NISTIR 89-3933

THE BASICS OF

CHEMICAL

INSTRUMENTATION

VOLUME I - SEPARATION

METHODS

Thomas J. Bruno

U.S. DEPARTMENT OF COMMERCE

National Inatltuta of Standards

and Tochnology

National Moasuromont Laboratory

Cantor for Chamlcal Tochnology

Tharmophyslcs Division

Bouldar, Colorado 80303-3328

U.S. DEPARTMENT OF COMMERCE

Robart A. Mosbachar, Secretary

NATIONAL INSTITUTE OF STANDARDS

AND TECHNOLOGY

John W. Lyons, Director

NIST
u
NISTIR 89-3933

THE BASICS OF

CHEMICAL

INSTRUMENTATION

VOLUME I - SEPARATION

METHODS

Thomas J. Bruno

U.S. DEPARTMENT OF COMMERCE

National Institute of Standards

and Technology

National Measuromont Laboratory

Canter for Chemical Technology

Thermophysics Division

Boulder, Colorado 80303-3328

February 1990

U.S. DEPARTMENT OF COMMERCE

Robert A. Mosbacher, Secretary

NATIONAL INSTITUTE OF STANDARDS

AND TECHNOLOGY

John W. Lyons, Director

Ill

ABSTRACT

This report is intended to provide an introduction to chemical

instrumentation to workers whose training lies outside the areas

of experimental chemistry. It is especially geared toward

scientific managers in government and industrial laboratories

who must interact on a daily basis with chemical professionals,

and who often have highly sophisticated analytical chemistry

laboratories under their jurisdiction. This first report deals with

instrumental separation methods. After an introduction to the

nature of mixtures, the topics of gas chromatography, high

performance liquid chromatography, supercritical fluid

chromatography and electrophoresis are discussed. These

sections are followed by an appendix covering calibration

methods. A glossary is provided in which terms not covered in

the body of the report are defined.

Key words: chemical instrumentation; chromatography;

electrophoresis; gas chromatography; liquid chromatography;

supercritical fluid chromatography; separation methods.

PREFACE

This work and its companion volume on spectroscopic methods

are intended to provide an introduction to chemical

instrumentation for workers whose fields lie outside of

experimental chemistry. This includes scientists and engineers

whose interests and endeavors are principally in theoretical

areas. The book is also intended to meet the needs of scientific

managers in government and industrial laboratories, as well as the technical staffs involved with corporate and government

patent law.

Theoretically inclined scientists and engineers often find

the need to collaborate with experimentalists in the testing of

theories and models. Since questions of chemical purity are

almost universal in experimental science, such collaboration will

often bring the theorist into contact with chemical analysis.

This book attempts to serve their needs by providing a simple

discussion of each major technique without an overemphasis on

the fine details of hardware. At the same time, however, the

text does not oversimplify the techniques. In this respect, we

attempt to cure the "black box syndrome," in which

theoreticians often do not have sufficient appreciation of the

complexities of analytical instruments and methods

development.

It is very common that middle and upper level managers

in government and industry have in their chains of command
VI The Basics of Chemical Instrumentation

some of the most sophisticated and costly analytical laboratories

in the world. It is also quite common that these managers have

little or no formal training in experimental chemistry. Many are

physicists or engineers, yet they must interact on a daily basis

with personnel who specialize in chemical instrumentation.

The managerial function will usually include presentations

to technical audiences and higher levels of management

(sometimes to Congress), preparation of proposals, and the

supervision of resource allocation. These tasks will often require

a basic familiarity with chemical instrumentation. Clearly,

these managers do not have sufficient time to read through the

excellent undergraduate or graduate level texts now available

that cover instrumentation. Indeed, these texts usually

accompany a one— or two—semester course of study, and contain

laboratory exercises and problem sets. Such a treatment may

seem a bit too detailed and intensive for an executive who is

continually pressed for time. This book will answer their needs

by providing a concise, conceptual discussion. Mathematical

expositions of each technique’s theory of operation are

intentionally kept to a minimum, since the desire is to convey

an intuitive "feel

H for the processes involved.

Another important audience for which this book is

intended is the community of attorneys involved in patent law,

a vital part of commerce. Many patent attorneys and counselors

have undergraduate degrees in chemical fields, but have little

time to remain current. This book affords such professionals a

basic understanding of modern instrumental techniques,

including approaches such as supercritical fluid chromatography,

a field which is relatively new.
Preface vii

The basic philosophy which is stressed throughout the

book is one of simplicity. The reader is urged to use the

simplest approach possible to solve a particular problem, while

still maintaining the required degree of accuracy. This involves

choosing not only the simplest instrumental method which is

suitable, but also the simplest calibration method which will

appropriately answer the analytical questions which are posed.

This philosophy was eloquently expressed by Russel B. Scott

(1902 — 1967), a world-renowned low temperature physicist at

the National Bureau of Standards, who once stated "If you can

do an experiment using a piece of copper tube and a few pipe

fittings, and still get the accuracy you need, then that is where

you stop."

The required level of accuracy is invariably determined by

the application for which the data are needed. In some cases, it

may be adequate to simply determine that a particular chemical

is present in a sample, while in another case, a quantitative

measure in the parts per trillion range may be an absolute

necessity. The difference in cost involved in qualitative

identification and parts per trillion quantitation is likely to be

quite significant. It is entirely appropriate and necessary to

include economic considerations in the decision—making process

when one specifies the required level of accuracy. In this text,

we address these questions explicitly.

The author is indebted to a number of people who helped

in important ways during the preparation of the manuscript.

Ms. Stephanie L. Outcalt, a chemical engineer at NIST, assisted

in research and figure preparation. Assistance with graphics was

provided by Wesley Breshears, Douglas Farmer, and Tony

0
Vlll The Basics of Chemical Instrumentation

Dang, all physical science aides at NIST. Ms. Carrie McCann

did an excellent job of preparing the camera—ready copy. The

financial support of the National Institute of Standards and

Technology, Center for Chemical Technology, is gratefully

acknowledged. The author must also thank Ms wife, Clare,

whose patience and support make writing an enjoyable

endeavor.

Boulder, Colorado T. J. Bruno

Note: Certain commercial equipment, materials, instruments,

and devices are identified in this volume in order to provide an

adequate description. Such identification does not imply

endorsement or recommendation by the National Institute of

Standards and Technology, nor does it imply that the

equipment, materials, instruments and devices are necessarily

the best available for the purpose.
LX CONTENTS

Page

ABSTRACT iii PREFACE v

1. THE NATURE OF MIXTURES

1.1 INTRODUCTION 1

1.2 INTERMOLECULAR FORCES 2

1.3 MOLECULAR GEOMETRY AND ISOMERISM ... 7

1.4 PHYSICAL PROPERTIES 11

SUGGESTED READING 16

2. GAS CHROMATOGRAPHY

2.1 INTRODUCTION . 17

2.2 GAS-LIQUID CHROMATOGRAPHY 31

2.3 GAS-SOLID CHROMATOGRAPHY 34

2.4 OPEN TUBULAR COLUMN CHROMATOGRAPHY . 41

2.5 SAMPLE INTRODUCTION . . 43

2.5.1 Flash Vaporizer 47

2.5.2 Splitter Injector 49

2.5.3 Splitless Injector 52

2.5.4 On—Column Injector 54

2.6 DETECTORS 55

2.6.1 Thermal Conductivity Detector . 55

2.6.2 Flame Ionization Detector ......... 59

2.6.3 Thermionic Detector 62

2.6.4 Electron Capture Detector 64

2.6.5 Mass Selective Detector 67
X The Basics of Chemical Instrumentation

2.7 DATA COLLECTION ............. 71

SUGGESTED READING .............. 74

3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

3.1 INTRODUCTION 75

3.2 LIQUID—LIQUID CHROMATOGRAPHY ..... 77

3.3 LIQUID-SOLID CHROMATOGRAPHY ...... 80

3.4 ION EXCHANGE CHROMATOGRAPHY ..... 80

3.5 SIZE EXCLUSION CHROMATOGRAPHY ..... 81

3.6 HPLC HARDWARE 82

3.6.1 Solvent Pumping Systems 86

3.6.2 Injectors ............... 91

3.6.3 Detectors * 91

3.6.4 Miscellaneous Hardware .......... 98

3.7 STATIONARY PHASES FOR HPLC ....... 100

3.8 SOLVENTS FOR HPLC 107

3.9 COMPARISON WITH GC .......... . 110

SUGGESTED READING Ill

4. SUPERCRITICAL FLUID CHROMATOGRAPHY

4.1 INTRODUCTION .............. 113

4.2 HARDWARE FOR SFC 120

4.2.1 Solvent Delivery Systems ......... 120

4.2.2 Injectors ............... 125

4.2.3 Detectors 127

4.3 MOBILE PHASES 130

4.4 STATIONARY PHASES ............ 134
Contents XI

4.5 COMPARISON OF SFC WITH GC AND HPLC . . 137

SUGGESTED READING 139

5. ELECTROPHORESIS

5.1 INTRODUCTION 141

5.2 PROTEINS 142

5.3 POLYNUCLEOTIDES 146

5.4 ELECTROPHORETIC SEPARATION 147

5.5 INSTRUMENTATION 153

5.6 SUPPORT MEDIA 156

5.7 DETECTION 163

5.7.1 Staining 163

5.7.2 Ultraviolet Absorption 164

5.7.3 Radiochemical Methods 165

SUGGESTED READING 169

APPENDIX I: CALIBRATION

1.1 INTRODUCTION 171

1.2 ABSOLUTE VERSUS COMPARATIVE METHODS . 172

1.3 SINGLE POINT CALIBRATION 174

1.4 TWO POINT CALIBRATION 177

1.5 MULTIPOINT CALIBRATION CURVES 178

1.6 STANDARD ADDITION METHODS ...... 181

1.7 INTERNAL STANDARD METHODS ...... 182

1.8 STANDARD REFERENCE MATERIALS 185

SUGGESTED READING 188

GLOSSARY 189

1

CHAPTER ONE

THE NATURE OF MIXTURES

1.1 INTRODUCTION

When we deal with analytical separation methods, we are

invariably dealing with samples which are presented as mixtures. These mixtures may be very simple, such as one

solute (or sample) dissolved in a solvent of our own choosing, or

quite complex, such as a potential hazardous waste sample over

which we have no control or prior knowledge. Most of the

mixtures we will consider for analysis in this volume will be

fluids or solutions, although gaseous samples are encountered as

well. In order to choose and properly apply a particular

instrumental technique, we will have to understand some of the

basic characteristics of fluid mixtures in general. This includes

some macroscopic features such as physical properties, as well as molecular and geometrical features on the microscopic level.
2 The Basics of Chemical Instrumentation Chap. 1

1.2 INTERMOLECULAR FORCES

The nature of mixtures is profoundly influenced by interactions

and forces which arise between the individual molecules which

compose the mixture. Indeed, it is largely the results of these

intermolecular forces that give us the observable bulk physical

properties of matter that we will discuss later. To effect a

separation of components of a mixture, we will need to

understand and take advantage of intermolecular forces and the

physical properties which result from these forces. This is not to

say that the start of each chemical analysis should begin with

the Schroedinger wave equation and quantum mechanics. We

will instead use our intuitive grasp of the microstructure of

matter to our advantage in desigmng effective separation

schemes using appropriate instrumentation. A comparison of

some important intermolecular forces and their energies with

representative chemical bond energies is presented in Table 1.1.

Dispersion forces (studied by London in 1930) which arise

among essentially nonpolar molecules contribute to both

attraction and repulsion. They are represented in Table 1.1 as the interactions occurring between simple fluids. At any given

instant, the electrons of a molecule (such as methane or carbon

tetrachloride) are in some configuration which will result in an

instantaneous dipole moment. A dipole moment occurs in a

molecule whenever the electrons and nuclei of the atoms are

arranged so that, at any given instant, part of the molecule has

a net negative charge and part has a net positive charge. This

instantaneous dipole moment can then electrostatically induce a

dipole moment in another molecule, resulting in a weak, short—
The Nature of Mixtures 3

TABLE 1.1 APPROXIMATE BONDING ENERGIES

Bond

Energy

kJ • mol -1

CH4 ...CH 4

Simple Fluids

0.5

CeHe—CeHe 1.6

C-H...0

Associating Fluids

11

C-H...N 13-21

S—trinitrobenzen e—mesitylene 40

C-C

Chemical Bonds

347

0—

H

465

range net attraction. This interaction is therefore alternatively

called the dipole-induced dipole interaction. The attraction can

be additive over several molecules, resulting in, for example,

short-lived nearest neighbor ordering. As the molecules

approach one another more closely, the weak attractive force

gives way to a net repulsive force. The susceptibility of a

molecule to induced dipole forces depends on the polarizability

of the molecule. A molecule has a high polarizability when it

contains groups with large, bulky electron clouds that can be

easily deformed. These dispersion forces will always be present

among molecules, regardless of the constituent atoms. They will

be the only forces which will occur between nonpolar simple

molecules such as the alkanes or the noble gases. Naturally,
4 The Basies of Chemical Instrumentation Chap. 1

molecules with more actively interacting atoms (such as oxygen,

nitrogen, and in some cases the strongly electronegative

halogens, for example) will display other interactions in addition

to dispersion.

A compound such as formamide, CHsNO, which has a

large permanent dipole moment, will show very strong

intermolecular interactions. The forces occurring between these

lands of molecules are called Keesom forces. These attractive

interactions will promote self association, causing the formation

of clusters of molecules. Because of this effect, these

dipole-dipole forces are considered to be orientation forces,

having a significant influence on long-range order (the scale is

large as compared to molecular size) and structure in a fluid.

Such highly polar molecules will be more strongly attracted to

one another than nonpolar molecules of comparable molecular

weight. Because of the strong interactions, mixtures of polar

molecules can be especially difficult and challenging to separate

using standard analytical techniques.

If we were to change one of the chlorine atoms of CCI4 to a

hydrogen, the formula and structure of chloroform, CHCI3,

results:

6

- 6

*

Cl

Cl ^>C—

H

«/
The Nature of Mixtures 5

where 6* and S~ represent fractional charges on the halogen

atoms and on the hydrogen. We now notice a significant change

in behavior due to the dipolar interaction known as the

hydrogen bond. This will occur when a hydrogen is bonded to

an atom which is in close proximity to an electronegative atom

(or group of atoms) such as oxygen, nitrogen or a halogen. The

hydrogen atom, being small and not shielded by a large electron

cloud, can interact with nearby electron—rich groups or

molecules. Thus, the hydrogen atom of chloroform will be

attracted to the oxygen linkage of an ether or the carbonyl of a

ketone, since they are relatively electron—rich. The hydrogen

bonding potential of a particular hydrogen is controlled by the

chemical neighborhood of the molecule. The electronegative

environment set up by the three chlorine atoms on the carbon

tends to unshield the hydrogen and make it "feel" more

positively charged to its neighbors. The hydrogen bond between

chloroform and a ketone would still be relatively weak, however.

A very strong hydrogen bond system exists in water, where a

relatively stable extended structure of four to five water

molecules can exist as a cluster at ambient temperature. No

such stable long-range structure exists in nonpolar fluids such

as CCI4 , in which dispersion forces are the predominant

interaction.

The hydrogen bond is relatively strong as intennolecular

forces go, having energies of 4 to 11 kJ/mol. These interactions

are highly directional in space, and may even be considered as being weak bonds. Hydrogen bonded systems will usually

present problems in the design of separations, especially those
6 The Basics of Chemical Instrumentation Chap. 1

involving chromatographic techniques. Peak asymmetry (which

we will discuss in detail) and the resulting quantitation

problems will usually have to be addressed in analyses of

systems such as these.

The stronger intermolecular interactions described above

can cause the formation of intermolecular complexes. A

complex is an observable, distinct pairing between molecules

which is higher in interaction energy and of a longer lifetime

than that which would be expected from random molecular

collisions. The complex is itself a discrete chemical entity,

having its own unique properties. For example, the well-known

complex formed between benzene and iodine has a unique

ultraviolet spectrum, having a band not present in the electronic

spectra of either pure benzene or pure iodine. This complex is

called a charge—transfer complex, since the movement of an

electron between the benzene tt—orbital system and the iodine

molecule is demonstrated. The hydrogen bonded clusters of

water molecules may also be considered a kind of complex.

Actually, complex formation can occur in any system containing

molecules which are net negative charge donors in combination

with negative charge acceptors. These complexes may be weak

and short lived, as in the case of chloroform with an ether, or strong enough to actually be isolated chemically. Complexation

will always have a significant effect on the properties of a

mixture and our ability to separate that mixture using

analytical techniques.

The forces which arise between ions in aqueous solution

are both very strong and long range. They will be an important

factor in many separations using liquid chromatography,
The Nature of Mixtures 7

especially when ion—exchange columns and ion—pairing agents

are used. Ions in solution will interact strongly with dipolar

molecules, causing ion—dipole interactions.

1.3 MOLECULAR GEOMETRY AND ISOMERISM

The physical arrangement of atoms in a molecule, which we

shall call the molecular geometry, can also have a profound

effect on the properties of a mixture. We shall consider two

examples of how differences in the molecular structure or

geometry of compounds can have a striking effect in one case

and a very subtle effect in another.

Ethanol and dimethyl ether are isomers that have the

chemical formula C 2H6 O, both with a molecular weight of 46.

The following table gives some of their relevant physical

properties:

Dimethyl Ether Ethanol

State Gas Liquid

Molecular Mass 46 g/mol 46 g/mol

Boiling Point -24.5° C 78.

5

e C

Melting Point -138.5° C 117.3° C

Clearly, these two chemicals have vastly different properties

despite having the same formula. The reason, as you may
8 The Basics of Chemical Instrumentation Chap. 1

suspect, relates to the previous section on intermolecular

interactions. The predominant interaction between molecules of

dimethyl ether is the London dispersion (or the van der Waals

interaction). While these interactions are attractive, the overall

effect is very weak. Ethanol, on the other hand, due to the

change in molecular geometry, has the specific interaction of

hydrogen bonding that we discussed earlier. As a result, it

actually exists in a long-range, self—associated network. The

effect of the hydrogen bond is so dramatic that it causes ethanol

to exist in the liquid state (at ambient temperature and

pressure) rather than in the gaseous state, as is the case for

dimethyl ether. We would not expect the problem of separating

a mixture of ethanol and dimethyl ether to be particularly

challenging on the basis of their physical properties.

Let us now turn our attention to two other isomers, each

having the formula C2F 3 CI 2 H and a molecular weight of

152 g/mol. These compounds are both representatives of the

newly developed ozone—benign alternative refrigerant fluids.

These compounds are of interest to many sectors of industry and

the environmental community, since they are not as long-lived

as the fully—halogenated refrigerants which are currently

thought to harm the atmospheric ozone layer. The following

table lists some relevant properties of these materials.

CF3 CHCI2 CCIF 2CHCIF

Boiling Point 27.1° C 28® C

Melting Point —107® C -78® C
The Nature of Mixtures 9

The slight difference in geometrical structure between these two

materials has a very subtle effect on their physical properties at

ambient temperature and pressure. Generally, the major

intermolecular forces to be considered for both are the dispersion

forces. As you may imagine, separation of these isomers for

analysis does indeed cause some difficulties, especially since

there is usually cross contamination of one in the other.

Our discussion of molecular geometry and isomerism

would be incomplete without a few words about stereoisomerism

and chirality. This is especially important in a book which

treats separation methods, since many of the more vexing and

rigorous analytical separations performed in recent years have

concerned stereoisomers. This is especially important in the

study of biomolecules for the pharmaceutical industry.

Stereoisomers are isomeric species which differ only in the way

the individual atoms are arranged in space. This can be

contrasted with the refrigerant isomers described above, in

which there were actually differences in the number of halogen

species on each carbon. Stereoisomers can be subdivided into

two groups, called enantiomers and diastereomers.

Isomers which are not superimposable upon their mirror

image are enantiomers:

I

vr>c—

y

/z

I

Y-—C<]¥

Z

mirror plane
10 The Basics of Chemical Instrumentation Chap. 1

This example shows a tetrahedral carbon atom with four

different substituents, W, X, Y, and Z. The solid lines indicate

a bond in the plane of the page, the dotted lines a bond beneath

the plane of the page and the filled triangles a bond projecting

from the page. If a molecule is superimposable upon its mirror

image, no isomerism exists. Molecules which cannot be

superimposed on their mirror images are termed "chiral" (from

the Greek work meaning handedness), while those that can are

called "achiral." Carbon atoms which contain four different

substituents are always chiral, and the carbon in question is

called the chiral center.

If we can isolate one enantiomer from the other, an

interesting optical phenomenon may be observed. The

individual enantiomers can rotate the plane of polarized Light to

the right or left, depending on their structure. In fact, one of

the isomers will rotate the plane to the right (the dextrorotary,

or isomer), and the other to the left (the levorotary, or

"

isomer) by the same amount. While the physics of this

interesting interaction need not concern us in the design of

effective analytical separations, we must have an appreciation of

the consequences of the structural features of these molecules.

