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Separation and Purification

Section:

Summary of Activities

July 1969 to June 1970

HONAL BUREAU ® STAT^A^SS

UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

NATIONAL BUREAU OF STANDARDS • Lewis M. Branscomb, Director

}-

NBS

-£

TECHNICAL NOTE 549

ISSUED JANUARY 1971

Nat. Bur. Stand. (U.S.), Tech. Note 549, 86 pages (Jan. 1971) CODEN; NBTNA

Separation and Purification

Section:

Summary of Activities - July 1969 to June 1970

David H. Freeman and Walter L. Zielinski, Jr. Editors

Separation and Purification Section

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

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NBS Technical Notes are designed to supplement the Bureau's regular publications program. They provide a means tor making available scientific data that are of transient or limited interest. Technical Notes may be

listed or referred to in the open literature.

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

(Order by SD Catalog No. C 13.46:549). Price 75 cents.

FOREWORD

The Analytical Chemistry Division was established as a separate division at the National Bureau of Standards on

September 1, 1963 3 and became part of the Institute for

Materials Research in the February 1, 1964, reorganization.

It consists at present of nine sections and 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, and in turn the

sections which they comprise, are charged with research at the

forefront of analysis as well as awareness of the practical

sample, be it standard reference material or service analysis.

In addition it is their responsibility to inform others of

their efforts.

Formal publication in scientific periodicals is a highly

important output of our laboratories. In addition, however,

it has been our experience that informal, annual summaries of

progress describing efforts of the past year can be very valuable in disseminating information about our programs. A word

is perhaps in order about the philosophy of these yearly

progress reports. In any research program a large amount of

information is obtained and techniques developed which never

find their way into the literature. This includes the "negative

results" which are so disappointing and unspectacular but which

can often save others considerable work. Of importance also

are the numerous small items which are often explored in a few

days and which are not important enough to warrant publication

yet can be of great interest and use to specialists in a given

area. Finally, there are the experimental techniques and

procedures, the designs and modifications of equipment, etc.,

which often require months to perfect and yet all too often

must be covered in only a line or two of a journal article.

Thus our progress reports endeavor to present this

information which we have struggled to obtain and which we

iii
feel might be of some help to others. Certain areas which it

appears will not be treated fully in regular publications are

considered in some detail here. Other results which are being

written up for publication in the journal literature are covered

.in a much more abbreviated form.

At the National Bureau of Standards, publications such as

these fit logically into the category of a Technical Note.

We plan to issue these summaries for all of our sections.

The following is the fourth annual report on progress of the

Separation and Purification Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

IV
PREFACE

This is the fourth annual report on the activities of the

eparation and Purification Section. The work of the Section

is devoted to those problems of science and measurement in the

separations field that seem most significant in terms of the

larger goals of the National Bureau of Standards. Energy is

divided between the long and short range, the basic and the

applied, and between the abstract need for the related

supportive knowledge and a more direct need for constructive

accomplishment

Three areas of interest have dominated the past year.

First, particle metrology has received attention in reply to

the growing awareness of need. The rules for characterizing

a particulate sample are incomplete and this section's

experience with ion exchange microstandards is suitable for

refocussing into a more general context. Second, liquid

chromatography is receiving a general and strong resurgence

of interest because of the recognition that recent instrumental

developments have opened vast fields for extended interest and

utility. This is somewhat of a difficult area experimentally

and the section is dedicating part of its effort toward creating

simplifications for others to use. Third, there is a widespread

and basic need for improved guidelines on the principles and

practices that should be used in preparing, characterizing and

containing chemical reagents of high quality. To illustrate,

the current general interest in supplying quality organic

chemical reagents is, at best, without pride. Some authorities

seem to encourage a lack of common sense in carrying out

elementary practices in these areas. Common to all of the above

is our constant finding of many problems for concern in the

separation and purification field and there is a growing

significance attached to the need to be as alert as possible

in trying to channel our energies as effectively as possible.

v
It has been necessary occasionally to identify commercial

materials and equipment in this report. In no case does such

identification imply recommendation or endorsement by the

National Bureau of Standards, nor does it imply that the

material or equipment identified is necessarily the best

available for the purpose.

David H. Freeman, Chief

Separation & Purification Section

vi
TABLE OF CONTENTS

PAGE

CHEMICAL MICROSTANDARDS 1

A. Introduction 1

B. Nanogram Standards 2

C. Microgram Standards 2

D. Thermal Stability 4

E. Humidity Effects upon Ion Exchange

Bead Diameters 4 P. Zeolites 8

ION EXCHANGE NETWORKS 11

A. Capacity Measurements 11

B. Network Characterization 14

MICROSCOPY 17

A. Quartz Spheres 17

B. Fine Test Dust 19

C. Cleaning of Microscope Slides 23

CHROMATOGRAPHY 2 5

A. Gas Chromatography of Aged Divinylbenzene ... 25

B. Liquid Chromatography 25

1. Liquid Chromatography Design 26

2. Steric Exclusion Chromatography

Selectivity 29

3. Liquid Chromatography Interactions 36

4. Detector Evaluation 37

a. Advantages 38

b. Disadvantages 38

5. Interactive Copolymer Gels 38

C. Standard Reference Materials 39

1. Clinical 39

a. Bilirubin 39

(1) Packing the Column 40

(2) Operational Modifications 40

(a) Column End-Fittings 40

(b) Sample Injection 4l

(c) Pulsation 43

(d) Reference Stream Recycling . . 43

(3) Precautions 43

(4) Bilirubin Chromatography 4 4

(a) Steric Exclusion

Chromatography 44

VII
TABLE OF CONTENTS (Cont'd.)

PAGE

(b) Gas-Liquid

Chromatography 46

(5) Summary 55

b. Glucose 56

2. Other 58

a. Organic Dyes 58

D. Polyaromatic Hydrocarbons 58

HIGH PURITY REAGENTS 60

A. Container Contamination 60

B. Light Scattering Studies 62

C. Pooled Reagents 64

D. Reagent Bank 68

PERSONNEL AND ACTIVITIES 70

A. Personnel Listing 70

B. Talks 70

C. Publications 71

REFERENCES 73

Vlll
LIST OF FIGURES

FIGURE NO. PAGE

1. Counterion stripping of Cr(III)

exchange beads 3

2. Exchange beads for microgram standards 3

3. System for relative humidity studies 5

4. Humidity cell 6

5. Exchange bead volume ratios as a function of

water activity in humidified air 7

6. Photomicrograph of irregular zeolite

particle 8

7. Photomicrograph of a cubic zeolite

particle 9

8. Linear relationship of sodium content as a function of particle volume in cubic

zeolites 10

9. Apparatus for measurement of ion exchanger

capacity 12

10. Infrared spectrum of sulfonated poly

(styrene/DVB) 13

11. Correlation of infrared absorbance to

titrated capacity 14

12. Relative absorbance as a function of DVB

isomer crosslinking in poly (styrene/DVB)

copolymers 15

13. Photomicrograph of quartz spheres taken

from a light microscope 17

14. Electron micrograph of quartz spheres 18

15. Electron microscope and vacuum evaporator .... 19

16

Photomicrograph of fine test dust taken

from a light microscope with bright and dark

field illumination 20

17. Electron micrograph of fine test dust 21

ix
LIST OF FIGURES (Cont'd.)

FIGURE NO. PAGE

18. Histogram of AC test dust 22

19. Cleaning of microscope slides 24

20. Steric exclusion chromatographic system 26

21. Liquid-solid chromatographic system 27

22. Proportional gradient generator 28

23. Gradient profile of n-hexane : chloroform 29

24. Gas chromatogram of mobile phase composition

during gradient generation 30

25. Steric exclusion chromatography of

organic solutes 31

26. Retention as a function of degree of

copolymer crosslinking 33

27. Photomicrograph of nitrated poly ( styrene/

DVB) beads under crossed-polars 34

28. Plate height vs. flow rate for solutes

having zero, partial, and total access to

the gel interior in SEC 35

29. Separation of a multifunctional solute

mixture on oxypropionitrile 37

30. Packing a gel column 41

31. Column teflon end fittings 42

32. Sample injection port 42

33. Pulsation dampening of the reference

stream 44

34. Steric exclusion chromatography of

bilirubin 45

35. Separation of gaussians with 5:95 relative

intensity ratios 47

x
LIST OF FIGURES (Cont'd.)

