A11101 QQ0fil7

i





A UNITED STATES DEPARTMENT OF COMMERCE

PUBLICATION

*=-! Bureau of Standards

FEB 6 1970

\

NBS TECHNICAL NOTE 509

National Bureau of StandartJs JUL 2 8 19724

/DO

Separation and Purification

Section:

Summary of Activities

July 1968 to June 1969

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS

UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

NATIONAL BUREAU OF STANDARDS • Lewis M. Branscomb, Director

NBS TECHNICAL NOTE 509

ISSUED FEBRUARY 1970

Nat. Bur. Stand. (U.S.), Tech. Note 509, 73 pages (Feb. 1970)

CODEN: NBTNA

Separation and Purification

Section:

Summary of Activities

July 1968 to June 1969

Edited by David H. Freeman

and

Walter L Zielinski, Jr.

Separation and Purification Section

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

NBS Technical Notes are designed to supplement the Bureau's regular publications program. They provide a means for 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. CI 3.46:509), Price 70 cents.

FOREWORD

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

September 1, 1963, 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 57 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.

in
Thus our progress reports endeavor to present this information which we have struggled to obtain and which we 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 third annual report on progress of the

Separation and Purification Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

iv
PREFACE

The year of activity in the pages to follow marks an

important transition in the growth of this, the Separation and

Purification Section. We now begin our fourth year. The past

year involved being on a frontier that was challenging, difficult, and rewarding.

The highlights are the accomplishments of which we are

proud. The Section completed its first certification of a Standard Reference Material, an ion exchange microstandard for

calcium in the 0.01 to 1 nanogram range. We shared in being

among the first groups, our own and one in Czechoslovakia, to

detect and measure crosslinking in organic ion exchange networks. We have obtained the best separation to date of the

divinylbenzene isomers. We studied copolymer swelling kinetics

on beads as small as 10 microns (urn) in diameter.

The frustrations we experienced provide some insight as to

problems needing more concerted effort. High pressure liquid

chromatography is potentially the most important single analytical tool for organic separations, but we have found the prediction of selectivity to be a subject of confounding difficulty

The provision of purified chemical reagents is in serious need

for a suitable container—one free from reactivity, diffusion,

brittleness, and impregnated particulates, among other problems.

There is a need for development of inorganic microstandard

matrices for better sample simulation, this is relevant for

geological and possibly for air pollution analysis. There is

uncharted territory concerning the chemical applications of

homogeneous solid solutions with mixed counterions that are

needed as microstandards

.

The most difficult challenge was the effort to introduce

at NBS a high pressure and high resolution liquid chromatographic discipline, and simultaneously apply it toward the

characterization of purity and the separation and measurement

of contaminants in clinical Standard Reference Materials. The

v
chromatography of bilirubin remains perplexing as an unsolved

problem of much concern.

The citation of any commercial materials and equipment in

this document is occasionally made only to specify procedures

which were used, and does not imply recommendation or endorsement by the National Bureau of Standards; nor does it imply

the use of such material and equipment as the only suitable

sources for the purpose at hand.

Mrs. Janice Hurst, our Section secretary, eased the task

of preparing this report and we gratefully acknowledge her

patient skills in translating penscript, some of it legible.

David H. Freeman, Chief

Separation and Purification Section

VI
TABLE OF CONTENTS

PAGE

I. ION EXCHANGE MICROSTANDARDS 1

A

.

Introduction 1

B. Preparation 2

C. Casting 3

D. Stripping 4 E. Analysis 5

P. Other Measurements 7

G. Measurement, Transfer and Packaging 9

H. Certification 10

II. ION EXCHANGE SUBSTRATES 13

A. Introduction 13

B. Preparation 13

1. Synthesis of Copolymer Beads 13

2. Sulfonation of Copolymer Beads 14

C. Characterization 18

1. Infrared Spectrophotometry of

Copolymers 18

2. Infrared Spectrophotometry of

Sulfonated Copolymers 24

3- Swelling Studies 25

III. COPOLYMER AND ION EXCHANGE MICROSCOPY ...... 30

A. Introduction 30

B. Characterization of Copolymer Beads 30

C. Characterization of Ion Exchange Beads .... 32

D. The Microscopy Laboratory Facility 34

IV. CHROMATOGRAPHY 35

A. Gas Chromatography 35

1. Divinylbenzene , 35

(a) Separations on Bentone-34 35

(b) Separations on Liquid Crystals .... 35

(c) Vinyl Toluene 38

2. Other 39

(a) Sulfolane 39

(b) 2-Methoxy Ethanol 40

(c) Ortho-Fluorobenzoic Acid 42

3. Gas Chromatography-Mass Spectrometry ... 43

(a) The Quadrupole Mass Spectrometer ... 43

(b) The GC-MS Interface 44

B. Liquid Chromatography 46

1. Introduction 46

vii
TABLE OF CONTENTS (Con't)

PAGE

2. Instrument Design ^7

3. Clinical Standard Reference

Materials - Bilirubin ^8

V. REAGENTS 53

A. Introduction 53

B. Ultra Pure Reagents Service 54

C. Ultra Pure Reagent Synthesis 56

D. Container Purification 56

VI. PERSONNEL AND ACTIVITIES 58

A. Personnel Listing 58

B. Talks 59

C. Publications 60

VII. REFERENCES 6l

LIST OF TABLES

TABLE NO. • PAGE

I. Network Equivalent Weight (E^) Measurements ... 8

II. Copolymer Beads with Known DVB Crosslinking ... 15

III. Gas Chromatographic Separation of DVB

Components in Mixed DVB 39

IV. Number of Units of Ultra Pure Reagents

Dispensed in FY 1969 55

viii
LIST OP FIGURES

FIGURE NO. PAGE

1. Sodium Ion Selective Electrode Used for

Monitoring Exchanger Effluent Stream 3

2. Analysis of Exchanger Loadings by

Activation Analysis 5

3. Effect of Bead Size Upon Calcium

Analysis by the X-Ray Microprobe 7

4. Clean Room Facility for Casting and

Packaging of Microstandards 9

5. Polyethylene Clean Room 10

6. First Micro standard Certificate:

Calcium H

7. Copolymer Sulfonation of Pre-Sized

Beads 16

8. Surface Sulfonation of Copolymer Beads 17

9- Preparation of 100% Pure Sulfuric Acid

by Slow Crystallization at 9 °C 17

10. Grating Infrared Spectrophotometer ...'.... 19

11. Infrared Spectrum of Polystyrene 20

12. Infrared Spectrum of Poly (s tyrene/

0.16 p-DVB) 20

13. Infrared Spectrum of Poly (styrene/

0.16 m-DVB) 21

14. Infrared Spectrum of Poly (styrene/

0.16 technical DVB) -. . 21

15. High Resolution Spectra of Polystyrene

and Poly (styrene/DVB) Copolymers 22

16. Absorbance Ratio of m-DVB Band Relation

to Mole Fraction of m-DVB (Base Line

Method) 23

17. Cal Comp Plot of Poly (styrene/0 . 08 p-DVB)

Unresolved and Computer Resolved Doublet .... 24

ix
LIST OF FIGURES (Con't)

FIGURE NO. PAGE

18. Preliminary Testing of Design Circuitry

for Automatic Digitizing Device 25

19. Capacity Relative to Adsorption for the

1408 cm1 ! Band of Sulfonated Copolymers .... 26

20. Mole Fraction of DVB Isomers Relative to

Swelling Fraction (}> [<J> = (Q-l)/Q, where

Q = swollen bead/dry bead diameter ratio] ... 27

21. Log-Log Plot of Swelling Time Relative

to Dry Bead Diameter Showing Homogeneous

and Heterogeneous Swelling 28

22. Photomicrograph Showing Heterogeneous

Swelling 29

23. Comparator Used for Measurement of

Particle Diameters and Particle Size

Distribution 31

24. Microscope with Ocular Micrometer,

Counting Cells, and Stop Watch for

Swelling Studies 32

25. Microscope with Micromanipulator and

Specimen Mounts for Microstandard

Particles 33

26. Separation of DVB Components on Bentone-34 ... 36

27. Separation of DVB Components on DHAB

Liquid Crystal 37

28. Analysis of Vinyl Toluene on DHAB Liquid

Crystal 40

29. Determination of Water in Sulfolane 41

30. Separation of Volatiles on Porapak Q h2

31. Quadrupole Filtering Schematic 43

32. The GC-Mass Spectrometer System 45

33. Schematic of GC-MS Interface 45
LIST OP FIGURES (Con't)

FIGURE NO. PAGE

34. Sample Inlet System for Septum Injection .... 48

35. Connection from Column Exit to Detector .... 49

36. Preliminary Ion Exchange Chromatographic

System 50

37. Schematic of Ion Exchange Chromatographic

System 51

38. Analysis of an Impure Bilirubin Sample

by Ion Exchange Chromatography 52

39. Microturbidimetric Measurement of

Particulate Matter Content 54

40. Effect of Boiling Nitric Acid in Removal

of Container Contaminants 57

XI
SEPARATION AND PURIFICATION SECTION: SUMMARY OP ACTIVITIES

JULY 1968 to JUNE 1969

Edited by David H. Freeman and

Walter L. Zielinski, Jr.