An equimolar enantiomeric mixture (50 percent of each isomer)

will not change the plane of polarized light, since the tendency

to rotate to the right and left is exactly balanced. This mixture

is called a "racemic modification." If there is a greater amount

of one over the other, optical activity may be observed in the

mixture. The direction of polarized light rotation in such a

mixture will depend on the predominating enantiomer.
The Nature of Mixtures 11

The physical properties of the individual enantiomers

which compose the mixture will be identical. This includes

density, boiling point, vapor pressure, refractive index, etc. The

only difference, other than the possibility of optical activity, will

be in the relative reactivity and interactions toward

enantiomeric reagents. This is fortunate, since it will allow us

to carry out separations of chiral materials using the

chromatographic methods discussed in this book.

Diastereomers are a class of stereoisomers which are not

mirror image isomers, as opposed to being simply not

superimposable. These isomers are much simpler to deal with

from a separation point of view. The physical properties of

diastereomers are always different, in many cases strikingly so. The chemical properties of diastereomers, while similar, will also

be different. It will be much easier to successfully apply the

methods of chromatography to mixtures of diastereomers than

to mixtures of enantiomers.

1.4 PHYSICAL PROPERTIES

Our discussion thus far has centered around individual molecules

in mixtures, and how microscopic properties of molecules

influence mixture behavior. In general, we do not have the

means to examine and analyze individual molecules, but only

large collections of molecules. The molecular and structural

features which we have discussed interact in a kind of

cooperative phenomenon to produce what we call the physical
12 The Basics of Chemical Instrumentation Chap. 1

properties of materials. These properties are quite different for

the gaseous, liquid and solid states of matter. From the

standpoint of analytical separations, a number of physical

properties are very important indicators. Several of these are

summarized in Table 1.2, which provides a listing of the

typically expected values of these properties for the different

states of matter. We will describe some important general

aspects of these physical properties here, but will leave detailed

discussions until the appropriate techniques are described.

Naturally, many other physical properties can be of value in the

design of effective analytical strategies. We have chosen to

focus on just a few of them to illustrate their importance.

The density of a material is a fundamental property which

is related to the closeness of the individual molecules (the

interatomic distance, shown in the last column of Table 1.2).

The presence of intermolecular interactions such as hydrogen

bonding will influence the magnitude of the density. The

density of gases tends to be relatively low; those of liquids and

supercritical fluids are higher (and are comparable in

magnitude), and the densities of solids are relatively high. The

dependence of density on pressure and temperature is very

striking for the fluid phases (gas, liquid, and supercritical fluid).

This dependence is depicted in the P—V—T (pressure—volumetemperature) surfaces which we will encounter in Chapter 4,

when we discuss supercritical fluid chromatography. We will

make use of density in many ways in chemical analysis, since

this property will allow the prediction and control of many

aspects of sample solubilization.

14 The Basics of Chemical Instrumentation Chap. 1

The vapor pressure may be termed a two-phase property,

since it is a gaseous pressure which always exists above the

surface of a condensed phase (liquid or solid) . For this reason,

no value range is provided for either the gas or supercritical fluid

state, since these are both single-phase systems. The vapor

pressure is a useful parameter in the development of many

analytical methods for liquid samples. Since vapor pressure is

directly related to the normal boiling point of a liquid, we can

use it as a design parameter for many chromatographic

separations. From a practical point of view, we will have to

consider the effect of the sample vapor pressure on sampling and

on the instrumentation we choose to apply.

The diffusivity, thermal conductivity, and viscosity are a

related set of physical properties which are called transport

properties. They describe the efficiency of either heat or mass

transfer that can be accomplished in a given state of matter.

The diffusivity is an important property to consider in all types

of chromatographic analyses (Chapters 2, 3 and 4), since it will

describe the tendency of a solute to disperse into the carrier and

stationary phase. The viscosity is also important in

chromatography, since in simple terms, it describes the

resistance of a fluid to flow. The thermal conductivity is

mentioned since instrumental methods often depend upon the

efficient transfer of heat to a sample, as in some injection and

detection techniques in chromatography. All of the transport

properties are dependent on temperature, pressure, and density.

Thus, by properly adjusting experimental variables, we can

often "tune" these physical properties to aid us in obtaining the
The Nature of Mixtures 15

desired analytical procedures and results. This is analogous to

the use of our knowledge of intermolecular interactions (such as hydrogen bonding) to choose chromatographic columns.
16 The Basics of Chemical Instrumentation Chap. 1

SUGGESTED READING

Molecular Complexes and Intermolecular Forces:

L. Andrews, Molecular Complexes in Organic Chemistry, Holden—

Day, San Francisco, 1964.

J. Rose, Molecular Complexes, Pergamon, Oxford, 1967.

M. D. Joesten and L. J. Schaad, Hydrogen Bonding , Marcel Dekker,

New York, 1974.

J. N. Israelachvili, Intermolecular and Surface Forces , Academic

Press, Orlando, 1985.

Isomers and Stereochemistry:

J. March, Advanced Organic Chemistry, 3rd Edition, John Wiley

and Sons (Wiley Interscience), New York, 1985.

Thermophysical Properties:

R. C. Reid, J. M. Prausnitz, and B. E. Poling, The Properties of

Gases and Liquids , 4th Edition, McGraw-Hill, New York, 1987.
17

CHAPTER TWO

GAS CHROMATOGRAPHY

2.1 INTRODUCTION

The term "gas chromatography" is a broad label which includes

many separate, distinct techniques and approaches. The

successful use of gas chromatography (GC) to solve an analytical

problem will require an understanding of the many differences

and tradeoffs in approach and instrumentation. We therefore

have a set of decisions and choices to make. We will have to

decide beforehand what exactly the questions are which we want

answered, and to what level of certainty. Only then can we

attempt to provide the answers using this technique (or any

analytical technique, for that matter). Our earlier discussion on

the nature of mixtures will play a vital role in guiding us to the

optimal (note that the word "correct" is avoided) combination

of hardware and method that will provide the answers we want.
18 The Basics of Chemical Instrumentation Chap. 2

Our discussion of gas chromatography will be very detailed for

two reasons. First, GC is the most important and economical of

all the separation methods. Second, many of the underlying

principles and approaches which pertain to GC also pertain to

liquid and supercritical fluid chromatography, which are

discussed in following chapters.

We must first understand that "gas chromatography"

broadly covers three major separation-analysis methods. All of

these methods share the common characteristic of a gaseous

mobile phase or eluent that carries the solute or sample through

the stationary phase, where separation occurs. While the eluent

gas chosen is usually helium, nitrogen or sometimes hydrogen 1

, one may choose many other carrier gases (or gas mixtures)

depending on the problem to be solved. We can gain a basic

understanding of the chromatographic process by considering the

series of "snapshots'® in Figure 2.1. Note that each frame of our

chromatographic movie is divided in half, one part marked M

(for mobile phase) and the other marked S (for stationary

phase). The moving carrier is always present in the mobile

phase part of the figure as it represents a steady flow of the gas.

The stationary phase contains an interacting (or active) species,

labeled D, which is affixed to a stationary base (hence the name

stationary phase).

The first frame of our movie shows the relatively

uninteresting situation of carrier gas flowing through the

stationary phase. The second frame shows the appearance of A,

hydrogen is a flammable gas which easily forms explosive mixtures with

air and thus must be handled with extreme care, preferably in an

explosion—proof laboratory.
Gas Chromatography 19

M

INTERACTION

(RETENTION)

COATING

SUPPORT

Figure 2.1 A "chromatographic movie" showing the processes which occur

in a column.
20 The Basics of Chemical Instrumentation Chap. 2

our sample. The nature of the sample, whether it is a polar

mixture or an inert gas, need not concern us just yet. We will

also put off the very important question of "just how did the

sample get in there?" Subject to both thermodynamic and

kinetic considerations, some of our sample will cross the

imaginary line and enter the environment of the stationary

phase, as in Frame 3. Depending on what kind of intermolecular

forces are present between A and D, an interaction of some kind

(weak, strong or in between) will occur, as seen in Frame 4.

Here is where our discussion of intermolecular forces becomes

important. The success of any gas chromatographic method will

very often depend upon our skill in using these forces as a tool.

The stronger the interaction between A and D, the longer that

interaction will last. We do wish to recover and detect our

sample, so we design the interaction to ultimately release A, as shown in frames 5 and 6.

In reality, our movie will be replayed many hundreds of

times as the sample is carried through the stationary phase by

the carrier gas. Although we have not mentioned temperature,

we have assumed implicitly that it has been constant during our

"chromatographic movie." As we might expect, raising the

temperature would be like shifting our movie into "fast

forward;" the A-—D interaction would in general be shorter in

duration, and the elution time faster. Just a brief comment on

the notation of Figure 2.1 may be in order. The A and D

moieties represent electron pair acceptor and donor species,

respectively, as in our discussion of intermolecular complexes.

In this figure we have chosen to make the stationary phase the

donor, but the reverse situation may be just as likely.
Gas Chromatography 21

So far, so good, but what about a mixture with, say, ten

components? An example might be a homologous series of

hydrocarbons. The strength of the interactions of each of the

individual components with D will be different, even if only

slightly. It would be difficult to imagine a mixture containing

components which all have identical chemical and physical

properties. Naturally, the more strongly interacting components

will form longer—lived interactions than those which are weaker.

These components will be "retained” in the stationary phase

longer, and will be released from the stationary phase later and

therefore detected later than other components. As we have

mentioned, temperature also makes a difference, and we will

always use it to our advantage in the design of a separation. In

general, the separation which will occur between two given

components will be greater at a lower temperature. While our

treatment so far is a drastic oversimplification, we will make do

for now and add details when needed as our discussion

progresses.

We can better understand the separation process by

referring to the hypothetical chromatogram shown in Figure 2.2.

The chromatogram is presented as a graph of detector signal

level (here represented as millivolts) as a function of time. Two

peaks have emerged, corresponding to two separated

components, a and b . These two peaks serve to illustrate three

important chromatographic parameters: efficiency, selectivity,

and resolution.

The efficiency of a column is a kinetic (rate dependent)

parameter which describes the column’s ability to separate

components sharply as a function of the sample residence time
22 The Basics of Chemical Instrumentation Chap. 2

Figure 2.2 An idealised chromatogram illustrating the concepts of

retention time and peak width.

in the column. The historical analogy between distillation and

chromatography resulted in means of expressing column

efficiency using chemical engineering parlance. The concept of

the "theoretical plate," or more simply, the plate number is

really a measure of solute zone spreading or dispersion. A
Gas Chromatography 23

theoretical plate can be thought of as an imaginary zone or volume element in which rapid equilibration of the solute

between the mobile and stationary phases can take place. For

the idealization of a Gaussian—shaped peak, the number of

plates contained on a given column is given by:

width (at half the peak height) expressed in units of time. The

larger the value for the dimensionless number n , the higher is

the column efficiency. An alternative expression for efficiency is

the "height equivalent to a theoretical plate," h , which is found

from:

where L is the length of the column. The efficiency of the

column increases as h decreases. It must be emphasized that

these simple equations are strictly applicable only to the

Gaussian curve. For asymmetric peaks the equations become

more complex and approximate, subject to many simplifying

assumptions.

There are many factors which affect the efficiency of a

column for the separation of a given solute. The major factors

are the mobile phase velocity through the column, the nature

and thickness of the stationary phase film (if any), the viscosity

n = 5.545 (t

r/w

1/2) 2

(2.1)

h = L/n ( 2 . 2 )
24 The Basics of Chemical Instrumentation Chap. 2

of the mobile phase, and column internal diameter. These

factors, along with parameters such as solute-solvent diffusivity,

can be controlled in order to optimize separations. The

situation can become complex, however, since most of these

factors are interdependent, many are temperature dependent,

and changing one parameter may cause a change in another.

The efficiency of a column can be predicted quantitatively

using the Van Deemter equation:

h = A + B/u 4- C'U . (2.3)

In this equation, A is a geometrical constant which describes the

multiple paths which carrier and solute can take in a packed

column (we will discuss the different column types in more

detail later). This is sometimes called the eddy current term

because of the electronic analogy of current flow inhomogeneity.

This term is minimized by keeping the particle size of the

packing as small as possible (approximately 200 /xm) without

causing an excessively high pressure drop across the column. As

we will see later, the value of this term can be made negligible

by using an open tubular column (or capillary column). The

constant B describes the relative diffusion of the solute in the

gas and stationary phase. It is most significant at low carrier

gas velocity (u in the equation above). The C term represents

the resistance to mass transfer of the solute at the

solute/stationary phase interface. Slow mass transfer, of course,

means longer analysis times. A plot of h versus carrier gas

velocity (Figure 2.3) illustrates the Van Deemter equation and
Gas Chromatography 25

i o —

20 40 60 80 100 120

u, cm/s

Figure 2.3 Plots of the results from the Van Deemter equation for

hydrogen and nitrogen.

one of its major consequences. The plot shows a broad

minimum which corresponds to the optimal carrier gas velocity.

Typical values for practical operating linear velocities range

from 8 to 12 cm/s, depending on the carrier gas used.

Selectivity is an essentially thermodynamic parameter

which refers to the ability of a column to isolate two (or more)

solutes into distinct bands, such as the peaks a and b on

Figure 2.2. The selectivity is a function of the relative
26 The Basics of Chemical Instrumentation Chap. 2

magnitudes of the intermolecular forces between the solute and

the stationary and mobile phases. This is where our discussion

of intermolecular forces comes into play. For example, a

relatively polar stationary phase will interact more strongly with

polar solutes than with simple alkanes. The choice of stationary

phase is therefore a very important step in the design of a

chromatographic analysis. Several retention index systems have

been developed to aid in the choice of stationary phase and

provide adequate selectivity. The scaling systems of Kovats, of

Rohrschneider, and of McReynolds have all attempted to

address this problem. The most extensive tabulation available

is for the system developed by McReynolds, which provides an

index of retention for a set of reference compounds on a given

stationary phase as compared with the nonpolar hydrocarbon,

squalane. The McReynolds constant system can provide a good

starting point in the choice of stationary phase.

The ultimate goal in a chromatographic measurement is to

isolate a sample into successive bands corresponding to the

individual components of the mixture. A measure of the

isolation between components is the peak resolution, R:

R =

Atr

( 2 . 4)

where Atr is the difference in retention time of two successive

peak maxima, and w^

2

and w^

2

are their corresponding peak

widths. Resolution is improved by increasing the peak

separation (i.e., improving selectivity) or by decreasing the peak
Gas Chromatography 27

widths (i.e., improving column efficiency). It is important to

realize that a given column may have good selectivity while

having poor efficiency, and vice versa. An optimized separation

will produce both well—separated and sharp peaks on the

chromatogram, and do so while minimizing the retention time.

Resolution is adequate when the degree of separation

allows the particular analytical problem being considered to be

solved. It may not be necessary to obtain optimum ’’baseline

resolved” peaks in every situation. One must factor in the

economic considerations of time and material costs. Quite often

reasonable quantitative accuracy can be obtained with three

percent overlap of two successive peaks. This corresponds to

peak maxima separation of approximately 2w for two

adjoining Gaussian—like peaks of equal size.

Now for some more choices and decisions. We will divide

gas chromatography into three convenient and practical

classifications: gas liquid chromatography, gas solid

chromatography, and capillary gas chromatography. There are

other classification schemes we could use, but this one will serve

us well for the present. While the instrumental details of the

three classes are unique and must be understood to properly use

the methods, many of the features are common to all three.

Figure 2.4 shows a ’'generic” schematic of instrumental

features common to all gas chromatographs. The carrier gas is

usually supplied from a high pressure (approximately 15 to

16 MPa, or 2200 to 2400 psi) cylinder equipped with a twostage pressure regulating valve. The pressure of this high purity

gas (most often 99.995 percent purity or better) is usually

regulated down to between 0.4 and 0.7 MPa (i.e., 60 and
lo

llowmeter

Iracnon

28 The Basics of Chemical Instrumentation Chap. 2

Figure 2.4 A schematic diagram showing the essential components of a

typical gas chromatograph.

mlogralor.

or

computer
Gas Chromatography 29

100 psi). A set of traps to remove trace quantities of water and

oxygen are installed after the regulator, which are themselves

followed by particulate filters. Trace water is effectively

removed with a short length of tube filled with 5A molecular

sieve, which is a synthetic zeolite (calcium alumino silicate).

Commercial traps are available which contain redox resins to

remove oxygen. These traps can also remove traces of carbon

dioxide and carbon monoxide. The filters which follow the traps

are usually sintered metal fitted disks having a 3 [mi pore size.

The gas stream is usually split at this point because many

common detectors require two streams, one for the sample and

one with pure carrier as a reference for property comparison.

We will discuss the more common detectors in detail later on. After the division of flow, the gas is passed through another

stage of regulation. This is done using precision pressure

controllers followed by needle valves (flow controllers), or by

using a set of mass flow controllers. For work requiring the

highest accuracy, these valves are maintained at constant

temperature in a separate oven. Pressure measurement is

usually provided by precision gauges or sometimes strain gauge

transducers having an electronic readout. The flowrate of the

carrier gas through the column is an important parameter which

we have under our control. Flowrate is often measured at the

column exit using a soap—film flowmeter, or in situ using a

rotameter or anemometer. Increasing the flowrate will generally

have much the same effect as increasing the column

temperature, but not always. Usually, the samples will elute

faster, and the peaks will be more closely spaced using a higher

carrier gas flowrate.
30 The Basics of Chemical Instrumentation Chap. 2

The sample line contains an injection device which

introduces sample into the carrier stream while causing minimal

flow disturbance or pressure pulsation. Sample introduction is a

very important and tricky part of the chromatographic system

hardware. The choice and method of operation of this device

will very often mean the success or failure of an analysis,

especially for quantitative measurements and in the analysis of

trace levels of components. We will spend a good deal of time

discussing the injector when we treat GC hardware in more

detail. Following the injector in the flow stream is the column

oven area. This temperature-controlled region is usually a

forced—air oven (only rarely is a liquid bath used, most often for

physicochemical meaasurement work) large enough to hold

several chromatographic columns, but not so large that it has an

inconveniently long thermal lag rime. This will allow the oven

to respond quickly to temperature adjustments. The

temperature should be stable to ±0.5* C for analytical work, and

should ideally be programmable. Temperature programming

allows the oven temperature to vary during an analysis,

according to some desired scheme. For example, we may

"program" the column oven temperature to begin at, say,

100 s C, but to increase to 150* C at the rate of 2* C per minute.

Not all gas chromatography is done at elevated

temperatures; occasionally one must resort to low temperatures

(—100 to 0

e C) to separate some stubborn mixtures, especially

those involving gases and volatile organics. This can be done by

pumping a cryogenic liquid (such as liquid nitrogen) into the

oven, or by permanently mounting a Ranque—Hilsch vortex tube

on the oven. A vortex tube is a unique refrigeration device
Gas Chromatography 31

which can produce temperatures as low as -40° C simply using a

source of compressed air.

Following the column oven are the detection devices. Now

our earlier discussion of bulk properties will come into play,

since we need an understanding of the behavior and properties of

our sample components to choose an appropriate detector. Our

generic gas chromatograph has two detectors; a thermal

conductivity detector and a flame ionization detector, the

responses of which may be logged on a computer or on an

electronic recorder/integrator. Most modern gas chromatographs are equipped with multiple detectors, in order to make

the instrument more versatile. We will discuss all of the more

common detectors in detail, including some of the more useful

optimization strategies.

2.2 GAS-LIQUID CHROMATOGRAPHY

Gas—liquid chromatography (GLC), as we will describe it, is

characterized by a stationary phase consisting of a liquid which

is coated or immobilized on an "inert " packed bed of supporting

material. The liquid coating is what we previously called D, the

stationary, interacting species in our "chromatographic movie."

This liquid may be materials such as a heavy hydrocarbon (for

example, squalane), a light polymeric liquid (such as polyethylene glycol), or a heavier silicone fluid (for example,

polymethyl siloxane). The choice of stationary phase liquid,

which we will discuss shortly, depends on the sample we wish to

analyze. The inert bed of support material is usually a
32 The Basics of Chemical Instrumentation Chap. 2

diatomaceous earth, although in some circumstances glass beads

or Teflon particles may be chosen. Diatomaceous earth, which

consists of the skeletal remains of small prehistoric algae called

diatoms, is about 88 percent silica. The materials used for

chromatographic supports are purified by acid washing, and the

surface is deactivated by a process called silanization in which

active sites on the surface are capped synthetically. While this

treatment cannot make the support surface perfectly inert, the

deactivated surface is very suitable as a support for the analysis

of all except the most polar of materials, such as alcohols and

organic acids.

Even the most highly deactivated surface will be affected

by the phenomenon of adsorption. Adsorption, which we will

discuss in more detail in the section on gas-solid chromatography, is a universal adhesion that occurs between the mobile

phase (both the carrier and sample molecules) and the surface of

the stationary phase. A number of different supports may be

chosen, differing mainly in density and available surface area. The choice, of course, depends upon the individual application,

but the variety of supports provides a degree of flexibility.