FIGURE NO. PAGE

36. Separation of gaussians with 10:90 relative

Intensity ratios 49

37. Separation of gaussians with 20:80 relative

intensity ratios 51

38. Detection of monochlorobenzene impurity

by SEC 53

39. Detection of monochlorobenzene impurity

by GLC 54

40. Detection of chloroform impurity by GLC 55

41. Ion exchange chromatography of glucose 57

42. Collection of air borne particles in and

outside a clean room facility 59

43. Nitric acid leaching of teflon containers .... 6l

44. System for steaming out polyethylene

bottles 62

45. Leaching of a contaminant from the wall

of an FEP Teflon container with 3:1 HNOo

HC1 63

46. Storage of ultra pure acids at -40 °C 67

47. Removal of trace impurities from 99-999

percent ammonia gas 68

XI
LIST OF TABLES

TABLE NO. PAGE

1. Capacity values of exchangers having

varied degrees of sulfonation 13

2. Analysis of aged divinylbenzene by GLC 25

3. Retention behavior of solutes as a function

of degree of copolymer crosslinking 32

4. Mass spectral detection of volatile

impurities in bilirubin samples 5^

5. Removal efficiency of latex spheres by

ultrafiltration 65

6. Removal of particulate matter from

inorganic salt solutions 66

7. Ultra pure reagents dispensed in fiscal

year 1970 69

Xll
SEPARATION AND PURIFICATION SECTION: SUMMARY OF ACTIVITIES

JULY 1969 to JUNE 1970

Edited by David H. Freeman and

Walter L. Zielinski, Jr.

This represents the report of activities for

the Separation and Purification Section for fiscal

year 1970. Studies on the properties of ion

exchange Standard Reference Material microbeads

is extended into the effect of humidity on bead

volume, thermal effects on bead stability, and

measurement of ion exchanger capacity. Zeolitic

particles are evaluated for their potential as

ion exchange microstandards . Preliminary investigations are completed for the quantitative

Infrared measurement of crosslinking in poly

( styrene/divinylbenzene ) copolymers. Light and

electron microscopy are used to help reveal the

heterogeneity in a test dust standard. Progress

is reported in the development and understanding

of analytical liquid chromatography with an

increased direction toward gel chromatography.

The selection of apparatus components , their

performance, and their application to examination

of the clinical Standard Reference Material,

bilirubin, is described. Particle contamination

in inorganic salts and in container walls are

examined, the former by ultrafiltration and

accurate light scattering measurements and the

latter by microscopic methods.

Key words: Air pollution; bilirubin; infrared

analysis; ion exchange; liquid chromatography;

purification; separation; styrene/divinylbenzene;

zeolites

1. CHEMICAL MICROSTANDARDS

A. Introduction

In our last annual report [1] we described the principles,

technique and the associated requirements for the preparation

of single bead ion exchange microstandards containing a known

minute weight of a given metal Ion. In this report we cited

1
the availability of the first "encapsulated" metal ion standard,

that of calcium (SRM 1800), on single cation exchange beads,

containing from nanogram to 10 picograms of calcium with a certified accuracy of within 10 percent. A detailed summary

describing the rationale, preparation and analytical calibration

of ion exchange bead microstandards has recently been published

[2]. The availability of such standards will serve to pique the

interest of investigators requiring small accurate amounts of

a given cation (or cation mixtures) in their research studies.

They have direct application to the calibration of instruments

employed in trace metal ion measurement; viz., neutron activation, the mass spectrometer, and the electron or laser microprobe

.

During the past year, Pb(II), Cu(II), Li, Cr(III), Na , Ni(III),

Fe(III), Mg(II), Zn(II), and U(IV) have been loaded onto ion

exchange beads. The difficulties associated with Hg(II) loaded

beads will be cited below in Section I.D.

B . Nanogram Standards

We had previously described the problems involved in

obtaining quantitative desorption of certain multivalent cations

(eg. Cr(III)) from cation exchange beads [1]. The difficulties

related to Cr(III) stripping have been resolved in the past

year. The details of these investigations have been submitted

for publication. In brief, it was found that Cr(III) is

removed from loaded beads with 98 percent recovery as chromate

in 30 minutes using a 1:4 mixture of IN NaOH:50% H

?

at pH 7-2

C. This result is compared in Figure 1 with three other

less effective procedures.

Microgram Standards

In order to extend the cation weight range to the microgram region, homogeneous, spherical ion exchange beads having

icle diameters of 100-200 ym need be employed (Figure 2).

beads are currently undergoing characterization to provide
1—I—I—

I—

-

10 15 TIME (hrs.)

Figure 1. Counterion stripping of Cr(III)

exchange beads.

Figure 2. Exchange beads for microgram standards

3
a working matrix for lead microstandards in the range of one

microgram and smaller.

D. Thermal Stability

The thermal stability of several batches of ion exchange

beads loaded with different cations was examined in vacuo

(10~ ym Hg) from 40-360 °C, using the solid probe of a quadrupole mass spectrometer. The only loss of metal ion occurred

with Hg(II) loaded beads. Here, traces of Hg vapor were

detected in the ion chamber as low as 60 °C, increasing

significantly from 80-360 °C. Similar effects were noted for

Hg(II) loaded zeolite particles.

E. Humidity Effects upon Ion Exchange Bead Diameters

The dependence of the swollen volume of an ion exchange

resin upon humidity is believed to be a thermodynamic property

and, therefore, path independent. The testing of this

hypothesis has not been thoroughly examined beyond the initial

studies of Boyd and Soldano [3] who first established experimental consistency with isopiestic water vapor pressure

equilibrations of various ion exchangers . At water vapor

pressures corresponding to water activities of less than 0.2,

slower swelling or deswelling rates are expected. In addition,

there is a possibility that repeated swelling cycles towards

higher humidities could be influenced by resin structural

changes . The experimental approach used in the present effort is

based upon the utility of microscopy to measure the diameters

individual ion exchange beads, and the use of recirculated

is bubbled through aqueous glycerol media in order to

the humidity level. The constant temperature humidification apparatus is shown in Figure 3. The microscope slide

is simply a conventional cell that is sealed with the

inside, and with ports at opposite lateral ends to

permit entry and exit of the circulated vapor. The air pump
Figure 3. System for relative humidity studies.

is simply that used in an ordinary fish tank. The air follows

the simple circle: pump/glycerol-water/cell . The cell is

shown in Figure 4

A constant procedure was followed with sodium form cation

exchanger beads (8 percent DVB) that were being independently

studied for their potential value as microstandards having

counterion mass up to one microgram. The humidification

procedure involved placing five or six beads in the cell,

cementing on a top cover slip, and allowing the humidified air

to circulate for a period of at least 24 hours.

5
Figure 4. Humidity cell.

The result of six repeated swelling cycles between water

activities of 0.5 and 0.1 showed volume ratios (swollen/

unswollen beads) of 1.139 ± 0.002 where the indicated standard

deviation for six beads (69 bead diameter measurements, in toto ) is about that which would be obtained due to measurement error

alone. In a similar experiment, the cycling was between 0.8

and zero water activity, the latter being obtained after careful

vacuum oven drying at 130 °C overnight. A larger variation was

found here in the volume ratio 1.499 ± 0.019 for five beads and

six swelling cycles (55 diameter measurements). The error

increase seems to be associated with measurement of the state

at increased humidities. To illustrate, the relative standard

deviation of reproducing a bead volume at water activity 0.8

was significantly higher (value of 0.007) than the value at a lower water activity (which ranged from 0.003 to 0.005). It

is possible that the highest water activity of 0.8 involved

coating the bead with a water film, which would be consistent

th the observations of an apparently higher surface reflectivity (seen but not measured).

6
Finally, six beads were taken through an excursion from

the dry state along the series of water activity: viz : 0.1,

0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.6, 0.4, 0.2, and 0.1.

Reproducibility was consistently apparent with relative

standard deviations that did not exceed 0.6 percent, except

for one point at . 4 activity. The averaged results are shown

in Figure 5. The end point at unit water activity was obtained

by measuring the usual swelling ratio in water (total immersion)

It is noteworthy that the results of low water activities do

show a trend headed above an intercept with the zeros origin.

Figure 5. Exchange bead volume ratios as a function

of water activity in humidified air.
This agrees with the corresponding isopiestic weight measurements reported by Boyd and Soldano [3]-

F . Zeolites

Some of the disadvantages inherent in organic ion exchange

beads, such as thermal instability, or the variation of bead

volume with water content, might be overcome by using inorganic

ion exchange materials. Zeolites are obvious candidates for

study. Their aluminosilicate framework affords a chemically

well-defined structure which retains its external shape independent of water content. The potential drawback to the use

of zeolitic particles in microstandard work is that they often

possess irregular shapes (Figure 6). Certain synthetic

<*s

>

Q O

Figure 6. Photomicrograph of irregular zeolite

particle

.

zeolites, however, are available, at least in part, in regular

geometric forms, and we have obtained octahedral and cubic

Darticles of synthetic fajasite from Dr. Howard Sherry of Mobil

search and Development Corporation, Princeton, New Jersey.