ABSTRACT

This is the annual progress report on the Separation and

Purification Section activities. The major task of developing

a certified ion exchange microstandard as a Standard Reference

Material is presented in terms of the preparative and characterizational work involved. Fundamental studies of ion

exchange substrates includes the application of quantitative

analytical infrared spectrophotometry to measure crosslinking

in the copolymer network, and to determine the degree of

sulfonation. Optical microscopy is applied to the study of

swelling kinetics for single copolymer particles. Analytical

gas chromatography is applied to the isomers of divinylbenzene

The recently acquired quadrupole mass spectrometer is described.

Liquid chromatography at high pressures and with high resolution has been begun recently. The activities of a project

dealing exclusively with ultrapure reagents is described

including the problems of contamination free storage.

Key words

Gas chromatography; ion exchange; infrared analysis;

liquid chromatography; purification; separation; styrene/

divinylbenzene; ultra-pure reagents.

xii
I. ION EXCHANGE MICROSTANDARDS

A . I ntroduction

A broad range of chemical entities exist in the form of

a single element, or combinations of elements, which can be

loaded onto ion exchange beads. Following suitable preparation, beads are isolated one at a time. For each given bead,

the mass of the element (counterion) in question is determined

by measurement of the bead diameter. Bead homogeneity is

likewise determined, so that the accuracy of the mass of the

element is also known within a well-defined, and rather small,

uncertainty. The happiest aspect to emerge from this effort

is an important and unique capability for the encapsulation

of quantities of matter that are otherwise unweighable . The

quantities are sufficiently small to permit novel experiments

and calibration of chemical measurement systems, especially

those designed for handling very small samples.

Several adaptations of this technique have evolved by

acceptance into other laboratories. Thus, the preparation of

micro sources of radioactivity has been carried out by Paul B.

Hahn at the Northeastern Radiological Health Laboratory

(HEW-PHS). Dr. Lynus Barnes (Analytical Mass Spectrometry

Section, NBS) has developed a method of standard addition that

permits quantitative mass spectrometric analysis at the nanogram

level. The combined co-calibration of the electron microprobe

for magnification and for elemental ratio (counterion to sulfur)

has been worked out by Robert Myklebust and Dr. Kurt Heinrich

(Spectrochemical Analysis Section, NBS). The results of all of

these studies will be made public at the forthcoming Eastern

Analytical Symposium (New York, New York, November 1969). The

observation that these and other disciplines are engaged in

extending the feasibility of using these standards, coupled

with the continuing improvement of detector sensitivity limits,

indicates their growing potential to a broad spectrum of

chemical microanalysis.

1
In the previous annual report [1], we outlined the

general capability of ion exchange micro standards . A full

description of their preparation and testing is planned for

publication in the near future so that a duplication of this

information will not be given here. The following section

will provide coverage of the highlights of the past year's

activities in this area. A separate project involves the

development of larger particles that are to be used in the

nanogram to microgram mass range, and these efforts are

described in Section II of this report.

B. Preparation

Ion exchangers have the obvious capability for exchanging

ions. The preparation of an ion exchange standard depends

upon two important requirements. The placement of the ions

must be done uniformly. Once placed, the ion composition

must be locked against accidental change. The former depends

upon access to an intrinsically homogeneous ion exchange

network. These conditions are not obtained without special

care in synthesis, and the proof of homogeneity involves an

experimental effort that we have approached comprehensively.

The preparative methods are monitored both for completion of ion placement and for possible contamination. In

column operations, we may start with a sodium form exchanger,

since this can be monitored using the sodium selective electrode,

shown in Figure 1. The prepared exchanger is tested for traces

of residual sodium content using flame photometry.
Figure 1. Sodium Ion Selective Electrode Used for

Monitoring Exchanger Effluent Stream.

C. Casting

The exchanger in its prepared form is washed with solvents

that deswell the network and the material is dried. The dry

exchanger is then immersed in a pure, inert and nonswelling

liquid, such as cyclohexane. The bead clusters are dispersed

ultrasonically and a dilute suspension is cast onto glass

slides. Pretreatment of the glass microscope slides involves

exhaustive leaching to extract potentially exchangeable

Impurities from the glass surface. while glass is not the

best possible choice of container, as we have reported in a recent summary [2], It does offer the advantage of providing

just enough electrostatic attraction to hold small beads in

place once they are carried by the hydrocarbon liquid onto

the surface. This was tested in some detail since we had to
show that such beads, once cast onto a surface and then sold

as Standard Reference Materials , would remain in place. In

routine tests, the beads adhered to the glass despite repeated

drop tests from an altitude of three meters above the floor.

D . Stripping

To function as a microstandard, the adsorbed element

must be released from the matrix of the ion exchange bead.

If the matrix is not to be destroyed per se , the problem is

one of eluting (stripping) a micro ion exchange system;

understanding that the procedure would be the same as for a macro system. In the case of nanogram quantities on micronsize beads either too large a volume of extracting agent or

too lengthy an extraction process would hamper the utility

of a microstandard and the potential for loss would likewise

become extreme.

For simple monovalent ions, stripping presents no

difficulty; water alone Is sufficient, so much so that high

room humidity can cause loss through leaching. With multivalent

ions stripping becomes more difficult; the ions are adsorbed

so strongly that a small amount of water cannot effect complete

removal.

Other possible methods of elution include washing with

complexing agents, or oxidation-reduction of the ion while it

is adsorbed on the resin.

The first microstandard where stripping became a problem

was chromium, adsorbed as Cr (III). Nelson, et_ al. [3] have

indicated that either concentrated or dilute hydrochloric

acid could effect stripping. Qualitative tests showed

however, that though results could be achieved using concentrated acid, the process was a lengthy one with complete

extraction not assured. EDTA showed the same behavior.

More satisfactory results were obtained by oxidizing the

adsorbed chromium with alkaline hydrogen peroxide. In a

matter of seconds the yellow color of the chromate ion could

4
be seen in solution. Preliminary quantitative measurements

indicate complete removal of the chromium from the resin.

E . Analysis

Activation analysis provided a significant test for the

five kilogram sample of 5-25 um beads that were used in the

initial effort in the development of ion exchange microstandards. The results are shown in Figure 2. The experimental

I0

4 ,=r

I I Mill I I INI 1—I I I I MM I I I I I l~E

Q.

UJI—O

Q

O

CO

I0 3 -

10*-

i i i mi I 'I I I I I I MM II i i i in

10 10 10 10 CALCULATED BEAD VOLUME (/im3

)

ioFigure 2. Analysis of Exchanger Loadings by

Activation Analysis.
particulars will be discussed in detail elsewhere, but it has

become obvious that the measured sodium content revealed a direct uniform variation of counter! on content with the

microscopically measured bead volume. The variations do not

extend appreciably beyond the anticipated measurement error.

-4

It is significant to note that these results involve 10 -fold

smaller quantities of matter than reported previously when this

type of test was first announced.

The electron microprobe analysis of the Ca-form ion

exchange beads showed a stable Ca/S ratio for the respective

K x-rays. The microprobe results as reported [4], imply

suitability of the beads as correspondence standards between

signal ratio and absolute elemental ratio.

The effect of particle size upon microprobe x-ray signal

was quite informative. The result for Ca is shown logarithmically in Figure 3- The results for S were identical in

trend, as expected. The slope of two indicates the

relationship

(Ca) or (S) « d

2

(1)

where d is particle diameter. This is the same as the

predicted behavior for an electron (or x-ray) opaque bead

in which penetration toward the bead core is slight. In

terms of the need to measure possible bead heterogeneity

however, shallow penetration is desirable for the following

reason. Given the independent network heterogeneity

determination provided by activation analysis, the major

possible source of heterogeneity should be traceable to

surface loss by ion exchange. Electron microprobe analysis

therefore, offers a significant contribution by narrowing

its search to this important region of the bead.
14.0

12.0- CALCIUM vs SULFUR

CALCIUM

Ka

INTENSITY

2.0 3.0 4.0 5.0 SULFUR K a INTENSITY

7.0

p

Figure 3. Effect of Bead Size Upon Calcium Analysis

by the X-Ray Microprobe

.

Other Measurements

It should be noted that other contributions to this

project have been carried out and will be reported elsewhere

The determinations of (a) ion exchange capacity, (b ) matrix

element measurements and (c) the anhydrous exchanger density

have been performed with the cooperation of R. A. Paulson

(Microchemical Analysis Section, NBS). Measurements of

counterions using polarographic [5] and flame photometric

methods have also been carried out [6].
One aspect of this interlaboratory work is the common

feature that the equivalent weight of the exchanger network

(excluding counterions) should be independent of the

counterion form i. The exchanger equivalent weight E (g/eq)

is the sum of the network (E,,) contribution and counterion

(E-j. = Ey.E.) contribution, where y. is the equivalent fraction

and E. is the equivalent weight of the particular counterion.

Note that for y. = 1, E. = E T . In Table I is a listing of ° 1

3 1 I

to various independent values of E„. Any one of these determinations is considered accurate at the 0.5 percent level,

and the differences are in all probability, real. The

variations should be a composite indication of the measurement error in addition to the real physical or chemical

difference. Sample heterogeneity, to be sure, tends to be

ruled out by single particle assays.