The liquid is easily coated on the support by dissolving the

liquid phase in a slight excess of a suitable solvent and adding

the support to the mixture. The solvent is then removed in a

rotary evaporator, leaving the support coated with the liquid.

This may seem unusual, since the coated support will look and

feel quite dry. What we mean, of course, is that there are layers

of molecules of the liquid on the support, and these layers are

liquid-like in their structure (remember the representative

properties given in Table 1.2). The coated support is poured
Gas Chromatography 33

into a tube which will serve as the column. The tube can be

made of copper, stainless steel, nickel, glass, Teflon or some

more specialized material. The diameter of this tubing can

range from 0.64 to 0.16 cm in diameter, and from 2 to 10 m in

length. Packed columns are relatively inexpensive and simple to

make up in the laboratory. The many possible choices of liquid

coatings are both a blessing and a curse. It is possible to design

a column to perform some very specific separations, but the

sheer number of possible coatings can be bewildering.

The chemical nature of the coated liquid imposes a

restriction on the operating temperature range. At lower

temperatures, many liquids will become very viscous or even

solidify. At higher temperatures, the liquid will "bleed" from

the support as a result of the increased vapor pressure. This is

clearly unfavorable, since not only does the separation power of

the column decrease, but also the detector response will become

noisy due to the continual passage of extraneous material.

Detector fouling can also result from column bleed, which can

cause significant instrument down-time. Some liquids will even

decompose at higher temperatures, also leading to detector

fouling. An understanding of the bulk and molecular properties

of the sample will guide the analyst toward a good choice of

stationary phase. Quite often, however, one or more "survey

runs" with several columns at several temperatures may be

needed to arrive at an optimal combination of liquid phase and

operating conditions for a particular analysis.

An important characteristic of gas-liquid chromatography

is the notion that the physical processes involved are actually

solution processes, with the sample being absorbed or dissolved
34 The Basics of Chemical Instrumentation Chap. 2

by the stationary liquid phase and later released through a

"vaporization” step. All of the thermodynamic treatments

applicable to solutions can be applied to gas-liquid

chromatography, including considerations of enthalpy, entropy,

equilibrium constants and activity coefficients. In fact, gasliquid chromatography has been used for many years as a tool

for the study of solution thermodynamics. One can actually

measure thermodynamic properties of solution, such as enthalpies and entropies of solution and vaporization, activity

coefficients, excess functions, and transport phenomena. Thus,

for gas—liquid chromatography, our "chromatographic movie” of

the previous section describes the repeated solvation and

evaporation of the sample into and out of the liquid phase.

2.3 GAS—SOLID CHROMATOGRAPHY

Most of the instrumental requirements of gas—solid chromatography (GSC) are very similar to those of gas—liquid

chromatography. The main difference is in the nature of the

stationary phase, and the processes which occur in that phase.

For clarity and convenience, we can divide the kind of

stationary phases available into three types: adsorption

columns, size exclusion columns, and porous polymer columns.

Naturally, many of the same physical processes occur in all of

these stationary phases.

The first kind of column we will consider is the adsorption

column. We stated earlier that adsorption was a universal
Gas Chromatography 35

adhesion process which will always occur at the boundary of any

liquid or solid with another medium such as a gas. The

difference between adsorption and absorption (or dissolution) is

depicted humorously in the now-classical cartoon shown in

Figure 2.5. The process is considered universal in that all solids,

whether porous or nonporous, metal or nonmetal, hard or soft,

will adsorb any gas, polar or nonpolar, on its surface. The

attraction may be caused by physical interactions (such as van

der Waals forces) or chemical interactions (possibly involving

electron transfer). Adsorption is an exothermic process; when a

gas is adsorbed on a solid, heat is liberated. Elementary

thermodynamic considerations therefore tell us that as the

temperature of a given system is increased, the amount of gas

adsorbed by the solid at a given pressure will decrease. We

should keep this in mind since, as we have already seen,

temperature is an important chromatographic variable which we

must control.

The most common packings used to prepare adsorption

columns are silica, alumina, carbon blacks (especially the

graphitized versions), and the synthetic zeolites (commonly

called molecular sieves). The column diameters are typically

the same as are used for the packings of gas-liquid

chromatography, but the column length is often less. All the

packings require heat activation prior to use, to drive off

material which is already adsorbed. The time and temperature

at which activation is performed very often will affect the

chromatographic behavior of the packing, so this step requires

careful attention. These adsorbents can be used successfully in

the separation of lower molecular weight saturated, unsaturated,
36 The Basics of Chemical Instrumentation Chap. 2

Figure 2.5 The difference between ABsorption and ADsorption .

aromatic, and halogenated hydrocarbons, as well as many

permanent gas separations.

Adsorbent packings are very easy to prepare in the

laboratory since the coating steps required for GLC columns are

not required. Alternatively, as with the more common

GLC columns, they can be purchased commercially made up and

ready to install in the chromatograph. The adsorbents can be

used at higher temperatures than most other chromatographic

stationary phases, since there is practically no danger of

decomposition or bleeding. The absence of bleeding is very

favorable from a detection standpoint, since the column will not

contribute "impurity noise" to the signal baseline. These phases

can also be used in subambient temperature work, since there is

no viscosity or solidification effect as there can be with coated
Gas Chromatography 37

liquid phases. With the one exception of the carbon black

packings, the adsorption phases are much more resistant to

degradation by trace amounts of oxygen in the carrier gas than

other phases. Naturally, carbon is subject to attack by oxygen,

especially at elevated temperatures.

The user must be aware of some pitfalls associated with

the use of adsorbent columns. Significant nonuniformity may

exist between different lots of silica and alumina adsorbents.

Many of the columns will adsorb some samples so strongly that

release will not occur without reactivation of the material at

high temperature. An example is the nearly permanent

adsorption of carbon dioxide by most of the zeolite molecular

sieve materials. They will also adsorb trace quantities of water

that may be present in the carrier and sample matrix, thus

changing the retention characteristics of the packing during

operation. Since the adsorption isotherms of these materials are,

in general, nonlinear, changing the size of the sample will change

the retention time on a given column. This will also cause peak

asymmetry (such as tailing) and sometimes incomplete sample

recovery from the column. Also, since the adsorptive surfaces

are of high energy, many polar and moderately polar samples

will show excessively long retention times on these columns.

Adsorption columns can be modified by coating the

material with a small amount of a nonvolatile liquid or an

inorganic salt. The most energetic sites on the surface of the

adsorbent will preferentially bind to these modifiers. As a

result, these sites will be unavailable to participate in retention.

This technique is sometimes called gas-layer—adsorption

chromatography.
38 The Basics of Chemical Instrumentation Chap. 2

The next type of column we shall consider is the

size—exclusion column. The packing for this column is the same

synthetic zeolite or molecular sieve which we discussed earlier as an adsorption column. The structures of these crystalline

aluminosilicate materials are unique in that they contain

cavities of a definite size, which can be controlled by the

elemental composition of the zeolite. The cavities are roughly

the same size as many smaller molecules such as water and the

permanent gases. Solute molecules in this size range which can

penetrate the internal pore structure will experience an increase

in retention time due to entropically enhanced adsorption. The

popularity of molecular sieve columns originally resulted from

their ability to separate oxygen and nitrogen rapidly under

reasonable chromatographic conditions. The most commonly

used packings are the 5A (calcium alumino—silicate, having an

average pore size of 0.5 nm), 13X (sodium alumina-silicate,

with an average pore diameter of 1.0 nm), and the 3A

(potassium alumino—silicate, having an average pore size of less

than 0.5 nm). Molecular sieve 4A (another sodium aluminosilicate, with an average pore size of less than 0.5 nm) has also

found chromatographic application.

Another type of molecular sieve is the carbon molecular

sieve, which is prepared from the pyrolysis of poly(vinyli dene

chloride). This material is very inert and nonpolar. The sieve

structure results from the cross-linking of individual, small

crystallites of carbon. This material has a high affinity for

hydrocarbons; in fact, water will elute before methane under

most chromatographic conditions. It is very useful for the
Gas Chromatography 39

separation of light inorganics, and has been particularly valuable

with mixtures containing water solutions of formaldehyde.

The final solid packing type that we will consider for

application in GSC is the porous polymer phase. These packings

are prepared using the method of suspension polymerization, in

which monomers and crosslinking agents undergo reaction in an

inert solvent. The more popular porous polymer phases are

copolymers of styrene and divinylbenzene. These polymers are

members of the Porapak series or the Century series of

commercially available packings. The porous polymer phases

are quite useful in the separation of polar compounds such as

alcohols, carboxylic adds, amines and amides, aldehydes and

ketones, and many organic and inorganic gases. The suspension

polymerization technique produces a sponge—like structure

having a uniform pore size which can be controlled by adjusting

the reaction conditions. Externally, the packing resembles

microscopic beads. The chromatographic behavior of the

packings is a function of the chemical nature of the polymer,

surface area and particle size in addition to the pore size. In

general, however, retention times increase as the pore size is

decreased.

Although we have chosen to discuss porous polymers under

GSC, there is some doubt as to the mechanism of retention on

these phases. At lower temperatures (say, below 100 e C),

adsorption appears to be the principle retention mechanism. At

higher temperatures, the polymers behave more like a coated

liquid phase (that is, the surface structure becomes liquid—like).

Under these conditions, a partitioning of the sample in the
40 The Basics of Chemical Instrumentation Chap. 2

polymer appears to occur, as was depicted in our

" chromatographic movie" of Section 2.1.

As with the other GSC phases, activation or conditioning

(at approximately 200° G) is required prior to use. This is done

in order to drive off any residual monomer or solvent, and to

remove any material which may be adsorbed on the surface. All

of the polymers have relatively high maximum operating

temperatures (at least 250° C), and introduce little impurity

noise to the detector. A somewhat different porous polymer

material is Tenax—GC, which is a linear polymer of p—2,6—

diphenyl—phenylene oxide. This material has a maximum

operating temperature of 375 e C, but it can be run at 400 9 C

for short periods. It is useful in the separation of high boiling,

polar compounds such as diols, methyl esters of dicarboxylic

adds, aldehydes and ketones, amines and amides, and phenols.

Short cartridges of Tenax—GC are also used as sampling

sorbents for the collection of volatile or gaseous samples. These

sorbent columns are espedally useful in environmental work,

and play a role in many standard procedures set by regulatory

agendes.

Some minor operational disadvantages may be experienced

in the use of the polymeric phases. It is sometimes difficult to

pack a porous polymer into a metal column due to the effects of

static charge. This is espedally pronounced in dry climates. It

is therefore often necessary to purchase columns already

prepared. Another problem occurs when some of the polymers

are used over a large temperature range. The polymer beads

tend to swell, thus changing the flow characteristics of a column.

This can lead to less than optimum detector performance.
Gas Chromatography 41

2.4 OPEN TUBULAR COLUMN CHROMATOGRAPHY

The final classification of gas chromatography we will consider is

capillary or open tubular column chromatography. This

technique centers around the use of columns consisting of long

(30 to 60 m), coiled, small diameter (0.25 to 0.05 mm inside

diameter) capillary tubes, usually made of fused silica coated

with a protective layer of polyimide. In recent years, fused silica

open tubes have been coated with a thin layer of aluminum in

order to extend the operating temperature, since the polyimide

will degrade at approximately 325° C. The stationary phase is

coated as a film on the inside surface of the tube, rather than a

coated packed bed of support particles. The flow heterogeneities

associated with packed columns are thus eliminated, along with

the A term (eddy diffusion) of the Van Deemter equation.

Capillary columns are available with efficiencies as high as 600,000 plates, in contrast to the typical maximum of 10,000 for

packed columns. Capillary column chromatography is useful for

all types of separations except permanent gas analysis. Of

course, preparative scale separations are also not suited to

capillary columns due to the very low solute capacity. The

technique is especially suited for the separation of complex

mixtures containing upwards of a hundred individual

components. In general, separations on capillary columns can be

performed at temperatures averaging 30“ C lower than would be

required using a liquid phase coated on a solid support.

The extremely high efficiencies of capillary columns make

stationary phase selectivity a less important consideration in the

design of an analysis. The most common liquid phases used
42 The Basics of Chemical Instrumentation Chap. 2

with open tubular columns are the methyl silicones and their

derivatives. An open tubular column coated with cross-linked

polymethyl silicone is essentially a boiling point separation

column (using primarily London dispersion forces as the

separation mechanism), while phenyl— and cyano—substituted

polymers provide for more specific polar interactions. These

liquids are coated to the desired thickness (typically 0.3 to 1 /mi,

although even thicker films are possible) and immobilized by

cross-linking the fluid to form a polymer network. The

silicone—based phases have high thermal stability, and can be

operated at temperatures in excess of 275° C.

The capillary column length is a relatively unimportant

parameter in the design of effective analyses. The parameter of

film thickness is very important, however. An increase of

0.3 /mi in film thickness can produce the same increase in

resolution as quadrupling the column length, without the

fourfold increase in analysis time that the longer column would

entail.

There are a number of disadvantages in the use of open

tubular columns which must be addressed. The manufacture of

these columns is a nontrivial process which cannot usually be

duplicated successfully in the laboratory. The commercially

available columns tend to be very expensive relative to packed

columns. Since the columns are so small in size, their sample

capacities are also quite small. To prevent column overload, a

flow splitter (to be discussed in more detail in the next section)

on the injection port is almost always necessary. The splitter

discards most (90 to 99 percent) of the sample injected from the

syringe. For trace analysis work, an injector capable of
Gas Chromatography 43

operating in M splitless mode" during injection is usually used.

These injectors are more complex and more expensive than the

simpler devices used with packed columns, and require a bit

more skill to use properly. An additional consequence of the low

sample capacity is the requirement for the most sensitive

detectors available. These are usually limited to the flame

ionization, thermionic, electron capture and mass selective

detectors. The small diameter of the open tubular column also

requires a higher degree of stability in the oven temperature

than is required for a packed column. The stationary phase

films in the columns are prone to oxidative damage, and

therefore require a constant flow of oxygen—free carrier at

temperatures very much above ambient.

2.5 SAMPLE INTRODUCTION

A major consideration in the design of effective GC analyses is

the method used to introduce sample into the carrier stream.

This involves the appropriate choice of chromatographic

injector, and the optimal operation of the device. If this initial

step is not handled properly, the outcome can be excessive

experimental error (reaching order of magnitude levels),

incorrectly assigned peaks, unexpected "ghost" peaks, or even no

peaks at all. We actually have a two—step process to deal with,

and we will discuss each step separately. The first step is to get

our sample into the appropriate injector, and the second is to

properly use that injector to apply the sample to the column.
44 The Basics of Chemical Instrumentation Chap. 2

The delivery of liquid samples to the injector is

conveniently performed using a graduated glass-barrel syringe

that usually has a total capacity of 10 /xL. When we use packed

columns, injection volumes may be as high as 5 /xL, while the

lower capacity of open tubular columns dictates volumes of

between 0.5 and 2 /xL. For the handling of volatile liquids and

solvents, it is possible to cool the syringe to -40° C using a

vortex tube, which supplies cold air to a glass jacket surrounding

the syringe. This approach can also aid in the analysis of

thermally sensitive mixtures. There are a number of pitfalls to

be aware of concerning the use of chromatographic syringes.

The influence of the needle volume on sample size, typically

0.8 /xL, must be considered. It is therefore desirable to

"bracket" the sample solution between two plugs of air or pure

solvent in the syringe in order to achieve the best

reproducibility. Some solutes can be adsorbed on the ground

glass parts of the interior of the syringe and thereby lost from

the sample. In addition, catalysis by this glass surface is not

unknown. Another difficulty, called sample discrimination,

occurs with all heated injectors such as flash vaporizers. This

will be discussed in more detail in the next section.

The application of solids directly into an injector presents

many problems. Fortunately, many solid samples can be

dissolved in a suitable solvent and handled the same way as a

liquid sample. When this is impossible, the best choice is an

encapsulation method. The sample is placed in a capsule which

can be opened inside of the injector. Capsules made of Wood’s

metal (which is melted at 60.

5

8 C), glass (which is mechanically
Gas Chromatography 45

crushed), or gold (which is pierced by a hollow "thorn") have

been used with varying degrees of success. The transfer of a gaseous sample to the chromatographic

injector must involve the consideration of temperature and

pressure since these variables have a profound effect on the

density (and therefore the number of moles) of a gas. We

learned this in the first chapter, when we discussed bulk

properties. The simplest and least expensive method to transfer

a gas to the injector is the use of a gas-tight syringe having a

volume between 0.001 and 50 mL. If the room temperature is

constant, a reproducibility of between 2 and 5 percent is

possible.

A better method of gas sampling involves the use of a

thermostatted sampling valve with a fixed volume sample loop

as the injector. Such a valve is schematically shown in

Figure 2.6. The valve consists of a stainless-steel body which

accomodates the necessary transfer lines, and a channeled

polyimide (usually) rotor which changes the flow path. In the

fill position, the gas to be analyzed is allowed to flow into the

sample loop. Enough sample gas should be allowed to flow

through the valve to ensure that any residual carrier gas is

swept from the loop and rotor. While residual carrier gas will

not show up on the chromatogram, its presence will impair

quantitative performance since carrier molecules will displace

molecules of sample. This will cause problems in achieving good

reproducibility among a set of replicate analyses. To assist in

carrier removal, some operators plumb a vacuum line into the

vent manifold. In the injecting position, the sample gas is swept
Load

46 The Baaica of Chemical Instrumentation Chap. 2
Gas Chromatography 47

into the column by the carrier gas. The valve is usually

maintained at an elevated temperature in a separate oven.

Temperatures of 150 to 250° C are normally employed to prevent

the adsorption of sample gas on the surface of the loop or rotor.

The rotors are usually made from a thermally stable polyimide

material which retains mechanical integrity over many cycles.

Using a sampling valve is the most accurate way to introduce

gaseous or very volatile samples into the chromatograph. The

total volume of sample injected is very reproducible since the

volume of the loop and rotor do not change from run to run. The injection process is very reproducible since the valves are

usually actuated using an electronically controlled solenoid or a

pneumatic cylinder. This provides for very rapid injection,

which minimizes the pressure pulse caused by the momentary

disruption of the carrier gas flow. The use of helium as the

pneumatic actuation gas usually provides the fastest switching,

especially if the pneumatic actuator is equipped with high—flow

pilot valves.

2.5.1 Flash Vaporizer

The most common type of chromatographic injector used

with all types of packed columns is the flash vaporization

injector, shown schematically in Figure 2.7a. The injector

consists of a heated cylinder containing a concentrically

positioned liner, usually made of glass or quartz. At the top of

the inlet is a self—sealing silicone rubber septum, through which

the syringe needle penetrates to introduce the sample. The

carrier gas inlet is usually at the base of the injector, and the gas
48 The Baaica of Chemical Instrumentation Chap. 2

septum

heater

quartz

liner

needle

guide

septum

retainer

stainless

steel body

quartz

sleeve

rarnar

inlet

to column

Figure 2.7a Schematic diagram of a flash vaporization injector.
Gas Chromatography 49

is warmed as it travels to the top, thus providing a preheated

flow stream. Flash vaporizers are relatively high volume

devices, sometimes having internal volumes as high as several

milliliters. They are usually maintained at a high temperature

(upwards of 200° C) to ensure complete vaporization of the

sample. This can be a disadvantage for a number of reasons.

Septum bleed, responsible for unassignable "ghost” peaks, is

always more severe at higher temperatures. This is aggravated

by the large volume of the injector, much of which may be

poorly swept by the carrier gas. In addition, flash vaporizers

cannot be used with heat sensitive samples. They all suffer from

a problem called "sample discrimination," which is especially

pronounced when the sample consists of a complex mixture of

components with widely varying boiling points. The more

volatile components of the sample will be more easily vaporized;

therefore the sample delivered to the column may be of different

composition than the one applied by the syringe. This makes

the flash vaporizer a marginal choice for the quantitative

analysis of complex mixtures. On the positive side, flash

vaporizers are low-cost devices and are easy to maintain. A

single unit can be used with packed columns made of different

materials and of different diameters.

2.5.2 Splitter Injector

Injection of samples into open tubular columns is a

somewhat more demanding process than injection onto packed

columns. As we discussed in the previous section, the sample

capacity of open tubular columns is much lower than that of
50 The Basics of Chemical Instrumentation Chap. 2

packed columns. This requires much smaller injector volume,

since sample volumes will rarely be larger than 2 /xL, and usually

closer to 0.5 /xL. To handle this situation, the splitter injector

(shown schematically in Figure 2.7b) was developed. This

injector consists of a heated liner (like the flash vaporizer) where

the sample is introduced by syringe and vaporized. A small plug

of glass wool or chromatographic support is located downstream

from this area in the split liner, to facilitate mixing of the

vaporized sample with the carrier. The flow of the mixture is

then split into two streams, one entering the capillary column

and the other vented through a back pressure regulator. Typical

split ratios (ratio of vented flow/column flow) are between 10:1

and 1000:1, controllable by the back pressure regulator. This

method of decreasing the column flow is done without affecting

the column pressure, and ensures that column overload does not

occur. Sample transfer to the column is fast, resulting in little

band broadening due to the injector volume.