8
The chemical homogeneity of the preferable cubic geometry

was evaluated by neutron activation at the NBS reactor, followed

2k by gamma radiation counting of Na decay and calculation for

sodium content. The particle volume was determined by measurement of at least two sides of each cubic particle using a light

microscope (Figure 7). The precisely linear relationship

between sodium content and volume of single particles is shown

in Figure 8. Hence, it should be possible to use such zeolitic

particles as calibration standards for sodium, directly. This

work is being prepared for publication.

Figure 7. Photomicrograph of a cubic zeolite

particle
Eni-W>O

c

rt Z

Figure 8. Linear relationship of sodium content

as a function of particle volume in cubic

zeolites

.

10
2. ION EXCHANGE NETWORKS

A. Capacity Measurements

The standard procedure employed for the determination of

the capacity (i.e., the concentration of available exchange

sites) of an ion exchanger involves titration of the hydrogen

counterion form of the exchanger with a standardized strong

basic titrant (eg. NaOH). The capacity value (meq/g) is then

obtained from the relationship

r

. me q NaOH

exchanger

where C is the capacity of the dry exchanger expressed in the

sodium form (R~Na ) . Best results are obtained if the exchanger

is allowed to stand overnight in excess base, followed by back

titration in the morning with standardized acid. Precautions

were taken to insure that alkali consumption by reaction with

atmospheric C0„, or with the container walls, was negligible.

The apparatus setup is shown in Figure 9- The precision of

the method was found to be excellent (4.580 ± 0.005 for three

replicates of a highly sulfonated sample). Some capacity values

determined for samples containing varying degrees of sulfonation

are given in Table 1.

Since the capacity of sulfonated poly (styrene/divinylenzene) exchangers is related to the concentration of available

ion exchange sites, and hence to the concentration of sulfonic

acid groups in the exchanger, it is expected that a relationship might exist between the degree of absorption of infrared

light (at a frequency attributable to the -SO ~ group) and the

capacity value, for such exchangers. Such a relationship

does, in fact, exist. Quantitative measurements were made

of the ratios of the depth of the absorbance band at 1408 cm

(attributable to sulfonation), to the depth of the absorbance

band at 1450 cm" (copolymer skeleton). Figure 10 shows the

-1

11
Figure 9. Apparatus for measurement of ion exchanger

capacity

.

position of these bands in the spectrum. The comparison of

the infrared absorption ratio to the titrated capacity is given

in Figure 11.

12
Table 1. Capacity values of exchangers having

varied degrees of sulfonation.

Sample Duration Dur of sulfonation (hours)

type 5

1 0.000

2 0.000

3 2.320

4 2.756

5 3.122

6 3.334

25 125

3.121

3.530

2.399

2.876

3.202

3.490

Samples were sulfonated using varied weight ratios of

nitrobenzeneH„S0k : copolymer

l4l

175

2 515

3 035

3 430

3 652

WAVELENGTH (MICRONS) 6 7 12 15

1. ........ ..1.,...

1 .. 1 ..1 ,1.1.1

3000 2500 2000 1800 1600 , 1400 WAVENUMBER (CM 1 ) 1200 1000 800 600

Figure 10. Infrared spectrum of sulfonated poly

(styrene/DVB)

13
2.0r2 4

CAPACITY (meq/gRNa)

(100% theoretical capacity = 4.8)

Figure 11. Correlation of infrared absorbance to

titrated capacity.

B. Network Characterization

The total characterization of a copolymer matrix requires

a knowledge of the intricacies of the topology of the crosslinked molecular strands, and a knowledge of the chemical

composition composing the copolymer bead. The experimental

measurement of the latter is possible by combined gas chromatography (to determine the molar composition of the divinylbenzene crosslinking reagent) and infrared spectrophotometry

Ik
(to measure the molar concentration of the divinylbenzene

isomers in the copolymer proper). The location of the

vibrational energies in the infrared spectrum for meta- and

para-divinylbenzene crosslinking in copoly ( styrene/DVB) was

reported last year [1]. During the past year, the quantitative

estimation of crosslinking has been worked out [4].

The procedure is based upon the determination of standard

curves for each type of crosslinking using styrene copolymers

prepared separately with the single DVB isomers. The standard

curves plot the absorbance ratio of the DVB isomer absorption

to that of a reference band attributable to the styrene chain,

as a function of DVB isomer concentration (expressed as X./

(1-X.), where X. is the mole fraction of DVB isomer used in the

1

s l

styrene-DVB reagent mixture). The relationships were found to

follow Beer's law linearity through 0.08 X. of either DVB isomer

The standard curves are shown in Figure 12.

A i/Asx

4 5 100 Xj /(l-Xj)

Figure 12. Relative absorbance as a function of DVB

isomer crosslinking in poly ( styrene/DVB)

copolymers

15
This study was extended to the infrared spectra of poly-

( styrene/DVB) prepared with a crude DVB reagent that is commonly

used in the commercial preparation of many copolymer networks.

The method requires the use of the standard curves cited above,

together with a knowledge of the original vinyl monomer composition of the technical DVB mixture. The latter information is

readily obtainable by GLC analysis using a nematogenic liquid

crystal stationary phase [1,5]. With this procedure in hand,

quantitative measurements of the moieties of the meta and para

isomers of ethylvinylbenzene and divinylbenzene in the copolymer

are attainable [4].

16
3. MICROSCOPY

In the past year, the microscopy facility in our Section

became involved in the examination of particles of 1 urn or less

Some of the more important recent undertakings, for example,

include the examination of (1) very fine quartz spheres, (2) a fine test dust sample, and (3) the particle contamination of

cleaned microscope slides.

A. Quartz Spheres

The scrutiny of quartz spheres was undertaken primarily

to obtain Information on their sphericity and size range. The

material was dispersed in cyclohexane by ultrasonic agitation

and then placed on a glass slide. Using light microscopy, the

best results were obtained by a 50X objective, with the sample

exposed to air. The photomicrograph (625X) In Figure 13,

~~H H

10 /im

Figure 13. Photomicrograph of quartz spheres taken

from a light microscope.

17
however, does not yield the desired information. One drop of

the prepared sample was then placed on a f ormvar-carbon coated

electron microscope grid and examined under the electron

microscope. Figure 14 is an electron micrograph (29,000X) of

Figure 14. Electron micrograph of quartz spheres

this material in which the particle diameters measured ranged

from 0.91 ym to O.56 ym. As indicated in this figure, the

sphericity of these particles was found to be excellent . Figure 15 is a photograph of the electron microscope which

allows direct observations to 80,000X and is equipped with

35 mm roll film and 2 1/4 in x 2 1/4 in. plate cameras for

electron micrography. Also shown in Figure 15 is the vacuum

evaporator which is used for carbon coating films on specimen

grids and metal shadowing of carbon replicas of materials that

are not suitable for direct examination in the electron

microscope

.

18
Figure 15. Electron microscope and vacuum evaporator

B. Fine Test Dust

In October of 1969 we received a sample for evaluation

identified as "AC Fine Test Dust." This material has been

considered as a standard particulate contaminant. It is

important, but likewise difficult, to measure trace particulate

contamination of gaseous or liquid systems . New standards are

required for the development of procedures which determine

the effect of the contamination. A prime requirement of any

material standard is chemical and physical homogeneity. Our

evaluation of the material indicated sufficient heterogeneity

so that its potential as a standard is somewhat marginal.

Because of the growing need for this type of standard, the

results of the study are given in the following.

19
The first indication of heterogeneity was provided with

the use of conventional light microscopy. The photomicrographs

in Figure 16 were obtained with the same dispersion of particles

from the test sample. These photomicrographs show a mixture

of opaque and transparent particles. Electron microscopy at

magnifications at 28,700 showed that the particle size range

_2 extended to below 10 urn. See Figure 17.

Figure 16. Photomicrograph of fine test dust taken

from a light microscope with bright and

dark field illumination.

20
Figure 17. Electron micrograph of fine test dust

A study of the particle size distribution was carried out

using both electron and light microscopy with measurements of

particle size (longest dimension) with the help of a Zeiss TGZ-3

particle size analyzer. The results are shown in Figure 18

where a differential histogram shows the apparently bimodal

distribution which peaks at 0.6 and at 2 ym. A very serious

limitation to measurement accuracy is involved in view of the

cross calibration between the light and the electron microscopy

in the overlapping regions in the vicinity of 0.5 to 1 ym.

Although it was not carried out, it should be possible to scale

the relative contributions of the two sized modes using a 5 ym

sieve

21
200 -

100 -

0.01 PARTICLE SIZE l/im)

Figure 18. Histogram of AC test dust.

