f-

•0 STATES fMENT OF 1ERCE

CATION

NBS TECHNICAL NOTE 589

Separation and Purification

Section:

Summary of Activities

July 1970 to June 1971
NATIONAL BUREAU OF STANDARDS

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snwtot mm of stans^^

NOV 12 $71

UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

(J ^ -NATIONAL BUREAU OF STANDARDS • Lewis M. Branscomb, Director

'' ^**^

NBS TECHNICAL NOTE 589

ISSUED OCTOBER 1971

Nat. Bur. Stand. (U.S.), Tech. Note 589, 64 pages (October 1971; CODEN: NBTNA

Separation and Purification

Section:

Summary of Activities - July 1970 to June 1971

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

<&a °f c%

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

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

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• .
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 60 different analytical comnetences

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 exulored 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 cover e.c

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 our sections.

The following is the fifth annual report on progress of the

Separation and Purification Section.

W. Wayne Meinke , Chief

Analytical Chemistry Division

IV
PREFACE

This is the fifth annual progress report of the Separation

and Purification Section. The guiding philosophy of the Section

is to concentrate our efforts on applications and improvements

of the separations field which are most important for the

achievement of national and scientific goals. This past year

started with major needs to expand and strengthen our capabilities in the practice and understanding of liquid chromatography.

We devoted all available energy to this.

The growth of interest in liquid chromatography has come

from the cooperative NBS-NIH program in clinical chemistry, of

the need with the NBS program aimed at ecological monitoring

known as Measures for Air Quality, and several other smaller

projects in the area of organic analysis. The chromatography

of bilirubin, which we did with some difficulty two years ago,

taught the frustration of working with an unstable compound

having low solubility properties. Our present responsibilities

provide a strong stimulation to our search for a way to predict

the conditions for successful chromatography. The advantages

of this are obvious.

Apparatus development was the important starting point

since we required considerable flexibility to change design

configurations, whenever needed. We were also faced with the

need for an estimated 10 or more liquid chromatographic units,

but a budget that would not permit this unless we built the

apparatus economically. We were thus forced to explore the

potential of do-it-yourself instrumentation. Fortunately,

the instrumentation problem with liquid chromatography is much

simpler than that in gas chromatography. The details of our

apparatus development work are described at length in this

report- -with the hope that it may stimulate those who would

enjoy the pursuit of liquid chromatography, but who are impeded

by the lack of design information.

v
The scientific understanding of the stationary phase is

the focal point for the efficient use of liquid chromatography.

There are many kinds of stationary phases , which makes this

subject extremely versatile and powerful in the carrying out

of efficient chemical separations. There are also many

opportunities to go wrong by non-productive trails with

improperly chosen materials. The conventional separating

materials for the organic chemist are silica gel and alumina,

two inorganic solids that are extremely difficult to predict,

control or reproduce in performance. In an attempt to

simplify the problem of organic separations we decided to

develop our own stable of stationary phases. Our reasoning

followed from the recognition that the powerful ability of ion

exchange resins for inorganic separations can be traced

specifically to the decision to locate a specific kind of

functional group upon an otherwise inert but porous network.

For organic compounds, such functionalization can be tied to

those interactions which lead toward complex formation. This

pathway has proved exciting, and progress was made in the

development of "interactive gel networks" which are reported

here in terms of both the synthesis and the preliminary

understanding of the theory.

The use of brand names are only for the purpose of

description and indicates that the equipment was used by this

laboratory and was found to be satisfactory. This does not

imply that these are the best or only available components

for the job.

The preparation of this report was expertly assisted by

Mrs. Janice Hurst, secretary to the Section, and this we

gratefully acknowledge.

Special Notice of Hazard to Users of Chloromethyl Methyl

Ether: see page 48.

David H. Freeman, Chief

Separation and Purification Section

VI
TABLE OF CONTENTS

PAGE

LIQUID CHROMATOGRAPHY 2

A. Description of Apparatus 2 1. Precolumn Components 2 2. The Reference Stream 7

3. Pressure Monitoring 7 4. The Analytical Column 8

5. Detectors 9

6. Flow Measurement 12

7. Comments 13

B. Parameters of Operation 14

1. Packing the Analytical Column 14

2. Mobile Phase 17

a. Depulsing 18

b. Flow Balancing 18

3. Column Efficiency 19

4. Fraction Collecting 21

5. Sample Introduction: Methods and

Problems 23

6. Measurement of Retention Volumes . 29

C. Peak Authentication 31

1. Spectrophotometric 31

2. Gas Chromatography-Mass Spectrometry .... 32

3. Proton Magnetic Resonance 36

4. Computerized Measurement of

Chromatographic Peak Areas 37

D. Column Packing Materials 37

1. Introduction 37

2. Steric Exclusion Chromatography 39

a. Measurement of Crosslinking 41

b. Performance 41

c. Limitations 41

3. Interactive Gels 41

a. Description 41

b. Synthesis 42

(1) Acidic Gels 42

(2) Basic Gels 42

GAS CHROMATOGRAPHY 49

A. Quantitative Analysis 49

1. Eicosane 49

2. Nitrilotriacetic Acid (NTA) 49

3. Tetramethylsilane (TMS) 50

VII
TABLE OF CONTENTS (Cont'd.)

PAGE

3. PERSONNEL AND ACTIVITIES • 51

A. Personnel Listing 51

B. Talks 52

C. Publications 52

4. REFERENCES 5 3

Vlll
LIST OF FIGURES

FIGURE NO. PAGE

1. General scheme of the liquid chroma to graph ... 3

2. Photomicrograph of 30-60 ym silica gel

used in resistance tubes 6

3. Components used in the construction of

resistor tube 8

4. Column inlet fitting 9

5. Column outlet fitting 10

6. Column components prior to packing 10

7. Connection of column to detector 12

8. The assembled liquid chromatograph 13

9. Schematic of depulsing and balanced

flow system 19

10. Illustration of balanced flow system 20

11. Liquid chromatograph including fraction

collector 22

12. Schematic of actuator circuit for

fraction collector 23

13. Concentration of collected fractions 24

14. Infrared spectrum of 10 yg of Cortisol

obtained by concentration 25

15. Inlet fitting for syringe injection 26

16. Two-way valve for sample introduction 27

17. Sample introduction with the two-way valve ... 28

18. Flowmeter for measurement of effluent

flow rates 30

19. Microdye assembly for preparation of

1 mm KBr pellets 33

20. Infrared beam condenser (4X) containing

a 1 mm KBr sample pellet 34

ix
LIST OF FIGURES (Cont'd.)

FIGURE NO. PAGE

21. Gas chromatographic data acquisition system ... 35

22. Thermal conductivity detector and pneumatic

valving system for GC/MS analysis 35

23. XY recording of a chromatographed sample

showing peak integrals 38

24. Interactive gel networks for organic

separations 43

25. Chloromethylation of copoly (styrene/DVB)

beads 45

26. Alkaline hydrolysis of chloromethylated

copoly (styrene/DVB) beads 46

27. Infrared spectrum of copoly (2-methyl- 5-

vinylpyridine/O .08 DVB) 47

28. Photomicrograph of copoly (2-methyl- 5-

vinylpyridine/O .08 DVB) 47

29. Mass spectrum of silylated NTA eluted from

a gas chromatographic column 50

30. Gas chromatogram of tetramethylsilane 50

LIST OF TABLES

TABLE NO. PAGE

1. Identity and sources for components

identified in Figure 2 4

22. Comparison of stationary phase types

used in different forms of liquid

chromatography 40
SEPARATION AND PURIFICATION SECTION: SUMMARY OF ACTIVITIES

JULY 19 70 to JUNE 19 71

Edited by David H. Freeman and

Walter L. Zielinski, Jr.

ABSTRACT

This is the annual progress report on the activities of

the Separation and Purification Section. During the past year

our studies in liquid chromatography, its application, control,

and systems design was continued at an accelerated rate.

Mobile phase flow was treated as an analogy to current in

direct current circuitry, following removal of liquid pulses

generated by a piston pump. A sophisticated, yet simple and

inexpensive liquid chromatograph design is fully described.

This design has been incorporated into all liquid chromatography studies being conducted in this Section. The operating

parameters of this system design are discussed. The development

of the underlying theory and synthesis procedures for interactive gels has led to the control and prediction of solute

retention, selectivity, and a magnified column capacity for

use in fraction collection and ancillary characterization of

solute impurities in clinical Standard Reference Materials.

A gas chromatographic method is described for the analysis of

nitrilotriacetic acid based upon the formation of its trisilyl

ester derivative.

