasant position of having

several hundred thousand observations connected to a precise standard

in an unequivocal manner. The chlorinity was defined in 1 902 as the mass of chlorine equivalent to the total mass of halogen in the mass of 1 kilogram of sea water. By 1937

the original stock of standard water was nearly depleted, and Jacobsen

and Knudsen [10] undertook the preparation of a new supply. They

recognized that the definition of chlorinity was dependent on the atomic

weight values and, therefore, subject to change with changes in the ac- cepted values. Since the purpose of chlorinity observations on sea water

is to describe the oceans and to compute the specific gravity from empirical relationships, Jacobsen and Knudsen recommended that the chemical

concept of chlorinity be discarded and be replaced by an oceanographic

operational definition CI ==0.3285234 [ Ag] %o or "the number giving the

chlorinity in per mille of a sea-water sample is by definition identical with

the number giving the mass with unit gram of Atomgewichtssilber just

necessary to precipitate the halogens in 0.3285234 kilogram of the sea- water sample." This gives rise to the unusual situation where the symbol

CI refers to a mass of silver.

It seemed impractical to change the data collected over nearly four

decades and the new definition was made to be numerically identical to the equivalence between silver and chlorine that had been assumed by

S0rensen in 1902. The specification to seven significant figures means

that this arbitrary definition will not need refinement. The ratio of

chlorine-equivalent to chlorinity of approximately 1.0045 is easily handled when problems requiring chemical concepts are considered. This

procedure of putting chlorinity measurements on a "silver standard" seems eminently sound.

The only point of real difficulty in the past seven decades has been the

limited use of sodium chloride as a standard by a few observers, as pointed out by Reeburgh [11], and these results can be converted to the
OCEANOGRAPHY 401

chlorinity definition if the assumed atomic weights have been stated by

the authors. However, there is an aspect in which some confusion has

arisen. In the review by Carritt and Carpenter [ 12] , we pointed out that many of the observations of ionic ratios showed discrepancies from constancy that were larger than might be expected from the estimated precision of the procedures and a statistical evaluation suggested that small

variations might be present in the oceans — similar to Dittmar's conclusions. These comments, in part, stimulated an extensive investigation of

specific gravity, relative conductivity, chlorinity and individual element

concentrations for 205 samples collected throughout the oceans. The first measurements on these samples were reported by Cox et al. [13] and it was concluded that the density (actually specific gravity) of sea water

samples could be estimated from conductivity measurements with less variation than from chlorinity measurements and this conclusion received

considerable publicity. Subsequently, as reported by Cox et al. [8], the

precision of the procedure for chlorinity measurement was found to be

less than had been thought. An alternate procedure was used to repeat the

chlorinity determinations and "comparison of the results shows that a

large part of the scatter previously reported in the relationships between

chlorinity and conductivity, and between chlorinity and density, was due

to analytical errors." This erroneous apparent failure of the constancy of major constituent

proportions has lead to suggested rejection of chlorinity as a primary measurement. For example, Horne [ 14] states "the electrical conductivity is a property which can be measured with very great accuracy both in the

laboratory and in situ . . . The concept of chlorinity has outlived its usefulness and should be allowed to die a natural death before it becomes an

impediment." It appears to me that Home was misled into confusing ac- curacy with precision and the "silver-standard" chlorinity will remain the

property of sea water which can be measured with a precision of 0.003

percent with the Standard Sea Water Service Volhard technique when

needed and with a definition that makes accuracy meaningful only in terms of the purity and isotopic composition of the standard silver. Until a technique for determining the cell constant of conductivity instruments

is devised, careful chlorinity measurements will serve a useful and

essential function in the study of the oceans and in the calibration of

conductivity instruments.

The requirements of extreme precision in oceanographic measurements

derive from the monotony of the distribution of properties in the oceans. Rather easily observed variations of the motions and saltiness occur in the

surface layers to depths of 200-400 m (5 to 10 percent of the volume). For
402 ANALYTICAL PROBLEMS

the remainder of the system, variations with time and position are rather

small. For example, some data being studied by W. Boicourt at the Johns

Hopkins University and the Woods Hole Oceanographic Institution are plotted in Figure 1 . The apparent change in S and O2 with time might be

construed as an oceanographic event. The change in S is approximately

0. 1 percent of the mean value and the change in O2 is approximately 4 per- cent of the mean value. Assurance that the precision of such measure- ments over the extended period of time was 0.01 percent for S and 0.4

percent for O2 is needed for confidence in interpretation.

36.50

if) 36.45

1 \ \ \ \ \ I \ \ I \ f I I I I I—

r

CM o

5.0 4.9

4.8

4.7 4.6 4.5 44

J__J \ \ \ L J \ \ I I I I I I \ L

J \ i \ \ \ I

MJ J ASONDIJ FMAMJ JASOND

1959 I960

I I I I I I MJ JASONDiJ FMAMJJ ASOND

1959 I960

Figure 1 . Time record of salinity and oxygen on the 1 8 °C isotherm at Panulirus Station (32°10'N64°30'W).

IV. Chemical Composition of Sea Water

Some approximate average values for the total concentrations of the

various elements in sea water are listed in Table 1. The elements have
OCEANOGRAPHY 403

Table 1. Approximate sea water chemical abundances.^

Element mg/1 Element mg/1

Sn 0.003

Na 10,600. Pb 0 003

Ms 1,300. u 0.003

S (SO4) 900. (2600) V 0.002

Ca 400. Mn 0.002

K 380. Ti 0.001 or Th 0.0007

Co 0.0005

N -LO. Ni 0.0005

0

.

Ga 0.0005

or fi0

.

Cs 0.0005

0 4 8 Sb < 0.0005

01 Q0

.

0.0004

FJ? L.O 0 0003

A U.D V 0 000*^

NT fNO ) U.O VZ^ INc 0yj 000"^ . \j\j\jo j\r 0\J .\J\J\JO OOO'^

SXIJ 0 19 Act 0 0003

P (POJ 0.07 (0.2) 0 0002

I 0.05 0 00011

Ba 0.03 w 0.0001

Al 0 01 0.0001

r e 0 01 YpAe 0 0001

Zn U.Ui Cr U.UUUUo

Mo 0.01 Sc 0.00004

Se 0.004 Be 0.00005

Cu 0.003 Nb 0.00001

As 0.003 Tl <0.00001

Modified from Goldberg [1).
404 >\NALYTICAL PROBLEMS

Table 1. Approximate sea water chemical abundances.^ (continued)

Element mg/1 Element mg/1

He 0.000005 2 X 10~7

Au 0.000004 no 8 X 10-8

La 3 X 10 hir 2 X 10-7

Ce 4 X 10""7 i m 4 X 10-8

Pr 2 X 10-7 Yb 2 X 10-7

Nd 8 X 10"^ Lu 4 X 10-8

Sm 1 X

10-'^ Ra 3.0 X 10-^^

il/U 4 X 10-8 Pa 0 X 10-12

Gd 2 X

10-'^ Rn 9.0 X 10-15

^ Modified from Goldberg [1 1.

been grouped into four ranges of abundances, which might be termed

"major, minor, major trace and minor trace." Some very rough generalizations may be made concerning spatial variations in the abundances of the

elements. As indicated above, the major elements occur in nearly constant relative proportions and the variations may be of the order of a few

tenths of one percent. The minor elements show greater variation, with

the extent of variation correlating with the participation of each constituent in biological processes. Bicarbonate ion, molecular oxygen, sil- icate ion and nitrogen as primarily nitrate ions show the greatest variation.

For the other minor constituents, the variations may be approximately 1 to 1 0 percent. The major trace elements are mostly metals and many are associated in biological processes. Phosphorus as phosphate shows ex- treme variation but the others may show variations of a factor of two to

six. The data for the minor trace elements are so sparse and uncertain that no impression of the real variability can be derived from the existing infor- mation. These rough estimates of the variations may serve as a guide in developing analytical methods.
OCEANOGRAPHY 405

V. Distinctive Aspects of Sea Water Analysis

In contrast to most chemical analyses that are made for some immediate purpose, analyses of sea water provide data that may be

referenced repeatedly. The data that have been accumulated over an ex- tended period of time and observations by more than one group are combined to produce descriptions of various ocean regions. While there are many examples of research carried to completion by a single individual

during a period of a few years, much oceanographic research is based on

pooled data. Questions are posed after the data have been collected, and

the analyst is not provided with a clear estimate of the required accuracy

and precision of the measurements. Because of this aspect, the compromise that must be made between accuracy, precision, cost, speed and

ease in choosing an analytical method should be weighted toward accura- cy and precision. For example, it is justifiable to build a mass spectrograph for dissolved gas analyses if the accuracy or precision of the measurements would be improved over those obtainable with commercial in- struments. Sample integrity is particularly significant. Water samples from the ocean are several hours old when they arrive on the deck of the research

vessel. Contamination of the sample with materials from the walls of the

sampling equipment is possible. Samplers must be lowered open and in- adequate flushing at depth may lead to contamination with surface waters,

a potentially serious problem for materials with a strong vertical gradient

such as radionuclides supplied to the ocean by atmospheric fallout. Hydrophobic coatings have only recently been used to decrease reaction

of the sampler surfaces with the sea water. Once on deck, reduced pressure and increased temperature may lead to

loss of gases and special samplers are necessary for samples that are analyzed for gases. A striking temperature and pressure effect has been

recently demonstrated for ion exchange equilibria of such ions as potassium in the interstitial waters of sediments and a large part of the apparent

enrichment with potassium in such waters has been shown to be a sampling artifact. Transfer of sample aliquots to smaller containers provides increased

surface areas relative to the aliquot volumes. Adsorption of dissolved and

particulate material on the walls of the sample container provides an en- riched environment for the rapid growth of bacteria. Biological processes can change the sample composition within a few hours. Rapid freezing has

I been shown to stabilize the phosphate and nitrate content for several

' months but changes in ammonium content have been observed with
406 ANALYTICAL PROBLEMS

Storage at —20 °C. Shipboard analysis is obviously desirable and, at a minimum, the sample must be processed to some stable form. Plastic containers have been used with serious evaporative losses through low density polyethylene bottles and nearly complete uptake of phosphate on the

walls of polyethylene bottles. Most of the organic constituents and the

"heavy" metals occur at micromolar or less concentrations and sampling

contamination or losses must be evaluated in detail for each procedure.

Transport of samples to shore laboratories aggravates the sample in- tegrity problem, but shipboard analysis poses problems in equipment and

analytical soundness. Shipboard laboratories are subject to various amplitudes and frequencies of motions, ranging from vibrations from

reciprocating engines to the pitching and roUing of the whole ship. Simple

manipulations involved in analytical procedures take on new dimensions

when they are performed with one hand while seasick and using the other

hand for physical stability. Measurement techniques have been limited to volumetric titrations, spectrophotometry, fluorometry and electrometric

determinations using soHd electrodes. As better ships have been made

available, more elaborate equipment such as mass spectrometers for nitrogen- 15 tracer experiments are being used successfully. Most of the

routine methods that are used on shipboard are spectrophotometric

because of the reliability of the equipment and the simplicity of manipulations. These procedures are adaptable to automatic equipment and a modified six channel AutoAnalyser was used on a recent expedition to the Black Sea.

VI. Some Examples of Analytical Problems

The major problems that may be found in using the chemical oceanographic data are inaccuracies and the lack of adequate tests of procedures

for the purpose of making realistic accuracy statements. As noted above,

many oceanographic questions could be approached using relative values

if adequate standards could be devised. Mangelsdorf and Wilson [15]

have developed a technique of difference chromatography using ion

exchange membrane cells as a sensor that is responsive to small variations

in the composition of solutions passing through the column. Using Stan- dard Sea Water as an arbitrary reference, they have been able to examine

several hundred samples for variation in the proportions of sodium, magnesium, calcium, potassium, sulfate and chloride. The precision of their measurements range from 0.1 percent for magnesium to, on occasion.
OCEANOGRAPHY 407

0.02 percent for potassium, i.e., the technique is suitable for oceanic

research. The results for potassium, which had not been studied previously with precise techniques, showed that the variations in the relative

concentrations of potassium are approximately 0.1 percent. Measurements by their technique which require stability of fractions of a microvolt

in the detecting circuit cannot be considered routine, but the high data rate associated with the multicomponent response of the system makes it at- tractive for oceanic surveys based on large numbers of samples. The

limitations are that all the samples must be examined with reference to the same standard and only relative values are obtained.

A. Magnesium

Other studies of the major constituents have been based on "absolute"

measurements, using supposedly known solutions as standards or reference solutions for tests of gravimetric procedures. The tediousness

and low data rate of the methods for the major constituents have

prevented real delineation of variations in the oceans. The results for magnesium may be used to illustrate the problem.

Some of the magnesium analyses of sea water are summarized in Table

2. The work of Culkin and Cox was based on 66 samples collected

throughout the oceans and is the most comprehensive of the recent work.

However, there appears to be a systematic error in the titration procedure

used by Culkin and Cox, which is that the photometric end point that they

used is approximately 1 percent beyond the equivalence point. This point

is discussed by Manella [16] and the results of calculations to evaluate

the possible error are shown in Figure 2. Correction of the results of Culkin and Cox for this probable error would change the Mg/Cl ratio from

0.06692 to 0.06629, in close agreement with Manella's result of 0.06626.

The other titrimetric results probably have end point errors of 1 percent

or more. The results of Billings et al. were corrected approximately from

0.0675 mg/1 per °/oo to 0.0658, assuming a density of 1.025 g/cm^.

As may be seen in Table 2, it is difficult to come to any conclusions re- garding possible differences in the magnesium content of various parts of

the oceans. The data reported for the Pacific Ocean average roughly 1 percent higher than data for samples from the Atlantic Ocean, but neither

Dittmar nor Culkin and Cox, who studied samples from both oceans, found systematic differences between these oceans. It appears that there

are unsuspected errors in the analytical methods as used by the various

authors.

428-999 O - LT - 72 - 27
408 ANALYTICAL PROBLEMS

,9 -

99 100

% EDTA ADDED

104

Figure 2. Theoretical titration curve for the analytical system Mg-EDTA-EBT used by

Culkin and Cox (1966).

Billings et al. report a 10 percent temporal variation in the magnesium

to chlorinity ratio for a station near Bermuda. Samples from the same area

studied by Manella did not show variations in the ratio larger than 0. 1 percent, and it may be inferred that there are substantial errors in the atomic
OCEANOGRAPHY 409

Table 2. Obsen^ations of magnesium—c hi or inity ratio. Reference Locality Method Mg/Cl

Dittmar (1884) Various 1 0.06801

Thompson and Wright (1930) N. Pacific 1 0.06695

Voipio (1957) Baltic 2 0.06693

Voipio (1959) Barents 2 0.06742

Pate and Robinson (1961) Pacific 4 0.06689

Culkin and Cox (1966) Various 3 0.06692

Szabo (1967) Atlantic 6 0.06650

Tsunogai. et. al. (1968) N. Pacific 5 0.06676

Billings, et. al. (1969) Atlantic 7 0.0658

Manella(1971) Atlantic 8 0.06626

1. Gravimetric as magnesium ammonium phosphate.

2. Titrimetric difference. Total alkaline earths determined with EDTA

standardized against calcium salt with visual end point.

3. Titrimetric difference. Total alkaline earths determined with EDTA

standardized against calcium salt with photometric end point.

4. Titrimetric difference. Total alkaline earths determined with EDTA

as the primary standard and visual end point.

5. Titrimetric difference. Total alkaline earths determined with EDTA

standardized against zinc metal with visual end point.

6. Titrimetric difference. Total alkaline earths determined with EDTA

standardized against magnesium metal with visual end point.

7. Atomic absorption.

8. Magnesium isolated with ion exchange and titrated with CDTA standardized against magnesium metal with photometric end point.

absorption procedure, even though the precision with standards was 1.5 percent.

Finally, several of the reports listed in Table 2 claimed precision and.

presumably accuracy, of a few tenths of one percent. It appears probable

that there were determinate errors of approximately 1 percent in these

results. The error is just large enough (10^^ g Mg in the oceans) to affect work on the colHgative properties of sea water and the ionic balance.
410 ANALYTICAL PROBLEMS

B. Strontium

There are not a large number of studies of the strontium content of the oceans, but strontium may be used to show the character of the data for a minor constituent. The first quantitative estimates were by Webb [17],

who reported 13 mg/kg with an emission spectrographic method in the

late 1930's. Flame photometry was popular in the late 1940's and measurements by Odum [18] and others led to values of 8.2 to 8.5 mg/kg.

Application of atomic absorption in the 1960's by Angino et al. [19] led

to values of 7.2 to 7.5 mg/kg. One explanation would be that the strontium content of the oceans is decreasing approximately exponentially with

time. However, Carr [20] has shown that the procedure of calibrating the

atomic absorption measurements with standards prepared in sodium

chloride solutions with a total salt content different from that of the samples produces systematic error. Comparison of internal standards or matched synthetic standards with the previous standards showed the ef- fect to be approximately 1 5 percent and the results of Angino et al. differ from the older ones by an amount that may be accounted for almost entirely by the improper standardization.

Turekian and Schutz [21] used x-ray fluorescence on freeze-dried sea water samples from all the oceans and found differences of 4 percent

between the Atlantic and Indian oceans, but their precision was 2 percent.

They report an average value of 7.5 mg/1 of strontium at a chlorinity of

1 9.00^/00 but feel their values are low because the "values probably have

a systematic error in the standards." Andersen and Hume [22] have

analyzed samples from the Atlantic using flame photometry following

concentration with an ion-exchange procedure. They report an average

value of 7.5 mg/1 and feel their results are accurate with a precision of 0.5 percent. It appears that the Sr/Cl ratio in the oceans may vary by a few

percent and clear description awaits application of methods with accuracy

and precision of 0. 1 percent.

C. Dissolved Oxygen

The number of measurements of dissolved oxygen on samples from the oceans is second only to the chlorinity or salinity estimates. Most of the

determinations have been made using the Winkler [23] . titrimetric method, for which the manipulations are disarmingly simple. Usually the

novice is given the job of carrying out the procedure and a precision of
OCEANOGRAPHY 411

roughly 1 percent is fairly easily achieved. There is a large variation of

dissolved oxygen in the ocean, with values ranging from 8 ml/1 at the sur- face at high latitudes to near zero in many cubic kilometers at inter- mediate depths in the Pacific Ocean. The dissolved oxygen concentrations are related to the intensity of biological processes and the rates and

patterns of physical motion, and are of general interest.

It might be expected that methods with a precision and accuracy of a few percent would be entirely adequate for delineating the distribution of

dissolved oxygen in the oceans. However, many interesting aspects of the oceans might be revealed by small variations in the dissolved oxygen concentrations. For example, exchange between the atmosphere and ocean

occurs with small gradients and disequilibrium of more than approximately 5 percent is rarely observed. Errors of 2.5 percent would change the

flux estimate by a factor of 2 and the errors in the dissolved oxygen concentration data make oxygen balance computations very uncertain.

Measurements of dissolved oxygen in the North Atlantic Ocean during

the middle 1930's have been compared with measurements made during

the 1950's with the result that there was an apparent change in the deeper

waters of the North Atlantic during the two decades. The apparent rate of

change would be compatible with the hypothesis that the deep waters were saturated with dissolved oxygen around 1810, a period of extreme

cold. This leads to the radical idea that the North Atlantic had been mixed

and saturated abruptly at that time and that there had been a slow

decrease since that time. Subsequent study of the procedures used suggests that rather than a catastrophic overturn in the North Atlantic, the

spectacular turnover was in personnel on the expeditions. The Winkler

method was modified to be more similar to that described in standard

texts for iodometric estimation of copper in metaUic samples. The as- sociated errors from air oxidation of iodide are negligible for decinormal

solutions of iodine that are used in copper assay, but many percent for

millinormal solutions that are involved in oxygen analyses of sea water. Similar discrepancies between British and American expeditions may be

accounted for in terms of the excessive acidity required in the use of

dichromate as a standard substance.