The density of the AC Test Dust sample was measured in a Linderstrom-Lang density gradient column with the use of

ethylene dibromide and bromoform. The average density of the

sample was near 2.8 g/cm , although this is only a rough

estimate. Approximately 99 percent of the particles float

on bromoform (2.9 g/cm ) . The remainder is composed of black

particles of higher density. These are easily removed by

centrifugation. A magnet placed into contact with the suspended

sample will remove the heavier magnetic particles whose composition includes iron as a major constituent.

A qualitative analysis of AC Fine Test Dust by emission

spectroscopy was carried out for us by Mrs . Virginia Stewart

of the Spectrochemical Analysis Section. Major elements

22
include Al , Ca, Pe, Na, K and Si. The minor elements include

Ag, Ba, Cr, Co, Mg, Mn, Ni , Pb , Sr, Ti, V and Zn.

An x-ray diffraction examination was carried out by

toward E. Swanson of the Crystallography Section at NBS

n he results showed that the sample was composed primarily of

.lpha quartz and orthoclase feldspar (K, Al, Si~Oo) in

ipproximately equal amounts. A small percentage of mica was

ilso noted (perhaps 5 percent). These findings approximate

;he analysis of a powdered alkali granite or rhyolite, without

appreciable chemical weathering.

The conclusion is that this sample is heterogeneous in

several ways. First, there is wide distribution in opacity,

lensity, and composition. The bimodal particle size distri-

)ution creates a serious impediment to the achievement of a leaningful size frequency analysis. The presence of magnetic

(articles represent an obvious opportunity for biased calibra-

;ion of metallic equipment to which the magnetic particles

rould be attracted.

Recommendations for other possible materials that would

offer better properties as a standard would be well guided on

the basis of taking a material with well defined physical and

chemical properties. Various geologically occurring materials

can be prepared synthetically, thus minimizing the problems of

heterogeneity which seem to be associated with the present

sample

C. Cleaning of Microscope Slides

The direct use of microscope slides which have not been

immaculately precleaned is unsuitable for the preparation of

slides of chemical microstandards . The slide cleaning procedure

used by our laboratory is illustrated by the photomicrographs

in Figure 19. Dark-field transmitted light microscopy was used

for the micrographs shown in this figure. Micrographs #1692

(250X) and #1693 (500X) illustrate a contaminated slide that

was supposed to be clean. Micrograph #1709 (500X) shows the

23
Figure 19. Cleaning of microscope slides.

surface of commercially available "precleaned" glass slides,

while #1710 (500X) shows the same slide after application of

cyclohexane followed by evaporation to dryness . Numbers 1711

and 1712 (500X) are two slides which have undergone rigorous

precleaning with boiling HNO~ and copious rinses in distilled

water with samples cast on them by our usual techniques. This

work was carried out in a clean room facility. The lines on

the micrographs are 10 ym spacings of a stage micrometer

obtained by double exposure of the micrograph.

2H
4. CHROMATOGRAPHY

A . Gas Chromatography of Aged Diviny lbenzene

It was of interest to our studies on diviny lbenzene

reagents to ascertain the composition of two samples of metaDVB , stored for over a year in a cold room maintained at 5 °C.

The procedure for the complete resolution of the meta and para

isomers of DVB on a nematogenic liquid crystal stationary

phase has recently been published [5l. The internal standard

procedure was used to determine the level of non-volatile

"polymeric" material present in the sample (eg. [6]). The

results are given in Table 2.

Table 2. Analysis of aged divinylbenzene by GLC

Relative percent in sample

Sample

No . m-EVB p-EVB m-DVB p-DVB naphthalene polymer

10.7

1.1 9.8

Internal standard was n-tetradecane . Column: 0.125" o.d.

by 10' s.s.j packed with 9% 4 ,4 '-dihexoxyazoxybenzene on

100/120 mesh AW-DMCS Chromosorb W (w/w) . Temperatures:

column 110 °C; injection port 180 °C; flame detector

210 °C. Carrier: 39 cm3/min He.

B. Liquid Chromatography

In recent years, a face-lifting process has occurred in

many phases of liquid chromatography. This has been due to

(1) the advancement of theory, (2) the design and development

of high sensitivity and low volume detectors, (3) apparatus

suitable for operation at elevated pressures, and (4) advances

in the development of column packing materials . During the

past year and a half, extensive efforts have been exerted in our

25

1 2.1 86.0 1.2

2 1.8 2.0 81.3 4.0
laboratory in striving for control of the extremely important

(but not always readily applied) discipline of high performance

liquid chromatography. The following sections will discuss

the progress of these efforts.

1. Liquid Chromatography Design . In last year's report

[1], criteria were cited for a desirable design of a liquid

chromatography system. Some components of the design may vary,

depending upon the requirements of the problem at hand.

Attention was given to the optimization of liquid chromatographic

systems for steric exclusion (gel permeation) chromatography.

Such a design in use in our laboratories may be seen in Figure

20. While the system was assembled for steric exclusion

chromatography s it has versatility such that the parameters of

Figure 20. Steric exclusion chromatographic system

26
column length and packing, temperature, flow rate, and type of

detection are easily changed. Certain modifications, such as

low dead volume teflon column end fittings, on-column injection,

pump pulsation dampening, and reference stream recycling were

investigated and incorporated into the SEC system. Chromatograms

illustrating the performance of the system are given in other

sections of this report.

In the past several months, investigation has begun in

liquid-solid chromatography, using silica gel as the stationary

phase, and a gradient mobile phase. When proper choice is made

of the mobile gradient profile and its composition, diverse

separations are possible for this system. A picture of the

chromatographic system for silica gel chromatography is given

in Figure 21, showing the use of the moving wire flame ionizaFigure 21. Liquid-solid chromatographic system.

27
tion detector. A simple gradient generator is shown in Figure

22. A composition-time profile of an n-hexane : chloroform

gradient may be seen in Figure 23. The data for this curve

was obtained by withdrawing aliquots from a side-arm on the

pump outlet with a syringe and analyzing these by gas-liquid

chromatography (GLC) using a Ucon Oil capillary column. A GLC

chromatogram of one of the data points is given in Figure 24.

Increasing the volume of the mixing chamber or using diminished

chloroform: hexane ratios in the solvent reservoir upstream of

the mixing chamber will result in a lower gradient slope.

Although the usual use of mobile phase gradients is from less

polar to more polar phases, the choice and manipulation of

Figure 22. Proportional gradient generator

28
5

QLO

li. O

cco

_lIo

o

2

O

I-o

<

or

a.

0.0

TIME ELAPSED (MINUTES)

Figure 23. Gradient profile of n-hexane : chloroform.

solvents is strictly at the discretion of the experimenter.

Moderated mobile phases (i.e., the addition of 1 percent or

less of a polar solvent to a non-polar mobile phase) after the

work of Maggs [7], Scott, and others is likewise under

investigation.

2. Steric Exclusion Chromatography Selectivity . The equations for resolution received development by Giddings

[8] and were applied to steric exclusion chromatography [9].

A chromatographic system may be made extremely efficient and

capable of eluting solutes having low band-spreading. Such a system however, is useless unless the column packing is capable

of separating the solute bands from one another. Various models

for selectivity are given in the excellent review article by

Altgelt [10]. Much work has been reported on the separation

29
-1

yj —.

. - |

i 2*rX

ru

!IC

• I

!

| ei

I

1

odo I P-, i 1 o i Pi i

i o 1

t -\ \

i X t

1 CJ |

i

j

i

1

i

j

1 j

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i

i

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•i—

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c

>H

1 1 1

" 1 *| ! - T

1 -r~ 1 i TIME

-^i

j1

I - 1

Figure 2k, Gas chromatogram of mobile phase

composition during gradient generation.

30
of high molecular weight polymers in the literature. On the

other hand, our interests have principally been concerned with

the separation of relatively low molecular weight solutes (M=700

and less). An example of steric separation of a solute mixture

of varying molecular weight is shown in Figure 25. Since the

average pore diameter of the gel particle may considerably

influence retention behavior, we investigated the role of DVB

crosslinking in styrene/DVB copolymers and the data is given

RUBRENE

533

BILIRUBIN

585

POLYSTYRENE

180,000

60

MINUTES TIME

Figure 25. Steric exclusion chromatography of

organic solutes.

in Table 3 and plotted in Figure 26. We have developed infrared

spectrophotometric techniques for the qualitative identification

and quantitative estimation of DVB content of styrene/DVB

copolymers, and this is presented in another section in this

report

31
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10,000

8000

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h-o

200

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M. W. REGION OF

SOLUTE EXCLUSION

M. W. REGION OF

FRACTIONAL PERMEATION

M. W. REGION OF

TOTAL PERMEATION

8%

DVB

5.5%

DVB 4%

DVB

1

10 12 14 16 18 ELUTION VOLUME (cm 3

)

20 22 24 26 28

Figure 26. Retention as a function of degree of

copolymer crosslinking

33
It is perhaps unfortunate that this technique lacks

adequate selectivity in the separation of solutes of close

molecular weight (actually, molecular volume). We have

initiated what we feel could become an important development

•in the use of gels for small organic molecule separations.