Key words: Clinical Standard Reference Materials; fraction

collecting; gas chromatography; interactive gels; liquid

chromatography design; nitrilotriacetic acid.
1 . LIQUID CHROMATOGRAPHY

A. Description of Apparatus

For the past several years, an extensive effort has been

undertaken in our Section tailored towards the development and

assembly of simple liquid chromatographic systems. A portion

of this effort was described in last year's report [1] . The

requirements for such systems are that they be inexpensive,

versatile, easy to assemble and troubleshoot , and that they

conform to state-of-the-art performance in liquid chromatographic analysis. This document will detail the components

which we have incorporated into a working system, and the use

of a functioning system for chromatographic separations. Our

work has essentially been directed toward the use of gel

packings, although other materials are equally applicable.

This section of this report will illustrate the various

materials used for the construction of a simple liquid

chromatograph. A major objective of the report is to encourage

an increased world-wide participation in studies involving

liquid chromatography at moderate pressures using simple

apparatus of excellent quality.

1. Precolumn Components . In order to assemble an

apparatus for the best performance of liquid chromatography,

certain system requirements must be met. It is the aim of

this section of the report to explain the specific design

employed by this laboratory in the construction of a liquid

chromatograph. It is hoped that some of the problems encountered in our earlier systems may be avoided by anyone desiring

to enter the field of liquid chromatography for the first time.

A rack or mounting frame of some fashion is required to

support the column and other components of the system. From

the operators view point it is desirable to have the components

mounted in a manner which presents the least difficulty for

injecting samples and making minor adjustments. The general
scheme of the liquid chromatograph is provided in Figure 1

and Table 1.

SEE FIGURE

DETECTOR

RETURN

LINE TO

RESERVOIR

FI6

T2

H6 TO WASTE,

J>* COLLECTION

T2 OR FLOWMETER

Figure 1. General scheme of the liquid chromatograph
Table 1. Identity and sources for components

identified in Figure 2.

Code Major Components Source

A Glas-Col heating mantle 1

B Round bottom flask, S/T 24/40, 500 cm 3 1

C Condenser, water-cooled, S/T 24/40 1

D Controlled volume miniPump, #196-31 2 E Stainless steel air tube, 6 mm o.d. 1

G Connector tube, 3 mm o.d. stainless 1

steel

H Silica gel filter tube, 6 mm o.d. 1

stainless steel

I Silica gel resistor tube, 3 mm o.d. 1

stainless steel

J Pressure gauge, 0-300 lb/in 2

, 1

5 lb/in divisions

K Cheminert sample injection valve, 3

#R60SV, with ring stand mounting

bracket, #R60MB

L Sample loop 3

M Luer syringe, glass 1

N Sample vial 1

P Precision bore column; 0.250" ± 0.0003" 4

i.d., 1/2" nominal o.d., tooled on

both ends 0.250" (tolerance of o.d.

flange + 0.005", - 0.000")

Q Liquid flow detector 5

Code Fittings Source

Fl 400-2-2-316, Swagelok male elbow 6

F2 401-A-2-316, Swagelok male adapter 6

tube to pipe

F3 400-3-316, Swagelok union tee 6

F4 400-C-316, Swagelok cap 6

F5 400-R-2-316, Swagelok reducing union -6 F6,7 200-6-316, Swagelok union 6

F8 200-4-316, Swagelok union cross 6

F9 100-R-2-316, Swagelok reducing union 6

F10 200-7-4-316, Swagelok female connector 6

Fll TEF107, Tube end fitting, and 107A3 3

coupling

F12 107B9, Female luer adapter 3

F13 TEF107, Tube end fitting 3

F14 687-053-0014, Stainless steel coupling 4

with 688-358-0014 insert

F15 200-6-1-316, Swagelok reducing union 6

F16 100-6-316, Swagelok union 6
Table 1. Cont'd.

Sources

1 Standard sources

2 Milton Roy Company, P.O. Box 12169, St. Petersburg,

Florida 33733

3 Chromatronix, Inc., 2743 Eighth Street, Berkeley,

California 94710

4 Fischer § Porter Company, Warminster, Pennsylvania

18974

5 Laboratory Data Control, P. 0. Box 10235, Riviera

Beach, Florida 33404, and review article: J. Chem.

Educ. 47, A549 (1970); ibid., 4_7, A675 (1970).

6 Crawford Fitting Company, 29500 Solon Road,

Solon, Ohio 44139.

The first component of the liquid chromatograph proper is

the reservoir (B) for maintaining a reserve of the mobile phase.

In our work we have found that a round bottom flask of 500 ml

or 1,000 ml capacity with a standard taper ground glass joint

and a simple water cooled reflux condenser is adequate. The

round bottom flask is placed in a "Glas-Col" heating mantle (A)

and a conventional powerstat is used to control the current for

sufficient heat to give a very gentle reflux (C) action. It is

not advisable to boil the solvent, but rather to supply only

enough to heat to drive off the dissolved gases in the liquid.

From the reservoir a Teflon tube (Tl) 1/8 in o.d. by 1/16 in

i.d. is attached to a liquid metering pump (D) which is used

to drive the mobile phase through the packed column. The two

pumps most commonly used for this purpose are the reciprocating

piston type and the high volume syringe type. Both types

produce pressure pulses on each pumping stroke. The piston

types normally operate at 15 to 35 strokes per minute and the

syringe types operate at 1 to 4 strokes per hour depending on

flow rates used.

For continuous operation the piston type pump is more

desirable if the pressure pulses can be made negligible. A

method has been devised by this laboratory to dampen the

pulsation to a level that is not detectable by the liquid

chromatographic monitors [2] in use. A flow resistive network
analogous to a simple electrical circuit is employed. Further

details of this approach are covered in section l.B.2.a. of

this report. The construction and assembly details of the

depulsing system are covered below.

The pump is used with our depulsing system, for driving

mobile phases through columns at operating pressures of less

than 500 lb/in 2. An important element of the depulsing network consists

of a 12 in length of 1/4 in o.d. standard wall stainless

steel tubing (E) . One end is capped so as to trap air in the

tube and the other end is fitted to the pump with a tee (F3)

perpendicular to the liquid flow stream. On the pumping stroke

the air is compressed and on the filling stroke the air expands,

analogous to the charging and discharging action of a capacitor.

A resistive element (H) is placed in the line to increase the

discharge time constant of the "capacitor," consisting of a length of 1/8 in o.d. standard wall stainless steel tubing

packed with 30 to 60 ym particles of silica gel. Figure 2 is a

Figure 2. Photomicrograph of 30-60 ym silica gel

used in resistance tubes.
photomicrograph of the silica gel with a 10 ym scale to show

the size distribution of the particles.

2. The Reference Stream . The amount of resistance

required in the reference stream (I) for balancing its effluent

flow with that of the analytical column is determined by the

equation given in section l.B.2. of this report. The required

length of steel tubing is filed flat and smooth across the

ends and all burrs are removed. A 1/8 in diameter 400 mesh

stainless steel screen disc is placed inside a 1/8 in stainless

steel union. One of the connectors containing a screen is

attached to the end of the tube and dry 30 to 60 ym silica gel

is added. The silica gel is uniformly packed in the tube by

tapping to a density of 0.52 g/cm 3 [2]. In order to verify

proper packing of the tube it is advisable to weigh the resistor

element before and after filling and calculate the packing

density. Small scissors are used for cutting the wire gauze

circles. The addition of silica gel to the tube is greatly

facilitated by a small plastic wash bottle. Figure 3 shows

the components used in construction of the resistor.

3. Pressure Monitoring . In order to monitor the stability

of inlet pressure in the liquid chromatograph during operation

a pressure gauge (J) is required at the head of the column. In

our work we have found that expensive gauges or pressure transducers are not required for pressures of less than 500 lb/in 2

. A simple bourdon tube gauge of the type normally used to

monitor air or steam pressure when completely filled with

the mobile phase has been found to be completely satisfactory

for this purpose. Care must be taken to displace all air from

the gauge by liquid; otherwise erroneous and erratic pressure

readings will be obtained. The gauge is attached to the liquid

flow line by a 2 to 3 ft length of 1/8 in o.d. Teflon tube to

monitor the pressure at the head of the column. The gauge will

also indicate system malfunctions as well as being useful to

estimate flow through the system. A sudden rise in pressure
r \

'v , *

W

Figure 3. Components used in the construction of

resistor tube.

usually indicates a clogged line and a drop in pressure indicates a leak in the system. A pressure versus flow plot is

useful for estimating flow rates and pump settings for a particular experiment.