Splitter injectors are more complex and costly than the

flash vaporizers used for packed columns. All splitters will cause

sample discrimination to some extent, although the problem can

be minimized more easily than with the flash vaporizer. The

elevated temperatures make the device unsuitable for use with

thermally labile samples. Obtaining reproducible data with a

splitter cam be problematical, since the split ratio which is set by

the operator before a run is not the ratio which is obtained when

sample appears at the splitter. The actual split ratio which

results is a complex function of sample volume and boiling point

range, solvent characteristics, injector temperature, syringe

technique, and the magnitude of the pressure pulse produced
Gas Chromatography 51

(N.O.

=

Normally

Open,

N.C.

=

Normally

Closed).

Courtesy

of

Hewlett-Packard

Corporation.
52 The Basics of Chemical Instrumentation Chap. 2

upon vaporization. Many of these variables are hardly

reproducible or predictable. For the best results, one should

reproduce the injection process precisely from run to run,

especially in terms of liquid volume injected and the time taken

for injection. It is also advisable, where possible, to use internal

standardization as the calibration method (a complete discussion

on the calibration process is provided in Appendix I).

2.5.3 Splitless Injector

An alternative injector for open tubular columns is the

splitless injector (Figure 2.7c), which is similar enough in

external construction to the splitter that a single injection port

can be modified to provide both split and splitless modes. As

with the splitter, small (0.5 to 1.0 /xL) volumes of liquid are

injected, making it attractive for trace analysis. This injector

uses a straight glass or quartz liner in place of the split liner,

and a solenoid valve diverts the purge flow during injection so

all carrier and sample present in the liner is deposited into the

column. The injection process is therefore much slower (the

rapid vaporization step required for the other injectors is not

needed) than that used in split mode, and is usually done at

lower temperatures. This makes the splitless injector attractive

for the injection of thermally labile samples, those samples with

components which elute near the tail of the solvent front, and

samples that are very dilute. The lower temperature is used to

refocus the sample volume, which is then deposited as a whole

into the column. An alternative to this thermal refocusing is to

make use of the so-called "solvent effect" in which the solvent
SAMI’I.K

Gas Chromatography 53 m o 5 «

<T3 to W 3O s o O "O

•+* QJ

lj ce

<u

O«—«

.s o

>>

®

—• 2 si s E

32 fc< a o ® Z * II q

S5a1 a • 0 J g O E a to to o o

55

O

II: s O “g ^ 10 s s

=s

J

la
54 The Basics of Chemical Instrumentation Chap. 2

plug is used as a secondary stationary phase to retard and

reform the solute components at the head of the column.

The splitless injector, while relatively simple in

construction and easy to maintain, can be difficult to operate

and optimize. The use of cool refocusing or the solvent effect

usually requires careful adjustment of the chromatographic

pneumatics, a trial—and—error process which can take longer

than the analysis itself. The sample size must be reproducible

within experimental error for the most precise quantitative

work. Septum bleed can be a serious problem even with lower

operating temperatures, since any septum impurities present in

the liner during splitless injection will be deposited on the

column.

2.5.4 On—Column Injector

An injector which can be adapted for both packed and

larger diameter open tubular columns is the cool on-column

injector. Using this device, the sample is syringe—deposited

directly into the packing or inside the capillary column. An

on—column injector is inherently simple, and is often found as a

homemade device modified from a flash vaporizer. Injection is

done quickly with the temperature maintained at or below the

boiling point of the solvent. Sample volumes are usually 2 /iL or

less. This method of injection is well suited to trace analysis, or work involving dilute solutions, since all of the sample is

deposited on the column. The effects of catalytic or thermal

degradation on the sample are minimal, as is sample

discrimination. It is generally the most precise injection
Gas Chromatography 55

method, and is relatively easy to implement and optimize,

especially with packed columns.

The application of on—column injection to the smaller

diameter capillary columns can be impossible, however.

Another disadvantage is that involatile material can be

deposited at the head of the column, accumulating over time.

Also, this injector does not lend itself well to automation.

2.6 DETECTORS

The function of the detector is, of course, to provide a

recordable, reproducible, quantitative response to the presence

of separated components as they emerge from the column exit.

We would like the response of the detector to produce a signal

that is proportional to the quantity of each component, and

linear over the concentration range of interest to us. Some of

the detection devices used in gas chromatography are based on

technology dating from the 1880’s, while others depend on

advances that are barely five years old. The nature of the

sample, the required level of sensitivity and the mode of

chromatography we wish to employ will play major roles in our

choice of detector.

2.6T Thermal Conductivity Detector

The first detection device we will consider is the thermal

conductivity detector (TCD), which is based on very old

measurement technology. This detector measures the change in
56 The Basics of Chemical Instrumentation, Chap. 2

a bulk transport property of the gas stream, and is therefore

universal in response. There are two basic TCD varieties

available, hot wire based and thermistor based detectors.

The most commonly used TCD is the hot wire version or katharometer, shown schematically in Figure 2.8, along with the

relevant Wheatstone bridge circuit. The carrier gas is passed

over short sections of coiled wire made from gold or rubidium

sheathed tungsten. Two cell compartments contain wires

bathed by the sample stream, while an additional two

compartments accept the flow from a reference column. The

wires in these four compartments form the legs of a Wheatstone

bridge circuit, with the chromatographic integrator or recorder

in place of the electrometer. The wire is electronically heated to

between 300 and 450° C, but the continuous stream of carrier

serves to remove much of the heat from the cell. High thermal

conductivity gases such as hydrogen and helium are especially

efficient heat removers. When sample elutes from the column

and enters the sample part of the detector cell, there will be a

differential change in the rate at which heat is removed from the

two sets of hot wires. This is due to the different (usually

lower) thermal conductivity of the sample relative to that of the

carrier. This leads to an increase in temperature of the wires in

the sample stream, and consequently an increase in their

electrical resistance. It is this electronic measurement of

resistance which provides the output. Alternatively, the wires

can be maintained at a constant temperature (regardless of the

presence or absence of sample) using a feedback circuit. The

response signal is then the measurement of the marginal change

in current applied to the wires which is needed to maintain the
Gas Chromatography 57

(a)

Filament

Current Control

(b)

Figure 2.8 A schematic diagram of the essentials of a thermal

conductivity detector, showing a typical cell configuration (a), and a

control and sensing circuit (b). (Courtesy of the Gow—Mac Instrument

Company.)

isothermal condition of the wires. This eliminates the

temperature gradient at the wire supports that occurs when the

wire temperature is allowed to change.
58 The Basics of Chemical Instrumentation Chap. 2

A hot wire TCD is prone to oxidation damage, and

therefore must not be operated without the constant flow of

carrier gas. The sheathed tungsten wires are susceptible to

attack by sulfur-containing compounds, and extensive use with

halogenated compounds win require special nickel alloy wires.

In addition, the volume of gas required for the TCD is relatively

high, even for the newer micro-scale cells. This usually limits

the usefulness of the hot wire TCD for applications with packed

columns only, where flow rates on the order of 20 to 40 mL/min

are possible.

The other type of TCD is one based on thermistor

elements instead of hot wires. The thermistors are

semiconductors in which the resistance decreases sharply with

increasing temperature. This type of TCD has a somewhat

higher sensitivity, but at a cost of operating temperature range.

The thermistors will fail at temperatures much above 150* C;

thus the detector cannot be used with high boiling point solutes.

The thermistor TCD is, however, very useful for work on

gaseous samples. The thermistor elements are much less prone

to oxidative damage than the hot wires, but they tend to be

sensitive to reducing agents. This usually precludes the use of

hydrogen as the carrier gas. Since the thermistors are small, it

is often possible to build detectors of much lower internal

volume than the hot wire based cells. Tins allows the use of

thermistor detectors with larger bore capillary columns.

When carriers other than hydrogen or helium are used

with a TCD, there is the possibility of obtaining negative peaks

on the chromatogram. This is because many materials have
Gas Chromatography 59

higher thermal conductivities than the other common carrier

gases such as nitrogen and argon. This is a problem since

integrators and chromatographic computer software usually

cannot process negative peaks. The TCD is a flow sensitive

detector which cannot be used easily for analyses that require

the column temperature to be programmed. The temperaturedependent viscosity of the carrier gas will change the flow rate

to the detector and thereby cause severe baseline drift, even if

mass flow controllers are used on the carrier pneumatics. While

the sensitivity of the thermal conductivity detector is somewhat

low as compared to other detectors, it does offer some unique

advantages which make it very popular. The TCD will respond

to water, carbon dioxide, carbon monoxide, and permanent

gases. It is in low cost, easy to operate, and requires very little

maintenance to provide good performance.

2.6.2 Flame Ionization Detector

The flame ionization detector (FID) is as popular for

routine analysis as the TCD. In fact, it is rare to find a gas

chromatograph which is not equipped with both of these

detectors. It is relatively simple in construction, and is easy to

operate and maintain. The FID may be considered universal in

response for organic compounds. A typical flame ionization

detector is shown schematically in Figure 2.9. A hydrogen — air

flame is maintained above the tube which delivers carrier and

sample from the column. The flame itself, in the absence of an

organic sample species, contains few ions and has a high

electrical resistance. When a sample containing carbon—
60 The Basics of Chemical Instrumentation Chap. 2

Figure 2.9 A schematic diagram of a flame ionisation detector. (Courtesy

of the Gow—Mac Instrument Company.)
Gas Chromatography 61

hydrogen bonds leaves the column and enters the flame it is

burned, and a cascade of ions is produced. This increases the

electrical conductivity of the flame by many orders of

magnitude. A collector electrode above the flame is maintained

at a potential of 180 to 300 V above that of the sample delivery

tube. This system of electrodes, in combination with a sensitive

electrometer, measures the flame conductivity and produces the

response signal.

The FID is one of the most sensitive and fast responding

detectors available. It can easily sense the presence of 10 _11

g

of

hydrocarbon, and the response will be linear over a wide

concentration range. The high sensitivity makes the FID very

suitable for use with capillary columns, where very small

amounts of material are analyzed. The device has high baseline

stability and shows little or no sensitivity to changes in the

carrier gas flow rate. It can therefore be used in temperature

programmed applications without the viscosity—induced

disruptions which plague the TCD. The dead volume of the

detector is very small, since the sample is released directly into

the flame.

Since any heated metal object will be a source of thermal

electrons, the FID performance is optimized at lower detector

temperatures. The body of the detector is usually heated just

enough to prevent sample or water condensation. Water is a

product of combustion of the hydrogen—air flame, and must not

be allowed to drip back into the base of the detector, since it

will cause temperature fluctuations and may even extinguish the

flame.
62 The Basics of Chemical Instrumentation Chap. 2

The FID will not show a response to water, carbon

dioxide, permanent gases and most inorganic species. This is a

drawback, although very often the chromatogram is "cleaner”

due to the absence of these peaks. When the FID is used with

samples dissolved in chlorinated solvents, soot formation in the

flame can be a problem. Soot can also form from a low level

bleed of silicone liquid phases, resulting in an insulating deposit

of silica in the detector. The response of the FID to species

containing heteroatoms (organic nitrogen and phosphorus

compounds, for example) can be unpredictable. It is sometimes

difficult to keep the flame lit when eluting these materials. For

this reason, most detectors can be operated with the igniter

energized and continuously glowing. The potential of thermal

electron interference makes this a last resort, however.

Several important operational disadvantages of the FID

must be considered. Since a hydrogen flame is used in the

detector, a slight but finite explosion hazard exists. An FID

would not generally be usable in an explosion—proof laboratory

(as defined in Class A, Group b of the National Electrical Code).

The detector is destructive to the sample and cannot be followed

downstream by another detector. It is not usually easy to

determine the carrier gas flow rate at the exit of an FID; one is

often forced to disconnect the column exit for this measurement.

2.6.3 Thermionic Detector

A modification of the FID is the thermionic detector

(Figure 2.10), a device which is specific for nitrogen or

phosphorus compounds. It is similar in construction to the FID,
Gas Chromatography 63

Collector

Ceramic

beau with

heater coil

Flame tip

Figure 2.10 A schematic diagram of a thermionic detector. (Courtesy of

Varian Associates.)

except that a small pellet of alkali metal salt is held in the

vicinity of the flame. The salts of potassium, rubidium, cesium

and sodium have been used. The detector is very sensitive for

certain compounds, approaching the level of the electron capture
64 The Basics of Chemical Instrumentation Chap. 2

detector (see following section). The use of this detector is

confined mainly to the analysis of pesticides.

2.6.4 Electron Capture Detector

The electron capture detector (ECD) is certainly a unique

device, since it has been called the best and the worst detector

available for gas chromatography. The love-hate relationship of

analytical chemists with this detector is no doubt the result of

the extreme sensitivity of the ECD. While it is a

chromatographers’ dream come true for some ultra-trace

analyses, the device can be a nightmare to optimize.

A simplified schematic diagram of an electron capture

detector is shown in Figure 2.11. There has been surprisingly

little change in the design of these devices ever since the first

viable unit was made in 1958. Naturally, significant advances

have been made in the sensing and control electronics since that

time. A radioactive source ionizes the carrier gas as it leaves

the column. The carrier used must be either pure nitrogen or argon that contains between 5 and 10 percent methane, since

these gases are most easily ionized. The carrier gas must be

ultra—pure, because any contaminants will interfere with

optimum operation. The radioactive source most used today is

a 83Ni foil, although some older models use adsorbed tritium,

3 H. The ionization of the carrier gas caused by the /3-radiation

produces a high background level of current measured across the

anode and cathode. A sample eluting from the column enters

this region, and will participate in ionization processes. If the

sample contains atoms that will absorb electrons (such as
Gas Chromatography 65

Figure 2.11 A schematic diagram of an electron capture detector.

(Courtesy of Varian Associates.)
66 The Basics of Chemical Instrumentation Chap. 2

halogens , nitrogen or organometallic moities), the background

ion current will drop sharply. It is this dramatic decrease in the

current which provides the high level of response from the ECD.

The response sensitivity of the ECD is strongly dependent

on the sample itself, and increases with the electronegativity of

the functional groups. Some "ideal" cases (for example, the

analysis of sulfur hexafluoride) have been reported in which an

ECD provided a response to 10 ~ 14 g/s of sample. The response

to an aliphatic hydrocarbon will be very slight, however. The

response will also be related to the operating temperature, which

is usually maintained at a level just high enough to prevent

sample condensation. The presence of trace quantities of

interfering species (such as oxygen, which is an electron

absorber) will also affect sensitivity. Even the careful control of

these factors does not guarantee reproducible results between

separate ECD’s, however. It is very important to use nonTM

bleeding stationary phases, such as the cross-linked methyl

silicones. Septa used in the injectors should be heat—conditioned

to minimize bleed, and the pneumatic lines should be baked out

to drive off adsorbed impurities.

As can be seen, it is a tricky business to optimize the

performance of the electron capture detector. Even in the ideal

situation, disadvantages persist. The linearity of the detector

over sample concentration ranges is comparatively low, being

two to four orders of magnitude. Once can compare this with

the eight—decade linearity of the flame ionization detector. This

is not a fatal flaw, however; the main consequence of the short

linear range is the need for a more careful and extensive

calibration (see Appendix I).
Gas Chromatography 67

2.6.5 Mass Selective Detector

The routine use of a mass spectrometer as a detector for

gas chromatography began the trend toward what have come to

be called hyphenated techniques (GC—MS). This combination is

one of the most powerful analytical techniques available. This is

because mass spectrometry is the single most useful tool for

organic structure elucidation and compound identification.

It is difficult to consider the mass spectrometer (or mass

selective detector, MSD, a term that will often be used

interchangeably) in the same vein as the TCD or FID. The cost

and complexity of the combination requires us to consider the

GC—MS instrument as a system unto itself. A schematic

diagram of such a system is provided in Figure 2.12. Only a

brief outline of the mass spectrometer will be given here, since

this topic is covered in Volume II of this text. The mass

spectrometer consists of a vacuum chamber pumped to a level of

1.3*10~3 to 1.3*10"4 Pa (10’5 to 10 -6 Torr). This can be

accomplished using a turbomolecular pump or a diffusion pump,

both of which must be backed by a mechanical rough pump

which produces 0.13 Pa (10‘3 Torr). Inside the vacuum chamber

is the ion source, the mass filter and the electron multiplier

detector. The ion source usually consists of an ’’electron gun”

filament which ionizes the sample eluting from the

chromatograph. Actually, only a small fraction of the sample is

ionized. Most of the sample leaving the column will be pumped

away without ever encountering an electron from the source.

Some instruments allow the use of chemical ionization reagent

gases as an alternative to electron impact ionization. The ion
68 The Basics of Chemical Instrumentation Chap. 2

Q. 0)

Figure

2.12

A

schematic

diagram

showing

the

essential

components

ofa

GC—

MS

system.
Gas Chromatography 69

source also contains a series of electronic lenses which accelerate

and focus the positive ions which are formed by the electrodes.

The beam of ions is passed to the mass filter, which is most

often of the quadrupolar type. The electron multiplier amplifies

the electron current signal, and a computer system is required to

process this signal into the familiar mass histogram format.

Usually, the output consists of a total ion chromatogram, which

is displayed as peaks as a function of time, just as the output

from any GC detector.

The tremendous advantage of the GC—MS system is that

one can choose any peak on the chromatogram, and at the punch

of a button, have a mass spectrum of that peak. The computer

allows manipulation of spectra, such as the addition,

subtraction, and averaging of spectra, and the searching of

measured spectra against libraries. This is of great value in the

qualitative identification of eluted samples. These libraries can

be built up by the user or purchased from the manufacturer.

The commercial libraries are usually based on the 49,000—entry

library developed jointly by the U.S. National Bureau of

Standards (now the National Institute of Standards and

Technology), the U.S. Environmental Protection Agency (EPA),

and the Mass Spectrometry Data Center of the Royal Society of

Chemistry (England).

The mass selective detector can be used in one of two

modes: mass scanning mode or single ion monitoring mode.

When operating in scan mode, the MSD will record complete

mass spectra over the molecular weight range selected by the

operator. It is usual to keep the molecular mass range which is

scanned as small as possible without sacrificing needed
70 The Basics of Chemical Instrumentation Chap. 2

information, since a finite time is require to sweep the spectrum.

This means if a large molecular weight range is scanned, there

will be relatively fewer spectra obtained per second than if a

smaller range is scanned. Single ion monitoring is used

primarily for high sensitivity quantitative analysis. Instead of

scanning over hundreds of molecular mass units, one (or several)

individual molecular mass values are selected for continuous

intensity measurement. When the MSD is used in this fashion,

the sensitivity approaches that of the electron capture detector.

The MSD is one of the most expensive detectors available

for the gas chromatograph. It can easily cost three or four times

more than the chromatograph itself. It is relatively complex

and requires some degree of training in order to be used

effectively. The interface between the chromatograph and the

high vacuum of the MSD requires some discussion as well. The

low mass flow rate of a capillary column can be accommodated

directly by most MSD pumping systems, but packed columns

usually require the use of a jet separator or some other device to

divert and vent the bulk of the carrier flow. A jet separator,

shown on the inset of Figure 2.11, draws off most of the column

flow in low vacuum. The solute molecules, being much heavier

than the carrier, remain roughly focused at the center of the jet

and are drawn into the ion source of the MSD by the high

vacuum. The use of a jet separator results in some degradation

of performance of the overall GC—MSD system. The major

manifestations of this are lower sensitivity and resolution.

Another consideration in the operation of a MSD is

maintenance which, relative to other GC detectors, is hardly

routine. Pumping system service, source cleaning, filament
Gaa Chromatography 71

replacement, and electron multiplier replacement must be done

periodically. This is usually an all-day job requiring extreme

care, ultra—clean conditions, and a very steady hand.

2.7 DATA COLLECTION

As shown in Figure 2.4, the output from whatever detector

is chosen is fed into a data collection system which can consist of

a strip chart recorder, a digital electronic integrator, or a

computer driven with special peak—processing software. A strip

chart recorder is not usually used in analytical applications of

GC, but is still quite useful in physicochemical work in which

the main experimental parameter is the retention time or retention volume. For analytical work, the use of a dedicated

electronic integrator is now standard. The purpose of the

integrator is to measure the area under each chromatographic

peak, since this quantity is most easily related to the quantity of

sample. The relationship between the peak area and sample

quantity or concentration is obtained by the calibration method,

discussed more fully in Appendix I. The analog output of the

detector is converted to a digital signal by the integrator, and is

internally manipulated as area slices in units of voltage, as depicted in Figure 2.13. Most of the modem integrators allow

calculation of concentration directly after the appropriate

calibration method is chosen and the standard values are stored

in the internal memory. Calculation of peak height and peak

widths is also standard, thus making theoretical plate height

calculations a simple matter. Many integrators can provide
72 The Basics of Chemical Instrumentation Chap. 2

programming (usually in BASIC) and disk storage of

chromatographic data as options.