Principally, this involves the synthesis of styrene/DVB

copolymers containing chemical selectivity. The first such

derivative prepared was a nitro derivative in which nitration

occurred throughout the copolymer matrix. An example of

nitrated beads evidencing strain character is shown under

crossed-polars in Figure 27. Ayres and Mann earlier showed

that nitrated copolymers may be useful in the separation of

Figure 27. Photomicrograph of nitrated poly (styrene/

DVB) beads under crossed-polars.

polyaromatic hydrocarbons [11] via tt-^* charge transfer

complexation. The use of chemically modified copolymers

offering separations based on both solute size and dipole

Interactions are the non-ionic analog of ion exchange beads

34
The development of such copolymers containing other kinds of

functionality is of interest.

Attention was given to the evaluation of band spreading

in steric exclusion chromatogtaphy . Employing small diameter

beads (10-30 urn), the effect of flow rate on solute band

spreading (H) was investigated. Figure 2 8 shows such a plot

H(mm)

RUBRENE

(Partial Access)

BENZENE

(Total Access)

_4 + ^ POLYSTYRENE

(Zero Access

Figure 28.

i i 3 V (cm^/min)

Plate height vs. flow rate for solutes

having zero, partial, and total access

to the gel interior in SEC

35
for an excluded solute (polystyrene 180,000 MW), a solute

that is free to permeate all pores of the gel (benzene) and

is non-selectively retained, and solute of intermediate size

which permeates only a fraction of the pores (rubrene). In

the first case (PS180K), the internal volume of the gel phase

(V. ) is unaccessible and thus V. ., n v = 0. In the

i

y i, accessible

last case (benzene),5 V. .,, = V.. For rubrene,

1, accessible 1

' however, < V. .,, < V., and it is here that flow

1, accessible 1'

rate dependence is important, as can be seen from Figure 28.

Low plate heights are still achievable, but only at low flow

rates and at the cost of analysis time. This can readily be

understood from the plate height equation for steric exclusion

chromatography [9]

H = [4D

m

/3Rv] + [R(l-R)dpV/20D

m] +

Z ["1/O.5 A n d + D /oo.d

2 vl

|_ 1 p mipj

where the factor R(l-R) in the nonequilibrium term approaches

its maximum value of 0.25 for solutes where < V. ., -, 1, accessible

< V., and where H as a function of R(l-R) increases with

increased flow rate (R = the ratio of solute velocity to mobile

phase velocity). At high mobile phase flow rates, v in the

nonequilibrium term (middle term) contributes to increased

values of H.

3 . Liquid Chromatography Interactions . Liquid chromatography using a 2 mm I.D. stainless steel column packed with OPN

(oxypropionitrile ) "brush" particles [12] gave indications that

serious tailing problems can arise for carbonyl-containing

solutes. In situ silylation of the column packing did not

improve matters, and indeed, caused at least partial removal

of the "brushes" as indicated by the appearance of a yellow

colloid in the column effluent. A separation of a solute

mixture is given in Figure 29. This was only obtained at low

mobile phase velocities and with the use of a solvent moderator

36
- • -•

. .....Li

- - l.'.llL

2

; . . .

|

1

/

3

: :

I

\ 4

.inject j

1

....... . 1 L'!.'*. i

i L

'. v

.;l ; li

^F^_ — .

—F--

ZMJ NUTES - ' y •

—F-h —F—-*~ -j ; ~ -4^-

10 15 20

Figure 29. Separation of a multifunctional solute

mixture on oxypropionitrile

of 0.5 percent of methyl ethyl ketone. The relatively more

rapid separations of solute mixtures containing only one type

of functionality is reported on chemically modified pellicular

(solid core) particles [13] in which the diffusion phenomena

associated with porous silica particles is negligible and rapid

solute equilibrium between mobile and stationary phases occurs.

The capacity, however, of pellicular particles for solute

sample size is small.

4. Detector Evaluation . In the past year, we have employed

three different detectors: (a) differential UV absorption at

254 nm, (b) differential refractive index (RI) detection in

organic and aqueous mobile phases, and (c) pyrolysis-flame

ionization detection using the moving wire detector.

37
a. Advantages . The UV detector was the most sensitive

detector and proved to be extremely satisfactory for steric

exclusion chromatography of bilirubin and standard aromatic

_7 solutes. Detection of 10 g of bilirubin is readily accomplished with this detector.

The RI detector has sensitivity limits that vary with

the magnitude of the RI difference between solute and solvent.

The RI detector tends to have the advantage of being less

_5 selective than the UV detector. We could detect 10 g of

bilirubin.

Both the RI and UV detectors which we have utilized have

low volume flow cells in the order of several microliters,

relatively fast response, and high sensitivity for their

relative functions.

The pyrolysis flame ionization detector has a clear

potential for becoming the most useful detector for the liquid

chromatography of organic solutes. It is well suited for

gradient elution techniques.

b. Disadvantages . Both the RI and UV detectors which

we have employed in our investigations are somewhat sensitive

to effluent flow rate changes. The former is the most affected

and is difficult to use with flow and temperature-programming,

and mobile phase gradients. The moving wire flame ionization

detector performs in an excellent manner for each of these

techniques. Its major difficulties however , lie in poor

efficiency of transfer of the effluent mobile phase to the

wire (presently only about 2 percent), and in variations in

the pyrolysis efficiency relative to the solutes in question

(although a solution to this seems to have been provided [14]).

5. Interactive Copolymer Gels . The theory of restricted

diffusion in SEC, while a reasonable concept —and there are

indications by workers using controlled pore glass particles

[15] that such a phenomenon can exist —predicts the pronounced

tailing of solutes and a dependence of solute elution volumes

38
(V ) on flow rate. Minimal or no tailing is usually observed

in SEC, however, excluding adsorption, sample overloading, or

other extraneous effects [16]. The separations phenomena on

styrene-DVB copolymers can primarily be explained on the basis

of steric exclusion. It has been observed that the selectivity

of copolymer gels for molecular size separations of chromatographed solutes is often adequate for polymer mixtures, but

extremely poor for small organic molecules where solutes having

only small molecular weight differences have identical retention

volumes

The introduction of specific functional groups to the

copolymer matrix may broaden the basis for selectivity using

copolymer gels. Being non-ionic, their capability of swelling

in organic solvents will be maintained and an interactive gel

system can be attained. Such non-ionic modification of

silicious particles has resulted in the commercial "brush"

packings. The chemically-modified copolymer gels, however,

avoid the hydrolysis problems associated with silicious "brush"

particles and provide microscopic three-dimensional interaction

for solute molecules entering the gel phase. The first derivative prepared was a nitrated styrene-DVB copolymer in which

nitro groups are attached to the benzene rings of styrene

Optical examination of this derivative preparation under crossed

polars may be seen in Figure 27. The implication here of the

application of copolymer gels containing polar groups which

interact predominantly with permeating solutes offers a separations potential not possible with the unmodified styrene-DVB

gels conventionally used in SEC. Experiments will broaden in

the coming year in both derivative synthesis and chromatography

to ascertain the extent of this implication.

C . Standard Reference Materials

1 . Clinical

a. Bilirubin . In last year's report [1] we indicated

some of the preliminary difficulties associated with bilirubin

39
analysis using liquid chromatography. In the past year,

extensive work has occurred on this problem and substantial

progress has been made. The system ultimately chosen and used

throughout this work has been steric exclusion (gel permeation)

chromatography employing chloroform as the mobile phase and

styrene-DVB copolymer beads (d = 10-30 urn) as the column

packing.

(1) Packing the Column . The column may be packed

with preswollen beads In a chloroform slurry. However, difficulty may be encountered when an upright column is packed from

the top, due to the solvent density being greater than the

swollen gel particles. This results in a portion of the packing

migrating to the top of the column even under pressure, and the

situation becomes increasingly difficult with low crosslinked

copolymers. This unfavorable situation was resolved by preswelling the beads in a solvent mixture (benzene : chloroform of

2.38:1, v/v) having the same density as the copolymer (p = 1.06),

and packing the column from the bottom under pressure as

illustrated in Figure 30. Using this technique, the column is

rapidly filled in several minutes. The column is then inverted,

and packing is completed from the top in the conventional manner.

The column is then thoroughly purged with chloroform to remove

all traces of benzene as indicated by following the UV absorption

of the effluent stream. This procedure results in a savings of

four-six hours over the conventional method.