4. The Analytical Column . The heart of the liquid

chromatography system is the column (P) . It must consist of

an injection port (K, and Figure 4) for introducing samples,

it must be able to retain the packing in a stationary position,

and withstand the applied inlet pressures. The columns used

by our laboratory are commercially available precision bore

1/4 in i.d. heavy wall glass tubes with glass pipe flanges at

each end. Metal coupling units are used to secure a Teflon
N0.53 DRILLTHRU NO. 53 DRILL TO «_ .218 C BORE 1/4 DEEP

1/4 28-N.F. TAP TO FIT *C'

1/4-28 TEFLON PLUG

CHROMATRONIX

NO. TEF 107 TUBE

END FITTING

lb) Rj-RSBMAT. TEFLON

Figure 4. Column inlet fitting

injection port at the head of the column (Figure 4) and a Teflon fitting containing a porous retaining plug at the end

of the column (Figure 5) . The porous Teflon plug (nominal 5 ym

pore with 55 percent voids) is used to retain the column packing

in the end fitting. Figure 6 shows the components used to

assemble a liquid chromatographic column. Both of these Teflon

fittings were designed in this laboratory and are adapted to

the existing precision bore columns. Figures 4 and 5 show

plans for machining these fittings from Teflon.

Glass columns of 1/4 in i.d. of lengths varying from 4 in

to 4 ft have been used in this laboratory at operating pressures

up to 500 lb/in 2 with no difficulty. The technique of packing

the column is described in section l.B.l.

5. Detectors (Q) . The detectors we have principally used

for liquid chromatography are differential refractive index and

the differential ultraviolet absorbance monitors. In order to
I" DIA. TEFLON ROD

1/16 Dl A.

1/16" X 1/4" O.D."0" RING GROOVE

Figure 5. Column outlet fitting.

^p.

Figure 6. Column components prior to packing.

10
obtain maximum stability and sensitivity from these detectors a flow of the pure mobile phase is monitored as the reference

stream. The flow through both cells of the detector should be

similar. A direct method for obtaining equal flow in the two

streams would be to have two identical systems operating in

parallel, but this elaboration is not necessary. The analogy

of the liquid system to an electrical circuit is available

for the calculation of the resistance required to give balanced

flow through both the column and the reference streams (see

section l.B.2.b.). In order to calculate the resistance needed

for the reference stream a flow rate measurement at a specific

column head pressure is required. After the value is obtained

a resistive element is made up as described above and installed

in the reference line. The detectors used for liquid chromatography in this laboratory are the "RefractoMonitor" and the

"UV Monitor," Model 1250 (Laboratory Data Control). The

detector is always placed as near as is physically possible

to the end of the column. The column outlet fitting is

attached to the 1/16 in o.d. capillary tube leading to the

sample cell through a reducing union (F15) . This technique

results in a low sample dead volume between column and sample

cell. Figure 7 is a close-up photograph of this column-todetector connection. In many instances it is desirable to

monitor a liquid chromatograph with the UV and the RI detectors

connected in series. The shortest possible length of 1/16 in

o.d. stainless steel capillary tubing should be used in

joining the detectors. In our work we obtain better results

when the RI detector is nearest the column. The UV detector

is less affected by the pressure drop generated by the

additional length of capillary tubing and it has a faster

response time because of the higher sensitivity. Both of

these detectors have a 25,000 ohm source impedence and are

connected to 10 mV potentiometric recorders to give a visual

display of the outputs.

11
Figure 7. Connection of column to detector.

6. Flow Measurement . In order to obtain precise effluent

flow measurements from the liquid chromatograph , an ordinary

10 ml or 25 ml capacity calibrated burette is modified by the

addition of a glass tube side arm just above the stopcock.

When a flow measurement is made the stopcock is closed and a stop watch is used to time the flow between graduation marks

12
of the burette. A calibrated 1 ml pipette has also been used

for flow measurement in a connect-disconnect mode. Figure 8

shows a completely assembled liquid chromatograph as it is

operated in this laboratory.

ft

y

Figure 8. The assembled liquid chromatograph.

7. Comments . If high precision trace impurity analyses

are to be achieved by liquid chromatography it is imperative

that no contaminants be added by the system. In our efforts

to achieve this objective, all systems are assembled such that

only glass, Teflon, or stainless steel contact the stationary

and mobile phases. The necessary interconnections between

these three materials are provided by a variety of connectors.

For steel tubes and some of the Teflon tubing, "Swagelok" type

13
connectors are most useful. The main disadvantage to this

connector is that its use with many solvents causes the metal

to become quite dry and the threads will begin to gall after

several changes. The presence of fine silica gel is also a contributing factor to thread galling. Such connectors should

be used in the system where the least amount of change is

expected, and only be changed when absolutely necessary.

For operating pressures of less than 500 lb/in 2 "Cheminert"

fittings (Chromatronix, Inc.) have proven to be very useful.

These fittings are very versatile with 1/8 in o.d. and 1/16 in

o.d. Teflon tubing, and adapters are available for use with

pipe, steel tubing, glass tubing, and hypodermic syringes.

The difficulty with the use of this connector is its use of a rubber spring washer to make the seal. A synthetic rubber

washer should be selected which will be least attacked by the

mobile phase in order to avoid possible contamination of the

system. A special tube flanging tool (Chromatronix, Inc.) is

easily used for assembling these end fittings and is necessary

for preparing proper lengths of tubing and making repairs.

The injection valve used by this laboratory and some of

the problems related to sample introduction are discussed in

section l.B.5. of this report.

B . Parameters of Operation

The parameters of operation determine the output quality

of any analytical device. Herein are described the essential

characteristics for packing a column, the prediction and

control of mobile phase flow, the control of column performance

with respect to band spreading, and the general methods employed

in operation of the chromatograph and generation of suitable

data

.

1. Packing the Analytical Column . Any packing material

may have associated with it a quantity of fines or excessively

large beads that should be removed before good performance can

be obtained in a chromatographic column. These are best

14
separated by sedimentation or levitation in a liquid of slightly

lower density than the packing.

A decantation procedure is easily employed for removal of

fines. The sample is placed in a beaker large enough to provide

sufficient volume for the addition of liquid at least 10 times

the volume of the beads. Liquid is added and vigorously stirred

for a minute and allowed to stand undisturbed. The settling

time determines the sizing; at least an hour is needed for good

results. The fine material in the supernatant liquid is poured

off, and the procedure is repeated five times or more depending

upon the proportion of fines to be removed.

We find that packing a column with particles in the range

of 10 to 30 microns results in good column efficiency.

Gels used as packings are usually also contaminated with

residual reactants from chemical synthesis. The purification

procedure depends upon the particular type of problem. One

example is the use of soxhlet extraction. A simple washing

procedure may suffice. The sample is placed in a flask with

the solvent and stirred, or agitated ultrasonically , and

filtered through a Teflon micro-filter. Additional washes can

be used, with a final step of using the same solvent that will

become the mobile phase.

Interactive gels (section l.D.3.) which have been used

for a long time with a variety of compounds in large sample

sizes may become contaminated. It may be necessary to clean

these gels with an acidic wash such as a solution of 5 percent

acetic acid in chloroform followed by a basic wash of 5 percent

triethylamine in chloroform if the gel is basic in character.

The reverse order applies if the gel is acidic. In either

case, the gel is finally washed with mobile phase solvent.

The column packing procedure for polymeric gels is as follows. The filtered gel is slurried in the mobile phase

liquid. The slurry should be thick enough to flow as a uniform

mass when poured. In preparing the slurry, dispersion of the

beads is enhanced by ultrasonic agitation. This may be done

15
in a small filtering flask, the side-arm of which serves as a convenient pouring spout in packing the column.

Before filling the glass column, it should be made clean:

rinsing with acetone, followed by washing with a warm mild

detergent solution, scrubbing the interior with a long brush.

The column is rinsed with distilled water and allowed to dry

thoroughly.

The bottom fitting containing a porous Teflon plug is

placed in one end of the column, which is then clamped

vertically. The packing slurry is poured as quickly as

practical down the inside of the column, permitting air to

escape from the top. This initial pouring should fill the

entire column. The excess liquid is allowed to drain through

the bottom fitting.

At the end of a few minutes the formation of the bed at

the bottom of the column will become apparent. Bed formation

will occur at a decreasing rate. The supernatant liquid is

then removed by inserting an inert plastic tube into the top

of the column, and aspirating the liquid into a filtering

flask. It is extremely important to leave at least one half

centimeter of liquid above the bed. If liquid level is

lowered to the top of the bed, a zone will appear on subsequent

addition of slurry, indicating non-uniform packing. This zone

will form even if the top of the bed is stirred prior to

adding slurry.

The procedure of removing supernatant liquid and adding

slurry is continued as long as this is practical. The top

fitting with its connections to pump and pressure gauge is

then fixed in place, and pumping of the mobile phase is begun.