The reason that the dissolved oxygen data are uncertain is that solutions of oxygen in water have not been used as standards. The Winkler

method involves reacting the dissolved oxygen with manganous ion and

subsequent reaction of the oxidized manganese with iodide to produce

iodine, which is frequently estimated by titration with thiosulfate. The unstable thiosulfate solutions have been standardized with iodine that is generated by reacting iodate or chromate with iodide. The method may be
412 ANALYTICAL PROBLEMS

used with an accuracy of 0. 1 percent if the various errors are minimized,

as reported by Carpenter [24]. However, errors of many percent may be

easily introduced. A frequent error is associated with the volatility of

iodine, and it would be unfortunate if the oceans appeared to lose several

percent of its oxygen content simply because of the availability of magnetic stirring devices which aggravate the loss of iodine during titrations. With reference to the data plotted in Figure 1 , the apparent time trend

may be real and the correlation between the salinity and oxygen data

tends to reinforce that idea, but the nagging possibility of systematic er- rors introduced by changes in technique is difficult to eliminate.

A variety of methods for oxygen analysis has been introduced in the

past two decades. Gasometric methods have been restricted due to dif- ficulties in manipulations in shipboard laboratories. The gas chromatographic technique developed by Swinnerton, et al. [25] has proved to be

practical for shipboard use. The dissolved gases in a sea water sample are stripped into the carrier gas flow in a simple glass chamber and carried

directly into a conventional gas chromatograph for analysis. The field per- formance of the technique was studied by Swinnerton and Sullivan [26],

who concluded that the technique was more rapid and simple than previ- ous methods and the precision was comparable to other methods. They

found systematic differences of approximately 5 percent between the gas chromatographic results and analyses by the standard Winkler method

and note that "the discrepancies are at present unexplained." Further

comparisons using a revised Winkler procedure showed agreement

between gas chromatographic and titrimetric results of better than 1 percent. Some of the reasons for the previous discrepancy are discussed by

Carritt and Carpenter [27]

.

An attractive feature of the gas chromatographic technique is that results for total carbon dioxide, nitrogen and argon can be obtained with

modification of the procedures. Mass spectrometry also has the virtue of

versatility, with the added possibilities of extreme sensitivity. As shown

by several groups at the Scripps Institution of Oceanography, mass spec- trometric techniques can be used for the noble gases at extremely low

concentrations. In addition, information on the isotopic composition may

be essential in distinguishing between biological and physical processes in the ocean, as illustrated by Rakestraw, et al. [28] . The oceanographic applications of analyses for the several gases have been outlined by Benson

[29]. Mass spectrometric techniques appear to be most powerful tools for dissolved gas analyses, but suffer from economic and data rate considerations. It appears probable that such techniques will receive increased at- tention for use in specialized research projects.
OCEANOGRAPHY 413

Polarographic techniques for dissolved oxygen determinations have

received sporadic attention. Enthusiasts have tolerated the problems of

the dropping or streaming mercury electrodes under field conditions or

struggled with the poisoning of solid electrodes. The membrane solid elec- trode in either the polarographic or galvanic modes has received some

application (Carritt and Kanwisher/[30], and Mancy et al. [31]). Such

electrodes solve many of the poisoning problems, but have remarkably

large temperature coefficients, sensitivity to ambient fluid motion and

light, and changing response with time. My own personal experiences suggest that such devices might have application as thermometers, current meters, photometers or clocks and, in fact, an electrode has been used to sense weak motions in the deeper portions of the ocean. The principal application of membrane electrodes to oceanographic dissolved oxygen

determinations has been in the form of recording continuous vertical

profiles for the study of details in the distribution, which is an application

where even relative values are useful. The quality of the data collected

with dissolved oxygen electrodes depends critically on the skill of the observer and attention to frequent standardization, so that no generalization

about the accuracy of such techniques can be made.

The introduction of these various methods for dissolved oxygen analysis has added versatility to the oceanographic field, but will make it more

and more difficult to assess the probable accuracy of the data.

D. Zinc

The current state of the knowledge of the major trace constituents may

be illustrated by considering data for the zinc concentrations in the ocean.

Errors may be expected from both contamination and loss when constituents at the micromolar concentration level are sought. Robertson

[32] presents data on the role of contamination in trace element analysis

in sea water. As he shows, zinc is ubiquitous and may appear in the ter- minal analysis from the structural materials used in sampling, sample containers, solvents, and reagents. Adoption of a particular technique such as neutron activation attenuates some of the problems but does not eliminate

them as has been frequently overstated. No generalization concerning

losses from the sample can be made, except that many investigators have

neglected to study the significance of this factor for their particular experimental conditions. The availability of radioactive isotopes of so many of

the elements makes such oversights almost inexcusable. Many of the con-
414 ANALYTICAL PROBLEMS

tamination and loss effects are functions of sample size, surface to volume

ratios, etc. and would be detected if these variables were studied. Replication to produce computed internal blanks, both positive and negative,

would add considerably to the quality of the data. Dr. Derek Spencer at the Woods Hole Oceanographic Institution has

attempted to assess the intensity of physical and biological processes that cause spatial variation in the distribution of many of the major trace ele- ments in the ocean. Variations of perhaps a factor of two or so are expectable. Concentrations that have been reported in the literature vary by more than an order of magnitude. Is the knowledge of the oceans as represented by Spencer's model missing an important concept or intensity

of some process or are the analytical data misrepresentations?

Some insights into this problem can be found in the recent study by

Brewer and Spencer [33]. As they state in their introduction, . . . "the concentration levels of most trace elements in sea water has been, and still is, a matter of some controversy. This study was organized as an attempt to determine whether different laboratories, working independently, could

obtain the same values for these various trace elements in two samples of

sea water." The results from the various laboratories for zinc concentrations are shown in Table 3. Brewer and Spencer discarded four of these

values and computed a mean for sample 1 of 4.92 ^tg/1 and a standard

deviation of 4.60 /x,g/l and, for sample 3, the mean was 5.6 /xg/l and the

standard deviation was 5.3 ptg/l. Sample 1 was Pacific Deep water and

sample 3 was Caribbean Deep water. The most elemental question might

be concerned with the existence of a difference in the zinc concentrations

at the two positions, but such questions are clearly beyond the capabilities

of present practices. No information that would lead to a preference for a

particular method can be gleaned from these data. Equally disastrous

results were achieved with a few thousand dollars worth of equipment and

with devices costing millions of dollars. One of the possible reasons for differences in the results with different

methods could be that some techniques measure only the ionic form and

others the total zinc. The scatter is so large that such effects cannot be detected in the zinc data. The authors note that "the total methods gave significantly lower results for cobalt than the extraction methods."

One point seems worth noting. Brewer and Spencer imply that laboratories are being compared. In a similar vein, Carritt and Carpenter [27]

describe a comparison of modifications of the Winkler method for dis- solved oxygen. In fact, the analytical results obtained by different people

are being compared — some with perhaps years of experience and some

with almost none. While development of new methods will contribute to
OCEANOGRAPHY 415

Table 3. Results of intercalibration of analyses for zinc reported by

Brewer and Spencer [33].

SAMPLE 1 SAMPLE 3

T qK rvtJoUll MfifVir»fl fxg/kg

1501/28/07 0.02 5.2 Dithizone extract into 0.6 6.1 chloroform. Colorimetric

1 Q

i.O b.b

1619/28/05 4.5 6.7 Solvent extraction

4.5 5.4 APDC-MIBK. Atomic

4.7 absorption

0800/04/01 4.2 6.2 Neutron activation of dried

5.1 6.4 sea salts 3.5 6.2

1105/10/07 5.2 5.8 Solvent extraction APDC4.0 6.6 chloroform. Atomic absorp

o.U

"7 A / .U tion in methanol solution

1200/07/07 6.11 3.90 Adsorption chelex— 100—

3.58 Eluted HNO3 Solvent

extraction AFDL—MIBK

Y ra\7 Ti iioi»c»cr»Cknr>c» yv I cLy iiuuitJoL-tJiict; 1600/10/07 4.8 6.6 Solvent extraction APDC—

MIBK. Atomic absorption

1500/16/07 99.15 1.47 Solvent extraction APDC1.00 L86 MIBK. Atomic absorption

1.25 1.72

1200/06/06 0.65 2.95 Solvent extraction APDC1.85 2.95 MIBK. Atomic absorption

2.15 2.90

1650/20/07 3.9 5.2 Solvent extraction APDC3.3 3.9 MIBK. Atomic absorption

2.9 4.1

1740/08/08 4.2 5.5 Solvent extraction. 3.9 " 5.3 Dithizone Colorimetric

3.0 5.6
416 ANALYTICAL PROBLEMS

Table 3. Results of intercalibration of analyses for zinc reported by

Brewer and Spencer [33] (continued).

SAMPLE 1 SAMPLE 3

Lab Kesult Kesult Metnod

1253/23/08 4.8 44 Neutron activation of

b.z o.b freeze dried sea salts

4.7 5.2

0000/01/09 6.0 6.8 Neutron activation of

5.3 8.0 freeze dried sea salts 6.8 6.5

8/07/70 0.8 0.75 Neutron activation of

0.76 0.72 freeze dried sea salts 18/9/70 18.5 22.8 Adsorption chelex— 100

21.2 25.4 ElutedHNOg. Solvent

15.7 19.4 extraction APDCchloroform. Atomic absorption in isopropanol solution

1200/31/7 5.11 Direct with anodic

5.45 stripping voltammetry

U.89

progress in oceanography, substantial improvement in the data will not be

realized until oceanographic laboratories hire analytical chemists and an

inter-laboratory comparison might be better predicated on this feature.

VII. Current Needs

Much of the above has been directed toward illustrating less obvious

aspects of the application of analytical chemistry to oceanography. The

most pressing needs appear to be in the areas of determining the chemical

species containing the various elements. Consideration of inorganic and

organic complexing is popular but dependent on development of

techniques and tools such as specific ion electrodes, gas chromatography

and sensitive polarography. The micromolar concentrations of the in-
OCEANOGRAPHY 417

teresting compounds, both inorganic and organic, means that most

techniques must be pushed near the useful limit. For example, specific

electrodes for copper are not useful for the study of complexing in sea water because the copper concentration is too low. Synthetic laboratory

work with copper is not practical because the many possible organic complexing agents have not been identified and synthetic systems would lack

application to the real ocean. The most formidable and pressing need is the development of general use methods for organic compounds. We are not going to be able to understand the chemistry of the oceanic system

until there are considerable advances in the data rate for organic species. In terms of number of analyses, the biologically active elements will probably be dominant. Growth stimulation and toxicity are still only

poorly documented for most of the metals and organic compounds. Even

the long recognized nutrients are not adequately described, particularly

the various nitrogen containing compounds. Usable but tricky methods

for nitrate ion were developed several decades ago and the oceanic

system has been described primarily in terms of the effects of nitrate. In

the last decade, usable but very tricky methods for ammonium ion have

been developed and the importance of this form in the nitrogen cycle and

in algal nutrition has received increased attention. Recently, methods for urea have been developed and the first few papers show that this com- pound must be considered in describing the system quantitatively. Other

forms of combined nitrogen may be important but are not considered

because of a lack of analytical methods and methods development in this area would probably receive immediate application. Study of many of the

compounds that are presently determined by bioassay, such as vitamins

and metal complexing compounds, would benefit from the development

of methods with improved precision, simplicity and speed of analysis.

VIII. References

[1] Goldberg, E. D., The Oceans as a Chemical System, in The Sea, Vol. 2, Chapter 1, Wiley Interscience, New York (1963).

[2] Ryther, J. H., Geographical Variations in Productivity, in The Sea, Vol. 2, Chapter

17, Wiley Interscience, New York (1963).

[3] Riley, J. P., Historical Introduction, in Chemical Oceanography, Chapter 1, Academic Press, New York (1965).

[4] Dittmar, W., Report on Researches into the Composition of Ocean Water, collected by H.M.S. CHALLENGER, Challenger Reports, Vol. 2,51 pp. (1884).
418 ANALYTICAL PROBLEMS

[5] Forch, C, Knudsen, M., and S0rensen, S. P. L., Editors, Berichte Uber die Konstanten Bestimmungen zur Aufstellung der Hydrographischen Tabellen, D.Kgl. Danske

Vidensk. Selsk, Raekke, naturvidensk. og methem., Afd XH.I, Skrifter, 6, 151 pp. (1902).

Joint Panel on Oceanographic Tables and Standards, International Oceanographic

Tables, 118 pp., UNESCO Publication Center, New York (1966). Wooster, W. S., Lee, A. J., and Dietrich, G., Redefinition of Salinity, Limnol. Oceanog. 14,437-38 (1969). Cox, R. A., Culkin, P., and Riley, J. P., The Electrical Conductivity-Chlorinity Relationship in Natural Seawater, Deep Sea Res. 14, 203-20 (1967). Carter, N. M., Moberg, E. G., Skogsberg, T., and Thompson, T. G., The Reporting of Data in Oceanographical Chemistry, Proceedings of the 5th Pacific Scientific Conference, Canada, 2123-27 (1933). Jacobsen, J. P., and Knudsen, M., Urnormal 1937 or Primary Standard Seawater, In- ternational Association of Physics and Oceanography, Pub. Sci. 7, 38 pp. (1940). Reeburgh, W. S., Some Implications of the 1940 Redefinition of Chlorinity, Deep-Sea

Res. 13,975-76(1966).

Carritt, D. E., and Carpenter, J. H., The Composition of Sea Water and the Salinity- Chlorinity-Density Problems, in Physical and Chemical Properties of Sea Water,

National Academy of Sciences Washington, D.C., 600 pp. (1959). Cox, R. A., Culkin, P., Greenlaugh, R.,and Riley, J. P., Chlorinity, Conductivity, and

Density of Sea Water, Nature 193, 5 1 8-20 ( 1 962). Home, R. A., Editor, Marine Chemistry, 568 pp., Wiley Interscience, New York

(1969). Mangelsdorf, P. C, Jr., and Wilson, T. R. S., Difference Chromatography of Sea

Water, J. Phys. Chem. 75, 1418-25 (1971). Manella, M. E., "Observations on the Magnesium to Chlorinity Rado in Sea Water,"

(Master of Arts Essay, Johns Hopkins University) (1970). Webb, D. A., Strontium in Sea Water and Its Effect on Calcium Determinations, Nature 142,751-52 (1936). Odum, H. T., Notes on the Strontium Content of Sea Water, Celestite Radiolaria and

Strontianite Snail Shells, Science 114, 2 1 1 - 1 3 ( 1 95 1 ). Angino, E. E., Billings, G. K., and Andersen, N. R., Observed Variations in the Stron- tium Concentration in Sea Water, Chem. Geol. 1, 145-53 (1966). Carr, B. A., Salinity Effects on the Determination of Strontium in Sea Water by

Atomic Absorption Spectrophotometry, Limnol. and Oceanog. 15, 3 18-20 (1970). Turekian, K. K., and Schutz, D. P., Trace Element Economy in the Oceans, Symposi- um on Marine Chemistry, University of Rhode Island, Occasional Publication No. 3, 41-90(1965).

Andersen, N. R., and Hume, D. H., The Strontium and Barium Content of Sea Water, Trace Inorganics in Water, Advances in Chemistry Series 73, American Chemical

Society, 296-307 (1968). Winkler, L. W., Die Bestimmung des im Wasser Gelosten Sauerstoffes, Chem. Ber. 21,2843-55 (1888). Carpenter, J. H., The Accuracy of the Winkler Method for Dissolved Oxygen Analysis, L/wno/. and Oceanog. 10, 135-40(1965).

Swinnerton, J. W., Linnenbom, V. J., and Cheek, C. H., Determination of Dissolved Gases in Aqueous Solutions by Gas Chromatography, Anal. Chem. 34, 483-85

(1962).
OCEANOGRAPHY 419

[26] Swinnerton, J. W., and Sullivan, J. P., Shipboard Determination of Dissolved Gases

in Sea Water by Gas Chromatography, NRL Report 5806, U. S. Naval Research

Laboratory, Washington, D.C. (1962). [27] Carritt, D. E., and Carpenter, J. H., Comparison and Evaluation of Currently Employed Modifications of the Winkler Method for Determining Dissolved Oxygen in Sea Water; a NASCO report, J. Mar. Res. 24, 286-3 18 (1 966). [28] Rakestraw, N. W., Rudd, D. P., and Pole, M., Isotopic Composition of Oxygen in Air Dissolved in Pacific Ocean Water as a Function of Depth, J. Amer. Chem. Soc. 73, 2976-79(1951).

[29] Benson, B. B., Some Thoughts on Gases Dissolved in the Oceans, Symposium on Marine Geochemistry, University of Rhode Island, Occasional Publication No. 3, (1965). [30] Carritt, D. E., and Kanwisher, J. W., An Electrode System for Measuring Dissolved OxygQn, Anal. Chem. 31, 5-9 (1959). [31] Mancy, K. H., Okun, D. A., and Reilly. C. N., The Galvanic Cell Oxygen Analyzer,

J. Electroanal. Chem. 4, 65-8 (1962). [32] Robertson, D. E., Role of Contamination in Trace Element Analysis of Sea Water,

Anal. Chem. 40, 1967-72 (1968). [33] Brewer, P. G., and Spencer, D. W., Trace Element Intercalibration Study, (Ref. No.

70-62, unpublished manuscript). Woods Hole Oceanographic Institution, Woods

Hole, Massachusetts (1970).

PANEL DISCUSSION

Analytical Problems in Oceanography

CHAIRMAN

Richard A. Durst, Chief, Electrochemical Analysis Section, National

Bureau of Standards

PANEL

Velmer A. Fassel, Professor of Chemistry and Deputy Director, Institute

for Atomic Research, Iowa State University, Ames, Iowa

Vaughan T. Bowen, Senior Scientist, Department of Chemistry, Woods

Hole Oceanographic Institution, Woods Hole, Massachusetts

Richard S. Juvet, Jr., Professor of Chemistry, Arizona State University,

Tempe, Arizona

Theodore J. Kneip, Deputy Director, Laboratory for Environmental

Medicine, New York University Medical Center, New York

James H. Carpenter, Professor of Earth and Planetary Science, Johns

Hopkins University (Speaker)

421
422 ANALYTICAL PROBLEMS

Bowen — Currently at the Woods Hole Oceanographic Institution,

chemicals that most people would consider pollutants are being used as tracers for oceanographic processes. For a long time, the artificial radioactivity from nuclear weapons test fallout has been used as a tracer. This type of approach is presently being expanded to using petroleum

hydrocarbons and chlorinated hydrocarbons, and it is hoped to use some

heavy metal pollutants as tracers for physical and geochemical studies of

movement in the ocean.

In the course of the work with artificial radioactivity, one of the most

serious needs for a standard reference sample was one that could be certified as a blank. This requirement came up over and over agaia Samples

of seawater consisting of 1 00 or more individual un'ts were produced rela- tively easily. Each 60-liter unit of seawater was of uniform and measurable content of strontium-90, cesium-137, plutonium, etc. But, when it came to providing a seawater facsimile that one could be assured didn't

have any of these constituents, serious problems were encountered. The

blank sample finally obtained, easily cost 10 times more per unit than did

the others.

On the other hand, in studies of this sort, and this is true whether one is motivated by pollution or tracer concern, it is almost always going to be

necessary to make sections of analyses outward from a point source or downward from a known plane source to as close as possible to zero concentration. The only way a large number of different analysts can be as- sured that they mean the same thing when they say zero is by being pro- vided with a blank sample, in much the same way as they are provided

with a standard sample.

The group at Wood's Hole is now greatly concerned as to how they can do this with chlorinated hydrocarbons. It is now felt that they have some

samples that are usable and are free from non-recently-biosynthesized

petroleum hydrocarbons. Hope was expressed that in the Standard

Reference Materials program of the National Bureau of Standards as is now being contemplated by a similar organization of the International

Atomic Energy Agency, serious consideration will be given to the provision of blank samples. Standard Reference Materials certified for all of the

major pollutants of present environmental concern, plus some that can be

anticipated in the future, are needed. Help is being received from other

agencies in the preparation of reasonably well-certified biological samples

for chlorinated hydrocarbons. However, this takes a great deal of work,

a great deal of expertise, a great deal of time, and a great deal of money.