(2

)

Operational Modifications

(a) Column End-Fittings — Our system

initially employed plastic end fittings which swelled

excessively in chloroform and other organic solvents resulting

in the complete blockage of flow at the column head, driving

the inlet pressure gauge out of calibration. This situation

was rectified by substituting machined teflon fittings

(Figure 31). These fittings have been 100 percent troublefree for the past year in continuous operating systems.

40
VENT

POROUS

I TEFLON

PLUG

T / IXL BANDED

I / \V( STOPPER COMPRESSED

AIR SOURCE

MAGNETIC

STIRRER

Figure 30. Packing a gel column.

(b) Sample Injection — Although we have used

both teflon switching valves with sample loops and stopped flow

for sample introduction, simple syringe injection through a septum has been used almost exclusively. This has particularly

been the case for SEC in which low inlet pressures (50-100 psi)

are employed. A low volume injection port containing a silicone

septum which is appropriate for sample injection directly onto

the column bed with the pump operating, is shown in Figure 32.

Sample sizes of 0.5 to 500 yl are readily used in this manner.

41
ooO > i

- ~m> i (JOJO

Figure 31. Column teflon end fittings

Figure 32. Sample injection port

42
(c) Pulsation — Mobile phase surges produced

by pulsating pumps severely affect the signal output from lowvolume, flow-sensitive detectors, unless provision for pulse

dampening is introduced into the system. On the sample side

of the detector, the column may or may not provide sufficient

dampening action of solvent pulsing. On the reference side,

pulsation in the detector is dramatic. We tried to remedy this

by the use of a stainless steel capillary coil; however, this

was prone to blockage at its inlet. A better solution is the

use of a three meter length of thin-wall teflon tubing (0.125

inch o.d. by 1.5 mm i.d.) which expands slightly with pulsation,

followed by a 5 cm stainless steel tube packed with 30/60 mesh

silica gel, both located upstream of the column. The steelteflon couple acts as a capacitive device which results in

lowering the gauge fluctuations from 13 psi to less than 2 psi

at 200 psi inlet pressure. The reference stream is adequately

dampened by a column of 30-60 ym silica gel, shown in Figure 33.

(d) Reference Stream Recycling — The consumption of mobile phase is avoided by return of the reference

stream effluent from the detector to the solvent reservoir.

This mode of operation permits overnight analysis as well as

assisting full time operation of the chromatograph

( 3 ) Precautions

(a) Swelling variations of a gel packing can

cause rupture of a glass column having a wall thickness as

great as 3 mm. Manipulation of mobile phase polarity should

be carried out in stainless steel columns. In the interest of

good chromatography, no mobile phase change should be introduced

which severely changes the bed dimensions.

(b) Flammable or toxic mobile phases (eg.

tetrahydrofuran) should be used with care, with adequate

provisions for ventilation, although no problem is encountered

with adequate precautions taken.

43
Figure 33. Pulsation dampening of the reference stream

(4 ) Bilirubin Chromatography

(a) Steric Exclusion Chromatography —

Chloroform has been found to be the only suitable mobile phase.

It affords sufficient solubility for bilirubin (ca. 0.4 mg/cm )

.

It is a non-reactive solvent for this compound. It is transparent in the UV at 254 nrn (the wavelength used by the detector)

It is also non-flammable. All analyses were performed at

ambient temperature, in the dark, using an aluminum foil jacket

around the column. A chromatogram of bilirubin is shown in

Figure 34 on a 4 percent DVB-styrene column. Reference to

44
REL. %

Minor peak: x — 4 0.2 Major peak: x-64 99.8

REL. %

Minor pea k: x — 8 1 . 3 Major peak: x-16 98.7

Figure 34. Steric exclusion chromatography of bilirubin

Figure 26 shows that bilirubin (MW 585) elutes near the top

of the useful portion of the standard curve . The fore-peak

impurity has been consistently found in bilirubin samples, is

larger in poorer grades of bilirubin, is not produced by the

system, and we are sure that it is real. Its identity is not

known to us and accurate measurement of its concentration in

45
bilirubin requires further work. Because of its position of

elution, its molecular volume is slightly greater than bilirubin, and its molecular weight is in the range of 500 < MW < 900.

The questions of gaussian distortion and peak integrity

are difficult to answer. The conditions of low resolution

chromatography have been simulated with a series of chromatograms that were calculated and plotted by a computer. The

series in Figure 35 shows consecutive plots of two gaussians

having a relative intensity ratio of 5 to 95 varied from 0.5a

to 6a separation in increments of 0.5c Two gaussians having

a 10 to 90 relative intensity ratio are given in Figure 36.

Finally, shown in Figure 37 is a similar series of two gaussians

having a relative intensity ratio of 20 to 80. The question

of peak integrity does not become suspect until at least a 1.5,

2.0, or 2.5c separation is achieved for a 20, 10, or 5 percent

intensity contaminant, respectively.

Shown in Figure 38 is an example of another bilirubin

sample which contains a lower molecular weight impurity.

While its retention lies below the useful portion of the gel

calibration curve, ancillary work (to be described below)

substantiates that this impurity is monochlorobenzene

.

(b) Gas-Liquid Chromatography — GLC is not

directly useful for bilirubin itself, owing to the high molecular weight, low vapor pressure, and susceptibility to thermal

degradation of bilirubin. This technique is extremely useful,

however, for the examination of volatile contaminants. Figure

39 shows a chromatogram of a bilirubin sample in chloroform.

The contaminant has been identified as monochlorobenzene by

mass spectrometry of the vapor from bilirubin placed in the

solid probe. Its level in this bilirubin sample was estimated

from GLC at 0.6 percent. A second volatile impurity was found

in several bilirubin samples from GLC analysis of bilirubin in

dimethylsulfoxide . This impurity, shown in Figure 40, has been

46
4.5

-4.0

3.5

8.0

Figure 35. Separation of Gaussians with 5=95

relative intensity ratios

47
3.0 1.5

1 1 1 1 1 1

8.0 -4.0 0.0 4.0 8.0

2.5 1.0

1 1 1 1 1 1 -4.0 0.0 4.0 8.0 -4.0

i 1 1 1 1 r 1 0.0 4.0 8.0

Figure 35. continued

48
5.0 3.5

1 1 1 1 1 1

8.0 -4.0 0.0 4.0 8.0

Figure 36. Separation of gaussians with 10:90

relative intensity ratios.

49
-4.0

3.0 1.5

1 1 1 1 1 1

8.0 -4.0 0.0 4.0 8.0

i

1 1 1 1 r

0.0 4.0 8.0

2.0 0.5

1 1 1 1 1 1

1 I

1 r

-4.0 0.0 4.0 8.0 -4.0 0.0 —

i

1 1

4.0 8.0

Figure 36. continued

50
5.5

1 1 1 1 1 1 -4.0 0.0 4.0 8.0 -4.0

4.0

~i 1 1 1 1 r

0.0 4.0 8.0

-4.0

Figure 37. Separation of gaussians with 20:80

relative intensity ratios.

51
3.0

1 1 1 1 1 1 -4.0 0.0 4.0 8 -4.

1.5

—i

1 1——:

1

0.0 4.0 8.0

2.5

n 1 1 1 1 r

1.0

-4.0 0.0 4.0

1 1 1 1 1 1

8 -4 4. 8.0

-4.0

1

1 1 1 r 0.0 4.0 8.0

Figure 37. continued

52
REL.%

Peak 1: x-2 1.0 Peak 2: x -4 9 8.5

Peak 3: x - 1 0.5

Cm'

Figure 38. Detection of monochlorobenzene impurity

by SEC.

identified by GLC-MS and MS solid probe techniques as chloroform

Its concentration was estimated by the standard addition

technique In GLC at 1-1.5 percent. The m/e peaks observed by

mass spectrometry for the identification of these two volatiles

are listed in Table 4.

53
BILIRUBIN

N

CHLOROFORM

Figure 39. Detection of monochlorobenzene impurity

by GLC.

Table 4. Mass spectral detection of volatile

impurities in bilirubin samples.

A. GLC/MS

m/e

118

117

83

48

47

Specie

+ CHC1

3

CCl

CHC1

CHC1

CC1

+

B. SOLID PROBE/MS

m/e

112

77

76-72

Specie

C

6

H

5

C1

C

6

H

5

proton

stripping

Volatiles: A-chloroform; B-monochlorobenzene

54
.D. M JS Q^

BILIRUBIN IN' DMIS O

Figure 40.- Detection of chloroform impurity by GLC

(5) Summary . Bilirubin may be readily analyzed

by steric exclusion chromatography under conditions assuring

its stability. Further liquid chromatographic effort is needed

for complete separation and identification of its high molecular

weight contaminant. The volatile impurities which may occur

in bilirubin samples are monochlorobenzene and chloroform.