Using appropriate software, a personal computer may be

used to process chromatographic data. The programs are quite

sophisticated, and allow deconvolution of very complex

chromatograms. Data from different detectors can be processed

and displayed in real time in separate windows on the computer

screen. The cost of a personal computer plus the necessary

chromatographic software is usually much higher than even the

best chromatographic integrator. Unless there is a compelling

reason for using a computer, the use of an integrator is to be

preferred on economic grounds. As was mentioned earlier, the

use of a computer with a dedicated software system is necessary,

however, when using the mass selective detector.
Gas Chromatography 73

WIDTH OF A SINGLE A/D READING

(AREA SLICE)

Figure 2.13 A schematic diagram illustrating the process of peak

integration for sample quantitation. (Courtesy of Hewlett-Packard

Corporation.)
74 The Basics of Chemical Instrumentation Chap. 2

SUGGESTED READING

Introductory Treatments

H. M. McNair and E. J. Bonelli, Basic Gas Chromatography.

Varian Aerograph, Walnut Creek, 1969.

A. Braithwaite and F. J. Smith, Chromatographic Methods, 4th

Ed., Chapman and Hall, London, 1985.

J. M. Miller, Chromatography: Concepts and Contrasts, John Wiley

and Sons (Wiley Interscience), New York, 1987.

J. E. Willet, Gas Chromatography. John Wiley and Sons (on behalf

of ACOL, London), Chichester, 1987.

P. A. Sewell and B Clark, Chromatographic Separations, John

Wiley and Sons (on behalf of ACOL, London), Chichester, 1987.

Detailed Treatments

C. F. Poole and S. A. Schuette, Contemporary Practice of

Chromatography, Elsevier, 1984.

R. L. Grob, Ed., Model Practice of Chromatography, John Wiley

and Sons (Wiley Interscience), New York, 1985.

E. Katz, Quantitative Analysis Using Chromatographic Techniques, John Wiley and Sons, Chichester, 1987.

M. L. Lee, F. J. Yang, and K. D. Bartle, Open Tubular Column

Gas Chromatography. John Wiley and Sons (Wiley Interscience),

New York, 1984.

Special Topics of Interest

C. J. Cowper and A. J. DeRose, The Analysis of Gases bv

Chromatography. Pergamon Press, Oxford, 1983.

R. J. Laub and R. L. Pecsok, Physicochemical Applications of Gas

Chromatography, John Wiley and Sons (Wiley Interscience), New

York, 1978.
75

CHAPTER THREE

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

3.1 INTRODUCTION

The systematic study of chromatography began with the work of

Mikhail Semenovich Tsvett (1872 — 1919). He described his

initial research on the application of adsorption phenomena to

the separation of biochemicals at a meeting of the Warsaw

Society of Naturalists in 1903. At this lecture, he described his

separations of plant pigments on more than 100 adsorbents in

funnels flushed with liquids. Thus, although most of the

chromatographic analysis performed today and reported in the

literature is done using gas chromatography, the very first work

done was in liquid chromatography.
76 The Basics of Chemical Instrumentation Chap. 3

The obvious difference between liquid chromatography and

gas chromatography is the use of a liquid as the mobile phase

carrier or eluent. We recall, from our discussion of gas

chromatography, that interactions of the solute with the carrier

gas were of little consequence. As long as the solute is present

as a vapor, it is miscible in all proportions with the carrier gas.

The gas does not actually participate in the partitioning process

with the stationary phase. With a liquid as the eluent, we have

the potential of intermodular interactions (such as hydrogen

bonding, complexation, and charge transfer, which were

discussed in Chapter 1) of the solute with both the mobile and

stationary phases. This adds another dimension of both

flexibility and complexity to this technique as compared to gas

chromatography. While gas chromatography depends on sample

volatization, liquid chromatography is done using samples in

solution at near—ambient temperature. This makes liquid

chromatography amenable to the analysis of heat-sensitive

compounds, macromolecules, natural products and biochemicals

which cannot be easily studied using GC methods.

Modem liquid chromatography is largely the result of

significant developments in instrumentation which boosted

efficiency and sensitivity as compared with the classical column

techniques. Advances in solvent pumps, injectors and detectors

and in the available stationary phases resulted in what we now

call high performance liquid chromatography (HPLC). We can

divide HPLC into four modes: liquid—liquid chromatography

(LLC), liquid—solid chromatography (LSC), ion—exchange

chromatography (IEC), and size-exclusion chromatography

(SEC, sometimes called gel permeation chromatography).
High Performance Liquid Chromatography 77

3.2 Liquid—Liquid Chromatography

The most important and commonly used modes of HPLC are the

varieties of liquid—liquid chromatography, in which the

stationary phase consists of a liquid or, more often, a species

having liquid—like structure, fixed on a support. This includes

the chemically bonded phases used in reverse phase HPLC,

which we will discuss later. Separation is achieved by the

partitioning of the solute between the liquid mobile phase and

the ’’liquid" stationary phase. Earlier adaptations actually

employed a liquid coating (water, for example) on silica, with

chloroform as the mobile phase. As you might imagine, much of

the water was stripped from the column by the mechanical shear

forces of the mobile phase acting on the liquid coating as it

passed through the packed bed. Modern adaptations generally

employ a liquid chemically bonded to a specially treated

support. Most of the supports currently used consist of a

specially treated silica having a tightly controlled particle size

distribution.

The partitioning process can be described by the

chromatographic parameter called the capacity factor, k:

k = (tri — tu)/^u (3.1)

where t ri is the retention time of a retained solute and t u is the

elution time of an unretained reference compound. The physical

meaning of Equation 3.1 can be understood by considering

Figure 3.1, in which two eluted solute peaks plus an unretained
78 The Basies of Chemical Instrumentation Chap. 3

A

® a

© £

Figure 3.1 An idealised chromatogram illustrating the concepts of

capacity and selectivity.
High Performance Liquid Chromatography 79

reference "peak” is shown. The capacity factor, being

dimensionless, could also be expressed in terms of the solvent

volume required for elution instead of the retention time. Thus,

the position of the unretained reference compound in a sense

represents the void volume of the column. The relationship of

the two retained peaks to one another is represented by the

separation factor, a, defined as the ratio of the capacity factors

(with the larger one in the numerator). By now you have no

doubt noticed the strong similarity of this discussion to our

description of the retention mechanisms of GC. The separation

factor gives us a means of judging the resolving power of a given

column/mobile phase combination. The capacity factor and

separation factor ignore the effects of peak dispersion. As with

GC, this is a function of column efficiency, which we describe in

terms of the theoretical plate height or total number of

theoretical plates on the column. The equation for the number

of plates on the column is very similar to the one used in GC:

N = 5.54 (V

r

/ w

1/2 )

2

(3.2)

where Vr is the volume of solvent required to elute the peak and

w^

2

is the width of the peak at one half of its height. Typical

HPLC columns available commercially are 25 cm long and have

25,000 to 50,000 plates, although some specialty packings and

microbore columns have considerably more. One way to

increase the separation factor is to increase the efficiency (as

described by the plate number) of the column. This can be done
80 The Basics of Chemical Instrumentation Chap, 3

by using smaller particles for the packing, using a slower mobile

phase flowrate, or increasing the column length. The other way

is to adjust the selectivity by using the solvent system to control

the partition process. It is this aspect of HPLC which requires

skill and experience. Indeed, the brute force method of

trial—and—error may be the only way to begin the optimization

process for tricky separations.

3.3 LIQUID-SOLID CHROMATOGRAPHY

Liquid—solid chromatography was the mode originally used by

Tsvett in his investigation of plant pigments. Separation is by

the reversible adsorptive interaction of the solute with a solid

adsorbent, and is dependent on the relative polarities of the

solute and adsorbent. The commonly used adsorbents are silica

gel, alumina, glass beads and polymer beads. Highly selective

and reproducible separations can be done using this mode. It is

most valuable when the sample is soluble in nonpolar or moderately polar solvents. The stationary phases used in LSC

separate solute mixtures into functional group classes. These

phases are also useful in the separation of mixtures of isomers

having similar properties.

3.4 ION-EXCHANGE CHROMATOGRAPHY

Modern ion—exchange chromatography (IEC), which is used in

the analysis of ionized or ionizable compounds, is the result of
High Performance Liquid Chromatography 81

nearly thirty years of technology. It is very useful in the

separation of biochemicals such as amino adds, nucleic acids,

and proteins by taking advantage of electrostatic (chargecharge) interactions. The ion exchange phase consists of a

polymer or silica support backbone to which the ion exchange

functional groups are attached. These bound functional groups

consist of an ion and a counterion, the latter being exchangeable

with an ion of the same sign in the solute. Ion exchange media

used to exchange cations bear negatively charged groups and are

most often sulfonate based. Ion exchange media used to

exchange anions bear positively charged groups, and usually

consist of quaternary ammonium groups. Complexing agents

such as citrate buffers or EDTA (ethylene diamine tetraacetic

add) are sometimes added to the mobile phase to influence the

separation.

3.5 SIZE EXCLUSION CHROMATOGRAPHY

Size exclusion chromatography is different in mechanism from

most other types of chromatographic methods we have

considered, since it is essentially noninteractive. Separation is

effected on the basis of size of the solute molecules, and is not

due to intermolecular forces or interactions. The column

consists of a stationary phase with pore structure of controllable

size. Smaller molecules, which can enter the pores, will be

retarded strongly while larger molecules which cannot enter the

pores will elute more quickly. Molecules of intermediate size

will, of course, experience moderate retention. The method is
82 The Basics of Chemical Instrumentation Chap. 3

used mainly for compounds having molecular masses above

2000 g/mol (up to 8*10 5 g/mol), such as polymers and larger

biomolecules. Solvent selection is relatively simple due to the

noninteractive nature of the separation mechanism. A single

solvent is required to dissolve the sample and does not

participate in partition. The method is fast, and high efficiency

separations are possible between narrow molecular weight

ranges. The technique is also applied as a physical method to

determine the molecular weight distribution (polydispersivity)

of polymeric materials.

The various "modes" of liquid chromatography are

summarized in Figure 3.2. This flow chart provides general

guidelines in selecting the appropriate method for a particular

analysis. Naturally, there may be situations in which more than

one approach can be used. For these analyses, one must

consider factors such as applicable hardware (for example,

detectors), available stationary and mobile phases, and the

economics of each method.

3.6 HPLC HARDWARE

We mentioned earlier that it is the great strides in instrumental

hardware which have resulted in modem high performance

liquid chromatography. Figure 3.3 shows a schematic diagram

of a "generic" HPLC instrument. A vessel of filtered, degassed

mobile phase solvent is supplied to a precision pumping system,

which has provisions for pressure and flowrate measurement and

control. It is important that the pump (1) produce a uniform,
High Performance Liquid Chromatography 83

Figure

3.2 A

flow

chart

providing

guidelines

for

applicable

techniques

of

liquid

chromatography.

(Courtesy

of

Millipore

Corporation,

Waters
84 The Basics of Chemical Instrumentation Chap. 3

Pulse

dampener

Forced air thermoslat Zero dead volume

solvent tiller

Figure 3.3 A schematic diagram showing the essential components of an

instrument used for high performance liquid chromatography.
High Performance Liquid Chromatography 85

controllable flow of one or more separate solvents, (2) resist

chemical attack from all solvents of interest, (3) allow easy

change from one solvent (or solvent system) to another, and

(4) be easy to maintain in the laboratory. The injection system

follows the pump, and serves to introduce sample to the carrier

stream. Injection must be performed such that the overall

HPLC system will experience minimal disruption in pressure or

flow. In addition, the injector must not introduce appreciable

"dead volume" into the system. The column containing the

stationary phase is most often made of 316 stainless steel

(because of the high corrosion resistance of the alloy) and is

usually 25 to 50 cm long, although newer microbore columns can

be up to 100 cm in length. Again, the minimization of dead

volume in the connections is a major consideration since dead

volume causes peak broadening and loss of efficiency. We will

discuss the stationary phases contained inside the column in

more detail later. It has become common in recent years to

control the column temperature using a forced air oven similar

to that used in gas chromatography, although the temperatures

employed are much lower.

The development of suitable detection devices has

contributed the most in putting the "HP" in HPLC. Analytical

chemists now have at their disposal sensitive, reasonably linear,

predictable detectors which contribute minimally to peak

dispersion. Although the vast majority of work is done using

ultraviolet (uv) absorbence and differential refractometry

detectors, devices based on fluorescence, infrared (ir) absorption,

electrochemistry, and mass spectrometry are commercially

available.
86 The Basics of Chemical Instrumentation Chap. 3

The data collection system is usually a computer equipped

with the appropriate peak processing software, or a dedicated

electronic integrator of the type used in GC (see Chapter 2).

Occasionally, however, the strip—chart recorder still finds a

place in HPLC work. It is not uncommon to have a fraction

collector following the detector, especially in exclusion

chromatography and in the study of biological samples. This

allows more extensive analysis to be done on individual

separated zones. A fraction collector will nearly always be used

in preparative—scale work.

3.6.1 Solvent Pumping Systems

The HPLC solvent pumping system consists of the solvent

reservoir, the pump hardware, and its controller. We mentioned

earlier the solvent should be free of dissolved gas and particulate

impurities. Dissolved gases, especially oxygen, can react

chemically with the stationary phase in much the same way as oxygen in the GC carrier gas can cause degradation of many

phases. Another possible consequence is the formation of

bubbles in the pump or detector. This will show up as an

unstable baseline on the integrator or recorder trace. It is

therefore standard practice to degas the mobile phase solvents

using low vacuum, gentle heating, agitation, the infusion of a

low solubility gas (such as helium), or a combination of these.

Sometimes sonolysis (the use of an ultrasound field of 35 kHz) is

employed to aid in solvent degassing.

Particulate impurities on the order of 1 to 5 /rm can cause

plugging of delivery lines and cause serious wear on seals and
High Performance Liquid Chromatography 87

moving parts (such as check valves, pistons, etc). It is therefore

necessary to filter all solvents to ensure the absence of particles

at least down to a size of 0.2 /mi. While many pressurization

systems have been used to pump the mobile phase, we will

discuss only those that currently find the widest application.

Most modern pumps can be operated in either constant flow or constant pressure mode. Pressure measurement, for readout and

pump control, is usually provided by a strain—gauge transducer

having a small internal volume. Filters (such as a 2 /mi pore

disk of sintered stainless steel) are installed on the pump inlet

and outlet to protect the components from wear.

The piston displacement pump, oil—driven diaphragm

pump and syringe pump are shown schematically in Figure 3.4.

The piston displacement pump (Figure 3.4a) consists of an

eccentric cam driven, spring-loaded piston which displaces a

solvent volume held between two ball—type check valves. The

check valves ensure the flow is in one direction, and that solvent

does not flow back into the reservoir. The flowrate from such a

pump is independent of the back pressure caused by the column,

and the pump will deliver fluid until the reservoir is empty.

The main problem with the device is that the flow is nonuniform, having a pulsating profile of the same period as the

piston stroke. Since this causes baseline noise, pulsing must be

alleviated as much as possible. This has been done by operating

two or three piston pump heads out of phase, by electronic pulse

compensation and by the use of pulse-dampening volumes

following the pump. It is never possible to completely eliminate

pulsing from piston pumps, but one can reduce it to a level

which does not adversely affect performance. The piston seals
88 The Basics of Chemical Instrumentation Chap. 3

(a)

Boil voiv«s S©iv«r»e ertomeaf

*

(b)
High Performance Liquid Chromatography 89

To waste

From reservoir

Solvent chamoer

Moving seal

Screw driven D'SCon

(C)

Figure 3.4 A schematic diagram of the more common solvent delivery

pumps used in HPLC. Figure 3.4a A piston displacement pump. Figure

3.4b An oil—driven diaphragm pump. Figure 3.4c A syringe pump.

(Reproduced with permission from Chapman and Hall, Inc.)

and check valves of this type of pump are subject to wear, and

must be replaced periodically. Piston seals are especially prone

to wear, and quite often particles of the seal are dislodged and

cause problems downstream in the system, such as at the

injector or column inlet.

The diaphragm pump (Figure 3.4b) is similar to the piston

pump in that it is a continuously operating displacement pump.

A flexible stainless steel diaphragm, usually driven hydraulically

using an oil, moves the solvent through the pair of unidirectional

check valves. The use of the diaphragm eliminates piston seals

that are wetted by the mobile phase. Thus, the problem of
90 The Basics of Chemical Instrumentation Chap. 3

particles of seal material causing malfunction of check valves

and plugging of columns is nonexistent. As with the piston

pump, the flow from the diaphragm pump is inherently

pulsating, and the same approaches to minimize the problem are

required.

The syringe pump (Figure 3.4c) is a simple volume

displacement device which produces a truly pulse-free flow,

since it is nonreciprocating. The main drawback is that it

cannot operate continuously. The piston delivers the volume in

the solvent chamber, then the chamber must be refilled by

opening the reservoir valve and withdrawing the piston. This

can be a disadvantage, especially in preparative scale work.

Syringe pumps are ideal for microbore columns, however, where

the mobile phase consumption is low and recycle time is

minimized.

It is possible to incorporate "solvent flow programming"

into an instrument with a moderate increase in pumping system

sophistication and cost. Flow programming is analogous to

temperature programming in gas chromatography. This can be

done by increasing the flowrate during the course of an analysis.

A more effective (although more difficult and expensive)

approach is to change the solvent composition during the

analysis. This allows the polarity (and therefore strength) of the

solvent to be increased or decreased during the run. This

technique is called gradient elution, as opposed to isocratic

elution in which the solvent composition remains constant

during the analysis. Gradient formers are available which make

the solvent mixture upstream from the pump (low pressure) or

downstream from the pump (high pressure).
High Performance Liquid Chromatography 91

3.6.2 Injectors

While it is possible to use a gas-tight chromatographic

syringe to directly inject a solute through a septum onto the

column, this is rarely done because of the low maximum

operating pressure of such a system. This is despite the

inherently high efficiency of this type of on—column injection.

Most injection systems on HPLC instruments make use of rotary

sampling valves, as shown in Figure 3.5. The most common

valve injector employs a relatively high volume sample loop (0.5

to 1 mL) which is flushed with mobile phase from the pump

(Figure 3.5a). The valve is switched to the inject position and

the solute, usually in a solution of the mobile phase, is applied

using a syringe. The sample solution will displace some or all of

the mobile phase from the loop into the waste vessel, depending

upon the volume of sample solution injected. The sample in the

loop is carried into the column upon switching the valve to the

inject position (Figure 3.5b). A new variation especially for use

with microbore columns is the internal loop valve. A small

volume of sample solution is syringe-loaded to a milled groove

on the valve rotor, thus allowing the efficient injection of very

small samples.

3.6.3 Detectors

The most popular detectors used in HPLC are ultravioletvisible photometers or spectrophotometers. These devices

measure the degree to which a sample solution will absorb

ultraviolet or visible light. Chromophoric groups on molecules
92 The Basics of Chemical Instrumentation Chap. 3

(such as carbonyl, multiple carbon—carbon bonds, aryl groups)

will absorb energy in the uv region, corresponding to a jump in

electronic energy level. Chemical systems consisting of long,

conjugated multiple bonds tend to absorb radiation in the visible
High Performance Liquid Chromatography 93

region by a similar mechanism as the uv—active functionalities.

It is possible to measure this absorption of the radiation using a

photometer, and to select and measure its frequency using a

spectrophotometer. Ultraviolet detectors vary in sophistication

from a very simple photometer to a modified diode-array

spectrophotometer capable of recording the "on—the—fly" uv

spectra of the eluted peaks. This is useful for qualitative

analysis (since one can use uv spectroscopic correlation data)

and quantitative analysis (since the optimum frequency can be

chosen).

A schematic diagram of a typical uv detector and cell is

shown in Figure 3.6. A typical flow cell will have a total volume

of approximately 3 to 8 /zL. Fixed wavelength detectors excite

electronic transitions using (usually) the 254 nm line of a low

pressure mercury discharge lamp, with the other frequencies (of

lower intensity) being filtered out. Multiple wavelength

detectors, using a medium—pressure mercury lamp as the source,

provide excitation frequencies of 254, 280, 313, 334, and 365 nm.

Variable wavelength detectors, using both a deuterium lamp (for

the uv region) and a tungsten—halogen lamp (for the visible

region) in combination with grating or prism optics, can provide

frequency selection from 190 to 800 nm. The instrument

responds to the decrease in intensity of the radiation which

occurs as a result of absorption by the sample.

The sensitivity of the ultraviolet detector is in large

measure a function of the absorptivity of the sample. A sample

that strongly absorbs radiation will have a lower minimum

detectable concentration. Typically, one can detect uv—active

compounds down to the 1 ng level. The detector is relatively
94 The Basics of Chemical Instrumentation Chap. 3

Figure

3.6.