Yet all comparable analytical programs have shown this to be essential. Another point that should be discussed is the question of what should
PANEL DISCUSSION

be analyzed, specifically in the context of environmental concern. We

really don't know the constituents of any of the major environmental

systems such as seawater, fresh water, river water, sediment, soils, etc. We are still in the first stage of Dr. Butler's sequence of increasing so- phistication. We must continue to analyze all of these systems for the ele- mental constituents and must at the same time collect data, which is not widely being done, on inputs of new materials.

An interesting illustration is the present problem of the distribution of

Plutonium. It has been known for nearly two decades now that plutonium was being introduced into the environment, but it was believed that this was not going to be a matter of concern and we formed the habit of in- troducing it without determining how much was being introduced in any

place at any time. It is now found that Pu is a material of considerable concern, about which a great deal must be learned quickly and in some

cases rather dirtily. This information might very well have already been

known if we had been willing to look a little further ahead.

At this point though, the analytical chemist really has to rely on some- body else to tell him which of these systems are going to be worth analyzing at any given time. In detail and extensively, he has to go to the biologist, the ecologist, the geochemist, and so on. These people will give him

predictions which are based on data, but they are based on data as in- terpreted by value judgements. However, it often isn't going to be obvious, how extremely poor is the data on which much of this expression of concern is in fact based.

Although a considerable body of estimates of toxic levels exists for

various things, there are virtually no instances where the estimates of

toxic levels of trace elements are based on an actual measurement of toxic

concentration in the system. Normally, the toxic level is described simply

in terms of the amount that was added to a unit volume containing one or more organisms. It is now known that this can't be any more than an upper limit. It is also known that the rate at which an organism sees these

things is always critical. If a fresh water clam is exposed to only that amount of manganese

which is contained at any unit time in his tissues and if this is presented to him, all at once, as manganous ion in solution, it will kill him and kill him

quickly. On the other hand, the manganese which had accumulated over

a long period of time was in a biochemical form of extreme stability and

didn't bother him in the least. This is relevant to what was said this morning concerning mercury in some of the large fishes. They have accumulated this in the same sort of way, and it is, almost certainly, equally inert biochemically. When you do extrapolate from a level of concern by a
424 ANALYTICAL PROBLEMS

biochemist, you have to do this with some reservations and should put a

little pressure back on the biologist to look more carefully.

The speciation problem which has been discussed for the past several

days is a special instance of this. It isn't always obvious that we need to know the chemical species of toxic elements. In a considerable number of

cases, it can be shown that the rate of the uptake process by organisms or

of the geochemical removal process is slow relative to the rate of

equilibration among the various physicochemical species involved. It can

also be shown that the stability of the biological form or the geochemically

immobilized form is high compared to these. In that case, all one needs to know is the total concentration because the total concentration will be ef- fective in driving the reaction toward the combined form. It is necessary

that we know in some detail the process with reference to which the

measurements are to be made; otherwise the speciation question is of

considerable academic interest from the physicochemistry of the system,

but it can't be shown to be necessary to the environmentalist.

Some really weird, unforeseeable speciation problems are encountered.

One has just come up in connection with a major intercalibration experiment being done under the auspices of the International Atomic Energy

Agency. It was necessary for investigators from a large number of dif- ferent countries to be able to produce analytical data on radioactive waste

disposal, specifically from reactor wastes. It was decided that samples

had to be collected, homogenized, and distributed for this purpose. We

had a good deal of experience for doing this for comparable samples of

fallout radioisotopes of the same elements. We knew what kind of containers were suitable, we knew the pH to which the solution had to be

reduced, we knew how to take off those species which were adsorbed

onto the walls of the containers, and we plugged all of this information

into the experiment.

Scientists in Britain collected a large amount of rather highly radioactive water from the Irish Sea, put it into containers and distributed it. So

far nobody is reported to have been able to get the radioactive cerium, the

radioactive zirconium, or several of the other constituents out of the

plastic containers by any treatment whatsoever. The material is known to be there because the solutions are hot enough, that we can set the whole

two-liter container on a gamma spectrometer and see the peaks, but we

can't get thern out. Apparently the radionuclides in these samples are present in a series of chemical species which were completely unlike

those in the fallout with which we were experienced.

There is one other very curious case which presumably must be related

to speciation of some reasonably, abundant dissolved constituents. The
PANEL DISCUSSION 425

biogenous sediments on the ocean floor can be reasonably easily divided

into two major classes, one is the calcium carbonate sediments and the

other is the siliceous sediments consisting of diatoms or of radiolaria. Generally these two sediments do not coexist. Considerable areas are found where one or the other predominates. No measurable differences can be seen in the water which overlies one type of sediment from the

water which overlies the other type, and yet a fairly major phenomenon is clearly different in the two cases. This is a very beautiful analytical

problem and of considerable geochemical interest. There are definite problems when one physically collects a sample a

mile below sea level in a container that can withstand the pressure. There

is a principle that is common to all such samplers: one lowers an open

tube to the depth from which the sample is to come, then at depth one

closes the tube, and brings the sample back. Presently the one absolutely

unsolved disadvantage of this is that such a sampler sees all of the surfaceactive material between the surface and the point of collection, at which

point it is closed. We have no way of obtaining large water samples or sediment samples that are not comparably contaminated by surface-active

agents. We have built some rather large samplers for some other reasons, and it turns out that the larger the sampler, the smaller the ratio of surface

to volume, and the better the sample is for almost any purpose, if the sampler is made of suitable material. It should be noted that several devices

collect small samples of water at depth, for microbiological purposes,

without "seeing" the overlying water.

If one asks what kind of sensors can be used at what depths, and how

far can one go down with certain types of sensors, we know that salinity,

temperature, radiation and density sensors can go down to all depths. The

deepest I have seen an oxygen sensor used has been at about 1000 meters, some years ago. It is probably correct to say that there are no other

sensors that can be used successfully at any significant depth at all. We

expect this situation to change rapidly.

Fassel — The last sentence in Dr. Carpenter's abstract states that "For

the trace constituents including many of the heavy metals, inaccuracy by

current techniques and analysis has produced a nearly useless body of

data," I am in complete agreement with Dr. Carpenter's conclusion that compositional characterization at the ppm, fractional ppm, or ppb levels poses singularly difficult problems, but what are the problems? They stem

primarily from the fact that trace element technology is not a mature

science. Analytical problems posed by oceanographic research serve as excellent examples to illustrate this immaturity.
426 ANALYTICAL PROBLEMS

It is appropriate to recall that all of the elements in the earth's crust are found in the ocean, but only 13 occur at concentrations above 1 ppm, and

the majority are present at less than 1 ppb. In spite of the striking ad- vances that have been made in trace element determinations, an analyst

must still be extraordinarily successful to measure impurities at these concentrational levels with sufficient accuracy and precision to allow

meaningful statistical evaluations and correlations that are so necessary

to oceanographic studies. Even if the analyst develops the observational

capability to achieve accuracy and precision at these concentration levels the problem is only partially solved. The taking of the sample and its handling prior to the analysis must also be factored in. The risks of intolerable

contamination or the loss of significant impurities are present from time of

sampling, during storage, and up to the final measurement.

Unfortunately, the various sources of contamination or the processes

through which impurities may be lost (adsorption, incomplete separations,

volatilization) have not been adequately identified, nor are they usually

obvious. All too often the consequences of contamination or loss of impurities are ignored. The various pathways that contribute contamination or

loss must be identified more adequately and controlled more effectively

before trace element technology can be labeled a mature science.

Trace element technology is immature from another standpoint.

Whether we are concerned with the role of trace elements in solid state science, or evaluating their biomedical effects, or assessing the degree of

water or air pollution, or observing the transport of trace constituents in the ocean, there is a need for determining multiple trace elements in a

large number of samples. This need can, in principle, be satisfied by spark source mass spectroscopy for many types of samples. However, the cost of the hardware and staff competency are often prohibitive. Moreover,

the direct quantitative analysis of small amounts of liquid samples of the

type often encountered in biomedical and health studies, is beset with

many problems. Another popular way to perform multielement trace determinations is by neutron activation analysis, but here again the hard- ware expense may be prohibitive, the element coverage is somewhat

limited, and for some elements the time required for irradiation may be

unacceptably high. Presently the most popular technique for determining

trace elements is flame atomic absorption spectroscopy (FAAS), but as presently practiced, FAAS is not a multielement technique. The deter- mination of say thirty trace elements in an oceanographic water sample in- volves too many time-consuming mechanical operations.

There is needed, therefore an analytical technique that satisfies the fol- lowing criteria: (a) the analytical operations should be reasonably simple
PANEL DISCUSSION 427

in their execution; (b) the hardware requirements should be met with rela- tively inexpensive instruments; (c) sample manipulation and treatment

prior to analysis should be minimal; (d) simultaneous or rapid sequential

multielement determinations should be possible; and (e) the power of detection of the technique should be in the part per billion range. During the past six years we have explored an analytical system that

satisfies the requirement stated above to a degree higher than any other

analytical approach known to me. This technique relies on one of the oldest experimental methods for the determination of trace elements, namely, the observation of optical emission spectra, emitted not in conventional arc or spark discharges but by inductively coupled plasmas

operated at atmospheric pressures. Since this technique and its potential

advantages have been adequately described in the Hterature, I shall just

remind you that the observed powers of detection of plasma emission

compare favorably to and in many cases are vastly superior to the best

atomic absorption values so far reported. The combination of this plasma

with a direct-reading optical spectrometer provides the analyst with the

capability of determining simultaneously as many elements as there are channels in the spectrometer.

Juvet — Analytical chemists should be able to contribute in this field by

suggesting modernization of the measurement methods currently used

from the buret-titration type to something of equal or better accuracy. For

example, have coulometric methods for chlorinity been tried where the

value of the faraday is very accurately known? Using instrumental

methods such as this, where one has only to push a button and have the answer printed out, should be very desirable since shipboard conditions

would be much less a factor in the results.

It would also seem possible using present day knowledge to design sensors to analyze these materials at depth rather than taking hours to bring

them to the surface to be analyzed. Certainly analytical chemists could be

of major assistance in the design of such instruments. Important instrumental design considerations which must be kept in mind in oceanographic studies include not only corrosion and erosion effects of the ocean

and marine organisms but also the tremendous pressure encountered at

' the ocean depths. Pressure increases at the rate of about 1 atmosphere for

I every 33 feet of depth in seawater. Since the deepest known area in the

I ocean, the Mindanao Trench, has a reported depth of 37,782 feet, a pres- sure equivalent to well over 16,000 Ib-in"^ must be withstood. One

published article was noted in which activation analysis was used to analyze the mineral content of the sea floor. In the case, the neutron
428 ANALYTICAL PROBLEMS

source was 200 micrograms of califomium-252. The gamma rays

produced by the irradiated sea floor indicated the elemental content. Kneip — I would like to make a few remarks concerning the concentration of heavy, or transition, metals. Statements were made today and

other days that the heavy metals become concentrated. Unfortunately,

this statement is misleading. We have all learned that the pesticides are concentrated up through food chains to the point where the top predators

are being lost because of this concentration effect. When someone says

that the heavy metals are concentrated in the food chain, the immediate

impression is that the same sort of phenomenon takes place; the higher one goes in the food chain, the higher the concentration.

By and large, this is not true for metals. In fact mercury is an exception

because we are dealing with a heavy metal which is converted to a metalorganic compound and it concentrates in the food chain because of the

specific transfer of the organic material. It is stored differently from the

pesticide. There is a methylmercury-protein complex in muscles, in addition to storage like the pesticides in fat. Thus to use mercury as a

representative example of heavy metal concentration factors in biological

systems is misleading.

The Institute of Environmental Medicine of New York University

Medical Center has been specifically and rather intensely looking at manganese and cobalt in the Hudson River estuary in the vicinity of a nuclear

plant because of interest in the circulation of radionuclides that are released by that plant. There is a need to predict what will happen to fu- ture releases by additional plants being built in the region. The very first thing one finds out is that these transition metals are heavily concentrated

by plants, and this includes plankton and rooted plants. The higher forms

of life in the region, the fish for instance, do not have nearly as high a concentration of the transition elements as the plants. Not only this, but

response times are vastly different. The plants are responding in very

short time periods. These time periods have not been resolved but they

are less than a week for rooted plants, while for the entire system, i.e., waiting for the fish to come into equilibrium with everything else, will take

anywhere from a month to two months.

The sedimentary interactions have been spectacularly important in un- derstanding the system. It was found that manganese and cobalt for in- stance, regardless of their origin, transfer to the sediments while the water

in the region is fresh, but when the salinity reintrudes into the area, huge

quantities of manganese and cobalt are released into the water. The

sediments are the only reservoir in the system containing sufficient
PANEL DISCUSSION 429

manganese to supply the amounts that appear in solution. There is an immediate response of the plants to this manganese. They take it up, concentrations jump, and the whole system reverses itself when another

intrusion of fresh water comes through the region.

This is one of the confusion factors that adds to the fun in studies of the

estuary. It is not the same from day to day and, if you plan your experiments for certain conditions and the weather doesn't cooperate, you can

find yourself on a body of fresh water when you planned to have a certain

salinity which is normal for that time of year.

It is fascinating to contemplate what is happening to some of the other

heavy metals. All of these materials seem to go to the sediments, but

whether all of them will behave in a similar fashion is doubtful. Multivalent states do not occur in many of the other metals. The chemistry of

manganese is unique, so one may find that there is a storehouse for some

of the heavy metals in this region rather than simply a transfer point. It would be interesting to know whether manganese itself in undergoing this cycling is merely temporarily held in the region and is carried out to sea as

it is redissolved.

There are times and places when the team leader in these problems

should be an analytical chemist. It is very obvious from some of the

problems of understanding interactions of sampling, the sampling container, the need that originated the study, the analytical methods that are

available, and finally the interpretation of the results to get back the answer to that original need, that the analytical man is the only one who is going to put it all together. He is able to take some of the information from

one particular speciality and some from another, then supply the overall

look at the whole problem and become the team leader. We should not become so intent upon being a service in this field. We ought to try to get

into the heart of the problem and, where it is appropriate, take the lead.

Carpenter — The most exciting thing in this regard is that the flux of ele- ments from the sediments is not entirely controlled by molecular diffu- sion. Profiles are seen that can only be interpreted by the bubbling of

methane, motion of organisms, etc. So we are going to be stuck with the

familiar problem of how to understand "turbulent diffusion" while looking

at the flux of these metals from the sediments. In reference to the comment this morning about phosphate, a lot of phosphate is seen in the sedi- mentary record. Presumably there are chemical conditions where

phosphate does accumulate, so that if those conditions were understood

well enough, in some places where we would like the phosphate to remain
430 ANALYTICAL PROBLEMS

in the sediment, a way could probably be figured out rather than removing

it all. Perhaps some creativity in the direction of understanding what

should be done if we don't want the phosphate to come out might be very

helpful. In regard to sampling at great depths, one ambitious group has pumped

samples from several miles. The problem is that you sit there and try to convince yourself that the sample does not change with time, and you

begin to wonder about sample integrity. However, with expenses of

several thousand dollars a day for the ship, one gets rather impatient

sitting there trying to make up his mind whether the whole sampling ap- paratus has come to equilibrium yet.

If we ask whether there are any geological data on which one can

establish a baseline in the sea deposit, such as saltbeds that could be used

to determine the history of trace elements in seawater, the answer is yes,

at least to a useful approximation. Most of the data we have, unfortunately, were collected by a few people who chose emission spectrographic

methods and then tested them against other methods with considerable

frustration. There were factors of 3 or 4 differences. So to that degree, to factors of about 5, samples from sediments of all the world's oceans have

been analyzed. We have some ideas of sedimentation rates in these areas, so one can make an approximate geochemical balance that is probably not uncertain by more than a factor of 5.
Index

A

Abscisic acid, 208

Abscission, as related to fruit harvest, 208, 209

Absorption, atomic (see atomic absorption)

Absorption

spectrometric method, monitoring color of bleached oil, 229

spectroscopy, 44

translocation, and metabohsm of nutrients

in plants, 1 87 Acceptable methods, criteria for accuracy, 105

precision, 105

sensitivity, 105

specificity, 105 Accuracy, 104, 105, 151

analysis, 105, 151

clinical work, NBS-Clinical Interaction

Plan, 137

referee methods, 134

Acetic

acid, 194

anhydride, 192 Acetone,97, 143, 145

Acetylene, 261

Acid-base and blood gas measurements, 90, 9 1 , 93

balance, 91 Acid phosphatase, 97

Acid potassium phthalate, 120

Acrolein, in atmospheric samples, 260, 277

Activation analysis, 70, 345

determination of minor constituents, 47

Acylanilides, 217 Adenosine triphosphate (ATP), 217

Adhesive coatings, 262

Adsorption chromatography, 359

with activated carbon, 360

Adulteration of fats and oils, 197

Aerial surveillance, 321

oil spills, 321

optical measurements, 321 photography and remote sensing, 32

1

suspended matter, 321 water pollution control, 32

1

water quality surveillance programs, 321

Aerosols, scattering by, 309, 310

Aflatoxin

carcinogens, 204

carcinogens in milk, 206

estimating by TLC, 204

levels of, 204

structural formulae for, 205

Aflatoxin in barley, 205

contaminated meal. 205

corn, 205

grain sorghum, 205

oats, 205

peanuts, 204

soybeans, 205

wheat, 205

Aflatoxin problem, 237

Aflatoxins Mi and M2

carcinogens in milk, 206

contaminated meal, 205 Aging and disease processes of cells, 1 8

1

Agricultural chemical analysis of fatty acids, standards, 234

Agricultural chemicals, foams as carriers

for, 216

Agricultural commodities, 162 and foods, action of molds on, 204

Agricultural research needs

animal health, 197

animal products, 188 automated analysis, 226

automation and instrumentation, 226

biologically active substances, 201 computer-aided analytical chemistry, 226

elements and water, 162

fibrous constituents of plants, 181

lipids, 172 microorganisms in foods, 195 molecular properties of natural polymers, 223

nutrients and their availability, 182

pollutants, 219

proteins and related nitrogen compounds,

166

seed and plant constituents, 1 85 sensory characteristics of foods, 194 431
432 INDEX

Agricultural science problems

dispersed plastics in fats, 180

procedure to distinguish various fats and

oils, 179

sequential analysis of lipids in membranes

of various cells in nature, 180

Agricultural water use, criteria for, 308

aquatic life, 308

chlorides, 309

fish and wildlife, 308

fungicides, 309

herbicides, 309

irrigation waters, 309

pesticides, 309 pH acidity and alkalinity, 309

radionuclides, 309

rodenticides, 309

suspended solids, 309

temperature, 309

total dissolved solids, salinity, 308

trace elements tolerance for irrigation waters, 308 USPH drinking water standards, 309

water quality characterization, 308 Agrochemical residues, 242 Aid programs to developing countries, 170