Table I. Network Equivalent Weight (E„) Measurements

Counterion

ha

Na

+

Ca

++

Ca

+ +

Pb

++

Cu

+ +

Cu

++

H

+

% b^' Wt

10. 36

10 5

8. 91

9- 10

34. 6

14.

13 3

519

Eq. Wt.

( Gross

222

219

225

220

299

227

239

195

t

E

N

199

196

205

200

196

195

206

193

1 - f

I

t Note: For a single counterion E^ = E-j. (

j_ ) where f is

wt . fraction of that counterion.
G. Measurement, Transfer and Packaging

From independent efforts, we know that liquid water

contains a small amount of electrolyte. This can result from

water hydrolysis, or from reactions following the adsorption

of atmospheric gases such as carbon dioxide. For these

reasons, a microscopic amount of water condensation is sufficient to cause ion exchange, and that of course would tend

to ruin a given microstandard. To minimize this possibility,

we have maintained a practice of restricting the humidity to

less than 50 percent of relative humidity at temperatures

near 23° C.

The elimination of atmospheric dust is an equally

desirable condition. For that reason, we have carried out

our operations in a specially constructed clean room facility

(Figure 4) which Is used for casting and packaging operations

^%

I

* '4

\

Figure 4. Clean Room Facility for Casting and

Packaging of Microstandards

.
for the issuance of the ion exchange beads as Standard

Reference Materials. In Figure 5 is a photograph of a less

elegant, but similar facility in which the room has been constructed out of polyethylene. Inside the room is a laminar

flow clean bench. The air in the room is principally recirculated by an air dehumidifier/air conditioner.

H. Certification

The first microstandard to be completed is that for

calcium in the region of 0.01 to 1 nanogram. This exciting

and uniquely minute encapsulation of the alkaline earth

cation is prepared as castings upon the microscope slides.

A minimum of one hundred isolated beads are adherent to the

glass surface. The beads are readily picked up on a clean

fine-tipped probe.

It is of interest to include the first certificate which

has been issued for these individual ion exchange bead microstandards. The certificate is displayed in Figure 6.

Fig. 5- Polyethylene

Clean Room.

10
U. S. Departfbfi^ of Commerce

Mauric^Ji. Stans

Seip^ayy

A. V. National BiplSj&s^)' Standardsirector (Untxitint?

Standard Reference Material 1800

Microstandard Ion-Exchange Beads

Calcium Loaded

(10

11 to 10~ J grams)

This Standard Reference Material is intended as a source of minute, known

quantities of calcium. The calcium is attached to ion-exchange beads that are 5 to 25 microns (^m) in diameter. The matrix is composed of a homogeneously sul- fonated network of a cross-linked copolymer of styrene with 8 mole percent

ra-divinylbenzene. The calcium is electrostatically attached to the sulfonate groups.

The calcium ions can be displaced by addition of an aqueous electrolyte solution.

Any single bead contains an amount, m, of calcium, sulfur, carbon, hydrogen,

oxygen (and some water) according to the equation: wCa = PaY, where V =

ird:i/6 is the volume of the bead, and mCa and p Ca are the mass and partial density, respectively, for calcium. The partial density of calcium in a typical dry

bead is certified as: pca = 0.135 ± 0.010 g/cm3. The superscript (°) is used to denote reference to a dry resin bead. The value of p° and its error (standard

deviation) is computed from neutron activation analysis and frdm electron microprobe analysis of forty individual beads. The microprobe meAsurjements also

agree with the expected constancy in the calcium to s&lfun^&tio:1

Analysis of gross quantities of this matej

subscripted matrix elements p§ = 0.21 5 ,o£/£ 0? of the dry material ranges from 1.44k (wAknui

trtial densities of the

= 0.06. The bulk density

1.50 (maximum) g/cm3.

The preceding measuremerits^feFjb fcfie material after vacuum drying at 120 °C for 16 hours. If eiSjsoselfl fo^^ro weeks or more to ordinary atmospheres

with relative humidi^l^shj^a range 20 to 40 percent, the partial densities will

be reduced accogjiingUo tftfe amount of sorbed water. Then, the partial density of

each of the^giemMas/»fciwe bead must be adjusted by multiplying by the factor

0.87. In tfte^se^pFralcium, the corrected partial density is: p Ca (20-40% RH) = 0.117 ± o5p9 gVcm3

.

Washington, D. C. March 17, 1969

20234 W. Wayne Meinke, Chief

Office of Standard Reference Materials

Caution : Inadvertent contamination by water, i.e., condensation, is sufficient to cause the loss of calcium by ion exchange hydrolysis. It is therefore essential that

this microstandard be kept at less than 50 percent relative humidity.

(over)

Figure 6. First Microstandard Certificate

11

Calcium.
Measurements leading to the certification of this Standard Reference Material were carried out by D. A. Becker, L. A. Currie, H. D. Dixon, R. A. Durst,

K. F. J. Heinrich, E. C. Kuehner, R. L. Myklebust, R. A. Paulson, T. C. Rains,

and G. Schmuckler of the Analytical Chemistry Division.

The overall direction and coordination of the preparation and technical

measurements leading to the certification of this material were performed under

the chairmanship of D. H. Freeman.

The technical and support aspects involved in the preparation, certification

and issuance of this Standard Reference Material were coordinated through the

Office of Standard Reference Materials by T. W. Mears.

USE OF MICROSTANDARD

The present microstandard was prepared by casting a dispersion of dry, cal- cium-form, ion-exchange beads onto a clean glass slide. The beads are naturally

attracted to the glass surface. The same attraction occurs when the beads are touched gently with a probe, and this permits them to be moved from the slide to another place.

Diameter measurement : Under microscopic examination it will be observed

that the majority of the beads in this standard are spherical. The nonspherical

beads are readily identified and rejected on the basis of their appearance. Bead

diameter measurements are then practicable to an estimated total uncertainty of

± 0.1 micron (/*m). Such measurements are not difficult, but they do require

proper microscopic measuring apparatus and illumination technique (such as Kohler illumination with the use of an interference filter at «560-nm) . These

procedures are described in the literature (D. H. Freeman, "Advances in Ion

Exchange," Volume 1, Marcel Dekker, Inc., New York, 1966, chapter 5).

Storage: The microstandard beads must be protected against exposure to excessively humid atmosphere and, for practical purposes, it should be assumed

that the standard is destroyed if exposed to humidities exceeding 50 percent

relative humidity. Storage over a mild desiccant is recommended. Alternatively, the beads may be vacuum dried at 120 °C for approximately 16 hr in order

to duplicate the dry state to which the composition measurements refer. After

drying, the beads can be kept in the dry state by storing them over fresh phosphorous pentoxide, or they can be immersed in a dry hydrocarbon fluid. There is some loss in accuracy (but a gain in convenience) by working in atmospheres of

controlled humidity. For example, isopiestic equilibration will occur after approximately two to four weeks of storage over an aqueous calcium chloride

solution in the range from 0.2 to 0.4 water activity (approximately 7.2 moles

CaCl2 per kg of H2 for 30% relative humidity)

These standards can be used in a variety of ways. A single bead can be treated

with electrolyte to release the ion-bound calcium. Or, a bead can be observed

destructively as in the laser microprobe where the sample is vaporized and then

analyzed by emission spectroscopy. Single beads can be viewed by an instrument

such as the electron microprobe which is capable of identifying and measuring

relative amounts of the calcium or sulfur atoms contained by the beads or by

comparative samples.

12
II. ION EXCHANGE SUBSTRATES

A. Introduction

The . microstandards issued recently [1,7] are based on

the availability of stable, reproducible and accurately

characterized poly[styrene/divinylbenzene] beads. These

copolymer beads serve as precursors for a wide variety of

possible ion exchange derivatives. In the case of microstandards, we have employed sulfonated beads. The properties

of the gel network are dependent upon the careful control of

the synthetic conditions, including monomer composition.

Specifically, both monomers should be pure, and their concentrations should be accurately known. Other conditions,

such as the bead size-range and the polymerization rates are

equally important. The subsequent reaction on the network

(sulfonation) likewise requires careful control. It has been

shown that such conditions can be achieved [8],

The researcher faced with using ordinary ion exchange

materials may show small or large variations in the properties

of different batches of supposedly identical networks. To

illustrate, the ion-exchange capacity will vary from batch to

batch, and it seldom approaches its ideal value. In this area

in particular, simple definitive tests are lacking to evaluate

the substrate properties.

With such considerations in mind, three areas of investigation were undertaken; viz . the synthesis, the characterization,

and the structure of sulfonated poly[styrene/divinylbenzene]

and its parent copolymers. These studies are more fully

described below.

B. Preparation

1. Synthesis of Copolymer Beads

The first problem in this area lies in obtaining pure

monomers. Styrene presents no difficulty, but either metaor para-divinylbenzene (DVB) is difficult to purify [9].