Pressure is maintained well below that to be used in the

chromatography. As pumping continues, the bed will settle

and nearly clear supernatant liquid will appear at the top.

As before, this is removed, fresh slurry is added, and the

pumping is repeated until the column is completely packed.

16
When the column is completely packed, the system is

connected and the pressure is raised to the operating rate

of liquid flow. It is desirable to pump liquid through the

column for a substantial period before putting it into

analytical operation. This can be done overnight. If further

compression of the bed occurs during this period, the addition

of a small amount of slurry will be necessary.

Chloroform is often used as a mobile phase solvent in

liquid chromatography using copolymer gels. Some difficulty

is encountered upon packing columns longer than about two feet.

The beads tend to float because of the greater density of the

chloroform. When packing is done with solvent flow in the

vertical downward direction, at first the bed will form and

even grow in length. This growth will cease when the condition

arises where the beads flow in an endless elliptical motion

without adding to the bed depth.

This difficulty can be overcome and the column successfully packed by preparing the slurry with a mixture of 2.5

parts of benzene to 1 part chloroform. The lesser density of

this mixture will allow the beads to settle. After the column

has been packed, the benzene is washed out by pumping chloroform through the column overnight.

2. Mobile Phase . The selection of the appropriate mobile

phase for liquid chromatography is governed by the analysis

desired, the column material employed, and to some extent, by

the detector being used. The mobile phase liquid and the

stationary phase packing must be compatible. If one is performing standard steric exclusion (gel permeation) or interactive gel chromatography (see section I.D.3.), it is a requisite to use a mobile solvent which swells the gel,

allowing solute permeation to the gel interior to occur.

(In liquid- liquid chromatography, it is necessary that the

mobile and stationary phases have high relative inmiscibilities

For brush particle columns, very polar alcohols such as methanol

will hydrolyze the interactive sites from the silica.)

17
In regard to detectors, the flame detector requires that

at least a portion of the solvent adhere to the moving wire,

which limits applications for aqueous mobile phases. In the UV

detector using single wavelengths, it is imperative that the

mobile solvent has low absorbtivity at the measuring wavelength

to minimize energy losses in the incident light beams. The

refractive index detector requires that the index of the mobile

phase be dissimilar from that of the eluting solutes.

Furthermore, the refractive index change of the mobile phase

with change in ambient temperature (6n/<5T) should be small, to

avoid excessive baseline drift and cycling. This can be

controlled by thermostating the detector cell.

In a properly designed liquid chromatographic system, the

flow rate of liquid through the reference and analytical column

streams should be both balanced and predictable. This aspect

of system design is described below.

a. Depulsing . Liquid chromatographs operating under

inlet pressures below 1,000 lb/in 2 commonly have mobile phase

flow provided by piston pumps. Unless the pulsed mobile phase

flow leaving this type of pump is damped sufficiently, the

pulses will produce bothersome signal oscillations in highsensitivity, low-volume detectors. The system we employ to

minimize pulses to <1 percent at inlet pressures up to 300

lb/in 2

, is shown in Figure 9. The system is analogous to an

RC filter in an electrical circuit.

b. Flow Balancing . Flow balancing in the analytical

column and reference streams is achieved by placing an appropriate resistor in the reference stream which matches the

parallel resistance in the analytical column stream. The

latter is achieved by simple analogy to DC circuits. From

Ohm's Law, R = AP/I, where AP is the pressure drop across the

analytical column, and I, the effluent flow rate from the

column. Combining Ohm's Law with D'Arcy's expression for

laminar flow through circular porous conduits, it is possible

18
DEPULSING

SYSTEM

* COLUMN FLOW

* REFERENCE FLOW

Figure 9. Schematic of depulsing and balanced

flow system.

to calculate the length of a resistor tube of given crosssectional area, packed with hard porous particles (eg. silica

gel; whose permeability (k) has been previously established),

which matches the resistance of the analytical column. For

3 mm o.d. x 1.6 mm i.d. stainless steel tubes, the length (L)

of a tube (in cm) packed with 0.52 g/cm 3 with 30 to 60 ym

silica gel (Figure 3) (k = 4.86 x 10 9 cm 2

) necessary for flow

balancing, is given by

L = .5 x 10 l * R

c /n

where R is the analytical column resistance in lb/in 2 -min/cm 3 and n is the viscosity of the mobile phase. Once flow matching

has been established (Figures 9, 10) at a given inlet pressure,

it should remain so, independent of inlet pressure, provided

the analytical column resistance is a negligible function of

inlet pressure. Greater details of this work are given in

reference [2]

.

3. Column Efficiency . An increase in column efficiency

is synonomous with a decrease in solute band spreading. Prior

to an investigation of column efficiency are the requirements

(1) to minimize the dead volume of all extra-column connections

19
iW

—

vm „*

ww

..;,.' •-*- w

m

J

i

Figure 10. Illustration of balanced flow system

and plumbing and (2) to use small, concentrated samples of

solutes dissolved in the solvent used as mobile phase for sample

introduction into the chromatograph. An excess of external

plumbing volume does not contribute as greatly to solute band

spreading in liquid as in gas chromatography due to the 10 k slower diffusion rate for liquids than for gaseous solutes.

20
Minimization of extra column plumbing volumes is nonetheless,

important in obtaining high system efficiencies. The use of

small sample volumes is sometimes impossible when the solutes

have low solubilities in the solvent used or when solute

detectability by the detector being used, is low. Sample loops

are especially effective for solute introduction, rather than

syringe injection. When performed properly, this use of

injection valves is more accurate and more reproducible than

any other form of sample introduction.

The use of small uniform and spherical particles (eg.,

10 to 30 urn) as column packing in liquid chromatography

increases column efficiency. The use of small column diameters

has some effect, but due to the slower diffusion of liquids is

not as pronounced as in gas chromatography. Flow rate can be

quite important, particularly for gel packings where permeative

fractionation controls the separation [1] . In such cases,

extremely slow flow rates (to 0.1 cm/min) produce the highest

efficiencies. The construction of a van Deemter plot will

quickly establish the best choice of flow rates to use for any

kind of chromatography.

4. Fraction Collecting . The use of a fraction collector

with a liquid chromatographic column is important in order to

(1) establish solute stability during chromatography, (2)

perform a mass balance analysis on separate components with

reference to the measured mass of sample originally introduced,

and (3) for subsequent determination of the separated

constituents. Subsequent thin layer chromatography is

especially powerful as a complementary analytical tool.

Collection of fractions from liquid chromatography is easily

achieved on a chosen time scale, with conventional collectors

(Figure 11). Unfortunately, no commercial collector is available which permits variable time collection for fractions of

the same analysis. Knowing the profile of the chromatogram

it may be desirable to collect the entire valley between widely

separated peaks into a single collection tube. Similarly,

21
Figure 11. Liquid chromatograph including fraction

collector.

separated peaks can be collected into single tubes. This may

be achieved by storing appropriately placed signals in a computer whose memory is swept at a fixed frequency. When a signal channel is reached during the computer sweep, an actuator

circuit is tripped, advancing the collecting tube tray by one

tube location. The schematic of the actuator circuit designed

for coupling to our Fabri-Tek 1070 computer is shown in

Figure 12.

When only a very small amount of a sample is collected,

the appropriate fraction (or combined fractions) may require

concentration by solvent removal. A fraction concentration

system is shown in Figure 13. An example of the success of

22
IOK

6Ki

_n_n

WW68 K 1-®

DR435

RELAY rJU

10 K COIL f lii, 20 @0.7mADC5 jINOO

TO COLLECTOR REMOTE

10 lOMFDjjT1

2N4I24

I wwvI 5K

+5VDC

^iU^\j-5V

MDA920-A

200 MFD

Figure 12. Schematic of actuator circuit for

fraction collector.

suitable sample concentration is given under the section of

Peak Authentication in Figure 14 which shows the IR spectrum

of 10 yg of Cortisol using a micro-pellet and a beam condenser.

5. Sample Introduction: Methods and Problems . Injection

of the sample to be chromatographed is made with the system at

full operating pressure and stable conditions. Injection can

be performed in either of two ways: (1) by piercing a rubber

septum with a syringe needle, or (2) by means of a sample valve.

Each method has advantages and disadvantages.

Use of the needle and septum permits the injection of

small samples, 5 or 10 microliters, for example. Injections

may be made even at pressures of 200 lb/in 2 or higher especially

if a fixed-needle syringe is used. This type of syringe avoids

leakage at the luer fitting of demountable syringes which

routinely occurs at high pressures. At high pressures, penetration of the septum results in small beads being driven into

a fixed needle so that not only is it impossible to inject the

sample but there also is the problem of a clogged needle

23
Figure 13. Concentration of collected fractions.

resulting. Removal of the particles is difficult, and may

necessitate discarding the syringe.