A

schematic

diagram

of

an

ultraviolet

detector

used

for

BPLC.
High Performance Liquid Chromatography 95

insensitive to temperature and flowrate variations, and is linear

over four or five decades. Since it depends upon the

measurement of a solute property rather than a bulk property, it

is ideal for gradient elution methods. It is not affected by a

changing mobile phase composition unless, of course, the mobile

phase contains a chromophore. Chromophoric mobile phases are

generally not usable with the uv detector. The device is

susceptible to misinterpretation of refractive index changes of

the eluting stream as a peak. This can sometimes appear to be

an absorption, due to the increase in scattering out of the beam

path. The presence of air bubbles in the cell can also sometimes

appear similar to absorption, but this more often causes the

appearance of sharp noise spikes. Along with these minor

problems, the major disadvantage of this detector is its selective

response only to compounds which contain chromophoric groups.

It is nonresponsive to such important classes of compounds as alcohols and ethers, whose uv absorption is in the very high

frequency range.

Refractive index detectors (or more precisely, differential

refractometers) are the second most widely used detectors in

HPLC. They are universal in application since they respond to

the bulk property of refractive index of the eluting stream. The

response is provided by the change in total solution refractive

index caused by the separated solute in the carrier stream. The

refractive index detector is far less sensitive than the uv

detector, having a minimum detectable concentration in the 1 to

3 ppm range. The ultimate sensitivity of the device is

dependent on the magnitude of the refractive index difference

between the mobile phase and solute component.
96 The Basics of Chemical Instrumentation Chap. 3

While there are several different designs of refractive index

detector, the most popular approach is shown schematically in

Figure 3.7. The sample cell, which is illuminated by a

collimated, focused light source, consists of a glass chamber

which is divided by a diagonal glass plate. A plane mirror is

positioned behind the cell. The reference side of the cell is filled

with pure mobile phase before starting an analysis, and the

reflected light beams from the cell combining at the detector to

determine the baseline signal. When the refractive index in the

sample compartment changes with respect to the reference, the

position of the emergent beam will shift. The detector measures

this shift, which is then displayed on the recorder, translated in

the form of chromatographic peaks.

The refractive index detector is very sensitive to changes

in operating temperature and must usually be thermostatted to

provide optimal performance. It is also sensitive to changes in

mobile phase flowrate since flow changes will disrupt the

temperature control. It is not practical to use this detector with

gradient elution methods, since the mobile phase refractive

index is continually changing. Although the detector can

resolve refractive index differences as small as 10*10"8

, it is still

two orders of magnitude less sensitive than the uv detector.

There are other detectors available which are useful in

many special situations. The fluorimetric detector is a very high

sensitivity device applicable to fluorescent solutes, or to solutes

which can be rendered fluorescent by chemical derivatization.

This detector is often used in tandem with the ultraviolet

detector. Infrared absorption can be used to detect compounds

not amenable to detection by uv or refractometric methods. It
High Performance Liquid Chromatography 97

used

for

HPLC.
98 The Basics of Chemical Instrumentation Chap. 3

is analogous in concept to the uv absorption detector except for

the use of the vibrationally—active bands of the solute molecule.

The use of Fourier transform infrared spectrophotometry offers

the possibility of obtaining complete infrared spectra of the

eluted zones. This is extremely valuable for sample

identification, since the infrared spectrum is rich with structural

information, and the fingerprint zone of the spectrum contains

peaks which are usually characteristic of particular individual

compounds. Electrochemical detectors can provide sensitivities

higher than those obtained using uv absorption, and are useful

for solutes which are easily oxidized, in high conductivity mobile

phases such as buffer solutions. They are generally less costly

than a typical uv detector, but are difficult to use and optimize.

Mass spectrometry has been used with EPLC for a few years,

and some successful commercial systems based on a thermospray

interface (Figure 3.8) have been introduced. The mass

spectrometer is usually a quadrupolar mass filter, similar to that

used for gas chromatography. This approach, while providing a

wealth of information about the solute, is the most costly

detection system.

3.6.4 Miscellaneous Hardware

Most of the detectors used in HPLC are nondestructive.

This allows the use of secondary methods to be applied to eluted

fractions from the last detector exit. Automatic fraction

collectors can be used to collect the column eluent in a series of

vials, each of which accepts a specified quantity of eluent before

the next vial is positioned for collection. Fraction collectors
High Performance Liquid Chromatography 99

Hsuier

Figure 3.8 A thermospray interface from a HPLC into a quadrupolar

mass spectrometer. (Courtesy of VG Instrument Corporation.)

have been used for many years as part of size exclusion methods,

especially in biochemical applications involving protein

macromolecules.

An automatic sampling device can be added to an injection

system to provide unattended analyses. These accessories are

most useful for repetitive analyses of a large number of very
100 The Basics of Chemical Instrumentation Chap. 3

similar samples. They provide the advantage of highly

reproducible injection volumes, which can be important with

certain types of calibration methods. They tend to be

expensive, and are usually not used in labs which do significant

amounts of methods development work.

3.7 STATIONARY PHASES FOR HPLC

The majority of HPLC work is done today using the liquid—

liquid partition mode, and we will devote a good deal of

attention to the appropriate phases. We can divide LLC into

two categories: normal phase and reverse phase chromatography.

Normal phase HPLC involves the use of a relatively polar

stationary phase, and a relatively nonpolar stationary phase.

Elution from the column begins with nonpolar compounds,

followed by the more polar compounds in order of increasing

polarity. Reverse phase LLC employs a nonpolar stationary

phase, and a relatively polar mobile phase. Elution in reverse

phase LLC thus begins with the most polar compounds, followed

by the least polar compounds, in order of decreasing polarity.

For several reasons that we will discuss shortly, reverse phase

HPLC has become the more useful of the two approaches.

Virtually no work is done today using the mechanically

held liquids on supports, as mentioned in the introduction, and

we will not discuss this approach. The development of

chemically bonded phases, especially for use with reverse phase

chromatography, is responsible for revolutionizing the method.

A bonded phase is one in which a chemical functionality is
High Performance Liquid Chromatography 101

covalently attached to the surface of a silica support. An

example is the octadecylsilane (ODS, n—C16) phase, the most

widely used packing in HPLC:

This phase is usually made by reacting octadecyltrichloro—

silane with the surface silanol (Si—OH) groups present on the

silica. The surface is first modified with an hydroxylating

reagent to maximize the number of surface silanols available for

reaction. The silica used for bonded phase preparation is of

uniform particle size, usually 3, 5, or 10 /mi. The particles can

be pellicular (having a porous surface, with a solid nonporous

core) or microporous (in which the entire particle is porous).

The microporous particles are far more useful for bonded phases

since higher efficiencies can be obtained. Because of steric

hindrance (the physical interference of one molecule in close

proximity to another), it is not possible to cap each surface

silanol with an ODS group. Since the silanol group is very polar

and would interfere in the partition process, the surface is

further treated with trimethylchlorosilane to form an end cap:
102 The Basics of Chemical Instrumentation Chap. 3

The surface formed by this process has a brush—like

structure of hydrophobic, nonpolar hydrocarbon species. Solutes

which are hydrophobic will selectively partition into the

stationary phase brush, while more polar solutes will prefer the

relatively more hydrophilic environment of the mobile phase

solvent. The more polar the solvent, the weaker an eluent it

will be in reverse phase mode. Since the bonded phase is

sensitive to strongly acidic and strongly basic conditions, the

solvent pH should be maintained between 2 and 7. The

separation mechanism is the effect of the solvent "forcing" the

solute into the hydrocarbon—like stationary phase.

While the ODS bonded phase has been of great value for

general use, it is especially useful for solutes of low polarity

which are soluble in aliphatic hydrocarbon. More specialized

bonded phases have been developed in an effort to finely tune

HPLC separations. Bonded octylsilane (n—08, eight—membered

carbon chain) and methysilane (C—1) materials are useful for

moderate and higher polarity solutes, respectively. A chart

providing general guidelines for the use of these materials with

various solvent systems is shown in Figure 3.9. In addition to

these carbonaceous bonded phases, phenyl, diol, cyano, nitro,

and cynaopropyl functionalities can be bonded to a high

performance silica surface. These more specialized bonded

phases are useful for providing additional selectivity for certain

classes of compounds. Some of these phases, such as the phenyl,

can be used in both reverse and normal phase solvent systems.

An important recent development in HPLC stationary

phases has been the introduction of the chiral phases. These

phases are used to separate optical isomers (stereoisomers), or
High Performance Liquid Chromatography 103

Sample Column packing Mobile phase

Low/ moderate polarity (soluble in aliphatic hydrocarbons)

Bonded C-18 Methanol/water

Moderate polarity (soluble m

methytethyl

ketone)

Bonded C-8 Acetomtnle/water

High polarity (soluble in Bonded C-2 1 , 4-Dioxane/water

lower alcohols)

Figure 3.9 Common reverse phase packing materials and useful solvent

characteristics. (Reproduced with permission of Wadsworth Publishing

Company.)

so-called chiral compounds. These are compounds that have

structures which are not superimposable upon their mirror

images, as we discussed in Chapter 1. Separation of chiral

compounds has become increasingly important in the

pharmaceutical industry and with the emergence of
104 The Basics of Chemical Instrumentation Chap. 3

biotechnology and bioprocessing. It is not infrequent that two

enantiomers (optical isomers) will show drastically different

biological activity. One enantiomer may be a useful drug, while

the other may be poisonous or addictive. Although some

adsorbed chiral liquids on silica are effective in performing chiral

separations, the most useful chiral stationary phases are the

bonded Pirkle phases and the /3-cyclodextrins. Examples of

these are shown in Figure 3.10.

Stationary phases for liquid—solid chromatography (LSC)

usually consist of silica or alumina microporous particles having

a high concentration of surface silanol groups. The great

majority of work in modern LSC involves silica phases.

Separation of nonpolar and moderately polar solutes can be

obtained with high efficiency (for example, columns having

>40,000 plates/m are available). The most efficient columns are

made from particles of 5 /zm diameter. Pellicular packings are

available, usually in the 40 /zm diameter range. These packings

produce columns of lower efficiency and lower pressure drop.

The surface of the silica is highly acidic, and will cause tailing in

peaks of basic solutes such as amines.

Stationary phases for ion exchange chromatography consist

of a support matrix and an organic ion exchange functional

group. Several types of packings have been developed. The first

we will consider is a pellicular bead having a 30 to 40 /zm

diameter, coated with the exchanger film of 1 to 2 /zm in

thickness. These packings generally have low ion capacities

(defined as the number of exchange sites per unit mass of

packing), but are useful for large samples. Porous

surface—coated exchangers consist of a solid core coated with a
High Performance Liquid Chromatography 105

, CS 3 CH 3 0 H H 0 NO: 1—5 i —0—5 1—0—i i—l CHo) 3—:N- —y—(!: —:N (J—

K I I II 1h 3 1h 3 0

No,

a)

Figure 3.10 The structures of some useful chiral stationary phases for

HPLC. Figure 3.10a Pirkle phase. Figure 3.10b /3—cyclodextrin, both

the chemical structure and the overall shape. (Reproduced with

permission of D. W. Armstrong).
106 The Basics of Chemical Instrumentation Chap. 3

layer of porous microparticles that hold the exchanger. The

solid cores of these and the pellicular packings can be either

silica or a polymer. The third type of ion exchange packing is

the porous resin, based on styrene—divinylbenzene with the

exchanger on the surface. These particles can have large

channels throughout their volume in addition to the usual

micropores which we have discussed earlier. Packings having

the large channels are termed macroreticular, and have better

dimensional stability than classical microreticular beads.

Macroreticular packings that have particle sizes of 5 to 10 /mi

provide higher efficiency than the larger pellicular beads. In

general, cation exchange columns have a higher temperature

stability (sometimes as high as 150° C) than anion exchange

columns, which have operating maxima of between 40 and 80 s C.

Pellicular packings can be used only at ambient temperature.

Exclusion chromatography has been used for many years

to separate high molecular weight solutes such as proteins.

Most of these classical methods have used long glass columns

containing xerogel materials such as cross-linked dextran. More

details on gel materials can be found in the discussion of

electrophoresis presented in Chapter 5. These materials are

generally too soft for use in HPLC. The packings used in HPLC

consist of either semirigid cross-linked polymer gels or rigid,

porous glasses and silicas (called aerogels). The semirigid

packings are usually based on a styrene—divinylbenzene, and the

pore sizes available allow fractionation of solutes from 100 to

8*10 5 g/mol in molecular weight. The particles are 5 or 10 /ma

in diameter, and can be used at pressures of 41 MPa (6000 psi).

The aerogel packings consist of glass or silica containing a
High Performance Liquid Chromatography 107

solvent. Thus, it is not a gel in the strict sense, since it does not

collapse upon removal of the solvent. These packings are used

at a pH below 10, and can be used with organic or aqueous

solvents. A wide range of pore sizes are available and

separations of molecular masses of 1000 to 1.5 million g/mol can

be done using a series of columns. Hybrid phases incorporating

properties of both a xerogel and an aerogel are also available.

3.8 SOLVENTS FOR HPLC

The solvents used in HPLC must be given careful consideration.

The use of high chemical purity materials is a given. The

presence of particulates must be addressed using milliporous

filtration. Bacteria removal may also be a consideration when

using aqueous solvent systems such as buffers. We have already

discussed the effect of dissolved gases on pumps and detectors.

Dissolved oxygen can cause a specific problem when using a uv

detector at 210 nm, since the absorbance level can change in a

nonreproducible way. The solvents must be compatible with the

stationary phase, and vice versa. Acetone, for example, will

react in the presence of silica to form diacetone alcohol. High

basicity solvents can dissolve silica and will destroy a packing.

When choosing solvents, the primary consideration must

be given to solubility. The solvent or solvent system (in a

gradient elution method) must completely dissolve the sample,

and not allow precipitation in the column. This may seem like a

silly point to emphasize, but many samples such as lipids are

soluble only in very polar solvents, and the separation selectivity
108 The Basics of Chemical Instrumentation Chap. 3

that is obtained during an analysis may be poor. Protein

samples in aqueous solutions at a pH near the proteins’

isoelectric point can also present special problems in this regard.

Proteins tend to be least soluble at this pH (see Chapter 5).

The next factor one must consider is solvent polarity, for it

is the polarity (or solvent strength) which will provide the

separation with selectivity on a given column. As we mentioned

earlier, obtaining the optimal solvent system may require

several survey analyses. The eluotropic series (Table 3.1),

developed for LSC, is a scale of solvents arranged according to

their energies of adsorption on alumina and silica. These

energies are called the solvent strength parameters, represented

by e° . The solvent energy of adsorption on the stationary phase

is important because the solute and solvent act in a competitive

equilibrium to occupy sites on the solid surface. Solvents which

have a high value of e

e will tend to displace solute molecules

from the surface of the stationary phase. The stronger solvent

will thus cause the solute to elute faster, since the solute will be

bound by the stationary phase for a relatively shorter time. The

eluotropic series thus provides guidance in choosing the

necessary solvent strength. The scale can be used for reverse

phase applications with octadecylsilane, octylsilane, and

tetramethylsilane as well, but the order of solvent strength is

essentially reversed, with a few exceptions.

When one is choosing between solvents of very similar

polarity which are suitable for a particular application, the next

variable to consider is viscosity. Solvents having a lower

viscosity will cause a lower pressure drop through the column,

and result in higher permeability through the stationary phase.
High Performance Liquid Chromatography 109

TABLE 3.1a ELUOTROPIC SERIES FOR HPLC SOLVENTS

Solvent €° (S1O2 ) 6 ° (AI2 O3 )

Pentane

Hexane

Cyclohexane

Carbon disulfide

Carbon tetrachloride

1—

Chlorobutane

Diisopropyl ether

2—

Chloropropane

Benzene

Diethyl ether

Chloroform

Methylene dichloride

Methyl isobutyl ketone

Tetrahydrofuran

Acetone

1,4—Dioxane

Ethyl acetate

1—Pentanol

Acetonitrile

1—Propanol

Methanol

Water

0.00 0.00

0.00

-0.05 0.04

0.14 0.15

0.14 0.18

0.26

0.28

0.29

0.25 0.32

0.38 0.38

0.26 0.40

0.42

0.43

0.45

0.47 0.56

0.49 0.56

0.38 0.58

0.61

0.50 0.65

0.82

0.95

Large

TABLE 3.1b RELATIVE ELUOTROPIC VALUES FOR HPLC

SOLVENTS ON OCTADECYLS1LANE (ODS)

Solvent Eluotropic Value

Acetone 8.8

Acetonitrile 3.1

Dioxane 11.7

Methanol L0

Propanol—2 8.3

Tetrahydrofuran 3.7
110 The Basics of Chemical Instrumentation Chap. 3

Lower viscosity solvents will thus allow for faster separations.

Last but not least is the matter of economics. Since it is not

unusual to require in the range of hundreds of milliliters of

solvent for a separation, the most economical solvent system

should be chosen, as long as the important analytical questions

can be answered to the desired degree of precision. It is always

advisable to keep things as simple as possible, and this includes

the mobile phase. Don’t use a ternary system when a binary

will do; don’t use a binary when a single solvent will do; don’t

use a gradient elution method when an isocratic nongradient

method will do!

3.9 COMPARISON WITH GC

In general, the initial capital outlay and cost of operation of

HPLC instrumentation is significantly higher than for high

resolution GC. A cylinder of high purity carrier gas used for GC

is far less expensive than the high purity solvent and filtration

system needed for HPLC. In addition, the detectors available

for GC work are usually much more sensitive, linear, and

information—rich than those used in HPLC. Separations usually

require more time to optimize in HPLC than in GC, since there

are more variables to consider and control. While many

analyses require HPLC techniques, sometimes a choice between

HPLC and GC is possible for a particular analysis. In keeping

with the overall philosophy of this volume (keep things as simple as possible, while still obtaining the required

information), it is usually best to choose GC methods.
High Performance Liquid Chromatography 111

SUGGESTED READING

Introductory Treatments

R. J. Hamilton and P. A. Sewell, Introduction to High Performance

Chromatography , 2nd Ed., Chapman and Hall, London, 1982.

A. Braithwaite and F. J. Smith, Chromatographic Methods. 4th

Ed., Chapman and Hall, London, 1985.

J. M. Miller, Chromatography: Concepts and Contrasts , John Wiley

and Sons (Wiley Interscience), New York, 1988.

Detailed Treatments

C. F. Poole and S. A. Schuette, Contemporary Practice of

Chromatography , Elsevier, Amsterdam, 1984.

L. R. Snyder, J. J. Kirkland, Introduction to Modern Liquid

Chromatography, 2nd Ed., 1979.

A. M. Krstulovic and P. R. Brown, Reversed—Phase High

Performance Liquid Chromatography , John Wiley and Sons (Wiley

Interscience), 1982.

A. Pryde and M. T. Gilbert, Applications of High Performance

Liquid Chromatography. Chapman and Hall, London, 1980.

Special Topics of Interest

D. J, Runser, Maintaining and Troubleshooting HPLC Systems; A

Users Guide, John Wiley and Sons (Wiley Interscience), New York,

1981.

J. C. Berridge, Techniques for the Optimization of HPLC

Separations, John Wiley and Sons (Wiley Interscience), Chichester,

1985.

113

CHAPTER FOUR

SUPERCRITICAL FLUID

CHROMATOGRAPHY

4.1 INTRODUCTION

One will often hear that supercritical fluid chromatography

(SFC) is something of an intermediary technique between gas

and liquid chromatography. While this is true to some extent,

SFC has a character all its own, with a somewhat higher level of

complexity than either GC or HPLC. This stems from the more

rigorous operating conditions involved, and the large number of

parameters which must be understood and properly controlled.

In a nutshell, SFC is a chromatographic technique in

which the mobile phase is a supercritical fluid, that is, a fluid

maintained above its critical point. This region of fluid behavior

is illustrated in the P—T (pressure—temperature) phase diagram

provided for pure carbon dioxide in Figure 4.1. In the

supercritical region, indicated by the shaded area on the figure,

#
114 The Basics of Chemical Instrumentation Chap. 4

Pressure

(atm)

(°C)

Figure 4.1 A pressure—temperature phase diagram showing the positions

of the triple point and critical point, and the supercritical region for

carbon dioxide.

the vapor and liquid merge into a uniform phase. In this region,

no amount of applied pressure will cause the condensation of a

liquid phase. 2 The great usefulness of fluids in the supercritical

2This is not completely true. If a suitable interface (surface) is present, a

fluid at a supercritical temeprature and pressure can be made to condense.
Supercritical Fluid Chromatography 115

state results from their relevant thermophysical properties,

especially density, viscosity and diffusivity. A supercritical fluid

has a density which is very similar to that of a typical liquid,

about 0.8 to 1.0 g/mL. This gives the fluid the potential of

being a very effective solvent, since a relatively large number of

solvent molecules are in contact with a potential solute

molecule. The polarity of the supercritical solvent then largely

determines the extent of solubility of the surrounded solute

molecule, just as in the case of dissolution by a liquid. The

increasing solubility of solutes in supercritical fluids at

increasing densities is shown in Figure 4.2. Here, a plot of the

mole fraction of naphthalene (solute) in carbon dioxide is

provided as a function of temperature. As one increases the

pressure from 7 to 30 mPa (1015 to 4350 psi) at constant

temperature, the increased fluid density is able to accommodate

much higher concentrations of naphthalene.