13
Purification procedures, analytical methods and procurement

channels have been determined [10]. One experienced manufacturing firm in the copolymer synthesis field (Ionac

Chemical Co., New Brunswick, New Jersey), agreed to prepare

beads to meet our specifications* using standard methods [11]

with purified divinylbenzenes . Accordingly, a stock of poly

[s tyrene/DVB] copolymers was synthesized for analytical and

structural studies. The crosslinking was varied according

to isomer (meta- or para-DVB) type and concentration, with

all other synthetic conditions held constant. The copolymers

are listed in Table II. Among those listed is 8 mole-percent

meta-divinylbenzene crosslinked copolymer (poly[styrene/0 . 08

m-DVB] ) which on sulfonation is the substrate for the ion

exchange microstandards

In order to compare the pure copolymers with commercial

samples, a synthetic investigation is in progress with

Dr. A. H. Greer (Ionac Chemical Co.) to determine which of

several experimental variables relate to the swelling behavior

which has been observed. This closely coordinated synthetic

approach is expected to provide structural insight for the

evaluation of fundamental ion-exchange properties. Similar

approaches have been used by other workers [8,12].

2 . Sulfonation of Copolymer Beads

Careful control of copolymer sulfonation is a necessary

prerequisite in the development of reliable cation exchange

networks. One type of experiment using pre-sized beads suggests

that sulfonation may be a two-step process as indicated in

*These specifications require the use of pure DVB (supplied

by us) within + 2 percent concentration accuracy; styrene of

the same concentration accuracy; specified particle size

range; and the production of optically clear, strain-free,

spherical beads.

14
Table II

Copolymer Beads with Known DVB Crosslinklng

Ident. Mole % DVB

Number Comments Met a P ara

1-19 Poly(Styrene/DVB) 1.0

1-20 Poly(Styrene/DVB) 2.0

1-21 Poly(Styrene/DVB) 4.0

1-22 Poly(Styrene/DVB) 8.0

1-23 Poly(Styrene/DVB) 16 . 1-25 Poly(Styrene/DVB) 1.5

1-110 Poly(Styrene/DVB) 2.0

1-111 Poly(Styrene/DVB) 4.0

1-112 Poly(Styrene/DVB) 8.0

1-24 Poly(Styrene/DVB) 16 . 1-11 Microfine Poly(Styrene/DVB

)

8.0

1-126 Poly(Vlnyl toluene/DVB) 8.0

1-127 Solvent Modified 10% Xylene,

Poly(Styrene/DVB) 8.0

1-128 Solvent Modified 50$ Xylene,

Poly(Styrene/DVB) 8.0

1-129 Poly(Styrene/DVB) 5-6 2.4

Figure 7 . These data imply that the final few percent of

sulfonation occur in weeks as compared to hours for major

sulfonation of the beads. Other results have demonstrated

that oleum (HpSOj. + SCO under mild conditions is a rapid

sulfonating agent when the proper solvent system is used [13]-

For certain purposes, surface sulfonation Is desirable.

Figure 8 indicates the result obtained in such an instance.

High purity reagents and careful control of concentrations

are most essential. A technique for preparing pure (100

percent) sulfuric acid is illustrated in Figure 9.

A single literature reference (Pepper et_ al. [14]) has

indicated that ideal sulfonation (one sulfonic acid group

15
SULFONATION OF 50-100 MESH BEADS AT IO°C IN 30% /

I28M H2S04-S0 3 ); NITROBENZENE SOLVENT. /

/

/

64

32

TIME

(hrs) 16

8

10 20 40 60 80 90

% SULFONATION

98

Figure 7. Copolymer Sulfonation of Pre-Sized Beads

16
Figure 8. Surface Sulfonation of Copolymer Beads

Figure 9- Preparation of 100% Pure Sulfuric Acid

by Slow Crystallization at 9° C.

17
per aromatic residue) of the copolymer is attainable. Other

workers claim this achievement is not possible [15]. Employing

the same catalyst (silver ion) as Pepper, we have found that

Pepper's conclusion is valid, even though our sulfonation

system was not HpSOj,, but oleum.

This result permits a greater accuracy in preparing

beads of high capacity and low variability; a factor of great

importance in the preparation of beads for microstandards

in the micro- and nano-gram range.

C . Characterization

1. Infrared Spectrophotometry of Copolymers

Beads prepared with pure DVB (Table II) were used to

evaluate qualitatively and semi-quantit at ively the crosslinking

with respect to both DVB isomer type and mole percent from

IR spectral analysis. Polystyrene* was used as a reference

standard.

The infrared spectra were obtained from a grating

infrared spectrophotometer (Figure 10) . The instrument

was calibrated to within an error tolerance of 0.5 cm

using ammonia vapor and indene liquid for the region 910-

790 cm . The copolymer beads were pretreated by soxhlet

extraction for 16 hours with benzene and then vacuum dried.

The beads were pulverized at liquid nitrogen temperature in

steel capsule containing a steel ball. The procedure of

powdering the copolymer was carried out with an electric

vibrator (Wig-L-Bug, Crescent Dental Company, Chicago,

Illinois) for two minutes. Four mg of the powdered copolymer

*N.B.S. Standard Reference Material 705 (Narrow Range

Polystyrene )

.

18
Figure 10. Grating Infrared Spectrophotometer,

were then mixed with 3 00 mg of KBr and the mixture was again

vibrated for one minute at room temperature . The KBr wafer

was formed in a 13 mm die using 15 tons of pressure.

Although the full frequency range of 4 000 to 400 cm

was examined (Figures 11-14) , attention was focused on a band at 797 cm in poly(styrene m-DVB) copolymers, and on the

lower frequency band of a doublet near 830 cm in poly

(styrene p-DVB) copolymers (Figure 15) • Both vibrations are

due to out-of-plane aryl hydrogen bending, their intensities

suggesting the amount of crosslinked meta- and para-DVB

present, respectively. Reference to Figure 14 indicates that

both types of bridging are present in technical copolymers.

19
3 i .

i 1

i 5

WAVELENGTH (MICRONS) 6 7

. , i . . . . i . . , i . .

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1

1

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3000 2500 2000 1800 1600 , 1400 1200 1000 800 600

WAVENUMBER (CM')

Figure 11. Infrared Spectrum of Polystyrene.

3

i 1 . I\

t WAVELENGTH (MICRONS) 6 7 i 5

. 1.. . ? 10 12 15 2

100 no 100 !<

f\

/I S \ A J \ A i ' A

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g£ 20

in i

i 20 r

J . / r

|f I

1

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1 3000 2500 2000 1800 1600 „ 1400 WAVENUMBER (CM 1

) 1200 1000 800 600

Figure 12. Infrared Spectrum of Poly (styrene/

0.16 p-DVB)

.

20
3

i . iI

i

i

>

1

WAVELENGTH (MICRONS) 6 7

. . i , , . . i . , , i .

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(PERCENT) 8

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2

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fl

i Z 7

"" | 20 J 3 n SI a

II

1 I u

1 1 1 T^i

u I 1 L3 1 1 3000 2500 2000 1800 1600 , 1400 WAVENUMBER (CM'') 1200 1000 800 600

Figure 13. Infrared Spectrum of Poly ( styrene/

0, 16 m-DVB)

.

3

. 2 . 1

i

WAVELENGTH (MICRONS) 6 7 {1 9 10

, , 1 12 15 2i.i.i,

« Q

100 II IUU s*\

/

/ \ rs

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/" \l \ cw\ ^\ (A- A £ 3 h- V

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1/5

' z j <02 9n || 3 ) a f) l_ zu 1 V

j

r\

3000 2500 2000 1800 1600 , ,1400 1200 1000 800 600

WAVENUMBER (CM')

Figure 14. Infrared Spectrum of Poly ( styrene/

0.16 technical DVB)

.

21
POLY (STYRENE / 0.08 TECHNICAL DVB)

_L _L 1 -L _L J_ 920 900 880 860 840 820 800 780 760 740

WAVENUMBERS (cm-1

)

Figure 15. High Resolution Spectra of Polystyrene

and Poly ( styrene/DVB ) Copolymers.

22
Indeed, the technical DVB often employed In copolymer synthesis

usually contains less than 50 percent meta- and para-DVB (in

an approximate 2.5:1 ratio); where gas chromatographic analysis

reveals the presence of meta- and para-ethylvinylbenzene , at

least three other vinyl aromatic compounds, and two dozen other

impurities [16,17].

The relationship of infrared absorbance intensity

relative to an increasing concentration of meta-DVB in

authentic poly(styrene m-DVB) copolymers is shown in Figure 16.

No such simple relationship exists for para-DVB copolymers

however, owing to the overlapping of the aryl hydrogen out

of-plane band of para-DVB bridges with a band of a similar

vibrational mode due to the polystyrene chain. Computer

analysis of the digitized doublet indicated resolved bands

at 838.5 cm for the polystyrene band, and at 826.2 cm

for the para-DVB band. The correlation study of para-DVB

l 797

905

0.8

1 1 1

-

0.6 - -

0.4 ^* -

0.2

<. 1

-

0.05 0.10

MOLE FRACTION m-DVB

0.15

Figure 16 Absorbance Ratio of m-DVB Band Relation

to Mole Fraction of m-DVB (Base Line

Method)

.