When syringes with replaceable needles are used, clogged

needles are a less serious problem. With experience and

patience, a small wire can be poked down the barrel of the

needle to dislodge particles. The needles can also be placed

into an ultrasonic bath which, with luck, may dislodge jammed

beads

.

A serious disadvantage of the two element syringe compared

to the fixed needle type is the problem of leakage at the hub

of the needle. This is particularly troublesome at pressures

of about 200 lbs/in 2 or more for most needles, depending upon

24
100

80 ZLUu

u

z

<t 40 Z

<£ 20

3000 2500

WAVELENGTH (MICRONS) 6 7 10

100 —_ I | I

1 , 100

I , ±-l- — 1(0

1 ,,

J \

-V H

1

r

1' J r

8 ) r\ P\ f ( N t- ^7* \J

\ /\

\ / \

' f

I r

1

6 A $ V

A )

1 4 i —

2 )

- 3 7

\ —1

—1

1 —i

LI

2000 1800 1600 1400 1200 1000 800

WAVENUMBER (CM )

Figure 14. Infrared spectrum of 10 yg of Cortisol

obtained by concentration.

the fit of the needle at the luer. With samples of small size

this leakage may be unnoticed and time may be lost waiting for

the chromatography of a sample which, indeed, had never been

inj ected.

When larger concentrated samples need to be injected for

the collection of fractions, needle injections against even

moderate pressure is difficult due to the back pressure on the

relatively large plunger of larger sized needles.

Septums themselves also give rise to problems. Chloroform

and THF swell and cause deterioration of most materials from

which septums are made. We have been able, however, to get

long service from viton septums when used in the type of

injection fitting developed in our laboratories and illustrated

in Figure 15. With this fitting only a small area of the septum

is exposed to the solvent.

Physical stability of the septum is not the only consideration in the use of septums for injections. The solvent may

extract components from the septum which may show up as peaks

in the chromatogram. Such artifacts often appear in fixed

25
.995

332 BORE 3/8 DEEP TO FLAT BOTTOM

3/8 24-N.E 8TH'D. MACHINE CUT

TO FIT '8'

NO. 60 DRILL TO t. .218 C BORE 1/4 DEEP

1/4 28-N.E TAP TO FIT 'c'

1/4-28 TEFLON PLUG- CHR0MATR0N1X

NO. TEF 107 TUBE

END FITTING

3 _J «-5

32

—( 1

3_4

I 1- W

NO. 59 DRILL

3/32 X 45° C'SINK

MAT TEFLON

6 EA. REQ. r-24N.C. THD.

Figure 15. Inlet fitting for syringe injection.

relation to the injection time indicating that the penetration

by the needle is a factor in the extraction of the septum

material. We should note here, another artifact which we

observed due to sample injection by needle through a septum.

In an earlier type of injection- fitting it was necessary to

use long thin needles in order to inject on the bed. It was

customary to direct the end of the needle into the small hole

in the injection port by the fingers of the left hand and indeed

to support it this way while pushing it through the septum and

into the column packing. Apparently, enough organic material

26
rubbed off the fingers onto the needle to show up in the

chromatogram when a UV absorbance detector was used.

Injection valves of the type illustrated in Figure 16

have proven very satisfactory in our work. They are made of

materials chemically inert to most solvents, and can be used

with inlet pressures up to 500 lb/in 2.

Pump

Column

6

Sample vial-7

Sample,loopLOAD SAMPLE

Column

6

Pump

Sample vial-7

Sample loop

INJECT SAMPLE

Figure 16. Two-way valve for sample introduction.

The operation of the valve is indicated in Figure 17.

In the "load" position flow of solvent flows directly from

the pump, through the valve, to the column. A sample is drawn

from a vial into the sample loop by means of a syringe connected

to port #3 by a female luer fitting. Turning the valve to the

"inject" position switches the solvent flow from the pump,

through the loop attached to the valve, delivering the sample

into the column. Another sample may be injected at any time

without stopping the solvent flow by repeating the procedure.

27
Figure 17. Sample introduction with the two-way valve.

Sample loops are easily interchanged, rinsed, or filled, without

admitting air into the system. The size of the loop has been

varied from a useful lower limit of about 50 microliters for

analytical work, up to 10 ml for collection of sample fractions.

It should be noted that the size of the injected sample is

larger than that of the loop by about 0.1 ml because of sample

28
dead volume contained in the connecting tubing and in one of

the rotor passages.

It would seem that on-column injection, which is possible

with needle and septum and not with valve injection, should

result in less band-spreading. We have observed no significant

difference in this regard.

Sample introduction valves of the kind discussed here are

expensive, considerably more than a system composed of a needle

and septum. However, when one considers the long-range cost

due to attrition of needles and syringes , and even more

importantly, the down-time arising from problems of septum

leakage and malfunctioning of needles and syringes , the valve

method is the preferred choice.

6. Measurement of Retention Volumes . A considerable

portion of our work in liquid chromatography has involved

precise measurement of solute retention volumes . The retention

volume is simply the difference between the retention times of

the solute and a reference solute, multiplied by the flow

rate. For the purpose of flow measurement we have found convenient the use of a modified burette such as is represented

in Figure 18. The glass side-arm is sealed in place and connected to the column exit port of the detector by plastic tubing

A luer-lock stainless steel connector permits uncoupling of the

exit tubing for recycling the eluant to the solvent reservoir

during stand-by periods such as overnight or overweekend.

To measure the flow rate, the stopcock is closed and the

eluant flows up into the burette for a flow of 5 ml. During

one or more periods of the chromatographic run, the time for

a flow of 5 or more ml is measured with a stop watch. Flow

rates measured in this way are usually reproducible with a precision of a few tenths of a percent which is also a reflection of the stability of the overall hydraulic system.

Day to day data, however, indicate a precision of 5 percent

in retention volume measurements, although replicates of

within-day values will agree to ± 0.1 to 0.2 cm 3

. This may

29
Figure 18. Flowmeter for measurement of effluent

flow rates .

reflect changes that occur by compaction of the column packing.

For this reason, normalized retention volumes are used. These

are obtained by reference of the solute retention volumes to

that of selected reference standards which are used each day,

either individually or as internal standards to the samples.

30
C . Peak Authentication

The problems associated with the determination of the

identity of a peak may be magnified in proportion to the amount

of material required by the various analytical techniques

employed. For example, while 10 yg can be used for IR,

less than 1 yg for UV (provided sufficiently high extinctions

exist for the component in question) and mass spectrometry,

1 to 10 yg for gas and thin- layer chromatography, NMR spectra

requires 100 to 500 yg of material. The difficulty involved in

peak authentication is further complicated obviously, if complete separation from other sample components is not attained

during liquid chromatography. Further, to insure collection

of sufficient material of single sample components representing

less than 1 percent of the sample, large sample inputs (as high

as 100 mg or more) can result in some deterioration of

chromatographic resolution. Approaches used in the various

techniques we have employed are described below.

1. Spectrophotometry . UV and visible absorption spectra

on collected liquid chromatographic fractions are most useful

in the favorable case where suitable standards for comparison

are available. Qualitative IR spectrophotometry in contrast,

provides important information concerning the presence of

functional groups in the material. Furthermore, absolute sample

identity has a greater degree of confidence from IR spectra

matching with the possible exception of closely related structures, than does UV and visible spectra matching.

The procedure we have used for obtaining IR absorbance

spectra from liquid chromatographic fractions involves sample

concentration by evaporating the solvent, making a KBr pellet

from the residue, and examining its infrared spectrum. In

isolated . instances it might be possible to identify an eluted

compound by examining it in the infrared while still in solution,

but routinely, the concentration of sample is too small and the

absorptivity of most solvents is too great.

31
The technique of preparing a 13 mm KBr pellet from the

residue remaining after evaporation of the solvent is generallyrestricted to samples having a total mass of 1 mg or more. As

an alternate procedure, for samples in the range of 0.5 to 1 mg,

we obtained better spectra by adding 150 to 200 mg of KBr powder

directly to the solvent, prior to evaporation to dryness and

preparation of a standard (13 mm) pellet. In both methods

blanks were run representing a portion of column eluant collected during a segment of the chromatogram during which no

sample components were eluting. Figure 13 is a picture of the

rotary evaporator used for concentrating eluant fractions.

In the more difficult case where very small amounts of a compound is obtained from the chromatograph , a similar but more

careful technique is employed. Care must be taken to insure

that containers and reagents used in the preparation of the

KBr pellet are scrupulously clean. This technique involves

the addition of 1 mg of KBr powder to the tube containing the

eluted peak. The solvent is then carefully evaporated and the

KBr-sample residue is used to prepare a micro pellet (1 mm

diameter) using a micro die assembly (Figure 19) . The micro

pellet is then placed in a 4X beam condenser in the spectrophotometer and the spectrum is obtained (Figure 20) . A beam

condenser is essential when 1 mm KBr pellets are employed.