The viscosity of the supercritical fluid is very similar to

that of a gas. This is advantageous, since a low viscosity fluid

moves more easily through a chromatographic system

(consisting of small diameter transfer lines, tubing, valves, etc.)

without causing a large pressure drop. The low viscosity of the

supercritical fluid also makes for faster analyses as compared

with liquid carriers. The diffusivity of a solute is a mass

transport property that describes its tendency to diffuse into

another species. The diffusivity of solutes in supercritical fluids

is intermediate between that in gases and liquids. The

magnitude of this mass transport property gives SFC the

potential of producing shorter elution times than HPLC

methods. The behavior of the binary diffusion coefficient, D 12
X

N

(Mo!e

Fraction)

116 The Basics of Chemical Instrumentation Chap. 4

Figure 4.2 A solubility diagram for naphthalene in carbon dioxide

showing the enhanced solubility in the supercritical fluid region.
Supercritical Fluid Chromatography 117

(solute, 1, diffusing into solvent, 2) is calculated in Figure 4.3 as

a function of temperature, using the Stokes—Einstein equation.

The Stokes—Einstein equation is a mathematical relation

between D 12 and the solvent viscosity, temperature and solute

size (see Glossary). We can see that the diffusion process will,

in general, decrease with increasing density and increase with

increasing temperature.

A glance at Figure 4.1 tells us that SFC will involve high

pressures and temperatures, usually above room temperature.

While this operating regime is somewhat reminiscent of HPLC,

the behavior of a supercritical fluid can be far different from

that of a liquid. The density of a liquid is relatively insensitive

to small changes in pressure. A supercritical fluid, on the other

hand, can show a very strong density response to both pressure

and temperature. This is especially pronounced just above the

critical point (the so-called near—critical region) and can be

seen in Figure 4.4, in which a pressure—density plot (for several

isotherms), in reduced units, is presented for carbon dioxide.

Reduced units means that each value of pressure and density is

divided by the critical pressure and critical density, respectively.

There is a flattening on the plot as one approaches the critical

isotherm. This will be an area of concern when we discuss the

applications of equations of state later in the chapter.

The near critical region is an area of mathematical

singularity, and of instability and fluctuation in terms of the

thermodynamic properties of the fluid. This makes control of

density and other variables rather more difficult than in the case

of a liquid carrier. The near critical region is, however, also an

area of great interest to both laboratory and industrial scale
118 The Basics of Chemical Instrumentation Chap. 4

0 20 40

Temperature (°C)

100

Figure 4.3 A diagram showing typical diffusivities of solutes in fluids.
Supercritical Fluid Chromatography 119

Figure 4.4 A pressure—density phase diagram in reduced units showing

the characteristic flattening of the isotherms near the critical point.

0

80

0

70
120 The Basics of Chemical Instrumentation Chap. 4

separations. The main reason for this is that both operation

costs and capital equipment costs increase very rapidly as the

temperature and pressure requirements escalate. It is therefore

economically advantageous to work as close to the critical region

as possible.

4.2 HARDWARE FOR SFC

In many respects, the instrumentation required for supercritical

fluid chromatography is similar to both GC and HPLC

hardware. The main components are shown schematically in

Figure 4.5. A typical apparatus consists of the solvent delivery

system, the injector, the column and column thermostat, the

detector, and the data collection system. It is also possible to

incorporate a fraction collector at the exit of a nondestructive

detector, to allow off-line analysis of selected zones. The

detection device may be a sophisticated spectroscopic

instrument, which is capable of providing qualitative and

structural information about the sample, as well as a calibrated

response for quantitation. The data collection system for SFC is

similar to those used for GC and HPLC. It may consist of a

dedicated electronic integrator, or a personal computer equipped

with appropriate chromatographic software.

4.2.1 Solvent Delivery Systems

The mobile phase solvents used in SFC can be divided into

two categories from an instrumental point of view, those which
flame

ionization

Supercritical Fluid Chromatography 121

Figure

4.5 A

schematic

diagram

showing

the

essential

components

ofa

typical

supercritical

fluid

chromatograph.
122 The Basics of Chemical Instrumentation Chap. 4

are liquid and those which are vapors or gases at room

temperature and pressure. The solvents that are liquids at

ambient conditions can be delivered easily using certain

modified HPLC pumps. The mobile phases that are gaseous at

ambient conditions (carbon dioxide, for example) axe usually

condensed to the liquid phase before being pumped. This can be

done by circulating a chilled liquid (such as a glycol—water

mixture) around the pump head, or by using a Ranque—Hilsch

vortex tube to cool the wetted pump components using an air

stream chilled to —40° C. A low operating temperature will also

help prevent cavitation (bubble formation) in the pump head,

but may hasten wear of pump components such as seals, which

may become brittle.

The desirability of a pulse-free carrier stream, which we

covered in our discussion of HPLC, also pertains to SFC.

Although the pulsation problem can be slightly less serious than

in HPLC (due to the higher compressibility of a supercritical

fluid), a syringe pump or a pulse compensated piston pump

(followed by a pulse dampener) is usually chosen for SFC. The

pumps for SFC are pressure—controlled rather than flowcontrolled, as they are in HPLC. The flow rates required in

SFC are much lower than in HPLC, usually on the order of a

few fjL/ min. This is especially true for capillary column SFC.

The pump must be capable of delivering this rather low flow

rate precisely and reliably over long periods of operation, and it

is only recently that this has become possible.

Since density is the main operative parameter in SFC, it is

desirable to measure and control the density of the mobile
Supercritical Fluid Chromatography 123

phase. The pressure, which is controlled by the pump through a

pressure transducer, is related to the density through the

P—V—T surface of the fluid. The P—V—T surface is a threedimensional plot describing how the pressure, volume and

temperature of the fluid are related. The reduced pressuredensity plot presented in Figure 4.4 may be considered a

projection of the P—V—T surface in two dimensions. A

mathematical model (called an equation of state) may be fitted

to the surface to allow estimation of the density, given the

desired temperature and pressure. Equations of state vary in

complexity from the van der Waals equation (which is the least

sophisticated two parameter fit of experimental data) to the

Schmidt—Wagner equation (which contains thirty—two

adjustable parameters). The parameters for such an equation

are determined using nonlinear curve—fitting routines on large

quantities of reliable, critically evaluated experimental P-V—

T

measurements. The accuracy of the resulting fit depends greatly

upon the quality of the experimental data used for the fit.

Although the more complex equations can precisely

correlate the P—V—T behavior of a fluid, most are somewhat

inaccurate in the near—critical region. If we refer back to

Figure 4.4, we can observe the characteristic flattening of the

isotherms near the critical point. It is this feature of the surface

which is difficult to model mathematically, since the equation

must become non—analytic in this region. While a discussion of

the complexities of equation—of—state correlation and testing is

outside the scope of chemical analysis, the results and

consequences are of great importance to the practice of SFC.
124 The Basics of Chemical Instrumentation Chap. 4

In GC, we use temperature programming to enhance the

elution of less volatile solute components , while in SFC we use a

program of increasing density. A computer program

incorporating an equation of state for the mobile phase adjusts

the pump pressure (at constant temperature) to produce the

desired density program. The increasing density over time

increases the solubility of the less volatile solutes, and they are

drawn into the supercritical mobile phase by the difference in

chemical potential of the solute in each phase. The column

temperature can also be programmed in SFC, to increase the

volatility of the solute. It must be remembered that increasing

the temperature at constant pressure will result in a decrease in

density (or, alternatively, an increase in specific volume). This

will partially offset the advantage of the increased solute

volatility. The best approach would be to program both density

and temperature, which allows control over the solute volatility

and solute solubility in the mobile phase. The reader will note

the increased level of complexity already apparent in the

operation of a SFC instrument. The separation design and

control process is thus more exacting than either GC or HPLC.

The pressure is maintained through the column by a flow

restrictor placed at the column exit. This restrictor is usually a

length of fused silica tubing of small internal diameter. Both

the length and diameter will determine the back pressure

obtainable. Another type of restrictor employs a glass frit held

in a fused silica tube. Back—pressure regulators, used in early

SFC instruments, are rarely installed in current instruments,

although a new restrictor—back pressure regulator combination

offers some exit flowrate control.
Supercritical Fluid Chromatography 125

4.2.2 Injectors

SFC can be performed using both capillary columns and

packed columns containing chemically bonded phases similar to

those used in HPLC. The choice of column type will determine

the type of injection system that will be needed. All injection in

SFC is now done using variations of HPLC—type sampling

valves incorporating low volume sample loops or internal loop

valve rotors. Injection into a packed column can often be done

directly, while injection into a capillary column will require the

use of a flow splitter following the sampling valve because of the

low capacity of these columns. The flow splitters are not as sophisticated as those used in GC, which incorporate complex

pneumatics to control the split ratio. For SFC, splitters usually

consist of a branched transfer line equipped with a restrictor to

provide the flow -path for the vented stream. No satisfactory

variable—flow restrictor has been introduced commercially to

provide the operator with continuous control of the split ratio.

A recently developed combination of restrictor and backpressure regulator appears to have good potential, however.

Whether a packed column or a capillary column is used,

the sample is deposited into the loop of a high pressure injection

valve in one of several ways. Delivery can be done very simply

by using a syringe (of the type used for HPLC valve loading),

with the sample dissolved in a suitable solvent. Alternatively,

the sample may be delivered to the loop from an on-line

supercritical extraction cell (Figure 4.6). In the latter case, the

sample arrives at the injection valve already dissolved in the

supercritical mobile phase. This is useful for trace analysis as
126 The Basics of Chemical Instrumentation Chap. 4

Figure 4.6 A schematic diagram of an extractor—based injection system,

showing a possible manifold (a) and an extractor cell (b).
Supercritical Fluid Chromatography 127

well as physicochemical studies, where the presence of a solvent

in addition to the supercritical eluent is undesirable. The

availability of on-line supercritical fluid extraction as a sample

preparation technique is a very attractive feature of SFC. Other

injection techniques are also reported in the literature, but these

two methods are the most generally useful.

4.2.3 Detectors

Many detectors originally designed for use in HPLC and

GC have been modified for use in SFC. The two most popular

are the uv spectrometric detector and the flame ionization

detector. These detectors are described in detail in the HPLC

and GC chapters, respectively, and only the salient points of

modifications for SFC will be described here.

The optics and electronics of the uv spectrometric detector

are essentially identical to those used for HPLC. The main

difference is in the design of the flow cell, which must

accommodate much higher pressures. This is achieved by

minimizing the size of the wetted or pressurized components,

thus decreasing the loading (force per unit area) to a small level.

Flow cells usually have an optical path length of 2 to 5 mm,

with the larger size more suitable for routine analysis. The

smaller path length is needed when closely eluting components

are being analyzed, since the smaller cell will lower the detector

contribution to peak dispersion. It is not uncommon to

experience cell sealing difficulties, especially when the cell is first

used or after several weeks of down time. The cells usually

contain Teflon gaskets which tend to cold flow under load and
128 The Basics of Chemical Instrumentation Chap. 4

must absorb some of the supercritical carrier and swell slightly

in order to produce a tight seal. In general, this detector

provides good sensitivity and stability, but can sometimes suffer

from baseline drift during density programming. Naturally, the

ultimate sensitivity is dependent upon the absorptivity of the

sample, and the detector is only applicable to the analysis of

compounds which contain chromophoric groups. Quantitation is

dependent upon calibration involving the Beer—Lambert law, so care must be taken to ensure that all the requisite assumptions

of that relationship are satisfied.

The flame ionization detector can be modified for SFC by

allowing the exit restrictor to vent at the base of the hydrogen

flame (Figure 4.7). This is usually done using a fused quartz

capillary, since an electrically conducting transfer line would

disturb the operation of the FID. The detector is operated at a

high temperature (approximately 300 8 C), since baseline spiking

can occur if the mobile phase cools significantly upon

decompression. For packed column operation, the FID can be

fitted with additional exit tubes to vent off excess mobile phase.

An FID for packed column SFC use was introduced

commercially several years ago, with mixed results. The

detector is very sensitive and linear, and is not adversely

affected by a slightly pulsing profile in the mobile phase flow.

The main problem with the FID is that it cannot be used with

flammable mobile phase fluids. This includes nonflammable

fluids modified with small (1 to 2 percent) quantities of

flammable fluids, such as the very useful carbon dioxide +

methanol or carbon dioxide + methanol systems.
Supercritical Fluid Chromatography 129

for

6

use h^SFC

SChematlC dia8ram of a ^ame ionization detector modified
130 The Basics of Chemical Instrumentation Chap. 4

Mass spectrometers can be used as detectors for capillary

column SFC, since the flow into the ion source is relatively low

(0.5 to 5 L/min) and will not cause a harmful overpressure. The

instrumental details, are, in general, very similar to the

application of this detector in capillary GC. Using a mass

spectrometer with packed columns requires a more carefully

designed interface, and most often chemical ionization rather

than electron impact methods are used. Other possible

detectors include Fourier transform infrared spectrometric

methods, which allow "on—the—fly” infrared spectra of solute

components to be recorded. This is especially valuable, since

the infrared spectrum is rich with structural information useful

for qualitative analysis. Fluorescence, flame photometric and

thermionic detectors have been applied to SFC, but to a much

smaller extent than the other detectors which we have discussed.

4.3 MOBILE PHASES

By far the most popular mobile phase for SFC is carbon dioxide,

for a variety of important reasons. The critical temperature of

carbon dioxide is 31.3° C, which is convenient from an

operational point of view. It can be liquefied at just a few

degrees below room temperature, and flashed into the

supercritical state just a few degrees above. It is nontoxic and

economical, readily available at an adequate purity level, and

further purification is relatively easy if it is required. Carbon

dioxide is also nonflammable and nonexplosive, and will not give

a background response in a flame ionization detector.
Supercritical Fluid Chromatography 131

Other common fluids for SFC are pentane, hexane, sulfur

hexafluoride, isopropanol and methanol. Methanol is especially

useful as a mobile phase modifier. It is added to carbon dioxide

in small amounts (0.5 to 2 percent) to increase the overall

polarity of the mobile phase. Other fluids either used or

suggested as mobile phases for SFC are listed in Table 4.1, along

with their relevant critical properties.

The critical temperature of a fluid being considered for a

SFC analysis is an important methods development parameter,

since the mobile phase temperature must be maintained above

this value. The fluid must be stable at the required operating

temperatures, while in contact with the construction materials

of the chromatograph. These include silica and several varieties

of stainless steels. This is an important consideration, since, for

example, methanol decomposes near its critical point when in

contact with stainless steels of the 300 series (AISI designation),

and forms hydrogen, carbon monoxide, and formaldehyde.

The critical pressure of a potential carrier fluid is useful as an approximation of the solvent strength of the fluid. The

critical pressure is usually higher for more polar fluids, although

there are some exceptions. Thus, in general, the higher the

critical pressure, the higher the solvent strength of the mobile

phase. The critical density is also a good indicator of solvent

strength, since solvent strength generally increases with

increasing critical density.

When a homologous series of hydrocarbons is examined for

potential usefulness as mobile phases, some definite

chromatographic effects and trends can be observed. The lower

hydrocarbons (such as propane) provide high resolution
132 The Basics of Chemical Instrumentation Chap. 4

TABLE 4.1 SOME USEFUL FLUIDS FOR SUPERCRITICAL FLUID

CHROMATOGRAPHY

Fluid TcC C) Pe(g/mL) PC(MP;

Carbon dioxide 31.413 0.460 7.4

Ammonia 132.5 0.235 11.4

Nitrous oxide 36.5 0.450 7.3

Sulfur dioxide 157.8 0.520 7.9

Water 374.1 0.40 22.1

Methanol 239.5 0.272 8.0

Isopropanol 235.3 0.273 4.8

n—Pentane 196.6 0.232 3.4

n—Hexane 234.2 0.234 3.0

Dichloro—

fluoromethane 178.5 0.522 5.2

Trichloro—

fluoromethane 198.0 0.554 4.4

Chloro—

trifluoromethane 28.9 0.578 3.9

DichloroTM

tetrafluoroethane 145.7 0.582 3.6

DichloroTM

difluoroemethane 111.5 0.558 4.0

Benzene 288.9 0.304 4.9

Xenon 16.6 1.155 5.9

Toluene 320.8 0.29 4.2
Supercritical Fluid Chromatography 133

separations at a relatively low supercritical temperature. The

higher members (such as pentane) provide fast analyses at a

somewhat lower resolution, since they have a somewhat higher

solvent strength.

Unfortunately, the most operationally useful fluids for SFC

(carbon dioxide and n—pentane) are nearly nonpolar. For

example, carbon dioxide has about the same polarity as n—hexane. The lack of more polar eluents than these is one of

the major limitations of SFC. Many investigators have used

polar modifiers (sometimes also called entrainers, especially by

the engineering community), added in small amounts to the

nonpolar fluids to increase the effective polarity of the mobile

phase. The most common additive, methanol, was mentioned

earlier. The gradient elution method has also been used with

carbon dioxide and 1,4—dioxane. The use of a mixed mobile

phase introduces great complexity into the density prediction

problem which we discussed earlier.

While it is possible (although in many cases difficult) to

develop equations of state for nonpolar mixtures, there is no

reliable model for mixtures having a polar component. Most

equations of state for mixtures depend upon mathematically

combining the properties of the pure components, using a set of

equations called mixing rules. In the absence of reliable data on

the properties of pure modifiers under the conditions of interest

to SFC, the development of even a marginal equation of state

becomes very difficult. Even the most sophisticated approaches,

such as those which employ an extension of the principle of

corresponding states, have been only marginally successful.

These methods make use of a well-characterized reference fluid
134 The Basics of Chemical Instrumentation Chap. 4

with which to scale the properties of a fluid of interest, using

reduced parameters.

The use of supercritical ammonia has been suggested as a

possible experimental solution to this difficulty, since it has a

relatively high polarity. There are still problems of materials

compatibility and safety with this fluid, however.

4.4 STATIONARY PHASES

As we mentioned earlier, SFC can employ either capillary or packed column methods. While the basic chromatographic

theory will not change from column to column, the techniques

will change significantly. Before we discuss individual phases, it

would be advisable to first discuss the two approaches in general

terms. The choice between capillary and packed columns will

primarily depend on the solute mixture to be analyzed, but this

decision will have important instrumental consequences. A few

of these considerations have already been mentioned in our

discussion of injectors and detectors.

The important physical characteristics of capillary

columns are the column length, the stationary phase film

thickness, and the internal diameter of the column. As in GC,

capillary columns can provide high efficiency (in terms of total

theoretical plates per column) for the separation of complex

mixtures containing many components. They can provide 250

to 1250 more plates per centimeter than packed columns. The

stationary phase film should be as thick as possible without

itself becoming an unfavorable contributor to peak dispersion.
Supercritical Fluid Chromatography 135

The length of the column should be maintained as short as

possible, to allow for faster analyses. (The speed of an analysis

cannot, in general, be increased by increasing the carrier

velocity.) The pressure drop through the capillary is relatively

low, making these columns very suitable for analyses which can

be done at lower mobile phase densities. Capillary methods are

quite amenable to detection by FID and MS methods. Lower

flow rates are used for capillary column methods, which require

higher accuracy in solvent delivery and control. Flow splitters

are needed for operation with the smaller diameter columns.

The important physical characteristics of packed columns

are the column length, column diameter, packing particle size,

and particle surface area. There are more independently

variable parameters in packed column SFC, and therefore more

flexibility and also more complexity. The packings are usually

modified silicas or polymers. The pressure drop through the

column is very high, so packed columns are somewhat more

suitable for use with higher density mobile phases. They are

suitable for separations requiring low or moderate efficiency,

such as relatively simple mixtures having about ten peaks.

They are also useful in situations in which complete separation

of the mixture is not critical. Analysis is much faster on packed

columns than on capillary columns. Mobile phase modifiers

have a somewhat different and more critical role with packed

columns. Since the silica surface always contains unreacted

silanol sites, the polar modifier is very often needed to ’’tie up"

these sites, and to prevent excessive solute peak tailing. This

often requires the use of higher operating temperatures for

packed columns, since the mobile phase mixture must be
136 The Basics of Chemical Instrumentation Chap. 4

maintained in the supercritical state. The use of the FID with

packed columns is possible; however the presence of flammable

modifier will very likely impair performance. If the modifier

concentration must exceed approximately 1 percent, the FID

probably cannot be used at all. The MS can also be used,

especially in chemical ionization mode, provided that the

interface can handle the higher volume of eluent. The

ultraviolet and infrared detectors can be used in regions where

the mobile phase does not show a strong absorption. The recent

appearance of packed capillary columns has excited much

interest, since they offer a useful compromise between packed

and capillary columns.