These are not surprising since the former is utilized in the

initial extraction of bilirubin from hog bile hydrolysate, and

the latter is used in the final recrystallization steps. The

present use of steric exclusion chromatography has afforded us

a useful separations technique for a chromatographically

difficult material.

55
b. Glucose . Investigations of glucose purity for SRM

glucose were performed by ion-exchange chromatography and GLC

on a sample received from Section 310.07-

Ion exchange chromatography was performed on a cation

exchanger (10-30 ym bead diameter) at 450 psi inlet pressure

using degassed distilled water as the mobile phase and a differential refractometer detector. Fructose and mannose

were potential impurities in glucose . The chromatograms

indicated that such contamination (Figure 4l) was absent at

the 1 percent level. Glucose had a retention time of 20 minutes

in this system. The separation factors (a) for both mannose

and fructose relative to glucose were 1.10.

Gas-liquid chromatography was carried out on a 10 percent

SE-30 column at a helium flow rate of 100 cm /min and a

temperature program rate of 4°/min from 125 to 250 °C. Care

was taken in the silylation of glucose to produce reaction

directly with the solid material rather than in a pyridine

solution, to avoid anomerization. The use of pyridine in the

silylation results in the conversion of 15-20 percent of the

a anomer to the 3 anomeric form. Separate peaks for silylated

a- and 3-D glucose are observable on the chromatogram under

the above conditions with a relative retention of 3/a of 1.16.

Under appropriate silylation conditions, the glucose sample

of Section 310.07, was found to be a highly pure a-D glucose,

lacking contamination from other monosaccharides

.

56
GLUCOSE

FRUCTOSE

INUTES

Figure 4l. Ion exchange chromatography of glucose.

57
2. Other

a. Organic Dyes . A broad investigation has been in

progress by the Analytical Coordination Chemistry Section under

Dr. 0. Menis to determine a dye standard having a broad and

constant absorption useful for the calibration of spectrophotometers. A candidate dye (Neolan Black) was obtained from

this Section and subjected to chromatography on Sephadex G-10

using distilled water as the mobile phase under gravity flow.

A minimum of five component bands were observed using . 1 ml

of a 10 percent aqueous solution of the dye.

D . Polyaromatic Hydrocarbons

Polyaromatic hydrocarbons (PAH) occur in the air as

environmental toxins produced from the combustion of organic

materials such as coal and gasoline. Some of these substances

have been shown to be carcinogenic to experimental animals.

The analytical procedures which are usually employed include

column, thin-layer, and paper chromatography for separations,

and fluorescence and UV spectrometry for measurement, using

the fractionated crude benzene extract of a particulate filter

from a high volume air sampler. An example of contaminated

vs. clean air sampled outside and inside a clean room facility

is shown in Figure 42. The clean room air filter represents

particles greater than 0.2 urn in 11 m of air sampled at

8.4 1/min. The contaminated filter pad shows such particulates

in 15 m of air sampled at 10 1/min outside the clean room

enclosure. For further details, reference is made to Stern [17]

(esp. Chap. 20). We successfully chromatographed chrysene,

3 , 4-benzopyrene , and 1 ,12-benzoperylene on a pressurized liquid

chromatography system using an alumina column, and ethercyclohexane mixtures as the mobile phase in a procedure similar

to that used by NAPCA. Sensitivity limits of these compounds

approach 0.1 ug using a UV detector at 254 nm. It was observed

that an alumina column of controlled activity (water content)

was inappropriate for a continuous dynamic system which could

58
OUTSIDE INSIDE

Figure 42. Collection of air borne particles in and

outside a clean room facility.

be employed over long periods of time. A column of cellulose

formylate gave limiting results. While gas chromatography

has shown some degree of promise, no continuous chromatographic

system is presently functioning for the complete separation

and measurement of all PAH compounds found in air particulates.

The outcome of our preliminary ventures (including both liquid

and gas chromatography) suggest that appropriate conditions

demand sufficiently strong and selective PAH-stationary phase

interaction energies (eg. via stationary phase tt-tt* charge

transfer complex formation with the aromatic solutes), perhaps

coupled to gradient mobile phase elution.

59
5. HIGH PURITY REAGENTS

A. Container Contamination

The purity of reagents must depend to some extent upon

the inertness of their containers. In the case of ultra pure

reagents, the enhancement of trace contamination can arise from

chemical attack of the reagent upon the container. This factor

can be minimized by choosing a suitably inert container material

FEP Teflon and linear polyethylene are used for storing nitric

and hydrochloric acids, respectively. Although each of these

materials is inert to the contained acid, particulate matter

occluded in the container walls presents a possible source of

contamination. Rigorous chemical leaching is used to reduce

this effect.

Two-liter insulated Teflon bottles were filled with nitric

acid to approximately 5/6 of their internal volume, fitted with

condensers and maintained at gentle reflux (110 °C) for six

days. Following treatment, the bottles were steamed out for

two days, washed, and then dried overnight at 130 °C under

vacuum. The nitric acid leaching system is shown in Figure 43.

Polyethylene bottles (500 ml) were refluxed (70 °C) for

five days In a 12-liter round-bottomed flask with concentrated

hydrochloric acid. The bottles were then steamed out for one

day to remove any water-soluble materials in the container

matrix, and finally vacuum dried overnight at 55 °C. Since

steaming-out periods of at least 2 4 hours were used, a semiautomatic system was devised using each inverted bottle as a condenser. As illustrated in Figure 44, the inner tube allows

steam to flow into the bottle. The inverted bell jar catches

the condensate, which Is returned to the boiler through the

side arm, that also functions as a water trap to prevent loss

of steam through it

.

A series of independent experiments was carried out to

investigate the nature of extraction for and the contaminants

removed from FEP Teflon containers . Pieces were cut out of

60
Figure 43. Nitric acid leaching of teflon containers.

Teflon bottles, water washed, and refluxed for two days in a

3:1 concentrated nitric : hydrochloric acid mixture, followed

by reflux for three days in concentrated nitric acid. The

presence of hydrochloric acid served to remove entrained metal

oxides from the container wall. The leached Teflon pieces were

'then immersed in boiling water for three days, which was changed

daily. The samples were finally dried under vacuum. The

removal of a particle 390 ym from the surface of the wall is

shown in the series of photomicrographs in Figure 45 together

with the phenomenon of flower cavity production. The concentration of particle contaminants in the FEP Teflon containers

4 3

was observed to be 1-2 x 10 particles per cmJ of Teflon.

Leaching periods of up to five days were required to remove

61
Figure 44. System for steaming out polyethylene

bottles

.

large particles (200 ym) . The examination of particle composition by the laser microprobe revealed the presence of Fe s Ti 3 Ni , Cr, Na, Ca and Si. It appears that the contamination is

introduced during the manufacture of FEP Teflon bottles from

stainless steel and a talc. The Teflon pieces became opaque

after drying of the leached samples. The infrared absorption

spectrum of the Teflon prior to and following leaching however,

was unchanged.

B. Light Scattering Studies

The presence of particulate matter suspended in high purity

reagents presents a common kind of contamination. Studies were

62
START 1 DAY

2 DAYS 3 DAYS

Figure 45. Leaching of a contaminant from the wall

of an FEP Teflon container with 3:1

HN0 o :HCl.

63
initiated to evaluate the efficacy of the removal of particulate

matter by means of ultrafiltration. A microturbidimeter

described previously [1] was used to measure the efficiencies

of particle removal.

Standard suspensions of 10 ppm of 0.500 urn and 0.357 ym

latex spheres in distilled water previously filtered through

a 0.10 ym pore filter were used as feed solutions. The percent

removal of the latex spheres by various filters is shown in

Table 5. Prior to use, the filters were precleaned by washing

with distilled water, which had previously been passed through

a 0.10 ym filter, until the filtrates indicated 0.00 ppm

particulate concentration in the microturbidimeter.

A second set of experiments was conducted with inorganic

salt solutions prepared from fresh reagent bottles and distilled

prefiltered water. The level of particulates (ppm) in the salt

solutions prior to and following ultrafiltration through a 0.10 ym filter was determined in the microturbidimeter. The

results are given in Table 6.

The data indicate that quantitative removal of solution

particulate matter is attainable by ultrafiltration when a sufficiently small pore diameter filter is used, and that the

pore diameters cited for commercial ultrafilters are usually

nominally accurate. Spurry, et_. al. [18] have investigated

the mean radius of Nucleopore filters by electron microscopy

and reported the nominal pore radius to be approximately 8

percent larger than the actual mean radius.

C . Pooled Reagents

Different lots of commercially obtained ultra pure reagents

tend to present a bothersome problem due to the corresponding

variations in the impurities. This imposes a time-consuming

need for separate blank analyses for each reagent. By contrast,

a bulk lot of a given reagent implies a more stable situation.