Using this method a satisfactory identification was made of

10 yg of Cortisol (Figure 14)

.

2. Gas Chromatography-Mass Spectrometry . Chromatography

of a solute on a gas chromatographic column provides only a single piece of information, viz., a retention time, or more

preferably, a retention volume. Chromatographic retentions on

two dissimilar columns (eg., one "polar" and one "non-polar"),

provides a higher confidence level for qualitative analysis,

provided sufficient material is available for two injections.

A. J. P. Martin early pointed out that solute mobility is a function of the contribution of the various substructures composing the solute molecule. Employing a two- column concept

32
Figure 19. Microdye assembly for preparation of

1 mm KBr pellets

.

and differential retention index analysis, it is possible to

obtain information related to sample identity from gas chromatography alone, provided that the general structure class (IR.

spectrophotometry can assist here) to which the solute belongs

is known (eg. steriods, sugars, etc.), and further that tabular

retention index information has been prepared for compounds of

the solute class (including the candidate compound) . The GCdata acquisition system is shown in Figure 21. The point

remains that this approach is able to differentiate among a limited number of solutes, and is not very suitable for solute

identification when the number of possible solutes is large.

Information concerning sample identity is greatly improved

when a mass spectrometer is coupled to the chromatograph . Mass

spectral data coupled with the availability of an infrared

spectra of the compound will often suffice for sample

identification.

33
i

WQTQfi

r

Figure 20. Infrared beam condenser (4X) containing

a 1 mm KBr sample pellet.

The thermal conductivity detection system and pneumatic

valving arrangement (Figure 22) for GC/MS analysis is such that

the GC column effluent is vented to atmosphere until a sample

component begins to emerge. This is then fed into the spectrometer without introducing contamination. This system provides

that the chromatography study, the solute retention volumes,

and their respective mass spectra may all be obtained on the

same instrument. Use of retention values and mass spectral

information can assist in the authentication of the compound

of interest. Following identification, a solution is made up

using the same solvent as was used for the liquid chromatography

and a small amount of the suspect compound from the shelf.

34
Figure 21. Gas chromatographic data acquisition

system.

THERMAL

CONDUCTIVITY

CELL

GAS CHROMATOGRAPHIC ^ COLUMN

EFFLUANT

He REFERENCE —

STREAM

SWITCHING

VALVE

1/16" STAINLESS STEEL

TUBING

•• TO MASS SPECTROMETER

•• TO VENT

Figure 22. Thermal conductivity detector and pneumatic

valving system for GC/MS analysis.

35
This is analyzed in the GC under the same conditions ; the

retention times measured, and compared with the retention time

of the unknown to double check the identification.

The detection/valving system within the GC is important

from three aspects: (1) Our samples are frequently obtained

in a large amount of solvent. By using this system, the solvent

peak can be vented to atmosphere. This prevents a large solvent

background in the spectrometer which might obscure the mass

spectrum of the sample peak. (2) The ionization region of the

mass spectrometer can become contaminated and this is also

dependent upon the amount of sample put into it. By simple

venting of solvent peaks , time between spectrometer shutdown

and cleanout has been extended. (3) The thermal conductivity

detector in the GC allows one to obtain a gas chromatogram each

time a sample is run. If the MS is used both as a detector and

peak identifier, the chromatogram resulting from total ion

current signals are often inferior. The MS parameters for

obtaining an acceptable chromatogram are slightly different from

those used for producing an acceptable mass spectrum. When the

MS signal parameters are changed during a chromatogram, spurious

peaks were recorded, making the chromatogram unacceptable.

The mass spectrometer can be used in conjunction with the.

IR for non-volatile compounds also. If there is enough sample

after solvent evaporation, a small quantity can be placed in a segment of a melting point tube sealed at one end and examined

in the spectrometer using the solid probe sample entrance.

This allows the sample, positioned at the tip of the metal

probe, to be placed near the ionization region of the

spectrometer. The "probe controls" allow external control of

the sample temperature, up to 450 °C. Thus by careful heating

of the probe to the appropriate temperature, the sample can be

volatilized and a mass spectrum obtained.

3. Proton Magnetic Resonance . The approach taken here

involves the collection of sufficient material from the liquid

36
chromatograph by applying high sample inputs. Good NMR spectra

are attainable from 500 yg of collected material. We have in

this manner been successful, in a cooperative effort with the

Organic Chemistry Section, in obtaining the identity of a sample impurity present at less than one percent of the sample.

This capability depends upon the special preparative features

we have found with interactive gel chromatography.

4 . Computerized Measurement of Chromatographic Peak Areas . A signal averaging computer was found useful for the electronic

integration of chromatographic peaks. The electrical output of

our gas chromatographs were available at amplif icaions up to

15 volts. The interfacing was therefore no problem. For

liquid chromatographic amplifications a simple 100-fold amplification was done using a bread board operational amplifier

circuit

.

In Figure 23 is shown a three component chromatogram with

the integral superimposed. The integral is determined by

taking the difference from the computer of digital voltage

readings between the beginning of a peak where the slope of

the integral is zero, and just after the peak where the slope

changes from positive to zero.

D . Column Packing Materials

1. Introduction . Chromatographic separations occur

because of the intrinsically different chemical properties of

the components of a mixture, relative to the chemical properties

of the stationary phase. These properties are transformed into

corresponding elution velocities which determine the order and

rate of component appearance at the column exit as the components travel through the column. The properties of the

stationary phase in the chromatographic column is at the center

of the action. Accordingly, stationary phase understanding is

the key to successful liquid chromatography.

The selection of a successful stationary phase depends

upon the criteria which indicate success. With modern high

37
j

/

_jT_

r*

f

/u —

1

i ] ^ I

_ i_

i.

7_t

\ t

\ v r

\ V i ^

Figure 23. XY recording of a chromatographed sample

showing peak integrals

.

speed liquid chromatography it is a simple matter to demonstrate

a one-second separation of benzene and benzoic acid, although

the problem may be trivial. The solving of new problems is

approached in parallel reasoning to known solved problems,

or on the basis of intuition and experimental guesswork. The

rate of problem solving is clearly not a constant for all

scientists, nor for all problems.

There are six major factors which need to be put into

perspective in a discussion of stationary phase selection:

The physical properties determine mechanical performance: the

average particle size and its distribution, shape, porosity,

38
density, thermal stability, etc. The capacity determines the

maximum sample size that can be handled per unit cross-sectional

area of column; with surface adsorbants the capacity tends to

be small, and with gels it is generally much larger. The available selectivity refers to the intrinsic ability of the stationary phase to interact differently with different solutes, and

thus provide a reason to expect, at least in qualitative terms,

that separation should or must occur. The predictability of

solute elution volumes refers to the degree of accuracy involved

in achieving in practice a precalculated elution volume for

each solute component. The mobile phase moderateability is a concept which we use to indicate the benefit provided by

options of solvent composition or effect of temperature in

order to suppress excess solute affinity for the stationary

phase. The available efficiency is properly described in terms

of the number of theoretical plates per meter for a given column

geometry, flow rate and temperature; it is the composite result

of all chemical and physical factors that tend to diffuse the

chromatographic peaks and thus impede the achievement of good

resolution.

Table 2 shows a comparison of how we view the relative

strengths of the different stationary phases. This is partly

opinion. It should be noted that the availability of "high

speed" characteristics is omitted since our view of speed

includes the rate of problem solving, and this is adequately

handled by the predictability of chemical performance.

2. Steric Exclusion Chromatography . The range of

separations based upon steric exclusion (gel permeation)

principles alone are principally dicated by the porosity of

the gel and by the mobile solvent controlling the degree of

gel swelling. The upper analytical (solute exclusion) limit

is dictated by the largest solute structure which is just

totally excluded from the gel interior, and this represents

the elution volume of a "non-permeating" solute. The lower

limit for analytical separations is based by the largest

39
Table 2. Comparison of stationary phase types used in

different forms of liquid chromatography.

X >.

>s 4-> +-> M •H •H

•H i—

1

iH X i—

1

•H •H

+-> •H X) >n X H X> •H U rt > 03 u fi >s CD H -M p CD +-> M

P U 13 •H •H OJ U •H o u u !h CD T3 J-l •H ce CD t—

1

CD a Mh p^ T3

CD 5h CD m rt O

CO &H OS pq U 2

Liquid/Liquid

Chromatography

Gel Permeation

Chromatography

Liquid/Solid

Chromatography

Ion Exchange Gel

Chromatography

Interactive Gel

Chromatography

solute structure which can permeate essentially all interior

corridors of the gel phase. Solutes larger than the former

are totally excluded and have the same minimum retention

volume; solutes smaller than the latter type exhibit total

gel permeation and have the same maximum retention volume.