Air

ambient, 255

monitoring, 250, 255

nitric oxide, 255

pesticides in, 269

Airborne particulates, 268

Air pollutants

dusts, 219

inorganic gases, 2 1

9

organic gases and vapors, 2 1

9

polynuclear aromatic hydrocarbons, 219

Air pollution

control, 245

methodology, 259, 260

monitoring, 248, 250, 255 measurement needs, 247

Air quality, criteria for instrumentation, 246, 248

measurements, 247, 250

standards, 259, 260

Albumin, 97, 134 Aldehydes, 176,221,275

need for measurement in volatile or- ganics, 261

Aldolase, 116, 145

Aldrin, 305

Alfalfa continuous drying of, 166 Medicago sativa L., infected by, 204

Aliphatic

aldehydes in atmospheric samples, 260, 277

and aromatic hydrocarbons, 260

Alkaline

earths, 337

hydrazine method, nitrate assayed

colorimetrically by, 268

serum, 127

Alkaline phosphatase, 97, 98, 123, 135, 143, 144 enzyme standards, 125

First Report of the Association of Clinical Biochemists, England, 123 human, need to develop standard

reference enzyme material, 124 non-human serum, 124

spectrophotometric assay for, 126

standardization of, 123

Alkalinity and acidity, 310

Alkalinity (CaCOg units), 305

Alkali Flame Ionization Detector (AFID), 218

isomerization, 175

or enzymatic methods, methods of hydrolysis of proteins, 167

Alkyl mercury halides, 235

nitrates, 221

AUergins, tuberculin, 200, 220

Allowable error, Tonks, 133 Aluminum, 24, 308, 3 19 sample handling for analysis of, 3 19 zone refined, 24 Amalgamation, 266 Ambient air, 255

monitoring needs, 250

monitoring nitric oxide in, 255 American

Association of Cereal Chemists, 181, 205

Oil Chemists' Society, 181, 205

Society for Testing and Materials, 226
INDEX 433

Amino acids adducts, herbicide, biological systems, 212

and proteins, 97, 170, 177

during acid hydrolysis in the presence of carbohydrates, instability of, 167

rapid method for determination of, 167 sequence of individual residues, 223

Aminotransferase, aspartate, 123 Ammonia, 305, 3 11, 329 Ammonia nitrites, nitrates, 310 Ammonium ions, determination of, 341 Amniotic fluid, 98 Amperometric end point determination,

coulometry, 43 Amylase, 97, 142, 144 Amylose, 186 Anaerobic biological populations, 300

biosynthesis, 301

nitrate reduction, 301

sulfate reduction, 301 Analyses for sulfate and nitrate, 268

Analysis

airborne particles, 265

anion, 268

automobile emissions, 274

chlorine, 267 CHN,267

continual instrumental, 270

dissolved gases, 367

elemental, 266, 273

inorganic anions, 364

lead, nitrogen, and sulfur by photoelectron spectroscopy, 290

metals, 337

methane, 252

morphological, 265

neutron activation, 266

organic matter, 354

Orsat, 270

particle-size, 265

particulate matter, 261 , 266

polynuclear aromatic hydrocarbons, 268

spectrofluorometric, 268

thermal neutron activation, 266

trace, 266

Analysis and characterization, solid state, 68

Analysis in electronic materials, determination of oxidation state, 270

Analysis of aromatic hydrocarbons, 274

blood or biopsy samples, antemortem

methods, 189

chemicals in intact tissue at the cell level, 189

esters byGLC, 186

olefmic hydrocarbons, 274

oxygenated hydrocarbons, 275

paraffinic hydrocarbons, 274

plant hormones, 208

Analytical

chemist, characteristics of, 72

chemistry and the health field, 79

goal for chemical measurements in medicine, 136

microprobes, high resolution, 228

procedures, sensitivity limits of, 344

techniques, precision and sensitivity, 60

Analytical methodology

physical characteristics, 33

1

physiological characteristics, 331

pollution control, 331, 337, 354, 364, 367

Analyzer

chemiluminescent, 275

colorimetric, 260

coulometric, 274

cross-correlation nondispersive, 256

electrochemical, 275

flame ionization, 274

infrared fluorescent, 255

ion-probe scanning, 66

nondispersive infrared, 27 1 , 274, 275 on site, 265

oxidant, 252

ultraviolet, 27

1

Anderson sampler, 264, 276

Angular and wavelength measurements of

scattering, 28 Animal

health, agricultural research, 197

products, 192

products, imported, 198 Animal diseases

foreign, 197

identification and diagnosis of, 199, 200
434 INDEX

Animal disease-causing microorganisms, in- spection of meat at ports of entry, 198 Anion

analysis, 268

determination by emission spectroscopy, 34

interference, 27

1

Annual costs for operating chemistry ser- vices, tests volume and basic cost, 109 Anodic stripping voltammetry, 349 Antemortem methods, analysis of blood or biopsy samples, 189

Antibiotic chloramphenicol base, 184

Antibiotics metabolic breakdown products, 193

residues, 192

Antipyrine method for estimating fatness in

live beef, swine and sheep, 190 AOAC, 181, 184, 192, 204, 205, 226 AOAC, official recommended methods for determination of aflatoxin in barley, 205

grain sorghum, 205

oats, 205

peanuts, 204

soybeans, 205

wheat, 205 AOAC, procedures for assay methods

bromination, 192

fluorometric, 192

gravimetric digitonin, 192

Appropriations for the development of ad- vanced pollution measurement

techniques, 298

Aquatic ecosystems, interaction of major

elements, 299, 300

Aquatic environment

biochemical transformations of mercury compounds in, 341

biological and chemical interactions, 301

role of metals such as cadmium, chromi- um, copper, lead, mercury, molybdenum, nickel, zinc, 337

water pollution control, 299

Aquatic life, fish and wildlife, parameters

for water quality characterization, 306, 308

color and transparency, 306

dissolved matter, 307

dissolved oxygen, 307

floating matter, 307 pH alkalinity and acidity, 307

plant nutrients, 307

radionuclides, 308

settleable matter, 307

temperature, 307

toxic matter, 307

turbidity, 306

Areas where research is needed in Agricultural Science

electron affinity detectors for halogen

compounds, 209

flame photometric detector for phosphorus or sulfur, 209

infrared spectrometers, 210

microcoulometric or electrolytic conductivity detection for nitrogen compounds

such as ammonia, 210 more specific GLC detectors for halogen and nitrogen, 209 new methods of analysis such as plasma

chromatograph, 210

Aromatic and aliphatic hydrocarbons, 260

paraffinic and olefinic hydrocarbons in vehicle emissions, analysis of, 274

Arsenic, 89, 292, 305, 308, 331

trioxide, 120

Artificial radioactivity, Standard Reference

Materials, 422

Asbestos, 288, 292

Aseptically canned products, spoilage of, 196

Ashing, 181, 183 Aspartate aminotransferase, 123

Aspergillusflavus, 206 Assay of hides conductivity and capacitative measurements, 185

electrodialysis, 185

electrolyte contents, 194

microelectrode probing to determine

stratigraphic distribution of electrolytes within hides, 194

Assimilability of iron by humans and

animals, 185 Association of Clinical Biochemists, England, First Report, Standardization of Alkaline Phosphatase, 123
INDEX 435

Association of Official Analytical Chemists,

(see AOAC)

ASTM Manual on Industrial Water, 317

Atmospheric

aldehydes, 261 cadmium, zinc and lead, 80

particles, instrumentation for measuring, 261

photooxidation of chlorinated solvents, 259

pollutant, nitric acid formation, 260

reactions, 290 Atomic absorption

competences in clinical chemistry laboratory, 100

spectrophotometers, 89, 102

spectroscopy, 35, 46, 100, 102, 135, 136,

162, 163, 229, 266, 267, 276, 292, 343,

344,366,410

Atomic distribution model, 384 Atomic fluorescence methods, 154

spectrophotometry, 343, 344 Atomic oxygen, reaction of nitric oxide, 254

Attenuation, beta-ray, 264

A2 hemoglobin, 98 Auger spectroscopy, 69 AutoAnalyzer, Technicon, 101, 274, 323, 329 Automated

analysis, computer-aided analytical

chemistry, 226

determination of nitrogen by high-energy neutron activation, 169

devices, 167

fluorodensitometry, 205

nondestructive instrumental methods,

186

surveillance systems, water quality, 322

Automatic or manual sampling, 3 1

8

Automatic water quality monitoring

systems

characteristics of, 323

data acquisition and computer programs

for data handling, 330

electrochemical sensors, 323

electrochemical transducers, 323 Technicon AutoAnalyzer, 323, 328

Automating wet analytical techniques, 328

Automation and instrumentation in agricultural research, 226

Automobile exhaust chromatography, 276

chromatography, gas, 274

colorimetric techniques, 276

emission regulation, smoke, odor, 278

particulate matter in, 276 PCB'sin,210

polynuclear aromatic substances, 276 Autopsy

acute myocardial infarction, 1 17 expenses, 109 Autoradiography, 3

1

Autoxidation of

triglycerides, 177

unsaturated fatty acids, 176

Auxins, 207

Availability of metal micronutrients, 302

Available oxygen content of rivers, lakes, and

streams, continuous monitoring, 223

starch in com, 186 Azo heterocyclic hydrocarbons, 276 B

Bacillus coagulans, Bacillus stearother- mophilus, 197

Bacterial toxins, tetanus, diptheria, botu- linum toxins, 200

Bacterins, 199

Barbiturates, 97, 144 Barium, 305

chloranilate method, 270

Bamett, medically significant values, 133 Baygon, infused into foams, 216

Beall degerminator mill, 173 Benzene, extracts, 269

Benzene-soluble particulate matter, 276

Benzoic acid, 120

Berg-Barrett, x-ray technique, 55

Bertholide compounds, electronic materials

analysis, stoichiometry in, 36

Beryllium, 292

Beta and gamma cellulose, 1 82

Beta-ray, attenuation, 264

Bicarbonate, 98

Bile acids, 145
436 INDEX

Bilirubin, 97, 120, 142, 144

Bioassay, 207

Biochemical

analysis, 148 changes reflecting cardiac muscle damage, 1 16 form of trace elements, 423

organic phosphates, 196 oxygen demand, 222, 223, 310, 311, 356

transformations of mercury compounds

in aquatic environments, 341

Biochemical-physical-ecological model, 385

Biochemistry, 388

Biodegradability tests, 363

Biodegradable organic compounds, 356

Biological

activity in water samples, 320

and chemical interactions, aquatic en- vironments, 301

availability, iron bound to protein, ferric phylate, 183

metal micronutrients and the toxicity of

metals, 338 oxygen demand (BOD) and sulfide content, pollutants in effluent from the unhairing system, 193 phenomena, 385

removal of phosphate, 385

source of the samples of fats and oils, GLC andTLC, 179

systems, herbicides in, 2 1

2

systems, in vitro, 1 99

value of proteins, 168

variations, intra-individual, 133

Biologically active substances, 201

Biomedical research, 78,81

Birefringence, 37, 40, 52

crystal habit and index, identity of material, 34

Birth defects of genetic origin, diseases, 85

Bitter patterns to observe domains, 53 Blank sample, 422

Bleached oil, monitoring color, 229

Blood

gas, acid-base measurements, 90

glucose, 82

lead, 98 pH, PCO2 PO2 systems, 97

serum, for seriological diagnosis, 197

sugar, 97

volatiles, 98 Bormann anomalous x-ray techniques, 55

Boron, 242,305,308

Botulinum toxins, 200

Bound niacin in com, 1 69

Brewers' grits, 172 Broad spectrum antibiotic medium for growing Salmonella, 196 Bromate, determination of, 164 Bromide, 97

potentiometric membrane electrode, 325

residues in foodstuff, routine control, analysis for, 165 Bromination fluorometric, gravimetric

digitonin, methods, AOAC, 192 Bromine, 163,242

origin determination by particle size, 290 BSP

retention, 97

sweat test, 142 Bud break, 208

Buffers, transphosphorylating, 131

Bulgur, 169 Bureau of Mines, 277

B12 assays, 145 c

Cabbage, 184 Cadmium, 89, 98, 235, 305, 308, 325, 331, 337,338,351,352

Calcium

and magnesium, 310

carbonate, 193

in serum, 138

potentiometric membrane electrode, 134,

137, 144,325,352,413

Califomium-252,428

Canned hams, 189 Canned whole tomatoes, 196 Carbamates, 21 1,306

Carbanilate-base herbicides, 113,213

Carbides, 24

Carbon, 67

chloroform extract, 305

dioxide, 97, 134, 142,219,270

filter technique, 360

in selenium, 23
INDEX 437

Carbon monoxide, 219, 255, 275

analyzer, nondispersive infrared, 255 and submicrometer particulates, satellites for measuring long-lived pollutants on

global scale, 258

and sulfur dioxide in various stack gases, 272

emission standards for, 274

infrared correlation spectroscopy and

nondispersive infrared techniques, 258,

275,277

instrumentation for, 25

1

measurements, 277

methane, gas chromatographic technique, 252

monitoring capability for, 259 Carbon monoxide analysis, standards, 274

Carbonyls, 177

polynuclear ring, 268

Carcinogenic dimethylnitrosamine, 218

Carcinogens, 204, 205, 206, 33

1

in aflatoxin-contaminated meal, 206

Cardiac muscle damage, tests for, 116

Cardiovascular problems, 1 9

1

Carotene, 97

Carriers for

agricultural chemicals, foams, 216

insecticides, foams, 216

Carrots, 184 Cascade impactors, 262

Catecholamines, 145 Cathode-ray polarography, 347

Cations, interference, 270

Cellulose, 182

and/or lignin contents of plant materials, determination of, 1 8 1 , 1 82 Cement plants, stationary source of pollutants, 269

Center of symmetry, 52,53

Cephalin flocculation, 97, 142 Ceramics, 20

Ceruloplasmin, 97, 143 Cesium, 341 Chaconine, leptines, solanine (steroidal al- kaloids), 202

Characterization materials, 20, 22

definition, 21 Charge carrier concentration in a semiconductor, 25

Cheeses, 184, 196 Salmonella in raw and pasteurized milks, and dairy products, 196

cholesterol content of, 191 Chemical

analysis standards, agricultural, 234

composition, mass and size, analyzing

particles by, 261

composition, sea water, 401

engineering, technical and management

skill, development of, 97

ionization, mass spectrometer, 275 measurements in medicine, analytical goal

for, 136 method for methionine and cystine, rela- tive nutritive value of foodstuffs, 167 oxygen demand, 223,310,311,329,356,

357

parameters for water quality, 310

potential, 312

senses, 385

species of toxic elements, 424

sterilizers, 242

Chemiluminescence, 253, 387

measurements, 258

Chemiluminescent

analyzers, 275

emissions, 255

Chemistry

costs vs charges, 109

related research accomplishments, 83

services, tests volume, 109 Chemotaxis, 385 Chloramben (3 -amino-2 ,5 -dichlorobenzoic)

acid, 212

Chloramphenicol-resistant strain, 184

Chlordane, 305

Chloride ions by selective electrodes, 367

Chlorides, 97, 134, 144, 142,268,305,309,

325,329

Chlorinated hydrocarbons, 210,211, 422

pesticides, 269

solvents, 259

Chlorine

analysis, 267 demand, 310

emitted from combustion of chlorine-containing coals, 259

monitoring capability for 259
438 INDEX

Chlorine — Continued

origin determination by particle size, 291

Chlorine-containing materials, incineration

of, 259

Chlorinity, definition of, 397, 400

Chlorodibenzoparadioxin, 235

Chloroform, 192

Chlorophyll, optical measuremlents for fluorescence, 321 CHN analysis, 267

Cholesterol,' 97, 134, 143, 192

clinical analyses of blood and serum, 192

content of foods, 174, 191

creatinine and serum enzymes, 133

determination by Leibermann-Burchard

color reaction, 1 92

spectrophotometric determination of, 1 74

Cholinesterase, 97, 143 Chromatographic analysis, gas, 275 Chromatographic methods,

column, 268, 273

electrophoresis, 100

gas chromatography, 100, 268, 269

ion exchange, 100, 406

paper chromatography, 100

thin layer chromatography, 100, 268, 269

Chromatographic separation, 202, 389

colorimetric procedures for diethylstil- besterol, 192 Chromatography and colorimetric techniques, polynuclear aromatic substances, automobile exhaust, 276

Chromatoids, nonvolatile, 268 Chromium, 89, 241, 308, 337

(hexavalent),305,329

sampling, handling, 319

Circulatory system, composition of

slaughter animals, 190

Citrinin, 205

Classical methods in clinical chemistry, 100

polarography, 344

Classification of odor, 334

Clean Air Act, as amended in 1967, 246

Clean Air Act, 1970,246

Chemiluminescent reaction, 272

Clinical analyses of blood and serum, for

cholesterol, 192

blood pH measurements, central reference point for, 1 2

1

chemist's role, 101 enzymology, 110, 122 measurements. Standard Reference

Materials, 119, 120 pH measurements on whole blood, tristris HCl buffer for standardizing, 121

standards, need for improved, 1 19

utility of enzyme measurements, 1 14

Clinical chemistry

analytical chemistry in, 94

education, 152

laboratory, 94, 156

medical laboratory, organization and per- sonnel, 96 team effort, 107

Clinical chemistry laboratory, competences

in chromatographic methods,

electrophoresis, 100

gas, 100

ion exchange, 100

paper, 100

thin layer, 100

Clinical chemistry laboratory, competences

in classical and other methods

gravimetry, 100

quality control materials, 100

solvent extractions, 100

standards, 100

titrimetry, 100

ultramicrochemistry, 100

Clinical chemistry laboratory, competences

in electroanalytical methods,

conductance, 100

coulometry, 100

ion specific electrodes, 100 pH acidity and blood gases, 100

potentiometry, 100

Clinical chemistry laboratory, competences

in optical methods

atomic absorption, 100

flame photometry, 100

spectrofluorimetry, 100

spectrophotometry, 100

Clinical chemistry laboratory, competences

in other instrumental methods,

osmometry, 100

refractometry, 100
INDEX 439

Clinical chemistry, medical laboratory, in- strumentation, 101 blood pH, 103

conductivity meters for reagent grade water testing, 103 coulometers, 102

electroanalytical methods and instru- ments, 103

electrophoresis chromatography methods

and instruments, 103 enzyme-kinetic instruments, 102 flame photometry, 102

gas liquid chromatograph with flame

ionization, 103 methods and instrumentation, 102

optical methods and instruments, 102 PCO2, PO2 systems, 103

protein electrophoresis units and densitometers, 103

specific ion electrodes, 103

thin layer chromatography apparatus- Buckler, 103

Clinical chemistry laboratories, within a

hospital, inpatient services, private, re- gional, 109

Clinical hospital laboratories, extra costs of, 109

increase in volume, 95 , 142

precision of tests, 142

Clinical laboratories in the United States, 79

Coagulation, evaporation, precipitation, and

crystallization, dairy products, 191

Coals, 259

Coatings, cyclonic collectors, dustfall jars, adhesive, sampling devices for large

particles, 262

Cobalt, 308, 338 Coenzyme dependent enzymes, spectral ab- sorbance curves of reduced NAD and NAD, 113

Coffee, 240

Cold ashing procedures, 228

Collagen, keratins, silk, fibrous proteins, 224

Collagens, intramolecular heterogeneity of, 225

Collecting samples, filters, 261

Collection techniques, filters, impingers, evacuated flasks and condensation

procedures, 262, 270

Colorado potato beetle, 202

Colorimetric

analyzers, 260

methods, 183, 246, 260, 274, 275, 276, 277

Colorimetry, 102, 167,202

Color, 332

and transparency, parameters for water

quality characterization, 306

bleached oil, 229

development and colorimetric measurement, 305, 310, 328, 332 Column chromatography, 268, 273 and gas chromatography with mass spec- trometry for analysis of hydrocarbons, 277

fluorescence spectroscopy, 277 Combined GLC/mass spectrometry, computer storage and handling of acquired

data, 210, 211 Combustion of chlorine-containing coals, 259 Combustion sources, indicators of control of

acetylene, 261

ethylene, 261 Commission for Enzyme Diagnostics and

Standardization, 123 Communication with physicians, 108 Competences in clinical chemistry laboratory, 100 Compleximetric titrations, 341 Component-specific dyes, 167 Composition and mass measurements, 264

Composition of

live animals, 190 meat and dairy products, 190

sea water, 395 Composite samples, grab samples, 317

Compositional

analysis, 186

analysis for grain and oilseed grading, 1 86 changes in lipids, 176 Computer-aided analytical chemistry,

agricultural research, automation and

instrumentation, 226 Computer

analytical tool, 69

control of vegetable oil refining and

processing, 228
440 INDEX

Computer - Continued

programming, 101 programs for data handling, automatic water quality monitoring systems, 330

storage and handling of acquired data, combined GLC/mass spectrometry, 210

Computers (limited access use), clinical chemistry medical laboratory, 104 Conductance, pH, acidity, 100

Conductivity

measurements, 103, 194, 398

of sea water, 328, 397

semiconductors, 22 Conductometric, type of electrochemical

sensor, 324

Conformational analysis of fibrous proteins, 228

Conjugatable dienes and nonconjugatable

dienes in triglyceride esters, simple

esters, 175 Connective tissue, 190 determined by histometric point-counter, Constant proportions, principle of, 396