We have obtained 10 liter batch quantities of perchloric,

nitric and hydrochloric acids of ultra pure quality. The first

6*1
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p

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cd 43 cd

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Ph 66
two acids were packaged in FEP Teflon bottles, while the latter

was stored in linear polyethylene containers (see Section 5.A.),

The reagents are stored in a chamber maintained at -40 °C

(Figure 46), affording a projected reagent life of 18 months.

W^W

Figure 46. Storage of ultra pure acids at -40 °C

We have also prepared bulk quantities of high purity

ammonia solutions from 99.999 percent pure NH„ gas. During

preparation, candidate impurities in the ammonia gas, such

as carbon dioxide, organic vapors, and particulate matter,

were respectively removed by passing the gas stream over

solid potassium hydroxide, activated charcoal, and through

a 5 ym Teflon filter (Figure 47). To avoid contamination

67
Figure 47. Removal of trace impurities from 99-999

percent ammonia gas.

from the flow system, the polyethylene tubing, traps, and

adsorption container were thoroughly leached with 10 M HC1

,

EDTA in dilute NH^OH solution, and steamed out prior to usage.

D. Reagent Bank

Our Section supplies, on a "self-service" basis, large

quantities of ultra pure reagents to chemists in the Analytical

Chemistry Division, either in the form of single commercially

packaged units or in specially prepared bulk lots. For all

reagents in the bank, decomposition is retarded by storage at

temperatures as low as -40 °C. The 150 units dispensed during

fiscal 1969 (Table 7) covered 15 separate reagents and represented an increase of 3 percent over that of the previous year.

The fact that 90 percent of the units represented either common J acids or ammonia solution indicates the need for bulk quantity

availability of these reagents.

68
Table 7. Ultra pure reagents dispensed In

fiscal year 1970.

Acids Bases

CH COOH 2 NH„OH

HC1

HF

HI

HNO^

HCIO^

H^SO„

Salts

NaCl

BaCl

2

NaP

2 NH.OH

26 NaOH

9

1 Redox

30 Br

2

36

10 Buffer Salts

CH COONa

1 Acid Anhydrides

1 P 2°5

1

69
6. PERSONNEL AND ACTIVITIES

A. Personnel Listing

Separation and Purification Section

David H. Freeman, Section Chief

Janice M. Hurst, Secretary

Chromatography

Delmo Enagonio

Walter Zielinski

Patti Byrne (summer student)

Ion Exchange Micros tandards

Gerald Sleater

Mass Spectrometry

William Dorko

Network Structures

Rosalie Angeles (Guest Worker)

Particle Metrology

Herbert Dixon

Ultra-pure Reagents

Edwin Kuehner

Youth Opportunity Corps

Linda Gant

Jennifer Jones

B. Talks

1 • D. H. Freeman , W. L. Zielinski, Jr., and W. F. Rittner,

"Recognition of Crosslinking in the Infrared Spectra

of Poly ( styrene-divinylbenzene )

, " International Conference

on Ion Exchange in the Process Industries, London,

England. July 16, 1969.

70
D. H. Freeman , W. L. Zielinski, Jr., and W. F. Rittner,

"Characterization of Ion Exchange Networks: Theory,

Microscopy, and Spectroscopy," Gordon Research Conference

(Ion Exchange), Meriden, New Hampshire. August 18, 1969.

D. H. Freeman, "Ion Exchange Chromatography," 1969

Symposium on Liquid Chromatography, Montreal, Canada.

August 25, 1969.

D. H. Freeman , V. M. Story, and W. L. Zielinski, Jr.,

"Ion Exchange Networks: Chromatography and Heterogeneity,"

158th ACS National Meeting, New York, New York.

September 11, 1969.

D. H. Freeman, "Sub-microgram and Sub-nanogram Standards

for Chemistry," Anachem Conference, Detroit, Michigan.

September 18, 1969.

D. H. Freeman, "Ion Exchange Networks: Their Characterization and Applications as Sub-Picogram Standards,"

Northeastern University, Boston, Massachusetts.

October 20, 1969.

7. D. H. Freeman, "Ion Exchange Network Characterization,"

Pittsburgh Conference on Analytical Chemistry and

Applied Spectroscopy, Cleveland, Ohio. March 2, 1970.

D. H. Freeman, "Sub-Microgram Encapsulation Chemistry,"

Pittsburgh Conference on Analytical Chemistry and

Applied Spectroscopy, Cleveland, Ohio. March 2, 1970.

D. H. Freeman, "Selectivity and Resolution in Gel - HPLC,"

Washington Chromatography Discussion Group, Washington,

D. C. June 11, 1970.

10. W. L. Zielinski, Jr., "Bilirubin Gel Chromatography,"

Washington Chromatography Discussion Group, Washington,

D. C. June 11, 1970.

C. Publications

1. R. Angeles, M. H. Aldridge , D. H. Freeman, and L. A. Wall,

"Cryodegradation of Polyelectrolyte Networks," J. Poly.

Sci., Part B Polymer Letters, Vol. 7, 8, p. 609 (1969).

2. D. H. Freeman, Book Review: "Ion Exchange" Volume 2

(to be published).

71
3. D. H. Freeman, L. A. Currie, E. C. Kuehner, H. D. Dixon,

and R. A. Paulson, "The Development and Characterization

of Ion Exchange Beads as Microstandards ," Anal. Chem. 42 , p. 203-209 (1970).

4. D. H. Freeman and W. L. Zielinski, Jr., "Separation and

Purification Section: Summary of Activities July 1968

to June 1969," NBS Technical Note 509., (1970).

5. D. H. Freeman and G. Schmuckler, "Homogeneous Ion

Exchangers as Microstandards," XXXIX Conference of the

Israel Chemical Society, Jerusalem, Israel, 9/29-10/1/69,

p. 174 (1969).

6. W. L. Zielinski, Jr., D. H. Freeman, D. E. Martire, and

L. C. Chow, "Gas Chromatography and Thermodynamics of

Divinylbenzene Separations on 4 ,4 • -Dihexoxyazoxybenzene

Liquid Crystal," Anal. Chem. 4_2, p. 176-I8O (1970).

7. D. H. Freeman, S. Goldstein, and G. Schmuckler,

"Homogeneous Sulfonation of Styrene-Divinylbenzene

Copolymers with Oleum in Organic Solvents," Israel J.

Chem. 7, No. 6 (1969).

72
7. REFERENCES

[ 1] National Bureau of Standards Technical Note 509 , D. H. Freeman and W. L. Zielinski, Jr., Eds.,

February 1970.

2] Freeman, D. H., Currie , L. A., Kuehner, E. C.,

Dixon, H. D., and Paulson, R. A., Anal. Chem. 42 , 203 (1970).

3] Boyd, G. E., and Soldano, B. A., Z fur Elektrochemie

57, 162 (1953).

[ '4] Zielinski, W. L. , Jr., Freeman, D. H. , and Dorko, W. D.

manuscript submitted for publication.

[ 5] Zielinski, W. L. , Jr., Freeman, D. H., Martire, D. E.,

and Chow, L. C, Anal. Chem. 4_2, 176 (1970).

[ 6] Zielinski, W. L., Jr., J. Amer. Oil Chemists' Soc.

41, 249 (1964).

[ 7] Maggs, R. J., J. Chromatog. Sci., 7, 145 (1969).

[ 8] Giddings , J. C., "Dynamics of Chromatography, Pt . 1,

Principles and Theory," Marcel Dekker, Inc., New

York (1965).

[ 9] Giddings, J. C, and Mallik, K. L. , Anal. Chem. 38,

997 (1966).

[10] Altgelt, K. H., "Advances in Chromatography," J. C.

Giddings and R. A. Keller, Eds., Vol. 7, Marcel Dekker,

Inc., New York, p. 3-46 (1968).

c 11] Ayres, J. T., and Mann, C. K., Anal. Chem. 38_, 861

(1966).

[12] H£lcisz, I., Fifth International Symposium on Advances

in Chromatography, Las Vegas, January 1969

[13] Kirkland, J. J., and DeStefano, J. J., J. Chromatog.

Sci. 8, 309 (1970).

[14] Scott, R. P. W., and Lawrence, J. G., J. Chromatog. Sci.,

8, 65 (1970).

[15] Haller, W. , private communication.

[16] Ref. [9], P. 9.

73
[17]

[18]

"Air Pollution," 2nd Ed., Vol. II, A. C. Stern,

Academic Press, New York (1968).

Ed

Spurry, K. R., Lodge, J. P., Jr., Frank, E. R.,

and Sheeslay, D. C, Environ. Sci. and Tech. 3, ^53

(1969).

74
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