Solute sizes in between these boundary limits may be separated

from one another provided there is a sufficient difference in

their effective molecular volumes (ie. in their relative degree

of permeation) . This intermediate region is defined as the

analytical or working region of the gel in regard to sterically

controlled separations

.

The working region of the gel can be extended on the upper

end by increasing the average pore diameter of the gel. This

can be achieved by lowering the degree of crosslinking during

40
copolymer synthesis, or by choice of an appropriate polymerization solvent and synthesis conditions in which large "pores"

are introduced during gel synthesis.

a. Measurement of crosslinking . Two separate studies

with copoly (styrene/DVB) conducted in our Section (1) have

revealed the location of adsorption bands in the infrared for

para- and meta-divinylbenzene crosslinking, and (2) have shown

that measurement of the degree of each type of crosslinking is

feasible from IR absorbance measurements on both standard, and

commercial (containing meta- and para-ethylvinylbenzene)

styrene/DVB copolymers [3,4].

b. Performance . The use of steric exclusion chromatography systems for the separation of organic solutes has been

described in detail in last year's report [1] . One finding

which is worth repeating here is that the plate height of a partially permeating solute varies oppositely with the flow

rate. Thus, solute separations by steric exclusion chromatography are improved under slow flow rate conditions.

c. Limitations . There are important limitations

to steric exclusion chromatography. There is its intrinsically

poor selectivity that leads to lengthy analysis times for

multicomponent samples. This is required due to the narrow

analytical elution volume range available for separations.

In principle, such separations operate only on the basis of

solute size discrimination.

3 . Interactive Gels

a. Description . The introduction of an interactive

functional group into a high porosity gel replaces the phenomenon of separation by solute size with the phenomenon of

separations based upon solute-gel interaction. The use of

interactive gels for the separation of solutes having zero

permeation differences greatly extends the working regions

for separations on a gel system.

An introduction to interactive gels, the rationale for

their use, and their potential for analytical separations in

41
liquid chromatography is covered in the reprinted article

shown in Figure 24, which was recently published [5].

b. Synthesis . Various methods are available for

synthesizing interactive gels for liquid chromatography. One

consists of suspension copolymerization with at least one of

the monomers containing a specified interactive functional

group. Another method consists of derivatization of a copolymer

to introduce specific functional groups into the network. In

order to insure homogeneity, the copolymer beads are swollen

in an organic solvent. The swollen state needs to be maintained

throughout the synthetic conversion. In last year's report we

described the preparation of nitrated styrene-divinylbenzene

copolymer [1]

(1) Acidic Gels . A weakly acidic functional

group, namely, -CH 2 0H, has been introduced into the copolymer

matrix by a two-step reaction. The method consisted of chloromethylation of the copolymer beads swollen in chloroethyl methyl

ether, followed by alkaline hydrolysis of the chloromethylated

gel. The material is being evaluated in our studies on chromato

graphic separation of proton-accepting organic compounds.

Figures 25 and 26 show the set-ups used for chloromethylation

and alkaline hydrolysis, respectively. Another chromatographic

material was obtained by conversion of a hydrogen-form sulfonic

cation-exchange resin into the Ag form. On the basis of the

ability of silver salts, such as AgN0 3 and AgClOU, to form

charge- transfer complexes with benzene and polynuclear compounds

[6] , the material is being examined in our investigations on

polyaromatic compounds that occur in air pollution.

(2) Basic Gels . A proton accepting network, a crosslinked polyvinylpyridine , was prepared and found to be

effective for the chromatography of weakly and moderately

acidic organic compounds. This material, copoly (2-methyl- 5-

vinylpyridine/O . 08 divinylbenzene) , is now commercially available from Bio-Rad Laboratories. An infrared spectrum and a

42
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44
> C

Figure 25. Chloromethylation of copoly (styrene/DVB)

beads

.

45
Figure 26. Alkaline hydrolysis of chloromethylated

copoly (styrene/DVB) beads.

photomicrograph of these copolymer beads are shown in Figures

27 and 28, respectively. We are now conducting experiments in

our laboratory on the synthesis of tertiary amine gels by

reduction of quaternary ammonium anion-exchange resins. This

synthesis is based upon the reaction of LiAllU with quaternary

ammonium salts [7] as shown below.

RN (CH 3 ) 3 I LiAltU

THF RN(CH 3 ) 2 + CH„

Other methods of converting anion-exchange resins into a tertiary amine derivative based upon the Stevens and SommeletHauser rearrangements [8] are being studied. In the coming

year, gel modification by introduction of specific functional

groups into macroporous copolymer beads will also be

investigated

46
4000 3000

7 8 9 10 11 WAVELENGTH (MICRONS)

Figure 27. Infrared spectrum of copoly (2-methyl- 5-

vinylpyridine/0.08 DVB).

Figure 28. Photomicrograph of copoly (2-methyl5

vinylpyridine/0.08 DVB).

47
Special Notice of Hazard to Users of Chloromethyl Methyl Ether

Chloromethyl methyl ether (CMME) may typically contain

1 to 10 percent (w/w) of an extremely dangerous carcinogenic

impurity : bis chloromethyl ether (BCME) . Results published

by S. Laskin and coworkers [9] suggest that BCME may be among

the most dangerous of all known chemicals. This is certainly

true for the rat. Regardless, all users of CMME which may be

contaminated by BCME are warned to adopt totally effective

safeguards , to prevent inhalation or any other form of contact

with the liquid or with its vapors.

48
2. GAS CHROMATOGRAPHY

A. Quantitative Analysis

1. Eicosane . Samples of >99.9 percent pure n-eicosane

crystals, before and after high intensity gamma irradiation,

were obtained for analysis. The work was performed on 12

percent (w/w) W98 silicone on 80/100 mesh Chromosorb W, AW/DMCS,

using programmed temperature conditions. It was possible to

detect n-alkane impurities below 0.01 weight percent. The

crystalline n-eicosane was found to be unchanged by the irradiation, at least to within the tolerance of the measurements

(0 . 1 percent)

.

2. Nitrilotriacetic Acid (NTA) . This section participated

with others during the past year in the development of methods

for the measurement of NTA in water. The analysis involves

two parts to the chromatography. Anion exchange chromatography

is used first to free the NTA from complexed metal ions. We

have concentrated our efforts on the measurement of the uncomplexed NTA by gas chromatography.

Since NTA itself [N(CH 2 C00H) 3 ] is nonvolatile, a gas

chromatographic procedure would be impractical unless the NTA

is derivatized to a volatile compound. NTA can be simply

esterified using the silylating reagent "BSA"

0-Si(CH 3 ) 3 H 3 CC=NSi(CH 3 ) 3

to form the volatile tri (trimethylsilyl) derivative [9]. This

derivative was examined by GC using conditions similar to those

described in this reference. The peak assigned to the triesterified NTA was verified using a mass spectrometer as the GC

detector. The mass spectrum is given in Figure 29. To use the

es terif ication procedure the NTA must be dry and in the

protonated (3H) form.

49
100

COz

uj 60

LlI >

40

20

BASE

^M

SILYLATEO NTA (NITRILO TRIACETIC ACID)

il

N-(CH2 -C;f „..„ )

MASS NUMBER

RELATIVE INTENSITYION REPRESENTED

407 0.25 P 392 0.42 P-Me

364 0.36 P-3Me

WHERE P== PARENT

60 T

100 120 140 160 180 200 MASS NUMBER

I

' P |'

|

'

|

'

I

'

I

' 220 240 260 280 300

Figure 29. Mass spectrum o£ silylated NTA eluted

from a gas chromatographic column.

3. Tetramethylsilane (TMS) . This compound is widely

used as an internal standard for nuclear magnetic resonance

studies. In determining a procedure for trace NMR analysis,

the Organic Chemistry Section at NBS recognized evidence for

contamination. This was then verified in detail by gas

chromatography with the finding of two contaminant peaks at

2.7 and 0.1 percent. The gas chromatogram is shown in Figure 3-0

II i -

- —

- ——

'

'—

1 r

r*\ -i_ p—*- I'M TTy'^'T'

. UN J i i

i — PTMS 97.2% =

i-— 1

' CO

- - — - — H > CML

p

hist IMPU]RITY 0.19p —:

\—wZ . — . — . i—

. —

i

s

I *—»_ . C\ 1 \^>*^2nd i

1 iv/ii ;i iKv. if ii ,i %. . — —

.

Tf .