Consultative support services, clinical chemistry laboratory organization

chart, 100

Continual instrumental analysis, 270, 271 Continuous

fast drying of heat-sensitive materials, control of product moisture content in, 165

monitoring, available oxygen content of

rivers, lakes, and streams, 223

monitoring of enzyme kinetic activity, 131

refractometry, 229

sampling equipment, 3 1

8

Control of combustion sources

acetylene, 261

ethylene, 261

indicators of, 261

Control

product moisture content in continuous

fast drying of heat-sensitive materials, 165

protein content in mixed feeds, 168

technology, 269

weeds, 207

Cooked meats and dairy products,

cholesterol content of, 1 9

1

Copper, 89, 98, 242, 292, 305, 308, 319, 329, 331, 337, 338, 342, 349, 351, 352 Coprex technique, isotope dilution, deter- mination of minor constituents, 48

Coproporphyrin, 98

Correlation spectroscopy, 256, 258, 273

spectrometers for sulfur dioxide, nitrogen dioxide monitoring, 258

Cortisol plasma, 143

secretory rates, 145

Cortisone, urinary free, 145

Cotlove, et ai, tolerable analytical variability, 133

Cotton, 206

Cottonseed, 187

oil, 234

oil, cyclopropenoid fatty acid content of, 178 Coulometers, 103 Coulometric method, 372

titration of dissolved oxygen, 370

Coulometry, 100 amperometric end point determination, 43 Coumestans, 204

Coumestrol, 204

Counting, scintillation, 266

Creatine, 97

kinase, 98, 116, 143

kinase (CK)^ myocardial infarction, lactic dehydrogenase, (LDH)^, 112

Creatinine, 97, 120, 134, 142 serum enzymes, cholesterol, 133

C-reactive protein, 1 16

Criteria for air quality instrumentation, 248

Cross-correlation nondispersive analyzers, 256 Crude fiber content of feeds and cereals ,181

samples, treatment of, 1 8

1

Crude oils, fuel oils, source of, 386

Crystal habit, index and birefringence,

identity of material, 34

Crystallization, evaporation, precipitation, coagulation of dairy products, 191 CSF protein, 97, 142, 143, 144
INDEX 441

Cultural and serological techniques for Salmonella, 196

Cupferron, 342

Cupric, potentiometric membrane elec- trode, 325

Curie temperatures, 40

Cyanide, 3 10, 3 11 , 325, 329, 367

ions by selective electrodes, 367

Cyanoglucosides in foods, 202

Cyclic derivatives and oxidized dimers,

toxic compounds in deep fat frying with

vegetable oils, 1 17 Cycloheximide influenced by, 209

Cyclonic collectors, 262

Cyclopropenoid, 234

fatty acid content of cottonseed oil, 1 1

8

material present in oils and meals, deter- mination of, 1 1

8

material, reaction products formed during

processing and after ingestion of, 1 18 Cystine, 170, 171, 193 and methionine, chemical method for, 167

Cytokinins, 208

Czochralski technique, 37 D

Dairy products, 196

cholesterol in, 174, 191 evaporation, precipitation, coagulation,

crystallization, 191 Salmonella in, 174, 191 Damage to tobacco leaf, 2 1

9

Darkness of myoglobin pigmentation, 188 Data acquisition, computer programs for data handling, 330 DDT, 210, 305 Debye-Scherrer-HuU, patterns, 33 Decoration

dislocations, characterizing vacancies, 58

electron microscopy, density measurements, etching, determination of structure, 52 Deep fat frying with vegetable oils, nutritive value, 177

Defatted soybean meal, 168 Defect mapping, homogeneity in, 68

Defects, perfection, determination of physical structure, 49

Deficiencies of iron in diets of Americans,

food consumption surveys, 185 Degenerative — chronic diseases, 85 Degradation-products for synthetic

polymers, 240

Degree of saturation of fatty acids, 174 Dehydrator, 166 Dehydrogenase, lactic, 123

Denitrification, hypolimnion, 301 Densitometry, 178

Density, 52

particulate matter, 28

non-specific, physical parameters, water

quality, 3 10, 331

seawater, specific gravity, relative conductivity, 401

Deodorization, 229

Derivative polarography, 344, 347

pulse polarography, 347

Design of measurement systems, water pol- lution control, 302, 304, 309, 312, 316

Detection

chlorinated hydrocarbon pesticides,

PCB's, interference in, 210

oil spills, IR and UV multispectral

scanning, 321

Detectors

electron-capture, 260

flame ionization, 260

flame photometric, 253

response, flame ionization, 261

specific for nitrogen, 2 1

1

Detergents, 311

Deterioration products in fat, 177 Determinate error, random error, precision, 327

Developing countries, aid programs to, 170 Development of advanced pollution measurement tech- niques, appropriations for, 298

technical, chemical, engineering and management skills in clinical laboratory, 107 Diabetes mellitus, diagnosis of, 82

Dialysis, 199

Dibenzo-p-dioxins, 210

Dichromate, 357

Dieldrin, 305
442 INDEX

Dienes, determination of, 175

Diesel

emissions, nitrogen oxides in, 277

exhaust emissions, odors, 276, 277

exhaust emissions, polynuclear aromatic hydrocarbons, 277

Diesel fuels, 277

Dietary cholesterol and saturated fats, 174 Diethanolamine (DEA), 131 Diethyldithiocarbamate, 342

Diethylstilbesterol

chromatographic separation and

colorimetric procedures, also GC

procedures, 192 metabolism, 192

Differential polarography, 347

scanning calorimetry, 1 82

spectrophotometry, 256

thermal analysis, 36

Digestibility of feed protein, 172

of cellulose, perloline, 203

Digestibility and availability, 169

Digital Fat Controller, 190

Digitonin or tomatine precipitation, 192

Dilantin, 98

Dilatometric studies, 36 Dimethyl mercury, phenyl mercury, methyl- mercury, mercury discharges, 339

Dimethylnitrosamine, carcinogenic, 2 1

8

Diphtheria, 200

Diseases

birth defects of genetic origin, 85

degenerative-chronic, 85

infectious, 84, 85

metabolic, 84

neoplastic, 85

nutritional, 84

Diseases only partially understood (lacking

definitive cures and preventative measures) in 1970,85

Diseases which biomedical research and

public health measures have prevented

or controlled by 1970,84

Dislocations

characterizing vacancies, decoration, 58

x-ray techniques, etching studies, 55 Disodium

hydrogen phosphate, 120

phenyl phosphate, 189

Dispersed plastics

in fats, agricultural science problems, 180

in rendered fats, 179

Dispersive infrared

analysis, 274

measurements, 277

Dispersive matched-filter spectrometers, 256

Dissolved gases

in natural waste waters, 368

water pollution control, analytical

methodology, 367

Dissolved matter parameter for water quality characterization, 307

physical water characteristics, 3 10, 33

1

Dissolved oxygen

content of the oceans, 410, 41 1, 412, 413

coulometric titration of, 370

electrodes, dissolved oxygen content of

the oceans, 413

measurement, voltammetric membrane

electrodes, 326

parameter for water quality characterization 305, 307, 3 1

1

plant nutrients, toxic matter, 307 Winkler method, 313, 369, 370, 371, 372,

410,411,414

Distribution particle-size, 263, 264

Dithizone, 163,342

Domestic water supplies

color, 305

inorganic chemicals, 305

organic chemicals, 305

pesticides, 305

radioactivity, 306

turbidity, 305

water quality characterization, 305 Doped silicon, 67

Doriden, 98, 144 Dormancy in plant tissues, 209 Double crystal rocking curve photographs, 55

topographs, Lang, 28 Drug analysis

positive identification, 148

positive identification, false positives, 149
INDEX 443

Drugs by

GLC,98

TLC,98

Dry gangrene, tall fescue grass Festuca

arundinaceae, "fescue foot", 203 Drying of com, 1 86

Dustfall jar, 262

Dusts, polynuclear aromatic hydrocarbons,

air pollutants, 219

D-glucose, 120 E

Ecology, 388

Ecosystem, 236

Edible solanaceous crops, steroidal alkaloids present in, 202 EDTA, 341 Education

environmental analysis, 291

service and research, medical triad, 86

Effective distribution constant, 38

Effluent standards, stream standards, water quali- ty criteria, 303

unhairing system, pollutants, biological oxygen demand, sulfide content, 193

Eggs, poultry, fish, meat and dairy products,

main sources of cholesterol, 174, 191

Electric birefringence, 225

Electrical conductance, 310, 325, 33

1

twinning in piezoelectrics, 53

Electrically inactive impurities, 22

Electroanalytical methods

conductance, 100

coulometry, 100

ion specific electrodes, 100 pH acidity and blood gases, 100

potentiometry, 100

Electroanalytical

methods and instruments, clinical chemistry medical laboratory, 103

techniques, 254, 364

Electrochemical

analyzers, 275

methods, 151,387

sensors, automatic water quality monitoring systems, characteristics, 323, 324

transducers, 271,323

Electrode, ion-selective, fluoride, 259

Electrodialysis, surface specific ion elec- trodes, 194

Electrolyte contents, microelectrode probing in assay of hides, 194

Electron

affinity detectors for halogen compounds,

nitrogen compounds, areas where

research is needed, 209

capture detectors, 260

capture gas chromatography, applied to determination of chromium or berylli- um, 389

micrographs, 3

1

microprobe, 67, 289

microprobe analysis, 66, 67, 69

microscopes, 262

paramagnetic resonance, 149 probe analysis, mapping, 30

spectroscopy for chemical analysis

(ESCA), 225

spin resonance, 1 82

Electron microscopy, 28, 52, 69, 227

particle sizing, 265, 288

scanning, 69, 288

transmission, 288

Electron transport and phosphorylation, 217

Electronic

analysis, stoichiometry in, Bertholide compounds, 36

data processing calculators, (clinical chemistry medical laboratory), 104

materials, determination of major constituents, 36

materials, determination of oxidation

state, 44

research, development, stoichiometry in, 37

sense of electric axis, 52

solid state research, 19

Electrooptical techniques, 273

Electrophoresis, competences in clinical chemistry laboratory, 100

Electrooptic, second harmonic generation

coefficients, 26

Elemental analysis, 265, 267, 273

428-999 O - LT - 72 - 29
444 INDEX

Elemental pollutants in water, rapid screening procedure, 386

Elements and water, agricultural research, 162 Emission

automobile, 246, 274

hydrocarbons, 275

monitoring, stationary source, 248

stationary source, 273

stationary source measurement, 269

spectroscopy, determination of anions and halogens by, 34

spectroscopy, determination of minor

constituents, 45

spectroscopy, preconcentration, 35

spectroscopy, steel, 45

standards, 274

Encapsulation of insecticides in plastic, 215,

216

Encoding chemical information, 154 Endocrine section, volume and precision on

individual tests, 143 Endogenous and plant origin of fecal nitrogen, distinguishing between, 172

Endrin, 306

Engineering, development of technical, chemical, and management skills, 107 Enrich foods with iron, 1 85 Enrichment medium for growing broad spectrum antibiotic, enzymetrypsin.

Salmonella, 196 Environmental

analysis, education, 291

contamination by lead, health effects of, 79

health, 100

pollution caused by discharge of mercury

in the waste of Kjeldahl nitrogen deter- minations, 172

Protection Agency, 257, 375, 388

segment, metal dynamics in an, 339 Enzymatic hydrolyses, 171 Enzyme

analysis, 151 assays and reference materials, stan- dardization of. Standards Committee,

International Federation of Chemists,

Expert Panel, 123

assays (Scandinavian Association of

Clinical Chemists), 123

electrodes, 151

kinetic activity, continuous monitoring of, 132 measurements, clinical utility of, 1 14 methods, standardized, 126

procedure for determination of, 234 serum creatine kinase (CK), 1 17 standard reference material, need to develop human alkaline phosphatase, 124

standardization activities, 122

standardization, Europe, 123

standards in serum, 1 25

trypsin, nulk casein, 196 Enzymes

evaluation of reference materials containing, 125 lUB Commission of, 1 3

1

non-human, 124

Enzymology-kinetics, 100 Enzymology, standardization problems

peculiar to, 121 Epilimnion

aerobic conditions, 301

thermocline, hypolimnion, thermally

stratified lake, 300 Epoxy acids, 186

Equilibrium relative humidity, (ERH), 165 Erucic acid content, 186

Erythrocyte sedimentation rate, 1 16

Essential amino acids and proteins, rapid measurement of, 166

Establishing oxidation state, 44

Esters of sucrose, glucosides, polyglycerols, and other polyols, 178

Estrogen, 204

Estrogens, fractionated, 145 Etching studies, 28, 52, 55

Ethanol, 97

Ethyl-amino-ethanol (EAE), 131 Ethylene, 207, 208, 209

Euphotic zone, oceanic system, 394

Eutrophication of Great Lakes, 304

Evaluation of FA conjugates for the identification of animal diseases, 200

reference materials containing enzymes,

125
INDEX 445

Evaluation of— Continued

veterinary biologicals, 199 Excess air, moisture, gas flow velocity, 269

Exhaust

emissions, automobile, 274

hydrocarbons, carbon monoxide,

hydrocarbons in blowby gases, emission standards for, 274

Extensive properties, 3 1

2

Extraction of dormancy factors from the living tissue, 207

techniques for polar pesticide metabolites, 214

Extracts, benzene, 269

Exudative PSE pork, pale soft, 189 Eye appeal of foods, 195 F

False positives in drug analysis, positive

identification, 149

Fat

acidity, 187 added to meat products, 190

deterioration products in, 177

in com grits, 172

quality, 177

Fat-enriched feeds, 1 80

Fats

agricultural science problems, dispersed

plastics in, 180 and oils, determination of biological source, 179

oils, meat, dairy, egg products, baked

goods, cholesterol in, 174

Fatty acid determination in oilseeds, nondestructive method for, 173

mixtures, 175

Fatty Acid Producers Council, 180

Fatty acids and lipids, rapid measurement, 172

degree of saturation of, 174

profile, 234

nature of, by methylation and GLC, 174

nonvolatile, 268

Fecal

fat, 145

nitrogen, distinguishing between en- dogenous and plant origin of, 172

output nitrogen, 172 Feces samples, 166

Federal

laboratories, 79 Water Pollution Control Administration, 304

Feed, 165

protein, digestibility of, 172 Feeding studies, 169

Feedlot, pollutants caused by agriculture, 221 Feeds and cereals, crude fiber content of, 181

Ferric phylate, 183 Fescue

foot, dry gangrene, toxic alkaloids and

perloline in Kenwell variety of tall fescue, 203

toxicity, 203

Fibrillarsarcoplasmic protein, 190

Fibrous

constituents of plants, agricultural research, 181

proteins, collagen, keratins, silk, 224

proteins, conformational analysis of, 223

Field methods, automated, 265

Filterable residues, 3 10

Filters collecting samples, 261

collection techniques, 270

polystyrene membrane, 266

Finished leathers, estimating bound glu- taraldehyde, 194

Fish, meat, eggs, poultry and dairy

products, cholesterol in 191 Fish and wildlife aquatic life, 306, 307, 308

radionuclides, 308

recreation and aesthetics, 306

turbidity, color and transparancy, 306

water quality characterization, 308

Fixed nitrogen, oceanic system, living or- ganisms, 394 Flame

atomic absorption spectroscopy, 426
446 INDEX

Flame - Continued

emission, 35

emission for determination of minor constituents, 46

photometric detector measurement, 89, 209, 253 Flame ionization analyzer, 274

detector response, 261

detectors, 218, 260

Flame photometry

clinical chemistry medical laboratory, 100, 102

strontium content of the oceans, 410

Flat sour spoilage, 196

Flavor and odor

taste-panel evaluation of food products, 194

studies, 240

Flax, 187

Floating matter, aquatic life, fish and

wildlife, 307

Float zoning, 22

Flowering, development of, 208

Flow-through laser photofluorometer, 200

Flue gases, 269, 271 Fluorescence

spectrophotometry, 363

spectroscopy, column chromatography, 277

used in detection of chemical pollutants, 321

x-ray, 267

Fluorescent antibody

reagents, 199

technique, confirmation for Salmonella, 196

"cat eye" spots, 206

infrared technique, 275

substances, 184

Fluoridated water supplies, 366

Fluorides, 305, 329

electrode, ion-selective, 259

hydrogen, 259

particulate and gaseous, 269, 272

potentiometric membrane electrode, 325

water-soluble, 259, 272

Fluorimetry, 102, 387

Fluorine, 242

Fluorochrome, 199

Fluorometric

analysis, determination of minor constituents, 49

assay, vitamin Be components in foods, 183 method (AOAC), 192 method, folacin in foods, 1 83

Fluorometry, determination of the hor- mones, 208 Foams, carriers for agricultural chemicals and insecticides, 216

Folacin activity in human serum, 184 Folded laser beam, 256

Foliar pathogens estrogen, coumestrol coumestans, infection of alfalfa Medicago sativa L., 204

Folic Acid, 145 Food

and Nutrition Board of the NAS-NRC,

183 consumption surveys, deficiencies of iron

diets of Americans, 185

flavors, 240

industries, pollution caused by, 222

processing plants, sanitation in, 196

products, 183

products, microbial growth in, 195 Foreign animal diseases, 197

organisms, identification of, 1 98 Formal charge, real charge, oxidation state determination, 44

Formaldehyde, analyzed in atmospheric

samples, 260, 277 Formic acid, 194 Formulation of mixed feeds by, least-cost

basis, nutritional values of ingredients, 168

Forward-scattering, right-angle-scattering, instruments, 262

Fourier transform NMR methods, 1 50

Fractional elutriators, various impactors and long horizontal tunnels for highervolume samples, 262

Fractionation, particle-size, 264

Frankfurters, fat added to meat products, 190 Free fatty acids, 145 Free thyroxin, 145
INDEX 447

Fruit growth, 208

harvest, abscission as related to, 208

Fuel oils, instrumental neutron activation characterization analyses, 386

Fundal, infused into foams, 216

Fungicide, 242

Fungicides, rodenticides, pesticide, herbi- cides and insecticides, agricultural water use, 309 G

Galactose, 98

Gallium phosphide single crystals, linewidth

in, 41, 42

Gardona, infused into foams, 216 Gas

bubblers, 266

flow velocity, excess air, moisture, 269

titration, 272 Gas chromatographic

colorimetric methods, 275

procedures, measurement of odor causing matter in water samples, 334

techniques, monitoring, 246, 252, 258,

259, 272, 277

techniques, oxygen analysis, 412 Gas chromatographs, periodic monitoring,

grab sampling, 260 Gas chromatography, 100, 148, 153, 156, 192, 235, 240, 268, 274, 275, 360, 369,

389,416

Gas-exchange separation, 369

Gasometric methods, oxygen content of the oceans, 412

Gases, flue, 269

Gel filtration, 199 Geophysical measurements, 259 Germanium, 70

arsenide, 67

silicon and germanium arsenide, preconcentration techniques, 46

single crystal, map of, 25

Gibberellins, 207, 208

Glass

electrode, 324

filters for spectrophotometry, 120

Glasses, 20

GLC, 179, 192, 195, 206, 208, 209, 210,

211,214,215,218

Global scale, long-lived pollutants, satellites for measuring, 258

Glucose, 134, 142, 144 Glucose-enzyme specific, 98

Glucosides, esters of, 178 Glutamic pyruvate transaminase (GPT),

112

Glutaraldehyde, tanning of leather, 194

Glycosides, herbicides in biological systems, 212 Goetz spectrometer, 276

Gold-silver wires, 67 Grab samples, 260, 317

Grain

boundaries, twins, Schulz x-ray pictures, 53 sorghum, aflatoxins in, 205

Grating spectrometers, 256

Gravimetric digitonin method, (AOAC),

192 Gravimetry, 37, 100 Grease and oil, 3 1

1

Great Lakes, eutrophication of, 304

Griess-Ilosvay reaction for nitrogen dioxide, colorimetric, 254

Ground meat, fat added to meat products, 190 Grown crystal, stoichiometry, 40 Growth

regulating compounds, 209

stimulants, 307

Guinier, powder patterns, 3

1

Gulactosemia, 144 H

Halogens, determination by emission spec- troscopy, 34 Ham and loin muscle, pH measurements,

189 Hardness, 310, 329

potentiometric membrane electrode, 325 Haze, particles, 262 HBr in acetic acid, determining the amount

of cyclopropenoid material present in

oils and meals, 178 Health effects of environment contamination by lead, 79
448 INDEX