23
content in poly(styrene p-DVB) copolymers with infrared

absorbance intensity, will continue. The resolved doublet

curve is given in the Cal Comp plot of Figure 17. A device

for automatic digitization of infrared spectra is being

designed in our Section (Figure 18)

2 . Infrared Spectrophotometry of Sulfonated Copolymers

Using techniques similar to those described above, it

was found possible to quantitate the 1408 cm peak of the

spectra in partially sulfonated samples [18,19]. The

resulting curve of percent sulfonation versus absorption

(Figure 19) shows a non-zero intercept, as also noted for

m-DVB copolymers above (Figure 16 )

870 860 850 840 830

WAVENUMBERSfcrrr1 ) 820 810 800

Figure 17- Cal Comp Plot of Digitized Poly( styrene/

0.0 8 p-DVB) Unresolved and Computer

Resolved Doublet.

24
Figure 18. Preliminary Testing of Design Circuitry

for Automatic Digitizing Device.

3 . Swelling Studies

It is generally known that certain physical properties

of copolymer beads are relevant to the network structure,

although details are less than fully understood. With this

in mind, a homogeneity test on the copolymer that is simple

and precise is attractive. Microscopic measurement of the

swelling of individual copolymer beads was found to give

both kinetic and thermodynamic data. These data are consistent

in terms of the known DVB content and the swelling ratio of

swollen to dry volume, as shown in Figure 20.

Commercial polymers quite often show large scatter among

the individual beads (Figure 21) and poor correlation from

25
.0 -

0.8

0.6 -

A 1408

1490

0.4

0.2 -

— 1111/

>6 o

O y/^

—

Of

— —Intercept Calculated From Copolymer Data

o —

—

_L _L _L _L

—

20 40 60

% TOTAL CAPACITY

80

Infra- Red Absorption Ratios of Partially Sulfonated

Styrene-DVB Copolymers (in KBr)

Figure 19. Capacity Relative to Adsorption for the

1408 cm

-1 Band of Sulfonated Copolymers.

batch to batch. Transient deformations can occur at low DVB

concentrations (eg. 2 percent) and are visible during the

swelling process (Figure 22). More generally, a spherical

shell structure is expected but it was not observed here.

26
1.0

1

• m

1

Tol jene _

0.8

*P gc

•P

oc

oc

0.6 • m

•P m+p»

• m

0.4 —

•P

0.2 —

1 1

I _L

2

% DVB

8

Swelling Data in Toluene' Volume Fraction of

Swelling <£=(q-l)/q versus Mole % DVB

p=para , m = meta, c = commercial

Figure 20. Mole Fraction of DVB Isomers Relative to

Swelling Fraction $ [<j> = (Q-l)/Q, where

Q = swollen bead/dry bead diameter ratio]

27
Log(too) 120

1 I I T Heterogeneous

Sample 0-119

4-C/toluene

o

o o

6>

x*

XX*

xxx^c o / oo

Homogeneous

Sample I- II

I

4- m /toluene

1 J L 1.7 1.9 2.1 2.3

LOG BEAD DIAMETER (/im)

Figure 21. Log-Log Plot of Swelling Time Relative

to Dry Bead Diameter Showing Homogeneous

and Heterogeneous Swelling.

28
Figure 22. Photomicrograph Showing Heterogeneous

Swelling.

29
III. COPOLYMER AND ION EXCHANGE MICROSCOPY

A. Introduction

With the advent of the use of ion exchange particles as

carriers for chemical microstandards (see Section I), the

technological problems of observance and manipulation of

microscopic particles have demanded satisfactory solution.

It would be obvious that in order to satisfy such objectives, special equipment is required and special techniques

must be employed if reliable Ion Exchange Microstandards are

to be obtained.

This section describes some of the techniques and equipment which have been used in the microscopy laboratory to

obtain satisfactory results with microstandard particles.

No attempt however, is made here to explain treatment of the

materials before they arrive or after they depart this

laboratory

.

B

.

Characterization of Copolymer Beads

One of the principal functions of the microscopy laboratory

is the careful scrutinization of the various copolymer materials

as they are received from various sources. Microscopic

examinations are performed in order to determine their suitability and acceptance for further research or for use as ion

exchange base material.

Photomicrographs of the materials are prepared using either

normal transmitted or polarized light. A Control Number is

assigned to each material, and all pertinent information and

observations are entered into a Control Catalog for future

reference

.

The criteria normally used to reject materials from further

study are (1) the entrappment of foreign particles in the

network, (2) non spherical or broken particles, and

(3) particles containing voids (holes) in the network. Once

a material has been accepted for further study it is separated

30
into different size fractions by sieving. The size distribution

of each sieve cut is determined by photomicrography. The

micrographs are scanned with a comparator and measurements are

made of the largest and smallest particles to give the size

range of each cut (Figure 23).

Further characterization of the accepted material involves

the swelling of particles in various organic solvents.

Individual particles are selected and their diameters are

measured in air. The particle is immersed in an organic

solvent, the time for completed swelling is recorded, and the

swollen diameter is then measured.

From such data, comparisons are made with other copolymers,

crosslinking data are obtained, and decisions concerning ion

exchange conversion are made

.

Figure 24 shows a microscope equipped with an ocular

micrometer, a counting cell, and a stop watch for swelling

rate measurements.

Jb,

«- **"

Figure 23. Comparator Used for Measurement of

Particle Diameters and Particle Size

Distribution

.

31
m

%

I

f .. T

'* Mp >*; ^pl

i^Sktr

^> . ^^r ^^

Figure 24. Microscope with Ocular Micrometer,

Counting Cells, and Stop Watch for

Swelling Studies

.

C . Characterization of Ion Exchange Beads

In addition to copolymer samples, numerous commercial

ion exchange resins are received by the microscopy laboratory.

Each of these materials undergoes microscopic examination and

cataloging in much the same manner as for copolymers. The

material is then either accepted or rejected using the same

criteria previously stated.

If a resin is accepted it must undergo chemical analysis,

homogeneity testing, and density measurements to insure

suitability for use as a chemical microstandard carrier. Ion

exchange resins which meet specification for microstandard

particles are subjected to electron microprobe analysis,

mass spectrometry, activation analysis, and various chemical

methods prior to certification.

32
Ion exchange particles examined by the electron microprobe are initially dispersed in a 0.1 percent solution of

polyisobutylene in cyclohexane and cast on a polished aluminum

or graphite disc. In the case of mass spectrometry, individual

particles of measured diameter are selected and transferred

directly to a filament which is then placed directly into the

ion chamber of the spectrometer. For activation analysis,

individually measured particles are selected following irradiation, and are mounted on a scintillation disc for counting.

Figure 25 shows a microscope with a micromanipulator and

some of the various mounts used for the analysis of microstandard particles.

Figure 25. Microscope with Micromanipulator and

Specimen Mounts for Microst andard

Particles

.

33
D . The Microscopy Laboratory Facility

The microscopy laboratory is equipped to perform conventional light microscopy via the use of transmitted light,

incident light, phase contrast and fluorescence, with magnifications up to 1500 X.

In addition to the light microscopes an electron microscope is used to examine very small particles and to study

structure defects and contamination in copolymer and Ion

exchange particles. The use of the electron microscope permits

direct observations of these materials up to magnifications

of 80,000 X.

Accessories to the electron microscope permit the

stabilization of substances via a vacuum evaporator, the

shadowing of specimens, and sample sectioning and preparation

using a microtome . The photographic equipment employed for

the preparation of photomicrographs obtainable from the

laboratory's light microscopes consists of several polaroid

cameras, a 4" x 5" plate camera, and a 35 mm camera. The

electron microscope is equipped with both a 35 mm and a

2 1/4" x 2 1/4" plate camera. A photographic enlarger and a dry rapid print processor complement this equipment.

The present capability of the microscopy facility to

observe microscopic defects, measure bead diameters with high

precision, and perform transfers of loaded ion exchange

particles-, forms a necessary and unique function in the Ion

Exchange Microstandards program.

34
IV. CHROMATOGRAPHY

A. Gas Chromatography

1 . Pi vinyl benzene

Divinylbenzene (DVB) Is widely used to prepare crosslinked poly (styrene/DVB) copolymers which may be derivatized

(for instance, by sulfonation) to produce ion exchange resins.

The importance of conducting analytical separations of DVB

sample components is obvious in the potential extension of

such studies to preparative scale separations to obtain the

pure isomers of DVB for copolymer synthesis, as well as to

provide detailed information regarding compositional makeup

of commercial DVB.

(a) Separations on Bentone-34

Historically, most of the separations of DVB mixtures

have been performed on a column composed of a mixture of

85:15:5 parts by weight of Chromosorb W: Bentone-3^: Ucon

oil [19] s and examples of this are reported in detail in a previous NBS report [20]. Since this information was released,

efforts were made to simulate detailed separations on Bentone34 of the order reported by Hannah, et_ al. [16] on Carbowax

6000 and di-2-ethylhexyl sebacate. A chromatogram representative of our work is shown in Figure 26.

(b

)

Separations on Liquid Crystals

The term "liquid crystal" implies a misomer, but in

reality such compounds evidence specific spatial structure in

the liquid state. That is to say, a liquid crystal material

does not become isotropic directly upon melting, but rather

may exhibit molecular alignment in three principal stable

structural phases prior to becoming isotropic; namely the

smectic, nematic and cholesteric phase states. Liquid crystals

as liquid phases in gas chromatographic separations provide

an extra dimension of solute selectivity based upon the spatial

geometry of their particular liquid state. Such materials

35
cnI CDcO

-PC

(D CQCo

to pc

(Dcoa6oo

PQ>P

CmOco

•H

-P

ctf M

cti

VD

CM

QJM

•H

36
have been successfully employed recently for difficult isomeric

aromatic separations by Kelker [21] in Germany and Martire [22]

in the United States.