Figure 30. Gas chromatogram of tetramethylsilane.

50
3. PERSONNEL AND ACTIVITIES

A . Personnel Listing

Separation and Purification Section

David H. Freeman, Section Chief

Janice M. Hurst, Secretary

Chromatography

Delmo Enagonio

Walter Zielinski

Herbert Dixon

Patti Byrne (summer student)

Ion Exchange Microstandards

Edwin Kuehner

Mass Spectrometry

William Dorko

Network Structures

Rosalie Angeles

Particle Metrology

Herbert Dixon

Robert Young

Ultra-Pure Reagents *

Edwin C. Kuehner

Youth Opportunity Corps

Jennifer Jones

"* Transferred to Section 310.06

51
B. Talks

1. R. M. Angeles, "Decomposition and Dehydration of Various

Polyelectrolytes Derived from Polystyrene," Sixth Middle

Atlantic Regional Meeting, Baltimore, Maryland.

February 5, 19 71.

2. David H. Freeman, "Donor-Acceptor Liquid Gel Chromatography,

State University of New York at Binghamton, Binghamton,

New York. February 5, 1971.

3. David H. Freeman, "Acceptor Donor Chromatography," Naval

Ordnance Laboratory, Silver Spring, Maryland.

February 10, 1971.

4. David H. Freeman, "Hydrogen Bond Liquid Chromatography,"

Pittsburgh Conference on Analytical Chemistry and Applied

Spectroscopy, Inc., Cleveland, Ohio. March 2, 1971.

5. David H. Freeman, "Interactive Gel Chromatography: A

Breakthrough in Organic Analysis," University of Maryland,

College Park, Maryland. March 23, 1971.

6. David H. Freeman, "Introduction to Liquid Chromatography

(Experimental, Theory, Column Selection)," Washington

Chromatography Discussion Group, Washngton, D. C.

April 2, 1971.

7. David H. Freeman, "High Resolution Liquid-Gel Chromatography

University of Florida, Gainesville, Florida. April 16, 1971

C . Publications

1. G. A. Sleater and D. H. Freeman, "Rapid Desorption of

Chromium (III) from Cation Exchanger with Hydrogen Peroxide

Solutions," Anal. Chem. 42, No. 13, p. 1666-68 (1970).

2. G. A. Sleater, D. H. Freeman, D. H. Olson, and H. S. Sherry,

"Zerolite Microstandards ," (accepted for publication in

Analytical Chemistry)

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

Purification Section: Summary of Activities July 1969 to

June 1970," NBS Technical Note _549 (1971).

4. D. H. Freeman and D. P. Enagonio, "Interactive Gel Networks

for Organic Separations," Nature, Physical Science, Vol. 230

135-36 (1971)

52
4. REFERENCES

[1] National Bureau of Standards Technical Note 549 , D. H.

Freeman and W. L~. Zielinski , Jr. , Eds . , January 1971.

[2] Zielinski, W. L., Jr., Dixon, H. D., Byrne, P. J., and

Freeman, D. H., "Analogy of Depulsed Mobile Phase Flow in

Liquid Chromatography to DC Circuits," (to be published).

[3] Freeman, D. H., Zielinski, W. L . , Jr., and Rittner, W. F.,

Proceedings, International Conference on Ion Exchange in

the Process Industries , London, July 1969.

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

manuscript to be submitted for publication.

[5] Freeman, D. H., and Enagonio, D. P., Nature , Physical

Science, 2J50_, 135 (1971).

[6] Mulliken, R. S., and Person, W. B., "Molecular Complexes,"

Wiley- Interscience , New York, p. 271-76 (1969).

[7] Sandler, R. S., and Karo, W., in "Organic Chemistry,"

A. T. Bloomquist, Ed., Vol. 12, Academic Press, New York,

p. 325-6 (1968).

[8] Pine, S. H., in "Organic Reactions," W. G. Dauben, Ed.,

Vol. 18, Wiley and Sons, New York, p. 403-464 (1970).

[9] S. Laskin, M. Kuschner, R. T. Drew, V. P. Cappiello,

and N. Nelson, Archives of Environmental Health 23 , 135 (1971).

[10] National Bureau of Standards Technical Note 459 , D. H. Freeman, Ed., December 1968.

ft U.S. GOVERNMENT PRINTING OFFICE : 1 971 -Wl -326/92

53
formNBS-114A U-71)

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PUBLICATION OR REPORT NO. NBS-TN ^89

2. Gov't Accession No.3. Recipient's Accession No.

4. TITLE AND SUBTITLE

SEPARATION AND PURIFICATION SECTION:

ACTIVITIES - JULY 1970 to JUNE 1971

SUMMARY OF

5. Publication Date November j 1971

6. Performing Organization Code

7. AUTHOR(S)

David H. Freeman and Walter L. Zielinski, Jr

8. Performing Organization

10. Project/Task/ Work Unit No.

31 0900

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

11. Contract/Grant No.

12. Sponsoring Organization Name and Address

Same

13. Type of Report & Pejiod

Covered Inter MI

July 1970 to June 19'

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or litetature survey, mention it here.) This is the annual progress report on the activities of the

Separation and Purification Section. During the past year our studies

in liquid chromatography, its application, control, and systems design

was continued at an accelerated rate. Mobile phase flow was treated

as an analogy to current in direct current circuitry, following removal

of liquid pulses generated by a piston pump. A sophisticated, yet

simple and inexpensive liquid chromatograph design is fully described.

This design has been incorporated into all liquid chromatography studies

being conducted in this Section. The operating parameters of this

system design are discussed. The development of the underlying theory

and synthesis procedures for interactive gels has led to the control

and prediction of solute retention, selectivity, and a magnified column

capacity for use in fraction collection and ancillary characterization c solute impurities in clinical Standard Reference Materials. A gas

chromatographic method is described for the analysis of nitrilotriacetic

acid based upon the formation of its trisilyl ester derivative.

17. key words (Alphabetical order, separated by semicolons) Clinical Standard Reference Material

fraction collecting; gas chromatography; interactive gels; liquid

chromatography design; nitrilotriacetic acid.

18. AVAILABILITY STATEMENT

[3 UNLIMITED.

I | FOR OFFICIAL DISTRIBUTION. DO NOT RELEASE

TO NTIS.

19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

20. SECURITY CLASS

(THIS PAGE)

UNCLASSIFIED

21. NO. OF PAG

6U

22. Price

60 cents

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from the Bureau's work in physical measurement,

data processing, and development of test methods.

It will also cover some of the work in acoustics,

applied mechanics, building research, and cryogenic

engineering. Issued quarterly. Annual subscription:

Domestic, $5.00; $1.25 additional for foreign mailing.

TECHNICAL NEWS BULLETIN

The best single source of information concerning the Bureau's research, developmental, cooperative, and

publication activities, this monthly publication is designed for the industry-oriented individual whose

daily work involves intimate contact with science and

technology

for engineers, chemists, physicists, re- search managers, product-development managers, and

company executives. Annual subscription: Domestic,

$3.00; $1.00 additional for foreign mailing.

Applied Mathematics Series. Mathematical tables, manuals, and studies.

Building Science Series. Research results, test methods, and performance criteria of building materials, components, systems, and structures.

Handbooks. Recommended codes of engineering

and industrial practice (including safety codes) de- veloped in cooperation with interested industries,

professional organizations, and regulatory bodies.

Special Publications. Proceedings of NBS conferences, bibliographies, annual reports, wall charts,

pamphlets, etc.

Monographs. Major contributions to the technical

literature on various subjects related to the Bureau's

scientific and technical activities.

National Standard Reference Data Series.

NSRDS provides quantitative data on the physical

and chemical properties of materials, compiled from

the world's literature and critically evaluated.

Product Standards. Provide requirements for sizes, types, quality, and methods for testing various industrial products. These standards are developed co- operatively with interested Government and industry

groups and provide the basis for common understanding of product characteristics for both buyers and

sellers. Their use is voluntary.

Technical Notes. This series consists of communications and reports (covering both other agency and

NBS-sponsored work) of limited or transitory interest.

Federal Information Processing Standards

Publications. This series is the official publication

within the Federal Government for information on

standards adopted and promulgated under the Public

Law 89—306, and Bureau of the Budget Circular A-86

entitled, Standardization of Data Elements and Codes

in Data Systems.

Consumer Information Series. Practical information, based on NBS research and experience, covering

areas of interest to the consumer. Easily understandable language and illustrations provide useful background knowledge for shopping in today's technological marketplace.

NBS Special Publication 305, Supplement 1, Publications of the NBS, 1968-1969. When ordering, include Catalog No. CI 3. 10: 305. Price $4.50;

$1.25 additional for foreign mailing.

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