Heart isoenzyme of isocitric dehydrogenase

(IDC), 115 Heat

capacity determination, phase change, 36

inactivation of acid phosphatases, 189

sensitive materials, control of product

moisture content in, 165 Heat-processed foods, monitoring for op- timum flavor development in, 195

Heat-sensitive fatty derivatives, quantitative analysis of 178 Heavy metals, 311,428

Hemoglobin electrophoresis, 98

Hemoglobins, 97 Hen's egg essential amino acid pattern, 168 Henry's Law, 315

Heptachlor, 306

acylanilides, 217

carbanilate-base, 2 1

3

electron transport, 2 1

7

expoxide, 306

in agricultural water use, 309

in soil, plants or water 212

phosphorylation, 217

s-triazines, 217

substituted uracils, 2 1

7

Herbicide action, structural basis of, 217

Herbicides 2,4D plus 2,4,5-T, plus 2,4,5- TP, 306

Herbicide-surfactant mixtures, 215

Heterocyclics, Azo, 268

Hides and leather, 193 High-energy neutron activation, automated

determination of nitrogen by, 167

High-erucic acid oilseeds, 185 High purity

certified organic and inorganic standards,

the availability of, 1 19

metals, SRM 685, SRM 680 and 681, and

zinc, 67

palladium metal, 67 High resolution analytical microprobes, 228 High-volume sampler, 262

Histometric point-counter, connective tis- sue, 191 Hormones, determination of, 208

Hospital laboratories

costs, 193

in the United States, 79

Human

alkaline phosphatase, standard reference enzyme material, need to develop, 124

Nutrition Laboratory, 183, 184

source, stable enzyme materials, 132 Humans and animals

assimilability of iron by, 1 85

physiological effects of ingesting trans isomers of polyunsaturated lipids in, 174 Hydrocarbons, 221, 252

air quality instruments for, 246

analysis of, 272, 277

emission standards for, 274

monitoring capability for, 259

nonmethane, 260, 261

polynuclear aromatic, 268

Hydrochloric acid emitted from combustion of chlorine-containing coals, 259 measurement of, 260

monitoring capability for, 259 Hydrogen fluoride, 221, 259

optical techniques for measurement of, 260 Hydrogen sulfide, 221, 259

organic sulfur compounds, gas chromatographic technique for, 259

organic sulfur gases, sulfur dioxide, Kraft

mill effluents, 272

Hydrographic tables, 399

Hydrolysis procedures for vitamins, 170, 183 Hydroperoxides, 176, 177

Hydrosuffide, 193 Hydrothermal quartz, 33 Hydroxy acids, 186 Hydroxybutyric dehydrogenase, 116

Hydroxysteroids, 97, 145 Hypolimnion,

denitrification, 301

epilimnion, thermocline, thermally

stratified lake, 300

Hypolinmitic zone, 300

I

Identification diagnosis of animal diseases, 198, 199, 200
INDEX 449

Identification— Continued

fats and oils, 189

high-oil high-lysine high-protein com, 187

microscopic, 265

organic groups at an air/solid interface, 224

Immobilized enzymes, layered column of, 171 Immune absorption techniques, 1 7

1

Immunochemical detection of species adulterants, 171 Immunodiffusion for IgA, IgM, IgG and

fetoprotein, 98

Immunoelectrophoresis, 98

Immunological-type detector, 150 Inmiunology and genetics, 101 Impactors, cascade, 262

Impingers, 270

Impurities, electrically inactive, 22

Impurity

or dopant concentration in semiconductors, 25

range of interest in semiconductors,

preconcentration, refinement of source, 45

stoichiometry and strain variations, index

variation methods reveal, 26

Inactivate viruses, processing temperatures, 189

Incinerators, stationary source of pollutants, 269

Independent clinical laboratories in the United States, 79

Index and birefringence, crystal habit, 34

Indicators of control of combustion sources, acetylene and ethylene, 261

Indigenous plant constituents, 201

Indoleacetic acid, 208

Indoles, 208

Indospicine and j3-Nitropropionic acid in Indigofera, 202

Industrial

pollutant, residue contamination in agricultural products, PCB's, 210

wastewaters, 354

water, ASTM manual on, 3 17

Infant formula containing soy isolate, 203

Infection of alfalfa {Medicago sativa L.) by foliar pathogens, 204

Infectious diseases, 84, 85

Infrared absorbtion measurements, 23

absorptiometry, determination of minor

constituents, 49

analyses, 174

analysis, dispersive, 274

analysis, nondispersive, 264, 271, 274, 277

correlation spectroscopy, 258

emission, measurement of gases by use of, 273

fluorescent analyzer, 255 measurement for fat and moisture in meat,

191 or Raman spectroscopy, 229

reflectance analysis, determination of oil moisture and protein, 1 87

spectrophotometric methods, determining

the amount of cyclopropenoid material present in oils and meals, 178

spectrophotometry, 102

Ingesting transisomers of polyunsaturated

lipids in humans and animals,

physiological effects of, 174

Inhibitors, stimulants and plant growth hor- mones, 207

Inorganic

anions in water pollution, 364

gases, organic gases and vapors, air pollutants, 219

particles, 219

phosphates, 365

pollutants, 260

Inpatient services, clinical chemistry labora- tory within a hospital, 109

Insecticidal fumigant, 163

Insecticides, 242

encapsulated in plastic, 2 1

6

in domestic water supplies, 305

repellents, uniform application or adsorption of, 216

Insect population, control of, 235

Insects, sex attractants of, 385

Insoluble humin, formation of, 171

Instability of amino acids, during acid hydrolysis in the presence of carbohydrates, 167

test organisms, 184
450 INDEX

In-stack instrumentation, 270, 272

Instrumental

analysis, continual, 270

measuring capability, 89

neutron activation analysis, 386

techniques, remote, 270

Instrumentation

clinical chemistry medical laboratory, 101

criteria for air quality, 248

for measuring atmospheric gases and

vapors, 246

for measuring atmospheric particles, 261

for sulfur oxides, ozone, carbon monoxide, nitric oxide, nitrogen dioxide, 247

in-stack, 270, 272

point-sampling, 257

Insulin, 82

Integrating nephelometer, 262, 263

Intensive properties, 312

Interdisciplinary

approach to analysis, 238

research, 241

research, concepts of, 237

Interfacial angles, measurement of, 34

Interference

anions, 271

cations, 270

figures, 52

Interferometers, 256

Intermittent sampling, 270

Internal medicine, 100

International Atomic Energy Agency, 424 Enzyme Commission, 122

Federation of Chemists, Expert Panel, Standardization of Enzyme Assays and

Reference Material, Standards Committee of, 123

Intercelluar target systems, identified for herbicides, 217

Interstate Clinical Laboratory Improve- ment Act of 1967 (CLIA 1967), 1 19 Intramolecular heterogeneity of coUagens, 225

Iodine

butanol, extractable, 145 number, 174

values, 179, 186

Ion-exchange, 100

chromatography, 341

liquid chromatography, 153 membranes, 342

separation, 184

separation, fluorometric assay, vitamin Be components in foods, 183

Ionic balance, sea water, 409

strength, 315, 328

Ionized calcium, 98

Ion-probe scanning analyzer, 66

Ion probes, 58,289

Ion-selective electrode methods, 151 Ion selective electrodes, 100, 259, 361, 366, 367

Iron, 242, 329, 337, 342 bound to protein, 183 compounds, 185

(filtrable), 305

Irrigation waters, 309

Isocitric dehydrogenase, 115, 116

Isoleucine, 170, 171 Isotope dilution, 48, 67 mass spectrometry (IDMS), method for

total serum calcium, 136 lUB Commission of. Enzymes, 1 3

1

lUB 1964 Enzyme Commission Recommendations, 122J

Jackson Candle Turbidimeter, 336 K

Kenaf, pulps, papers, 182 Kenwell variety of tall fescue, 203

Keratins, silk, fibrous proteins, collagen, 224

Ketones, 275

Ketosteroids, 97

Kinetic mechanisms, 260

models, 384

Kinins, 207

Kjeldahl

method, 358

environmental pollution caused by

discharge of mercury in the waste of, 172
INDEX 451

Kraft

mill effluents, 272

paper mills, 254 L

Labeling

definition of foods, 174

transporting and specimen procurement,

sampling errors, 105

Labile amino acids, destruction of, 170

Laboratories

mobile, 260

shipboard, 406

Lactic dehydrogenase, 97, 98, 116, 123, 134, 143 Lactose and proteins, total solids, 191 Lakes, continuous monitoring for available oxygen content, 223 Lang and double crystal topographs,

techniques, 28, 55

Lanthionine, 193 Laser beam, folded, 256

microprobe analysis, 30 Raman spectroscopy, 257, 258

Lathyrus factors in certain legumes,

phytochemical substances, 202

Lattice parameter, 43 Layered colunm of immobilized enzymes,

171 LDH, 144

Lead-containing particles, 276

Lead-contaminated

air, 163

crops, 163 Lead, 89, 163, 235, 288, 290, 292, 305, 308,

325,337,338,342,349,352

concentrations in natural and waste

waters, 351

health effects of environment contamination by, 79

origin determination by particle size, 290 Lean meats, 192

Least-cost basis, formulation of mixed feeds

by, 168

Legal and administrative implications, water

pollution control, 374

Leptines, solanine and chaconine, 202

Lidar-type system, 258, 273 Liebermann-Burchard color reaction, cholesterol determination, 192

Life Sciences, National Academy of Sciences report, 86

Light

reflected from complex surfaces, 195

scatter measurements, 337

Ligninin-sulfonates, optical measurements

for fluorescence, 321

Lignins, herbicides biological systems, 182, 212

Lignocellulosics, 182

Lime-sulfide unhairing of hides, tanning in- dustry, sulfur-containing compounds,

193

Limiting amino acids, 167, 169

Limit of detection, analytical method, 327 LiNbOs, broad linewidth ^^Nb nuclear magnetic resonance, 39

Lindane, 306

Linewidth in gallium phosphide single

crystals, 41

Linolenic acid, 173

determination of, 175

Lipase, 97, 143

Lipid systems, 177

type and amount of, 174

Lipid-containing products, test for shelf life of, 176

Lipids

agricultural research, 172

compositional changes in, 175

fatty acids, rapid measurement, 172

Lipoprotein, 180

electrophoresis, 98

Liquid chromatography, 153

high pressure, 214

Liquid fumigants, testing of, 2 1

5

Liquid-nitrogen preservation of organism, 184 LiTa03,42

Lithium, 89, 98, 308

niobate, parametric material, stoi- chiometry, 37

Live animals

antipyrine method for estimating fatness

in, 190

composition of, 1 90
452 INDEX

Livestock and poultry industry, 197

Living

organisms, oceanic system, 394

tissue, critical phase of analysis is extraction of dormancy factors from the, 207 Li20-Nb203 diagram, 41 Loin muscle, ham muscle, pH measure- ments on, 1 89 Long horizontal tunnels as fractional elutria- tors, 262

Long-lived pollutants, satellites for measuring on global scale, 258

Long-path or optical measurements, 257, 269 Low

angle grain boundaries, mapping, 28

loss glasses, 49

molecular weight hydrocarbons, 277

Low-concentration-level geophysical measurements, 258

Low-fat goods, 192 Luncheon meats, cholesterol content, 191 Lysine, 169

losses due to heating, 169

Lysine-free e-amino content of proteins, 170 M

Macroglobulin, 97 Magnesium, 97, 1 34, 310, 342, 352, 407

analyses of sea water, 407

in blood, fluorescence measurement, 151 Magnesium-chlorinity ratio, 409

Magnetic and ferroelectric materials, solid state, 53 domains, 54 Major constituent, trace analysis, coulometry, 43

Malic dehydrogenase, 1 16 Mammalian toxicity, 2 1

3

Management skills, 107 Manganese, 242, 292, 308, 319, 337, 342,

349,423,428

(filtrable), 305 Manual procedures, 270

Manufacture of starch, 186 Map of germanium single crystal, 25 Mapping

birefringence, grown crystal, 40

decoration, 28 development of, 59, 67

electron probe analysis, 30

low angle grain boundaries, 28 methods, optical, 26

precision lattice parameter techniques, 28

resistivity probing, 25 Schulz technique, 28 Mass

concentration, quantitative measurement, 262

concentration, suspended particulate matter, 262,263

size, chemical composition, analyze parti- cles by, 261 Mass spectrometer chemical ionization, 275

detection of aflatoxin contamination, 206 Mass spectrometry, 148, 235, 240, 277,

363,369,389

analysis of dissolved gases, 412 Mass spectroscopy, 274

Material balance of a stream of wastewater, 317

identity of, 34

Materials

characterization, 20, 70

characterization, definition of, 2

1

electro-optic and second harmonic

generation coefficients, 26

polycyclic organic, 269

science, 20

Matter

particulate, 271

physiological response to, 261

stationary source pollutants associated

with, 269 Measurement

air quality, 250

air pollutants, 219

available methionine, 170

capability, need for in air pollution, 247 coenzyme dependent enzymes, activity

of, 113

fat and moisture in meat, 191 fluorescence for magnesium in blood, 151

gases, 250, 273

halides in sea water, 400

interfacial angles, 34

long-lived pollutants, 258
INDEX 453

Measurement — Continued

odor causing matter in water samples, gas chromatographic procedures, 334

of sulfur dioxide and nitrogen dioxide, 257

stationary source emission, 269

systems, water pollution control, 304,

309,312,316

volatile organics, aldehydes in, 261 Measurements

acid-base and blood gas, 90

dispersive infrared, 277

flame photometer, 89

geophysical, 259 mass composition, 264

mobile source emission, 274

nonurban rural, 259

reflectance or transmittance, 262

reliable, quantitative, 104 Meat

animal disease-causing microorganisms,

198

cholesterol in, 174, 177, 191 composition of, 190 enzymes, 177

monitoring optimum flavor development

in, 195

preservation of, 176

products, 177, 191

qualities, 188,225 wholesomeness of, 188 Mechanisms, kinetic, 260

Medical laboratory

methods and instrumentation, 101, 102,

103, 104

organization and personnel, 96

Medical technologists, 96, 100

Medical triad, research, education and ser- vice, 86

Medically significant values, Bamett, 133 Medicare, 1 19 Melanin, 98 Membrane filters, polystyrene, 266 Membrane electrodes, 3 13, 373

dissolved oxygen electrodes, 413

Mercury, 89, 162, 172,235,331,337

compounds in aquatic environments, 341

content, monitoring of fish for, 162

decontamination device, 163

discharges, phenyl mercury, methylmercury, dimethyl mercury, 339

waste, 172 Metabolic breakdown products, antibiotics, 193

diseases, 84

Metabolically active protein 188 Metabolism, diethylstilbesterol, 192 Metal

analysis for water pollution control, 337

binding ligands, 338 complex formation, 350

concentration, availability of metal

micronutrients, metal species, 302

dynamics in a environmental segment,

339,340

micronutrients, toxicity of metals to biological systems, 338

particulates, aerosol, 339

species, metal concentration, availability of metal micronutrients, 302

Metallic cation, analysis, 23 Metallo-ammonium or amine complexes,

182

Metals, 3 10

aquatic environment, 337,339

trace, 273 Metanephrine test, 98, 143 Meteorological range, 263 Methane

gas chromatographic technique, carbon monoxide, 252

instruments for low-concentration-level geophysical measurements, 258 Methanol chloroform, determination of fat content, 190 Methemoglobin test, 97

Methionine, 169, 170, 171 and cistine, relative nutritive value of

foodstuffs, 167 Method

and personnel interactions, 107 barium chloroanilate, 270

for total serum calcium, 136

nondispersive infrared spectrometric, 270

precision and accuracy standards, 104

sulfate determined by methylthymol blue, 268
454 INDEX

Methods

and instrumentation, clinical chemistry medical laboratory, 104 computers (limited access use), 104

electroanalytical 103

electronic data processing calculators, 104

electrophoresis chromatography, 103

gas chromatographic, 275

hydrolysis of proteins, alkali or enzymatic

methods, 167 manual, 104

protein by refractometry, 103

ring oven, 266

water pollution control, design of measurement systems, 309

Methoxychlor, 306

Methyl

bromide, 163

elaidate, 174

mercaptan, 193 orange alkalinity, 329

parathion, infused into foams, 216

Methylated sulfides, 22

1

Methylation and GLC, nature of fatty acids by, 174 Methylene blue active substances, 305 Methylmercury, dimethyl mercury, phenyl mercury, mercury discharges, 339

Methylmercury-protein complex in muscles, 428

Microbalance, piezoelectric quartz crystal, 264

Microbial growth in food products, 196

Microbilirubin, 97, 144

Microbiological assays, 183, 184

Microchemistry, 98, 144, 227

Microcoulometric or electrolytic conductivity detection for nitrogen compounds

as ammonia, 210

Microelectrode probing to determine strati- graphic distribution of electrolytes within hides, 194

Micronutrients and the toxicity of metals to biological systems, 338

Microorganisms, 339

in foods, 195 Micro pH, 143

Microprobe, electron, 262, 265, 289

Microscopic identification, 265 Microscopy

electron, 288

optical 33, 199, 288

scanning electron, 69, 288

Milk

casein, 196

concentrates, 196

of cows, fed aflatoxin-contaminated meal, 206

Mini-gasimeter, 176 Minor constituents, determination, 46

Mist, particulate, 27

1

Mixed feeds, control of protein content in, 168 Mobile

laboratories, 260

source emission measurements, 274

Mobility in semiconductor crystals, 22, 26 Moderate dislocation-free material, solid

state, 68

Modified Halphen methods, 178 Modulator and parametric oscillator crystals, 37

Moisture

content, 166, 187

gas flow velocity in flue gases, 269

in-stack gases determined gravimetrically,

270

spectrophotometric technique, 191 Molds, growth, water sampling of, 320

Molecular

absorption spectrophotometry, 344

fluorescence, spectrophotometry, 344

properties of natural polymers, 223

weight in the 1000 to 15,000 range, deter- mination of, 225 Molybdenum, 242, 308, 337, 338

Monitoring

air pollution, 248

ambient air, 250

capability, air quality standards, 259

needs, ambient air, 250

networks, 25

1

nitric oxide, ambient, air, 255

nutrient concentrations in Lake Erie, 328

offish for mercury content, 162 optimum flavor development in meat,

poultry, heat-processed foods, 195
INDEX 455

Monitoring — Continued

silicates and nitrates in sea water, 328

stack emissions, plumes, 258

stationary source emissions, 248

surface temperature, 166

systems, automatic water quality, 322, 328

toxic elements, 163 Monsanto impactor, 276 Mossbauer spectroscopy, 44 Motor vehicle emission, 247 Movement in the ocean, tracers for physical and geochemical studies of, 422

Multicomponent analysis, samples of unknown history, 2 1

1

Multi-element characterization analyses, 386

Multiple-channel radiometers, 256

Multiple Internal Reflection Spectroscopy

(MIRS),224

Multitoxin methods, 205 Muscle system, composition of, in slaughter

animals, 190 Mycotoxins, 204, 205

subclinical levels, 206

Myocardial infarction, 1 11, 1 12, 117 N

NASA, 258

National Academy of Sciences report entitled, "Life Sciences", 86 Committee for Clinical Laboratory Stan- dards (NCCLS), 119

Renderers' Association, 180

Natural and waste waters, 35

1

dissolved gases in water, 368

growth regulating compounds, 207

linolenate and linoleate in soybean oil, 175

waters and wastewaters, standard and recommended methods for analysis of

water, 3 1

1

waters, metals in, 339

Nature of fatty acids by methylation and GLC,174

NBS — Clinical Interaction Plan, accuracy

in clinical work, 136

Near-infrared spectrometry, 362

Nederbragt's method, 253

Neoplastic diseases, 85 Nephelometer, integrating, 262, 263 Nephelometric colorimetric procedures, 276