Separations of DVB components have been found to be quite

dramatic on liquid crystals, specifically in the separation

of the meta- and para-DVB, and meta- and para-ethyl vinylbenzene isomers (Figure 27). The liquid crystal employed for

this work was recry stallized 4

,

H

' -dihexoxy azoxybenzene (DHAB)

at a 3 percent coating on 60/80 mesh acid-washed, DMCS-treated

Chromosorb G in a 12 foot by 0.125 inch O.D. stainless steel

1 1 l 1 1 1

1 11 1 1 1 II I

I I I I I I I I I I I I I I I I I

l

' I

I

I I I I I I I I I I M I I I I I I I I I I I I I

I I I I I I I

"i 1 r

4 6 8

Minutes

1 2 10 12

Figure 27. Separation of DVB Components on DHAB

Liquid Crystal.

37
column. The analysis shown in Figure 27 was conducted at

127 °C. This liquid crystal melts at 81.0 °C into a nematic

phase which becomes isotropic at 128.2 °C [23]. In the

nematic phase state model, the liquid crystal molecules are

arranged as non-layered parallel rods, enabling para-substituted

aromatics to "fit" into and interact with the liquid crystal

nematic phase more easily than met a and ortho substituted

derivatives. Additional distinguishing features of analyses

of DVB samples on liquid crystals relative to Bentone-34

columns provide (a) a much lower analytical temperature, (b) a faster analysis time, and (c) a realistic scale up of metaand para-DVB separations to preparative scale gas chromatography

The a separation factor for meta- and para-DVB is better than

1.2 and a complete analysis can be conducted in less than 15

minutes. Complete preparative separation of all sample components is indicated by lowering the temperature further

within the 81-128 °C nematic range. The data in Table III

provides a comparison of analysis of a DVB sample on Bentone-34

vs. DHAB liquid crystal.

(c ) Vinyl Toluene

Vinyl toluene (VT) has been examined as a candidate monomer

in place of styrene for copolymer synthesis. Gas chromatographic analysis of commercial vinyl toluene, using a liquid

crystal stationary phase, has revealed the presence of the

para- (peak 5) as well as the meta-VT (peak 4) as major components, and six minor impurities (Figure 28). Peak area

computations reveal that the meta (70.9 percent) and para

(28.8 percent) isomers compose 9 9-7 percent of the sample.

This information is essential for following reaction pathways

using a VT monomer composed of the mixed isomers.

38
Table III. Gas Chromatographic Separation

of DVB Components in Mixed DVB

Component

fc R

a Relative

Retention Perc

A

1.6

ent

A

d

B

e

3.8

A

0.72

B c f

.62

B

18.2 0.59 1.8

16.5 4.5 0.66 .70 0.65 0.6 0.8

25.1 6.4 1.00 1.00 1.00 77-7 79-0

23.8 7-7 0.95 1.20 1.07 I8.9 17.4

m- Ethylviny lb enzene

p -Ethylvinyl benzene

m-Divinylbenzene

p-Di vinylb enzene

Naphthalene 35-3 11.2 1.4l 1.75 2.07 0.4 0.4

Adjusted retention time in minutes

Relative to m-DVB as 1.00.

Relative chromatogram peak area percentages

dBentone-34 (NBS)

e DHAB liquid crystal (NBS)

fCarbowax 6000 (Ref. 16).

2 . Other Chromatographic Studies

(a) Sulfolane

Sulfolane (tetrahydrot hiophene-1, 1-dioxide ) was investigated by the Electrochemical Analysis Section as a potential

solvent for EMF measurements. Despite its low molecular

weight, it was not detected as a peak by gas chromatography.

It was possible however to determine its water content at

0.6 percent by weight by the method of standard addition with

an absolute error of less than 0.1 percent. An unidentified

contaminant peak was also present at 3-7 times that of the

water peak. Samples were dissolved in benzene and chromatographed on 80/100 mesh Poropak Q at 200 °C (Figure 29).

39
I I I

i I I I I I I I 1 1 1 I I 1 I I I I I 1 I 1 I I I I I I 1 1 1 I I I I II I I 1 1 I

fl>z zr zr: — c :— © 1

' 2 - (2\ i ± § :zzzzzzz:£)z:' zzzzzzzzzzzzzzzzzzzzzzzzzzzzz

zzfi izzzzzzzzHg zzzzzzzzzzzzzzzzzzzzzzzzzzzzz:

rzzzzz: 8° = — 1 w

v '

1 1 ± :: :QX: : :

/s\

= ::= = ==(§)== = :== = : F=z===== zzzzzzlzzzzzz zzzzzzzzzzzzzzzzzzzzz:

==== = ==="""== = =============="=====±======

IF

- LV

^ .= V

/f! s^

zi': z zzzzzzzz z zzzzzzzzzzzzzzzzzzzzzzzzzzzzz:

: S- := (3) :F= : == zzzzzzzzzzzzz zzzzzzz:

= ? = F==:(2S:F"i"""""""="-----------: ^±^t:b4-|t=:z^:~:~:::

==^e5^|I EEe|^zzzz|z^zzz=#=z:

±±.z :: ± ± 3:

"T

2

1 4

Minutes

6 8

Figure 28. Analysis of Vinyl Toluene on DHAB

Liquid Crystal.

(b) 2-Methoxy Ethanol

This material, commonly known as methyl cellosolve, has

also been considered as a solvent for EMF measurements by the

Electrochemcial Analysis Section. Commercial samples were

evaluated by gas chromatographic analysis on 80/100 mesh

Poropak Q at 200 °C using a thermal conductivity detector at

a helium flow rate of 40 ml/min., for the presence of water

and volatile impurities. The peak area response factors

40
Benzene

Fig. 29 Determination

of Water in

Sulfolane

.

Sulfolane Peak

Not Obtained

Unidentified

Contaminant

Minutes

41
calculated separately for water and 2-methoxy ethanol were

1788 and 1080 mm /mg, respectively. Methanol, cited as a known degradation product of 2-methoxy ethanol, was not

detected. The water content of the samples was found to be

0.036 weight percent. A chromatogram showing the separation

of water, methanol, ethanol, and acetone on Poropak Q under

these conditions is shown in Figure 30.

- A. — 1 4. ::: ~:

:'j:~l

1 P

::=

._ .. , 1 H

2

Oo. ,

. ...L_ tr 1

o.,.L_ j

. 1 ...... —

— <JMeOK

= r :

1... , — dl

EtC>h11 — I A

- +*

, — ~ p"

\up

1

" !__

1 —

i_ ~ZZXI1

"

zi::; '-. <

- - t It

1

1

1

1 1 j 1 1 1

1

L

>

' 1 t -1

\i ._ .- -T 1

' _ | r -

' "7^ :—

<

/ B+- 1

Figure 30. Separation of Volatiles on Poropak Q

(c ) Ortho-Fluorobenzoic Acid

This compound is issued as a Standard Reference Material

(SRM 1^9) for microanalysis for fluorine. Gas chromatography

of the silylated acid by temperature programming from 100 to

300 °C on 10 percent UC-W58 silicone coated on 80/100 mesh

Chromosorb W using a flame ionization detector shows that

analysis was simple and rapid.

42
3 . Gas Chromatography -Mass Spectrometry

(a) The Quadrupole Mass Spectrometer

The quadrupole mass spectrometer is different from most

other mass spectrometers because it utilizes only an electrostatic field rather than the magnetic and electrostatic fields

of conventional magnet instruments to produce a mass spectrum.

In conventional spectrometers the magnets required to produce

an adequate magnetic field are necessarily large In contrast

to the quadrupole system.

The sample enters the ionization region of the mass

spectrometer as shown in Figure 31. The resultant sample

FOCYSING

ELECTRODES

GAS

SAMPLE'

V

%==^

ELECTRON BEAM

FOR IONIZATION

-COLLECTOR

FIRST STAGE OF

ELECTRON

MULTIPLIER

Figure 31- Quadrupole Filtering Schematic.

fragment ions are focused by the focusing electrodes and enter

the quadrupole portion of the spectrometer. The quadrupole

portion is composed of a mass filter which consists of two

pairs of precisely aligned metal rods. The rods are electrically connected, in opposite pairs, to radio frequency

(RF) and direct current (DC) potentials resulting in a hyperbolic electrostatic field. To produce a mass spectrum

the RF and DC potentials are increased uniformly in time from

zero to maximum potential. At a given time, ions of a

43
specific mass -to -charge (m/e) ratio are deflected as much

toward one rod as they are toward another. Ions of this

particular m/e value are initially excluded. As the voltages

sweep from zero to their maximum values, the entire mass range

(0 to 750 m/e) is scanned. The range thus starts at low m/e

values and increases linearly to high m/e values.

Ions which avoid deflection and traverse the field are

detected by an electron multiplier and produce an electrical

signal which is amplified, and monitored on an oscilliscope

.