Nephelometry, 37 Networks, monitoring, 25

1

Neutron activation analysis, 164, 242, 266,

344,386,427

Nickel, 292, 308, 337

Nitrate

ions by selective electrodes, 367

nitrite, organic nitrogen, 3 1

1

potentiometric membrane electrode, 325

reduction, anaerobic biological populations, 301

Nitrates

in sea water, 328

plus nitrites, 305

total organic nitrogen, 310

Nitric acid, 259

formation, atmospheric pollutant, 260

emitted from coals, 259

Nitric oxide, 275

ambient air, monitoring of, 255

atomic oxygen, reaction of, 254

chemiluminescence method, 253

photooxidation of, 260

reaction with ozone, 255

Nitrides, 24

Nitrites, nitrates, total organic nitrogen,

310,311,329

Nitrogen, 67, 188,311 automated determination of, 1 69 compounds^in urban particulate matter, 290

content of bacterial cells, 171

detector specific for, 2 1

1

dioxide standards, 274

phosphorus and carbon levels, 300

Nitrogen dioxide, 27 1 , 275

colorimetric, Griess-Ilosvay reaction for, 254

correlation spectrometers for, 258

instrumentation for, 25 1 , 277 measured with nondispersive ultraviolet analyzer, 277

monitoring capability for, 259
456 INDEX

Nitrogen oxides, 22 1 , 269, 275

colorimetric methods used for, 277

in diesel emissions, 277

standards, 274 NMR, (see Nuclear Magnetic Resonance)

Noncholesterol sterols, 192 Nonconjugatable dienes and conjugatable

dienes in triglyceride esters, determination of, 175 Nondestructive

analysis of plant constituents, 187

dyes, use in agricultural science research 167 method for determination of fatty acid in oilseeds, 173 methods of extraction, 208

test for flat sour spoilage in canned

products, 197 Nondispersive infrared analyzers, 225 ,271, 274, 275

instruments, investigations for carbon monoxide and nitric oxide, 255, 275

method, 258, 270

Nonfat dry milk, 196 Nonfood use of farm products, 185 Nonlinear optical materials, solid state research development, 37 Nonmethane hydrocarbons, 260, 261 Nonpolar lipids, 177

Nonsterile canned meats, 1 89

Nonvolatile

dicarbonyl, 177

fatty acids, 268

Non-congruent melting, 38 Non-human

enzymes, 124

serum, alkaline phosphatase, 124 Non-protein nitrogen, 97

Non-specific water quality parameters

chemical, 310

physical, 310

physiological, 310 Noxious weeds which pollute the environ- ment, 207

Nuclear magnetic resonance, 39, 148, 224, 388

studies, determination of minor constituents, 48

Nuclear quadrupole resonance, 44

Nucleotides, 196

Nutrient

concentrations in Lake Erie, 328 demand, tests used for pollution of, 3 1

1

Nutritional

availability of proteins and amino acids, 169

diseases, 84

value of grain products, 186

values of ingredients, formulation of mixed feeds by least-cost basis, 168 o

Objectives of analysis, water pollution control, 302

Obsolescence, technological, 107 Oceanic system, 394

Oceanographic Institution, Woods Hole, 422

Oceanography, 388, 393

historical review, 395

ionic ratios, 401

tracers for physical and geochemical stu- dies of, 422

Oceans, dissolved oxygen content, 394, 412

Ochratoxin, zearalenone and aflatoxin in com, 205 Odor, 305, 3 10, 332, 333

causing matter in water samples, measurement of by gas chromatographic

procedures, 334

characteristics, 333

diesel exhaust emissions, 277

domestic water supplies, 305

panels, subjective, 195 smoke, automobile exhaust emission

regulation, 278

types of, 333

Oesophageal-pharyngeal fluids, blood, serum, serological diagnosis, 197

Oilcholesterol in, 174

fluorescence, chlorophyll, phenol,

ligninin-sulfonates, Rodamine WT, 321

infrared reflectance, 178, 187 meals and cyclopropenoid material present in, 178

moisture and protein in soybeans, 187, 191
INDEX 457

Oil— Condnued

Pollution Act, 375

spectrophotometric technique, 178, 191

spills, detection of, 32 1 , 322

Oilseeds, 186, 187

nondestructive method for determination of fatty acid in, 173

Olefinic, paraffmic and aromatic hydrocar- bons in vehicle emissions, analysis of, 274

Onion, 211 Opacity of plumes, power plants, 273

Optical

densities, 182

electron microscopy, 265

emission spectroscopy, 67, 266, 267

instruments mounted in aircraft, 258 mapping by scattering of a laser beam, 28 mapping methods 26

measurements, aerial surveillance, photography and remote sensing, 321 measurements, long-path or remote, 257

methods, 343 methods and instruments, clinical chemistry medical laboratory, 102 methods, spectrophotometry, spectrofluorimetry, flame photometry,

atomic absorption, 100

microscopy, 288

techniques, 256

turbidity, 336 Optimum flavor development in meat,

poultry, heat-processed foods, monitoring of, 195 Orchard leaves, 120 Organic

chemicals, domestic water supplies, 305

gases and inorganic gases, vapors, air pollutants, 219

material in water, 223, 354

materials, polycyclic, 269

nitrogen, 311,358

peroxides, 221 phosphate insecticides encapsulated in

plastic, 216

phosphates, 306

phosphorus, 358

pollution, characteristics of, 355

substances in particulate matter, 268

sulfur compounds, hydrogen sulfide, gas chromatographic technique for, 259

sulfur gases, Kraft mill effluents, 272

wastes in effluents determined by BOD,

222

Organisms, stabilized, 185

Organizational chart, clinical chemistry

laboratory, 96, 98, 99, 100 Organophosphorus pesticides, 269

Orsat analysis, 270

Orthophosphates, 311, 329, 365

Oscillographic polarography, 347

Osmolality, 98, 142, 144 Osmometry, 100, 103

Ossification of bones, 188

Oxalic acid in spinach, 202

Oxidant

air pollution, tobacco leaf damaged by, 220

air quality instruments for, 246

analyzers, 252

Oxidation

lipids, 175

phospholipids, 177

products with performic acid, 167

state determination, formal charge, real charge, 44

state, type of species, water quality measurement, 302

Oxidation-reduction equivalents, 182

Oxidative deterioration of lipids, 176

Oxidative rancidity, 176 Oxidized

dimers and cyclic derivatives, 177 pork fat from adipose tissue, 176 Oxygen

in high purity silicon, 23, 67 membrane electrodes, 373

solubility, 3 15 Oysters, cholesterol in, 191 Ozone, 251

instrumentation for,251,253,258

monitoring capability for, 259

reaction of, with nitric oxide, 255 Rhodamine B, reaction with, 25 1 , 253

Ozone-ethylene reaction, 253 P

Paper chromatography, 100, 112, 268, 360
458 INDEX

Papers, kenaf, pulps, 1 82

Paraffinic olefinic, aromatic hydrocarbons,

vehicle, emissions, analysis of, 274

Parameters

for water quality characterization, 305 measured by Technicon CSM, 329

Particle analysis, 265, 289

Particle Atlas, 288

Particles aerosol, 264, 339

airborne particle-size and morphological

analysis, 265 by mass, size, chemical composition, 261, 264

haze, 262

lead-containing, 276

suspended, 262, 268

Particle-size, 289

distribution, 263,264

fractionation, 264

Particulate and gaseous fluorides, 272

Particulate matter, 27 1, 273, 288, 331

analysis, 261

auto exhaust, 276

benzene-soluble, 276

in air, thermal neutron activation analysis, 266

nitrogen compounds, analyses for, 290

organic substances in, 268

physiological response to, 262

stationary source pollutants associated

with, 269

Particulate total suspended matter, 263

Patterns, Debye-Scherrer-Hull, 33

Patulin, mycotoxins, 205, 206 PCB's

asphalts, rubber tires, paints, plastics, 210

industrial pollutant, residue contamination in agricultural products, 210

interference in detection of chlorinated hydrocarbon pesticides, 210

Pea soup, flat sour spoilage, 196

Penicillic acid, 205

Peptide linkages resistant to hydrolysis, 170

Perfection and defects, 49, 6

1

determination of physical structure, 52, 61 Performic acid, oxidation products with, 167

Periodic monitoring, 260

Perloline, 203

Peroxyacetyl nitrate, 260

Personnel, clinical chemistry medical

laboratory, organization and, 96

Perylene, separation of benzo (a) pyrene and benzo (e) pyrene, 268

Pesticide complexes and metabolites, 212

disposal, 21

1

herbicides, insecticides, fungicides, rodenticides, agricultural water use, 309

levels, economical field tests for, 2 1

1

metabolites, 213

residue, 212, 213

residue determinations, 388

residues, confirmation of identify, 209

residues in soil, 213

ultimate fate of, 2 1

4

Pesticides, 242, 268, 306, 309, 311, 364, 385

chlorinated, 269

domestic water supplies, 305

in air, 269

organophosphate, 269

qualitative and quantitative test for, 2 1

1

Petrography, 34

Petroleum refineries, stationary source of

pollutants, 269 pH, 144, 189

acidity and alkalinity, 309

acidity and blood gases, 100 and salt contents of pickled hides, 194

standardization, 125 Pharmacy, 388

Phase

change, heat capacity determination, 36

diagrams, 35

identification, x-ray diffraction, 3

1

Phenalen-l-one,269

Phenol, optical measurements for fluor- escence, 321

Phenols, 189, 268, 310, 311, 321, 329, 331 Phenyl mercury, methylmercury, dimethyl mercury, mercury discharges, 339

Phosphatase

acid, 143

alkaline, 123,143

alkaline, assay for, 126, 127
INDEX 459

Phosphatase — Continued

human, alkaline, standard reference en- zyme material, 124

Phosphate, biological removal of, 385

Phosphates, 134, 144,329,331,366

Phosphatide, determination of phosphorus

in, 183 Phosphide single crystals, linewidth in galli- um, 41 Phosphohexosisomerase, 116

Phospholipids, 177 Phosphorus, 183,242,300,305,413

natural ratio to nitrogen, 307

orthophosphate, polyphosphate and or- ganic phosphorus, 3 1

1

sulfur-containing pesticides and

metabolites, GLC of, 2 1

1

Phosphorylation, herbicides interfere with

electron transport, 2 1

7

Photoelectron spectroscopy, 290

Photography and remote sensing, 321

Photolysis of the N-nitroso linkage, 2 1

8

Photometer, Volz sun, 262

Photomicrographs, Atlas of, 265

Photooxidation of nitric oxide, 260

Photosynthetic organisms, 394

Physical and chemical composition of food, problem in determination of vitamins

in, 183

structure, solid state, 7

1

techniques including optical emission, spectroscopy, 266

water quality characteristics, 33

1

Physicians

communications with, 108

office laboratories, 79

Physicist-instrument designer, 100

Physiologic dual salt phosphate buffer, 125

Physiological age of the slaughter animal, 1 88

characteristics of water quality, 310, 331 response to particulate matter, 262

Phytochemical substances, 202

Phytotoxicity,217

Pickled cattlehides, specification parameters, 183

Piezoelectric tests, 52

Pigeon breast muscle, transaminase en- zymes, 1 12 Pigmenting power, 166

Pigs, stress susceptibility in, 1 89

Pitot tube, 269

Placental estriol test, 98

Plankton, rooted plants, 428

Plant

materials, cellulose and/or lignin contents

of, 181 metabolism, (see plant hormones)

nutrients, 307

Plant-damage characteristics, ethylene, 261

Plant growth

hormones, 207, 208

regulators, 207

Plant hormones

abscisic,207

auxins, 207

cytokinins, 208

ethylene, 208

gibberellins, 208

indoleacetic, 208

indoles, 208

natural growth regulating compounds,

207

plant pigment phytochrome, 207

Plant pigment phytochrome, 207

Plants, herbicides in, 2 1

2

Plasma

bicarbonate, 90

chromatograph, 210

Cortisol, 97

emission spectroscopy, 427

hemoglobin, 97

Plastics 210, 259

dispersed in fats, agricultural science, 1 80 Plumes

monitoring of, 258

stack gases in, 271

Plutonium, distribution of, 137

Polarographic

analysis, 35,43,49, 163,276,347,416

techniques for dissolved oxygen content

of the oceans, 413

Pollutants

agricultural, 219

air, measurement of, 2 1

9

in effluent from the unhairing system, 193

inorganic 260
460 INDEX

Pollutants caused by

agriculture, feedlot, 22

1

food industries, 222

poultry processing plant effluents, 422

Pollution nuisances, tests used for, 3 1

1

Pollution, tests for

specific nutrients, 3 1

1

toxicity, 3 1

1

Pollution problem-solving teams, 298

Polychlorinated biphenyls (PCB), 235

in industry, 210

Polychlorinated triphenyls, 210

Polyethylene, 179

Polyglycerols and other polyols, 178 Polymer composition, surface properties re- lated to, 224

Polymeric membranes, 373

Polymers, 20

Polynuclear aromatic hydrocarbons, 219, 268, 269,

276,277

ring carbonyls, 185 Polyphosphate, organic phosphorus, tests used for 3 1

1

Polystyrene membrane filters, 266

Polysulfide, 193 Polyunsaturated

fatty acids, 177

lipids, 174

Polyunsaturates, 234

Pork

carcasses, 189

pale, soft, exudative PSE, 189

Porphobilinogen, 98

Ports of entry, animal disease-causing

microorganisms, 198

Positive identification, drug analysis, 148, 149

Potassium, 89, 97, 134, 142, 144, 341, 342

Potassium chloride, 144, 120

Potassium dichromate, 120

Potassium dihydrogen phosphate, 120

Potentiometric

glass electrode, 324

measurements, 313 membrane electrodes, 324, 325

techniques, 352

Potentiometry, 100

ion-selective electrodes, 344

Poultry

cholesterol in, 191 heat-processed, 195

industry and livestock, 197

processing plant effluents, 222 Powder patterns, Guinier, 3

1

Power plants opacity of plumes, 273

sources of pollutants, 269

Precipitator, electrostatic, 264

Precision and accuracy, 60, 108, 109, 327, 328

lattice parameter techniques, mapping, 28

required of a referee method, 133 Preconcentration emission spectroscopy, 35

techniques, Ge, Si, GaAs, 45, 46

Pregnanetriol, 98

Preservation of meat and meat products, 176

of sample, 1 82 Primary

odors, 198

protein standard, 1 19 sensor characteristics, 326, 327, 328

Principle of constant proportions, 396

Private clinical laboratories, 109 Procedures

manual, 270

turbidimetric, 268

Production of fatty acids ,180

Promoting substances, 200

Prostatic acid phosphatase, 97

Proteins and amino acids, nutritional availability

of, 169 and lactose, total solids, 191 and related nitrogen compounds, agricultural research, 166

herbicide complex in biological system, 212

Lysine-free €-amino content of, 170

Protein bound iodine, 97, 143

chemistry, 100

content of dairy products, 168

differentiating between vegetable and

animal, 171

digestibility in wheat millfeeds, deter- mination of, 169
INDEX 461

Protein - Continued

electrophoresis units and densitometers, 102

fibrillarsarcoplasmic, 190 methods and instrumentation, 103 moisture oil, measured by spec- trophotometric technique, 191

Proteolytic enzymes, 171 Protons, neutrons, electrons, measurement

of, 167

Public Health Service, 304

surveillance, 321 water supplies, trace metals in, 338 Pulmonary disease, 92

Pulps, papers, kenaf, 182

Pulse and fast scan oscillopolarographic

methods, 163

Purification on

celite columns, 205

silica gel columns, 205

Purified protein derivative tuberculin PPD,

201

Pyroelectric constant, 53

Pyruvate, lactate, biochemical changes

reflecting cardiac muscle damage, 116 Q

Qualitative analysis, 34

Qualitative and quantitative tests for pesti- cides, 21

1

Quality and quantity of water of surface waters, U. S. Geological Survey, 304

Quality

control, solid-state materials, 66, 97

of surface water for irrigation, U.S.

Geological Survey, 304

parameters, domestic water supplies, 305

Quantitative measurements, 33, 104

chemical, 88

of heat-sensitive fatty derivatives. 178 TLC with ultraviolet fluorescence, 21

1

total cholesterol-free and ester-in foods, 191 Quartz, 49, 288

crystal, microbalance, piezoelectric, 264

R

Radiation, tunable infrared, 257

Radioactivation, 67

Radioactive

fallout, 424

waste disposal, 424

Radioactivity, domestic water supplies, 306

Radiochemical separation, 346

Radiometers, multiple-channel, 256

Radionuclides, 309

aquatic life, fish and wildlife, 308 USPH drinking water standards, 309 Radium, 306 Raman scattering, measurement of gases by

use of, 273 Raman spectroscopy, 224, 229

laser, 258

Rancid flavor compounds, 177 Random error, 327 Rapeseed oil, 173 "Rapid Rolodex Files," 108 Reaction

chemiluminescent, 272

nitric oxide with atomic oxygen, 254

nitric oxide with ozone, 255

ozone-ethylene, 253

ozone, Rhodamine B, 169

Reactive organic substances, 261

Real charge and formal charge, 44 Recommended standard and standard methods for analysis of natural waters and wastewaters, 3 1

1

Recording spectrophotometers, 102 Recreation and aesthetics, water quality characterization, 306 Redox potential, 324 Reduced NAD and NAD, 1 13 Reduction methods, 196

Referee methods, 133, 135 development, 132

goals, 134

Reference materials containing enzymes,

evaluation of, 125

Reflectance transmittance measurements, 262

Refractive index, 179 LiNb02,38

Refuse Act, 375
462 INDEX

Regener Method, 253

Regional chemistry laboratories, integral part of the medical community, 1 10 Regulatory laboratories, 234

Relative conductivity of sea water, 401

density, 179

nutritive value of foodstuffs, chemical method for, 167 Remote instrumental techniques, 270

optical instruments mounted in aircraft, 258

optical measurements, long-path, 257

stack instrumentation, 272

Renal calculi, 97 Rendered fats, dispersed plastics in, 179

Repellents or insecticides, uniform sprays,

application or adsorption of, 2 16 "Report of Inter-Society Commission for Heart Disease Resources," 174

Representative sample, sampling natural and wastewaters, water sampling pro- gram, 316

Research, education and service, medical

triad, 86 Research programs, support for, 86

Residues

antibiotics, 192 contamination in agricultural products, 210

in foods and crops, 216, 142

Resistivity

measurements, 25

probing mapping techniques, 25 Resonance linewidth measurements, 40

Reverse osmosis, 359 Rhodamine B, ozone reaction with, 253

Right-angle-scattering instruments, 262

Ring

carbonyls, polynuclear, 268 oven methods, 266, 292 Rodamine WT, optical measurements for fluorescence, 321

Rodenticides, 309 Rubber tires, PCB's in, 210

Rubidium, 341 Ruminant diets, 165 S

Safety in sampling processes, 3 1

8

Safflower, 172, 187

Salicylates, 97, 144

Salinity, 3 10

definition, 397, 398

sea water, 395, 396

Salmonella,

cultural and serological techniques for, 196 enrichment medium for, 196

in dairy products, 196 Salmon, cholesterol content of, 191 Sample

collection, 328,425

handling, 3 19

integrity, 401

preservation of, 3 1

8

Sampler, Anderson, 264

Samplers, tape, 262, 263

Samples, high-volume, 262

Sampling

at great depths, 430

equipment, continuous, 318

errors, 105

for large particles, 262

grab samples, composite samples, 317

intermittent, 270

natural and wastewaters, 316

of a wastewater from pipes, 317

of waters containing dissolved gases and

volatile constituents, 318

point, 257

safety in process of, 3 1

8

sites, water sampling, 317

solid state, 25

technique, 269

Sanitation in food processing plants, 196

Saponification value, 179

Satellites for measuring long-lived pollutants, 258

Saturated fats, dietary cholesterol and, 174 Scanning

analyzer, ion-probe, 66

electron microscopy, 182, 224, 288

Scattering

instruments, 262

of a laser beam, 28 Raman, 273

Schulz technique, mapping, 28

Schulz x-ray pictures, grain boundaries,

twins, 53
INDEX 463

Scintillation counting, 266

Seafood, cholesterol in, 1 9

1

Sea water

analysis, 401

chemical composition, 395, 401

conductivity of, 328, 397

ionic balance, 409

monitoring, 328 measurement of halides, 400

potassium, 407

salinity, 396

samples, salinity, 395

specific gravity, 401 Secondary sensor characteristics, 326, 328

Second-derivative spectrometer, 256 Second harmonic generation

coefficients, materials with large, electrooptic, 26

temperature dependence of, 40

Secretive system, muscle system and circu- latory system, composition of slaughter

animals, 190 Seed dormancy, 208

Seedless fruit, formation of 208

Selective ion electrodes, 103, 325, 331, 366,

367,416

Selectivity coefficient, 327

Selenium, 22, 241, 292, 305, 308

Semiconductor properties, 22, 25, 45

Sensitivity

analytical, 105

limits of common analytical procedures, 344

Sensor systems, need for development of, 331

Separation

of benzo(a) pyrene and benzo(e) pyrene and perylene, 268

methods, 150

techniques, 359

Sequence of individual amino acid residues, 223

Sequential analysis of lipids, 180

Serine, 170, 171

Serological diagnosis, 197 Serum

alkaline phasphatase method, 127

alkaline phosphatase, non-human, 124, 125

blood, clinical analyses of, 192

CK assays, 117

electrophoresis, 97, 143 enzymes, cholesterol, creatinine, 133 enzyme standards in, 125

glutamic-oxaloacetic transaminase (SCOT), 112, 114 ICD isoenzyme of liver origin, 1 1