A recording of the mass spectrum can be initiated at any time

by an oscillographic recorder contained within the unit to

photograph the spectrum displayed on the oscilliscope. The

system can produce a mass spectrum from m/e 10 to m/e 250 in

less than three seconds with unit mass resolution,

(b) The GC-MS Interface

The ability of the quadrupole mass spectrometer to

rapidly scan the m/e range of to 750 renders it especially

useful as a qualitative detector for organic vapors eluting

from a gas chromat ograph (Figure 32). The gas chromat ograph

however, operates at carrier gas pressures of a few atmospheres

while the MS operates at pressures in the range of 10 to

one atmosphere. In order to avoid swamping of the MS detector

with ionized bulk carrier gas (He), an interfacing assembly is

used to pump away most of the light GC carrier gas thereby

enriching the gas stream entering the MS in sample eluent

.

The interfacing assembly is shown in Figure 33'

Gas flowing into portion A proceeds to the capillary

portion D. In order to gain access to the MS, the gas must

traverse the distance between capillaries D and C. The

presence of a vacuum at this point removes most of the light

helium carrier gas molecules from their D to C trajectory.

Solutes of sufficient momentum (greater mass) eluting from

the GC, proceed to point C and enter the mass spectrometer.

44
Figure 32. The GC-Mass Spectrometer System

FRITTED GLASS

B C D

TO MASS

SPECTROMETER

\ It

\

TO VACUUM

FROM GAS

CHROMATOGRAPH

Figure 33. Schematic of GC-MS Interface

45
The utility of combined gas chromatography -mass

spectrometry will undeniably lend strong analytical support

to the programs of the Separation and Purification Section

in the areas of applied and basic research and development.

This instrumentation is presently being applied to the

identification of unknown components present in commercial

divinylbenzene j and will be extended to the identification of

isolated contaminants in clinical Standard Reference Materials.

B. Liquid Chromatography

1. Introduction

Recent developments leading to improved resolution and

faster analysis in ion exchange and liquid-liquid chromatography have resulted in a resurgence of interest in these

analytical techniques. Efficiencies and elution times in

optimized systems can be made to approach those commonly

experienced in gas chromatography. Column efficiencies in

liquid systems may be expressed by theoretical equations

analogous in form to the modified van Deemter equation for

gas-liquid chromatography. An important exception is that

the diffusion coefficient in liquids is four orders of magnitude smaller than in gases. The ability of a system to

provide resolution is governed by the smallness and size

uniformity of the particles employed as column packing material,

the uniformity of the packed bed, the thinness of the film or

ion exchange l^yer coating the particle, the smallness of

sample sizes introduced into the column (which creates a requirement for sensitive detectors), and the narrowness of

the column bore to minimize trans-column flow variations.

All of these factors are pointed toward minimizing the

theoretical plate height for a given column length and solvent

carrier flow rate. The use of very small particles (10-50

microns in diameter) produces a sizeable resistance to flow

such that, to insure that retention times of solutes are not

prohibitive, pressures on the order of 100-1000 p.s.i. are

46
used with thick-walled glass columns (for example: 0.25" I.D.

with a 0.125" wall). Pressures as great as 3000 p.s.i. or

higher require the use of stainless steel columns, where

column bore diameters as small as 2 mm are appropriate.

2 . Instrument Design

A liquid chromatograph suitable for liquid-liquid,

liquid-solid, and ion exchange is presently in use in our

laboratories. High inlet pressures require reliable pumps.

A precolumn or a presaturated carrier solvent reservoir is

used with liquid-liquid chromatography to presaturate the

mobile solvent phase, which should be adequately degassed to

avoid the escape of air bubbles at the low pressure end of

the column or in the detector. Pressures and liquid flow

rates are carefully monitored, and the feature of measured

column thermostatting is desirable. The injection port should

not allow the entrance of air upon sample injection, and a high-quality high-pressure valve with a changeable sample

loop (as used by Scott at ORNL ) has been concluded to be

nearly ideal. However, septum injection utilizing .a double

septum in the injection port has also been found to work well

at inlet pressures up to 800 p.s.i. (Figure 3^) • The connection from the column exit to the detector flow cell inlet

(Figure 35) and the flow cell itself should be as small in

volume as possible. Finally, the detector should be highly

sensitive to compositional changes in the column effluent,

having a low electronic noise level, fast response, and

preferably low selectivity to solute type. Although no

specific detectors are suggested, the differential refractive

index detector and the ultraviolet photometric detector

(Laboratory Data Control, Danbury, Connecticut), and the

microadsorption detector (under investigation at Oak Ridge

National Laboratories), are detection devices which we have

considered. Acknowledgment for considerable assistance

obtained in suggestions and informative discussions are owed

47
"iT"

'

Figure 34. Sample Inlet System for Septum Injection.

to Dr. Paul B. Hamilton (Alfred I. du Pont Institute,

Wilmington, Delaware), Dr. Charles Scott (Oak Ridge National

Laboratories, , Oak Ridge, Tennessee) and Dr. Jack Kirkland

(E. I. du Pont de Nemours, Wilmington, Delaware), among many

others

.

3 . Clinical Standard Reference Materials - Bilirubin

Liquid chromatography permits the analysis of underivatized high molecular weight, heat-labile compounds which may

not be successfully analyzed by gas chromatography. Numerous

and diverse clinical materials of a biological nature tend to

fall into this domain. The necessity for accurate separation

and measurement of impurities in such compounds forms a part

of our clinical Standard Reference Materials program. The

Analytical Chemistry Division is involved in the examination

of bilirubin samples obtained from various sources. The

bilirubin molecule contains two vinyl, two propionic acid,

four methyl, and two tautomeric hydroxyl residues, attached

48
Figure 35- Connection from Column Exit to Detector.

to four pyrrole rings and is clinically used as a diagnostic

indicator for jaundice. Bilirubin is unstable when under

exposure to light and heat, in certain basic solutions, and

is oxidizable under mild conditions to biliverdin. The

chromatography of this compound has been obtained on a preliminary system at an inlet pressure of 570 p.s.i.g. on an

aluminum foil-covered column packed with a 10-30 urn cation

exchange resin using a differential refractive index detector

(Figure 36). A schematic of this system is presented in

Figure 37- The analysis of an impure bilirubin sample obtained

on this system is shown in Figure 38. The carrier solvent

used in this chromatogram had the composition 0.02 M NHkOH-0.02 M

NHhCl. The stability of bilirubin in this solvent is not

49
Figure 36. Preliminary Ion Exchange Chromatographic

Sy stem.

ideal. Other solvents for the chromatography of bilirubin are

being considered. Its chromatography in dimethylsulfoxide

(DMSO), an excellent solvent for bilirubin, is under investigation. Spectrophotometric measurements in DMSO gave a comparable final reading (0.795 A) to the initial reading

(0.79^ A) over a period of forty minutes. The molar extinction

coefficient for bilirubin in DMSO was calculated from this

data to be 61.92 x 10 3.

50
n it

A. Hot plate supporting solvent reservoir

B

.

Pump

C. Sample inlet (syringe injection)

D. Pressure gauge

E. Analytical column

F. Detector (R.I. or U.V.)

G. Recorder

Figure 37. Schematic of Ion Exchange Chromatographic

System.

51
12 10

i 8

1 6

Minutes

2

~lO

Figure 38. Analysis of an Impure Bilirubin Sample

by Ion Exchange Chromatography.

52
V. REAGENTS

A. Introduction

The increasing sensitivity of instrumental analysis,

coupled with the rising significance of trace impurities in

wet chemical analysis, necessitates the availability of

reagents whose purity criteria far exceed those of the normal

"CP" or "reagent grade" classification. This is especially

true at the National Bureau of Standards where reagents with

impurities measurable in parts per billion must now be

available. When reagents in this purity range are needed in

the Analytical Chemistry Division it is the obligation of

this Section to make them available.

While formerly it was necessary to synthesize such

materials ourselves, the potential market in ultra pure

chemicals has encouraged commercial firms to expand in a direction to fulfill this need. The more common chemicals

can be purchased in quantity, while more specialized ones

can often be obtained by specific order. These aspects of

our reagent program are covered in Section B, below. There

are, however, instances when commercial sources cannot answer

our problems, and the synthetic work must be done by this

Section, as is discussed in Section C, below.

In addition to fulfilling the immediate needs for ultra

purity in the reagent field, research on certain aspects of

contamination removal has been undertaken. The use of a microturbidimeter to accurately measure particulate matter to

one part per billion In high purity reagents is shown In

Figure 39- Previous studies on reagent contamination via

entrained foreign materials available in container walls have

been continued and are reported in Section D below.

53
Figure 39 • Microturbidimetric Measurement of

Particulate Matter Content.

B. Ultra Pure Reagents Services

In recent years it has been possible to purchase from

commercial sources the more common reagents in ultra pure

form and to maintain a stock of these chemicals on hand. To

retard their decomposition during storage, the chemicals are

kept in freezers. A selection of such chemicals available

for immediate "self-service" distribution is kept at °C,

while a larger quantity is kept on reserve at -30 °C. Of

the 27 separate reagents stocked for direct use, 145 items

were dispensed over the past year (Table IV). This is an

increase of roughly 250 percent over the previous year.