5

iron, 1 16

nickel, 1 16

total lactic dehydrogenase, (LDH), 1 17 Service

laboratories, 1 19 research and education, medical triad, 86 Sex attractants of insects, 242, 385 SCOT, 144 measurement, 148

transaminase, 97, 1 16, 134, 143 Sheep, antipyrine method for estimating fat- ness in, 190 Shipboard laboratories, 406

Significance of parametric measurements,

ammonia, nitrites, nitrates, 310

calcium and magnesium, 3 10

cyanide, 310

dissolved solids, 310

metals, 310

phenols, 310

sulfides, 3 10

sulfates, 3 10

synthetic detergents, 3 10

total organic nitrogen, 310

Silicates, 366

Silicates and nitrates in sea water, monitoring for, 328

Silicon, 67, 70

Silk, collagen and keratins, fibrous proteins, 224

Silver, 305

Silver-copper wires, 67

Single crystals, 20

Size, mass chemical composition, particles by, 261

Skills, competences in clinical chemistry

laboratory, 100, 101 Skim milk, 192 cholesterol content of, 1 9

1

Slaughter animals, muscle system, circulato- ry system, secretive system, composition of, 19
464 INDEX

Smoke, odor, automobile exhaust emission

regulation, 278

Sodium,89, 134, 142, 144,413

origin determination by particle size, 290

Sodium chloride, 120 Sodium sulfate, 194

Soilpesticides residues, 213

plants, water, herbicides, 212 Solanum chacoense, 212

Solid-state materials, 20

analysis and characterization, 68

bulk quality control, 66

magnetic and ferroelectric materials, 54

materials, domains in a ferroelectric, 54

moderate dislocation-free material, 68

physical structure, 7

1

research and development, nonlinear opti- cal materials, 37

research and electronics, 19, 72

sampling, 25

Solid surface, 152

monitoring, 151

Solvent-extraction, 100, 342

Source emission measurement

mobile, 274

stationary, 269 Soybean

cereal products, vitamins in, 182

products, trypsin inhibitor content of, 203

roots, 213

trypsin inhibitor (STI), 204 Soybean oil, 173

natural linolenate and linoleate in, 175

partially hydrogenated, 175 Spark-source mass spectroscopy, 67, 70, 266,267

Speciation,237,424

Species

characterization, 346

identification, 388

specific antisera, 171

Specification parameters, pickled cattle- hides, 193

Specificity and accuracy, 105

Specific

gravity, sea water, 401

ion electrodes, (see ion-selective elec- trodes)

nutrients, 3 1

1

Spectral absorbance curves, 113

Spectrofluorimetry, 100,269

Spectrography, 67, 267

Spectrometers

correlation, 257

dispersive matched-filter, 256

grating, 256

second-derivative, 256

Spectrometric

method, nondispersive infrared, 270

techniques, 362

Spectrophotofluorometry, 269

Spectrophotometric and sonic methods for determining connective tissue, 191 assay for alkaline phosphatase, 126

equipment, 195 methods, 151 Spectrophotometry, 35, 100, 269, 344, 366

Spectrophotometers, atomic absorption, 89, 266

Spectropolarimetry, 208

simple, small, 102 Spectroscopy

absorption, 44, 101 , 344

correlation, 256

infrared correlation and nondispersive in- frared techniques for carbon monoxide,

25

laser, Raman, 258

optical emission, 67, 202, 266, 344

optical emission, blanks in the filter substrate, 266

photoelectron 290

spark-source mass, 266 Sperm fructose, 98

Spin-flip Raman laser, 257

Split-bum technique, 46

Sprays, 216

Stack

emissions, monitoring of plumes, 258

gases plumes, velocity, 270

remote instrumentation, 272

techniques of measurement for flue gases and vented gases, 269

Standardization

activities, enzyme, 122

efforts in service laboratories, 1 19
INDEX 465

Standardization— Continued

of enzyme assays and reference materials, 123

of ion selective electrodes, 1 2

1

of methods for the estimation of enzyme

activity in biological fluids, 123 problems peculiar to enzymology, 121 Standardized enzyme methods, 126

Standardizing clinical pH measurements,

121 Standard methods of

analysis for water and wastewater, 311, 376

clinical chemistry, 132 Standard reference enzyme material, 124

materials, 67, 119, 120,384,388,422

Standards

agricultural chemical analysis, 234

air quality, 260, 274

clinical analysis, 100, 151 method, precision and accuracy, 104

solid-state, 49

Standard sea water, 397, 406

service, 400, 401

Stationary source emission measurement, 269

emissions, monitoring, 248

requirements, 247

Statistical control methods, 101

Steel, emission spectroscopy, 45 Stem growth, 208

Sterochemical theory of odor, 333

Steroidal alkaloids, 2 1

2

present in edible Solanaceous crops, 202

Steroid analysis, 153

Sterols, 179

Stimulants and inhibitors, plant growth hor- mones, 207

Stoichiometry, 25,40,43

determination, 61

in electronics research and development, 37

lithium niobate, parametric material, 37

strain variations, impurity, 26

Storability indicators, 187 Stream standards, effluent standards, water

quality criteria, 303

Stress susceptibility in pigs, 189

Stripping voltammetry, 266

Strontium, 410

content of the oceans, 4 1

0

Strontium-90, 306

Structure, physical, determination of, 49

Submicrometer particulates, 258

Substituted uracils, 165

Sucrose, esters of, 178 Sugar esters, herbicide biological systems, 286

Sulfa levels, 97

Sulfate, 305, 329

analysis for, 268

determined by sizing, methylthymol blue method, 268

reduction, nitrate reduction, anaerobic

biological populations, 301

Sulfide, 193,310,311,325

content in effluent from the unhairing

system, 193

Sulfuricacid, 192, 194,288

Sulfur-containing essential amino acids, 167

Sulfur and phosphorus containing pesticides and metabolites, method for, 2 1

1

Sulfuric acid, total particulate sulfate, 264

Sulfur, 165,242

flame photometric detector for measure- ment of compounds of, 253

lead, nitrogen and nitrogen compounds in, 290

method for phorphorus containing pesti- cides and metabolites, 2 1

1

Sulfur dioxide , 257, 258, 259, 270, 271,

171,173

analyzer, 254

Sulfur oxides

instrumentation for, 25

1

stationary source pollutants associated with particulate matter, 269

Sulkowitz, 97

Sunflower, 189 Super conductors, 20

Support for research programs, 86

Surface

tension, 331

wetting behavior, 225

Surfactants, 364 Suspended

matter, 309, 3 10, 321

particles, 262
466 INDEX

Suspended — Continued

particulate matter, mass concentration,

263,264

Sweat test, 97, 142 Symmetry determination, 52

Synthetic

antigens, 200

detergents, 3 10 polymers, degradation-products for, 240

Systems

approach to research, 236

Lidar, 273 T

Tall fescue grass Festuca arundinacea, 203 Tanning

industry, 193

leather, glutaraldehyde, 1 94 Tape samplers, 262

Taste, 305, 3 10, 332, 333

Taste-panel evaluation of food products, 194 Taxophane, 306

Teaching of clinical analytical chemists, 150

Technical, chemical, engineering and management skills, development of, 107 Technicon AutoAnalyzer, 101 , 323, 328

Techniques

chromatographic, 4 1

2

colorimetric and chromatographic, 246, 260

electrooptical, 273

filters, 270

gas chromatographic, 246, 259

long-path, 269 measurement of flue gases, vented gases,

stacks, 269

optical, 255, 271

particle-sizing collection, 264

remote instrumental, 270

resistivity probing, mapping, 25

sampling, 269

spectroscopy, nondispersive infrared, 258

wet chemical, 266

x-ray fluorescence, 265

Technological

obsolescence, 107

shock, 107 Technology control, 269

Temperature effects, 309, 328 Temperature of processing, determination

of, 189

Testing of liquid fumigants, 2 1

5

Tests used for pollution of natural water

nuisances, 3 1

1

nutrient demand, 311

specific nutrients, 3 1

1

toxicity, 3 1

1

Test for shelf life of lipid-containing

products, 176

Tetanus, 200

Tetra alkyl lead, 221 Texture of Longissimus dorsi, 188 Thermal

neutron activation analysis, 266 power plants, stationary source of pollutants, 269

stratification, 167, 301 Thermalgravimetric analysis (TGA), 36 Thermocline barrier, 301 Thermodynamic activity of water, 1 65 Thin film, solid state materials, 20

Thin-layer chromatography, 100, 102, 148, 268,269,360

Thioglucosides in crucifers, 202

Thiosulfate, 193 Three electrode voltametric membrane elec- trode, 374

Threonine, 170, 171 Threshold odor number, 334 Thymol, 142

blue alkalinity, 329

turbidity, 97

Time-activity pattern of serum glutamic-oxaloacetic transaminase (SGOT), 1 14 Tissue lead, 98

Titrimetry, 100 TLC, 179,206,208,218

Tobacco leaf, damage to, 2 1 9, 220

Tolerable analytical variability, Cotlove, et

a/., 133 Tolerance limits and water quality stan- dards, 338 Tomatin in tomatoes, 202 Tonks allowable error, 133 Topographical analysis, 66
INDEX 467

Total

carbon, 3 10, 3 11, 358

cholesterol-fice and ester, in foods, quantitative measurement of, 191 dissolved solids ,305,308

lipids, 192

organic content of water, 357

organic nitrogen, 310

protein, 97, 134, 144 serum calcium by AAS, 135, 136

solids, proteins, and lactose, 191 Toxicity of metals to biological systems, 338

tests used for 3 1

1

Toxic

alkaloids, 203 compounds, 177

elements, chemical species of, 424

elements in food, 235

levels in trace elements, 423

matter, 307

metabolites, 204

Toxicology, 100

Toxins, 200, 205

Toxoids, 199 Trace amounts of volatile organic compounds,

240

analysis, 43, 266

analysis at the ppb level and below, 61 contaminants in pesticides, 210

metal concentrations, natural and waste

waters, 351

metals, 273, 331, 337

metals in water supplies, 338

minerals, 182

organic matter, 33

1

Trace elements, 241

biochemical form of, 423

concentration levels in sea water, 413, 414

growth stimulants, nitrogen, phosphorus,

vitamins, plant nutrients, 307

in foodstuffs, 242

neutron activation analysis, 413

technology, 426

tolerance for in irrigation waters, agricultural water use, 308 Transaminase

acute myocardial infarction, 1 1

1

enzymes, 1 12 Tranducers, electrochemical, 27

1

Transient intermediary metabolites, 196

Transition metals, 428

Transimission electron microscope, 69, 288

Transmittance measurements, 262

Transphosphorylating buffers, 1 3

1

Transport mechanisms across cell membranes, 181 Traps, 26,47 Treatment and control, water, 304

Tree

characteristics, determination of, 208

physiology, 208

Tremorgens, 205

Triene, 175

Trifluoroacetic acid, 269

Triglycerides, 98, 143, 175, 179

autoxidation of, 177 Tris(hydroxymethyl)aminomethane, 118

Tris-tris HCl buffer, standardizing clinical pH measurements, 1 19 Trypsin inhibitor, content of soybean

products, 203

Tryptophan, 167, 170, 171

acid-stable derivative of, 168

Tuberculin, allergins, 200

Tunable

dye lasers, 154

infrared radiation, 257 Tungsten-molybdenum alloy, 67

Turbidimetric procedure, 267

Turbidity, 262, 305, 306, 329, 336

domestic water supplies, 305

Twins, Schulz x-ray pictures, grain boundaries, 53 Type

and amount of lipid, 1 74

of monitoring systems, 328 Types of

foods, diversity in physical and chemical

composition, 183

odor, 333

tests performed, future growth trends, volume of tests performed, 95

species, water quality measurement, 302

Typical time-activity patterns, alterations from, 117

Tyrosine, 170
468 INDEX

u

Ultimate fate of pesticide, 214

Ultrafiltration, 199

Ultramicrochemistry, 100

Ultraviolet analyzers, 27 1 ,277 assay for SCOT, 113

transmission, measurement of gases by

use of, 273 Uncreamed cottage cheese, cholesterol content of, 191 Unsaturated

acids, 186

fatty acids, 176 Uranyl ion, 305 Urban

measurements, 259

particulate matter, nitrogen compounds

in, 290

Urea, 97,120,142,168

adduction, 177

nitrogen, 158

Uric acid, 97, 120, 142, 158 Urine

electrophoresis, 98, 143

lead, 98

sugar, TLC, 97

Urobilinogen test, 98 Uroporphyrin test, 98

U.S.Corps of Engineers, 375

Geological Survey, water quality data, 304 USPH water standards, 309

UV-spectrometry, 362V

Vacuum fusion, determination of minor constituents, 49 Vacuum spark mass spectroscopy, 23

determination of minor constituents, 46

Valine, 170, 171 Vanadium, 308

origin determination by particle size, 290

Vapors, instrumentation for measuring at- mospheric gases, 250

Varieties of grain, 186

Vegetable and animal proteins, differentiat- ing between, 171 Vegetable oil, 173

refining and processing, computer control

of, 228

Vehicle emissions, 274

Velocity of stack gas, 270

Vented gases, techniques of measurement

for, 269

Veterinarian, 197

Veterinary biologicals, 198, 199,200

Viable bacteria, estimation of total, 196

Viral products 199

Viscosity, 182

nondestructive test for flat sour spoilage

in canned products, 197

Visual color observation, 44

Vitamins

chemical methods for, 183

hydrolysis procedures for 183

in soybean and cereal products, 182 Vitamin B12, 183 Vitamin 36,183,184

VMA,98, 143

Volatile matter, radioactivity, 331

organics, aldehydes in, measurement of, 261

organic compounds, trace amounts of, 240

Voltammetric membrane electrodes, 324

Voliammetry, stripping, 266

Volz sun photometer, 262 w

Warburg

apparatus, 176

respiration technique, 364 Wastewater

dissolved gases in, 368 from pipes, sampling of, 3 17

material balance of a stream of, 3 17 standard and recommended methods for analysis of natural waters and, 3 1

1

Waste mercury, 172
INDEX 469

Water

containing dissolved gases and volatile constituents, sampling of, 3 1

8

content, 165

hardness, 341

soluble fluorides, 259, 272 thermodynamic activity of, 165

treatment and control, 304 Water pollution control

analysis for metals, 737

analysis for organic matter, 354

analytical methodology, 33

1

dissolved gases, 367 Water pollution control, design of measure- ment systems,

aquatic environment, 299

intensive versus extensive measurement, 312

parameters for analysis, 304

programs, 374

objectives of analysis, 302

sampling, 316 Water quality surveillance programs, 320

aerial, 321 automated systems, 322

manual, 320

public, 321 Water Quality Act, 303 Water quality

aquatic life, fish and wildlife, 306, 307, 308

characteristics, physical, 331

characterization, agricultural water use, 308,309

criteria, stream standards, effluent stan- dards, 303

data. Public Heahh Service, Federal Water Pollution Control Administration, U. S. Geological Survey, 304

domestic water supplies, 305 measurement, oxidation state, type of

species, 302

monitoring systems, automatic, 322

parameters, non-specific, 305, 309, 3 10

physiological characteristics of, 322

recreation and aesthetics, 306

standards, 375

standards, tolerance limits and, 338 Water recycling, 387 Water samples

changes due to biological activity, 320

in situ analyses, 320

handling of, 320 Water sampling, sites, 3 1

7

Water sorption isotherm, 165 Water supplies. Public, trace metals in, 338 Water surface, photography and remote

sensing, 321 Water use, agricultural, 308, 309

Well water, 21 1,212

Wet-milling operation in miniature, 186 Wet chemical techniques, 266

Wheat, 205 Wheat millfeeds, 169 Whey, 196 White blood count, biological changes

reflecting cardiac muscle damage, 116 Wholesomeness of meat, 188 Wide-band NMR, 182

Wide-line NMR, 186

nuclear magnetic resonance (NMR), 173 Winkler

method, dissolved oxygen, 410, 414

test,313, 315, 369,370,412

Woods Hole Oceanographic Institution, 422 Wool keratin, relating surface properties to polymer composition of, 224 X

Xanthophy11 pigments, 166

Xylose, 98

X-irradiation, 3

1

X-ray

analysis, electron microprobe, transmission electron microscopy, scanning

electron microscopy, 1 27

hydrostatic density, 41

crystallographic analyses, 150

studies, 28

techniques, 55 X-ray diffraction, 224, 265, 289

determination of structure, 52

phase identification, 3

1

X-ray fluorescence, 267

strontium content of the oceans, 410

techniques, 35 , 1 64, 265
470 INDEX

X-ray fluorometry, 266

X-rays, 190 Z

Zearalenone in com, 205, 206

Zinc, 89, 242, 292, 308, 33 1 , 337, 338, 341, 349

and lead, atmospheric cadmium, 80

concentrations in the ocean, 413

sampling handling, 319

Zoning, 22

U. S. GOVERNMENT PRINTING OFFICE : 1972 O - LT - 428-999
FORM NBS-IKA (1-71) U.S. DEPT. OF COMM. 1 . PU BLIC AT ION OR RE PORT NO. 2. Gov't Accession BIBLIOGRAPHIC DATA NBS-SP-351

3. Recipient's Accession No.

4. TITLE AND SUBTITLE ANALYTICAL CHEMISTRY: KEY TO PROGRESS

ON NATIONAL PROBLEMS

5. Publication Date August 1972

6. Perfocmiog Organization Code

7. AUTHOR(S) W. Wayne Meinke and John K. Taylor, Editors

8. Performing Organization

9. PERFORMING ORGANIZATION NAME AND ADDRESS NATIONAL BUREAU OF STANDARDS DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Taslc/Work Unit No.

11. Contract/Grant No.

12. Sponsoring Organization Name and Address Same as number 9.

13. Type of Report & Period Covered

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant bibliography or literature survey, mention it here.) This book is the formal report of the proceedings of the 1971 Summer Symposium in Analytical Chemistry held at the National

Bureau of Standards, June 16-18, 1971, and cosponsored by the Analytical Chemistry Division of NBS , ANALYTICAL CHEMISTRY, and

the American Chemical Society's Division of Analytical Chemistry.

It contains six invited papers by subject matter experts comprehensively reviewing urgent research problems for which advanced

analytical techniques need to be developed in the important areas

of agriculture, air pollution, clinical chemistry and biomedicine,

oceanography, solid state research and electronics, and water

pollution. Summaries of related discussions by leading analytical

chemists serving as panel members or in attendance at the Symposium

are included. Hundreds of important analytical problems retarding

progress in these important areas are described and extensive

references are given to permit a deeper insight into the problem

areas. Accordingly this volume should not only stimulate interest

in important problems but should provide a valuable guide for highly relevant analytical research for some time to come.

17. KEY WORDS (Alphabetical order, separated by semicolons) AgriCUltUre^ air pollutionj

analytical chemistry; biomedicine; clinical chemistry; electronics;

oceanography; solid state; water pollution.

18. AVAILABILITY STATEMENT 19. SECURITY CLASS

(THIS REPORT)

21. NO. OF PAGES Pn UNLIMITED. UNCL ASSIFIED 481

1 1 FOR OFFIQAL DISTRIBUTION. DO NOT RELEASE

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20. SECURITY CLASS

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22. Price

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