54
Table VI. Number of Units of Ultra Pure

Reagents Dispensed in FY 1969«

Acids Bases

CH COOH 2

HBr 2

HC1 19

HF 8

HI

HNO- 24

HCIO^ 19

H o S0,, 9

NH^OH 21

NaOH 1

Redox

Salts

CaCl

2

4

LiBr 2

KC1 3

NaCl 5

BaCl

2

2

BaF 1

Mg(N0

3

) 2

2

NaF 3

Na

2

CO^ 2

Br

2

1

h

LiF 1

KI 2

Na

2

S

2 0^ 1

Csl 3

Nal 1

Buffer Salt s NH^Cl 3

CH COONa 3

Na

2

SO^ 1

In addition, special requests for specific reagents

needed by people in this or other Sections at NBS have been

met through contact maintained by us with commercial manufacturers. For example, the difficulty in obtaining leadfree phosphoric acid for mass spectroscopy was solved by

advising its preparation from re-sublimed phosphorous pentoxide

A sample of ULTREX P^ (J. T. Baker Co., Phillipsburg, New

Jersey) was used in this instance.

55
C

.

Ultra Pure Reagent Synthesis

For the Section's Microstandard program a need arose for

ultra pure inorganic chemicals which could be loaded on ion

exchange beads. Spectrographically standardized reagents

(Johnson, Matthey Co., Ltd., England) are used for this purpose.

However, it is not always possible to purchase directly the

chemical compound desired; the requisite compound must then

be synthesized from some other available ultra pure compound.

In preparing the ion exchange microstandards of chromium and

magnesium only the insoluble oxides were obtainable. Therefore,

the suitable chemical forms, chromium (III) perchlorate and

magnesium chloride were prepared from the respective oxides.

D

.

Container Purification

In a previous report [1] it was noted that the constant

iron contamination of re-distilled hydrochloric acid was

traceable to Iron-containing particles embedded in the polyethylene receiver. The removal of such entrained container

contamination became a matter of necessary concern.

The same problem was encountered using Teflon PEP bottles.

Here however, the inertness of the container material permitted

drastic chemical methods for contamination removal from the

container, even for contamination existing beneath the container

surfaces. Figure 40 illustrates the result of using boiling

nitric acid in removing such entrainments

.

56
Photomicrograph of opaque particle

embedded in wafer of Teflon PEP.

Particle is positioned ca . 70 microns

below surface. (125X)

Photomicrograph as per above, but

after 60 hours of immersion in boiling

nitric acid. (125X)

Figure 40 . Effect of Boiling Nitric Acid in Removal

of Container Contaminants.

57
VI. PERSONNEL AND ACTIVITIES

A . Personnel Listing

Separation and Purification Section

David H. Freeman, Section Chief

Janice M. Hurst, Secretary

Ion Exchange

Rosalie Angeles (Part time)

Lawrence Morgenthaler (Summer faculty participant

from Georgetown University)

Robert Young (Summer student participant from

Georgetown University)

Chromatographic Separations

William Dorko

Delmo Enagonio

Walter Zielinski

Sister Laura Turbini (National Science Foundation

Summer Institute for Teachers

of Physics and Chemistry)

Particulate and Microscopic Studies

Patti Byrne (Guest Student Worker, University of

Delaware

)

Jane Doyle

High Purity Reagents

Edwin Kuehner

Gerald Sleater

Microscopic Standards

Herbert Dixon

Vincent Story

Youth Opportunity Corps

Linda Gant

Sylvia Prather

58
B. Talks

1. D. H. Freeman , W. F. Rittner, "Studies of Ion Exchange

Resin Structure," 156th National Meeting of the American

Chemical Society, Atlantic City, New Jersey. September

11, 1968.

2. D. H. Freeman, "Particles in Solution by Laser-Light

Scattering," Scientific Apparatus Makers Association,

National Bureau of Standards. September 17, 1968.

3- D. H. Freeman, "Microstandards for Activation Analysis,"

1968 International Conference on Modern Trends in

Activation Analysis, National Bureau of Standards.

October 8, 1968.

4. D. H. Freeman, "Ion Exchange Resin Standards: Characterization and Applications," Howard University, Washington,

D. C. November 21, 1968.

5. D. H. Freeman, "Chemical Microstandards," American Microchemical Society, Hoboken, New Jersey. December 12, 1968.

6. D. H. Freeman, "Chemical Microstandards," American

Chemcial Society, 4th Annual Middle Atlantic Regional

Meeting, Washington, D. C. February 13, 1969-

7. D. H. Freeman, "Chemical Microstandards," 20th Pittsburgh

Conference on Analytical Chemistry and Applied Spectroscopy,

Cleveland, Ohio. March 5, 1969.

8. D. H. Freeman, "Studies of Ion Exchange Networks,"

Georgetown University, Washington, D. C. March 19, 1969-

9. W. L. Zielinski, Jr., "High-Resolution Liquid Chromatography," Washington Gas Chromatography Discussion Group,

Rockville, Maryland. May 15, 1969.

10. D. H. Freeman, "Light Scattering Measurements of Sub-PPM

Concentrations of Liquid-borne Particulate Matter,"

1969 ASTM Convention, Atlantic City, New Jersey.

June 23, 1969.

59
C. Publications

1. D. H. Freeman, "Separation and Purification Section:

Summary of Activities July 1967 to June 1968, " NBS

Technical Note 459, (1968).

2. D. H. Freeman, Book Review: "AnalaR Standards for

Laboratory Chemicals," Anal. Chem. 4_0, 75A (1968).

3. D. H. Freeman and E. C. Kuehner, "Containers for Pure

Substances," (Chapter 30) in Purification of Inorganic

and Organic Materials , Marcel Dekker, Inc., New York,

N. Y., 299-306 (196b).

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

"Recognition of Crosslinking in the Infrared Spectra of

Poly ( styrene/divinylbenzene )

, " accepted for publication

in the Proceedings of the International Conference on

Ion Exchange in the Process Industries.

5. D. H. Freeman, Book Review: "Reagent Chemicals," Anal.

Chem., 41, 87A (1969).

60
VII. REFERENCES

[ 1] National Bureau of Standards Technical Bote 439 3 D. H.

Freeman, Ed., December 1968.

[ 2] Kuehner, E. C. and Freeman, D. H. , "Purification of

Inorganic and Organic Materials j" M . Zief, Ed., Marcel

Dekker, Inc., New York, N. Y., 1969, Chapter 30, p. 297-

[ 3] Nelson, F., Murase, T., and Kraus, K. A., J. Chromatog .

_, 13, 503 (1964).

[ 4] Freeman, D. H. , Currie, L. A., Kuehner, E. C, Dixon,

H. D., and Paulson, R. A., "Chemical Micro standards ,

submitted to Anal. Chem. for publication.

[ 5] National Bureau of Standards Technical Note 505 3 J. K.

Taylor, Ed. , 1969-

[ 6] National Bureau of Standards Technical Note 504 3 0.

Menis, Ed., 1969.

[ 7] Provisional Certificate 1800, Microstandard Ion Exchange

Beads, Calcium Loaded, N.B.S., 1969 (see Fig. 6).

[ 8] Wiley, R. H. and Venkatachalam, T. K., J. Poly. Sci. 3 4 3 1982 (1966).

[ 9] Ref. [8]; and Ref. [1], p. 19.

[10] This Report; and Ref. [11].

[11] Winslow, F. H. and Matreyek, W. , Ind. Eng

.

3 43 3 1108

(195D.

[12] Boyd, G. E. and Soldano, B. A., Z. Electrochem. 3 57 3 162 (1953).

[13] Ref. [1], p. 6.

[14] Pepper, K. W. , J. Appl. Chem. 3 l 3 124 (1951).

[15] Wheaton, R. M. and Hatch, M. J., in "Ion Exchange,"

Vol. II., J. Marinsky, Ed., Marcel Dekker, Inc., New

York, N. Y. , 1969, p. 200.

[16] Hannah, R. E., Cook, M. L. and Blanchette, J. A., Anal.

Chem. 3 39 3 358 (1967).

[17] Zielinski, W. L. Jr., personal communication.

61
REFERENCES (Con't)

[18] Freeman, D. H. and Aiyar, A. S., Anal. Chem. 3 39 3 ll4l

(1967).

[19] Wiley, R. H., Allen, J. K., Chang, S. P., Musselman,

K. E. , and Venkatachalam, T. K. , J. Phys. Chem. 3 68 3 1176 (1964).

[20] Ref. [1], p. 21.

[21] Kelker, H. and von Schivizhoffen, E., "Advances in

Chromatography 3 " Vol. 6, J. C. Giddings and R. A. Kelker,

Eds., Marcel Dekker, Inc., New York, N. Y., 1968, p. 247.

[22] Martlre, D. E., Blasco, P. A., Carone , P. F. , Chow, L. C.

and Viclni, H., J. Phys. Chem. 3 72 3 3489 (1968).

[23] Chow, L. C. and Martire, D. E., J. Phys. Chem. 3 73,

1127 (1969).

* V. 8. GOVERNMENT PRINTING OFFICE : 1 970—392-2^9/1 1

1

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