The most common stationary phases used with capillary

columns are the cross—linked polymethyl siloxanes, favored for

their inertness and homogeneity. Many variations based on the

siloxane backbone have been synthesized, including liquid

crystalline polymers. For packed column work, most of the

phases have been based on silica, either bare or chemically

bonded. The bonded phases which are most commonly used are

the octadecylsilane and octylsilane materials used in HPLC,

although the more polar cyano, amino and diol packings have

also been used.

As with GC, SFC can be used to perform physicochemical

measurements. Using an uncoated, long capillary column, the

binary interaction diffusion coefficient, D12, can be measured

using the method of Taylor and Aris. The diffusion coefficient

represents the tendency of the solute to be transported into the

supercritical fluid phase, and is therefore useful in analytical

methods development in work on the design of supercritical fluid
Supercritical Fluid Chromatography 137

extraction processes. Other physicochemical parameters

experimentally accessible using SFC are partial molal volumes

near the critical point, partition coefficients, and the

polydispersivity of polymeric materials.

4.5 COMPARISON OF SFC WITH GC AND HPLC

As we have observed from the above discussion, the theory and

practice of SFC can, in general, be much more complex than

either GC or HPLC. The cost of an SFC system is also very

high, even when compared to HPLC. There are, however,

instances in which SFC will provide an elegant solution to

problems not readily solved using GC or HPLC. One important

example is in the analysis of thermally labile compounds. These

materials cannot withstand the elevated injector temperatures

which are often needed to volatize the sample. Compounds such

as natural products, biological compounds, and pharmaceuticals

often fall into this category. Another example is higher

molecular weight compounds such as polymers. These materials

often cannot be analyzed by GC due to their very low vapor

pressure. Analysis by HPLC is often not possible due to sample

insolubility. In the past, one usually had to pyrolize the sample

and analyze the products by GC. The enhanced solubility

afforded by supercritical fluids can often provide the key to

these difficult analyses.

Despite the unique advantages of SFC, there will be

significant areas of overlap for the application of this technique

with GC and HPLC. In these instances, it will usually be best
138 The Basics of Chemical Instrumentation Chap, 4

to choose the simplest approach which will provide the desired

answers to a particular analytical problem. In most cases, this

will mean GC if possible, followed by HPLC. If an analysis can

be done using gas or high performance liquid chromatography

rather than supercritical fluid chromatography, it would be

foolish to choose the more costly and complex SFC.
Supercritical Fluid Chromatography 139

SUGGESTED READING

R. M. Smith, Ed., Supercritical Fluid Chromatography. Royal

Society of Chemistry, Burlington House, London, 1988.

B. A. Charpentier and M. R. Sevenants, Supercritical Fluid

Extraction and Chromatography . American Chemical Society,

Washington, DC, 1988.

S. Ahuja, Ed., Ultrahigh Resolution Chromatography . Chapters 10

and 11, American Chemical Society, Washington, DC, 1984.
-
141

CHAPTER FIVE

ELECTROPHORESIS

5.1 INTRODUCTION

Electrophoresis is a technique used for the separation of ionized,

(often) colloidal—sized molecules under the influence of an

applied electric field. The method has historically been most

useful among biochemists and biologists, but the emergence of

commercial—scale biotechnology and the increasing industrial

applications of bioprocessing has increased the popularity and

importance of this technique in the chemical and engineering

communities. Although the most common application of

electrophoresis is in the analysis and characterization of ionized

species such as proteins in aqueous solution of buffers, the

method has also been applied on the preparative scale for the

purification of larger quantities of such compounds. The

primary application of this technique is for the separation of
142 The Basics of Chemical Instrumentation Chap. 5

proteins and polynucleotides. A short discussion of the nature

and structure of these compounds would be helpful before we

explore how electrophoresis is used in their chemical analyses.

5.2 PROTEINS

The name "protein" is derived from the Greek word proteios, meaning first. Proteins are, in short, the substance of life, since

they provide the motive power for all animal species. They are

large polymeric molecules which comprise the structure of skin,

muscle, nerve, blood, antibodies, enzymes and hormones found

in any living cell.

Protein polymers are made up of monomeric units of

G—amino carboxylic acids (actually, the L—, or levorotary

stereoisomer), or more simply, amino acids. The general

structure of an amino acid can be represented as:

0

II h 2n—ch—coh

I a

with the amine and the carboxyl groups both bound to the same

carbon in what is called the alpha configuration. The "R" group

represents a chemical moiety or side chain which is responsible

for the differences among the amino adds. The amino acids are

crystalline at room temperature and decompose upon melting

when subjected to elevated temperatures. They are relatively
Electrophoresis 143

insoluble in nonpolar solvents such as the alkanes, but are more

soluble in polar solvents and can be very soluble in water,

forming solutions having properties similar to salt solutions.

The extent of solubility depends upon the nature of the group

represented by "R." Amino acids are dipolar ions, and can have

either a positive or a negative charge:

0 0

ii „

ii . +H3N—CH—-COH H 2N—CH——CO

1 1 R R

(a) (b)

The pH of the solution will determine the charge that the

ion carries. At a relatively low pH level, the carboxylic acid

groups will not be ionized, and the amine group will be

protonated due to the relative excess of hydronium ions (H 3 0

+

) in the solution. This will leave the protein with a net positive

charge. At a high pH, the carboxyl group will be ionized due to

the interaction with the relative excess of hydroxyl ions, and the

amine group will not be protonated. This will leave the amino

acid ion with a net negative charge. At intermediate pH levels,

some amine groups will be protonated and some will not. This

depends on the acidic strength of the particular molecule. There

will be one particular pH value at which the amino acid will

have no net charge. This point is called the isoelectric point, at

which the amino acid exists as a dipolar ion, having an ionized

carboxyl group and a protonated amine group. The amino acid
144 The Basics of Chemical Instrumentation Chap. 5

will usually be least soluble at this pH level, with the solubility

increasing if the solution is made more acidic or alkaline.

Amino acids have interesting geometric features. Every

amino add (except the simplest, glycine) has at least one chiral

center. As we discussed in Chapter 1, the presence of chirality

(nonsuperimposability of a molecule upon its mirror image)

leads to the phenomenon of optical activity, the rotation of the

plane of polarized light which is passed though a solution. In

fact, all amino acids which occur as part of biological systems

are levorotatory (L, or " preceeds the name of the amino acid)

and rotate the plane of polarized light to the left. Organisms

are quite fussy about the optical purity of the amino acids which

make up the biological structure, and one of the most important

functions of the liver is the destruction of dextrorotatory (D, or

precedes the name) amino adds and thdr polymers.

Proteins are polymers built up from amino acid monomers.

The polymerizations result from the reaction of the amino group

of one amino acid with the carboxyl group of another, forming

what is called the peptide bond, occurring upon release of water:

0

J.

r

H

H 2N—CH-€—-OH

I R

0

II + HTMN—CH—COH

J

0

n r 1

i 0 i -h 2 q ,11 , ———* H 2 N—CH—C—NH-—bH~~QH

L _ _ J

r

N—terminus peptide C—terminus

bond
Electrophoresis 145

The amino and carboxyl groups of the individual amino acids

are used in the formation of the peptide bonds. This leaves only

the terminal amino groups (the N—terminus) and the terminal

carboxyl groups (the C—terminus) intact. These terminal

groups on the protein are subject to ionization in the same way

as on amino acid units, with a strong dependence upon solution

pH. The individual amino acids making up the polymer are

called residues. The polymer is classified by the number of

residues which comprise the polymer. Thus, if a polymer

consists of three amino add residues, we refer to it as a

tripeptide. Polymers of molecular weights up to 10,000 g/mol

are termed polypeptides, and those above this figure are what

we call the proteins. While the peptization reaction is the

principle means of protein formation, some amino add residues

will allow additional covalent bonds to be formed. Cysteine,

which contains a thiol (—SH) group, is important due to its

ability to form disulfide bridges in the protein with other

cysteine units. This forms very stable cross-linked proteins.

These proteins are often found in areas outside of living cells

such as the bloodstream or digestive tract of complex organisms,

where the chemical environment is relatively severe. Proteins are divided into two broad classes, fibrous and

globular. Fibrous proteins are long, thread-like molecules often

containing many intermolecular hydrogen bonds. They tend to

be sparingly soluble or insoluble in water. Globular proteins are

quasi—spherical in shape and tend to contain intramolecular

hydrogen bonds; they are generally more soluble in water.

Because of the large size of the protein molecules, the solutions

are often very similar to colloidal suspensions. The two general
146 The Basics of Chemical Instrumentation Chap. 5

categories are further subdivided according to solubility and

reactivity of the protein. For example, albumins are water

soluble proteins which are denatured (irreversibly precipitated

or coagulated) by heat. Globulins are proteins which are

insoluble in pure water, but soluble in dilute salt solutions.

5.3 POLYNUCLEOTIDES

Polynucleotides are similar in concept to proteins, although they

are quite different from both a chemical and a functional point

of view. Research into nucleic acids and polynucleotides is one

of the most active areas of scientific pursuit, since these

compounds are the basis of heredity.

Polynucleotides are polymeric molecules which contain a

long chain backbone that differs only in length from one

polynucleotide to another. In proteins, the polymer backbone

was the polyamide chain. In polynucleotides, a polyester chain

forms the backbone. The ester is derived from phosphoric acid

and the alcohol portion of a sugar. A heterocyclic base is

attached to the first carbon of each sugar. The sugar-base

combination is called a nucleoside, and the base—sugarphosphoric acid combination is called a nucleotide. The general

structure of a polynucleotide is made up of these repeating units:

base 0 base 0

sugar—0—P—0—sugar—h—P—0

-* n

i i
Electrophoresis 147

The two most well—known polynucleotides are RNA and DNA.

In RNA, the sugar is D—ribose, while in DNA the sugar is

D—2—deoxyribose (Figure 5.1a). The bases found in RNA are

adenine, guanine, cytosine, and uricil, while those in DNA are

adenine, guanine, cytosine, thymine and 5—methylcytosine

(Figure 5.1b). Polynucleotides differ from one another in the

relative proportion of these bases and their sequence of

occurrence on the backbone. RNA consists of a single polymeric

strand, while DNA exists as a double strand in the well—known

"double helix" structure, stabilized by hydrogen bonding.

Polynucleotides are similar to proteins in that they have

characteristic ionization behavior. When polynucleotides are

dissolved in aqueous solution, they will always form the anion.

The single stranded polynucleotides are soluble only in neutral

or basic solutions, and therefore the phosphate groups are never

completely protonated. The double stranded polynucleotides

shield the potentially ionizable base molecules by the steric

(space—geometry) hindrance and the involvement in hydrogen

bonding. DNA molecules are also only soluble in basic solution,

and exist in aqueous solution as the negatively charged specie.

5.4 ELECTROPHORETIC SEPARATION

Electrophoresis is a technique which uses an electric field

applied to a solvent matrix or medium to cause the separation of

charged colloidal particles by the mechanism of differential

migration. The method was first introduced by the Swedish

chemist Arne Tiselius in the doctoral thesis he presented in
148 The Basics of Chemical Instrumentation

\ H 2

H Adenine

0

0 hn^ N:h

I H Urac i

NH'

'NTH

vCH

H Cvtosine

Guanine

0

0 HN/ N:—ch3

Thymine

Figure lb

NHo

\r;

.CilCll

5-Methylcytosine

Figure 5=1 The chemical structures of DNA and RNA (a

chemical structures of the nucleotide bases (b).

Chap. 5

and the
Electrophoresis 149

1930. This work brought him a Nobel Prize in 1948. Although

the method has been extensively used and studied for over fifty

years, a detailed and quantitative theory remains somewhat

elusive. A highly simplified view of the process is shown in

Figure 5.2:

Starting point

Cathoae

Potential difference

Power supply

Anode

©

0—

0

Figure 5.2 A schematic diagram depicting the electrophoretic process.
150 The Basics of Chemical Instrumentation Chap. 5

The length of the arrows on the ions indicate the relative effect

of the field on singly and multiply charged ions. The solvent

matrix, which is placed between an anode and cathode, is a very

important component in the design of an electrophoretic

separation, and will be discussed in much greater detail later on. For the present, we will simply assume that it is an electrically

conductive material which will dissolve the samples without a

significant danger of precipitation. If the sample is applied to

the center of the solvent matrix, and the current is turned on,

the migration of cations to the anode and anions to the cathode

will be observed.

The force of the applied electric field will accelerate the

charged molecules until it is balanced by the opposing force of

viscous drag produced by the molecules of the matrix or the

solution. As you might expect, the effect of the applied field

will be greater on a multiply—charged ion than on one which

carries a charge of only plus or minus 1. The ultimate velocity

at which the molecule then moves toward the electrode is called

the "migration velocity. n The migration velocity is proportional

to the applied electric field:

v = uE

where v is the migration velocity, E is the strength of the

electric field, and u is a constant of proportionality called the

electrophoretic mobility (which, by definition, takes the same

sign as that of the ion). If many simplifying assumptions and
Electrophoresis 151

approximations are made, the viscous drag force of a particle in

a medium can be described in terms of Stokes’s law:

F = 6 7r r 77 v

where r is the radius of the particle (which is assumed to be

spherical), and 77 is the viscosity of the medium. Thus, the

magnitude of the viscous drag force is proportional to the

viscosity of the medium and the size of the molecule.

Conversely, the migration velocity is inversely proportional to

the viscosity and molecular size.

We have made many assumptions in the above discussion

to simplify the rather complex process involved in

electrophoresis. For example, we have assumed that the

particles which make up the sample are spherically shaped

molecules. In reality, this is never the case, although many

molecules such as globular proteins have an approximately

spherical shape. The drag forces will increase as the molecular

shape becomes less spherical. We have also assumed that the

medium exerts no effect other than viscous drag. This is clearly

not the case, since the medium is a conductive, ionized system

capable of participating in electrostatic interactions with the

sample. As the sample moves through the medium, its ions will

become surrounded by a shield of solvent ions of opposite sign.

This shield will decrease the effect of the applied field, and the

sample ion will move through the medium more slowly than we

might otherwise expect.
152 The Basics of Chemical Instrumentation Chap. 5

With these considerations in mind, we can summarize the

factors which influence the movement of ions in the

electrophoretic system. The migration velocity (and therefore,

the speed of analysis) will be directly proportional to applied

field strength and the net charge on the migrating molecule.

The migration velocity will be inversely proportional to the

viscosity of the medium, the size of the migrating molecules, and

the extent of departure from sphericity of the migrating

molecules.

The temperature of the electrophoretic system is also an

important consideration, since the viscosity of the medium and

buffer solution pH are temperature dependent, as well as the

current and voltage across the medium. It is therefore advisable

to minimize changes in temperature variations caused by the

"Joule heating" by the applied field during an analysis. Other

undesirable effects due to heating are sample band distortion

and diffusion, buffer solution evaporation, and the appearance of

convection currents in the medium. The heat dissipation on the

surface of the medium will clearJy be different from that in the

interior, and some temperature nonuniformity will always be

present. Since a higher field strength produces a faster

separation, a compromise is needed to obtain an optimally

designed experiment. It is generally best to use as high a field

strength as possible, but not so high as to incur serious

consequences from poor heat dissipation. A high field strength

is particularly important in the separation of smaller molecules,

since separation may not occur at all at lower levels. The use of

a constant current or constant voltage power supply is standard
Electrophoresis 153

in modern electrophoresis, and this helps to reduce temperature

fluctuations.

Since both the sample species and the buffer solution are

ionic, the possibility of undesired interactions is always present,

as we mentioned earlier. This interaction usually produces a

sample ion which is surrounded by a cage of ions of opposite

charge. The larger effective size of sample will lower its

electrophoretic mobility. It is therefore desirable to use solution

ionic strengths and buffer concentrations which are as low as

possible. This will minimize the number of cage-forming

counterions. This use of low ionic strength solutions must be

considered in the light of sample solubility, however, as we

discussed earlier.

5.5 INSTRUMENTATION

The original electrophoretic technique developed by Tiselius was

called the moving boundary method, which was used extensively

in protein research until about 1950. This was a liquid solution

method, in which proteins were differentially concentrated in

glass columns below the anode and cathode. Separation was

observed by bands of "schlieren," wavy patterns in the solution

produced by differences in refractive index of the compounds

separated. This technique is rarely used today because it suffers

from extremely low band resolution.

The most important and widely used electrophoretic

technique used today is the zone method, in which the separated
154 The Basics of Chemical Instrumentation Chap. 5

components are distributed into discreet "zones" in a stabilizing

medium. The media, which we will discuss in detail later on,

can be a high grade filter paper, a cellulose acetate film, or a gel

made of starch, polyacrylamide, or agarose. The process of zone

electrophoresis is shown schematically in Figure 5.3, in which

sample is applied to the top of the medium, and separation into

zones occurs as the current is allowed to flow for up to several

hours. Note that on this figure, the zones are all moving toward

the anode. This does not always happen, of course, since the

migration direction is dependent upon the sample ionization

behavior.

A variation of zone electrophoresis is the steady state

technique called isoelectric focusing. Recall in our earlier

discussion of amino adds, we discussed the formation of the

dipolar ion, in which the amine group was protonated, and the

carboxyl group is ionized. This dipolar ion is stable only at a

specific pH called the isoelectric point. In isoelectric focusing, a

stable pH gradient is set up in the medium, such that as one

moves along the medium, the pH will either increase or decrease.

If a polypeptide or protein is applied to such a medium, and the

current is applied, the zones will move along the gradient until

the pH corresponding to the isoelectric point of the ion is

reached. At this point the zone will move no further. As we

noted earlier, a polypeptide or protein may not be very soluble

at the isoelectric point, and may even precipitate in the

medium, which can sometimes be a major disadvantage.

Nonetheless, the method has a high resolving power, and can

even be used on the preparative scale.
S3

mple

applied here

Electrophoresis 155

lumpaiu

oo

o

c

CO

Figure

5.3 A

diagram

illustrating

the

progress

ofa

separation

by

zone

electrophoresis.
156 The Basics of Chemical Instrumentation Chap. 5

5.6 SUPPORT MEDIA

Filter paper (high quality, 96 percent alpha—cellulose) was the

first medium used for zone electrophoresis. It was introduced

for this application in the early 1950’s, and was popular because

it allowed simple and low cost separations to be performed

relatively quickly. The paper is saturated with a buffer solution,

and then bent over a plastic holder with its ends dipping into

buffer solution compartments, one containing the anode and the

other the cathode. A bell jar or similar cover is then placed over

the system to exclude dust and minimize evaporation. Since the

passage of current will result in the production of heat, the

apparatus is usually located in a refrigerator or "cold room." A

relatively low field strength of about 20 V/cm is used with

paper, which is sufficient for larger polypeptides and proteins.

The use of paper as the medium is relatively rare now because of

some important disadvantages. Thin paper produces sharper

bands, but is more prone to tearing and must be handled with

extreme care. Thick papers have been used mainly for

preparative scale separations, but the resolution is somewhat

lower. The chemical structure of the paper itself can also pose a

problem, since proteins will interact with the free hydroxyl

groups which are always present on its surface. This is

analogous with the problems associated with the free silanol

groups on the surface of HPLC packings, and the result here is

the same. The interaction will cause peak broadening and

tailing, ultimately leading to a significant loss in resolution.

Another serious problem is the electro-osmotic effect, caused by

the tendency of the molecules on the paper surface to take on a

o
Electrophoresis 157

negative charge in the presence of water. It is probable that the

small number of carboxylic add groups on the cellulose

structure ionize, forming hydronium ions, H30 +

. The

hydronium ions are very mobile and move toward the cathode

under the influence of the applied electric field, thus disturbing

the transport of the protein or polypeptide ions of interest.

Cellulose acetate, introduced in the late 1950’s, was a

significant improvement over cellulose filter paper. The

hydroxyl groups of cellulose are treated to form the ester, and

are thus unavailable to interact in an unfavorable way with the

sample molecules. Cellulose acetate dissolves in many solvents,

which makes sample recovery easier than with filter paper. The

medium is used in the form of a thin membrane, and the

apparatus and methods employed are similar to those involving

filter paper. Unfortunately, cellulose acetate will also show an

electro—osmotic effect due to the ionization of the carboxyl

groups, however some commercially available membranes show

only a very small effect due to methylation of the anionic

groups. Cellulose acetate is still used as a medium in many

clinical procedures, especially those involving small samples.

The analysis times are short, with separation occurring in one or

two hours, thus partially offsetting the relatively higher cost of

the material (as compared to paper).

Most of the modern research—level electrophoresis is done

with gel media. A gel is a two-component system (consisting of

a disperse phase and a continuous phase) with a semi—solid

structure, having some properties similar to colloidal systems.

This semi—solid form of the system may be distinguished from

its semi—liquid form, which is called a sol. The gels used in
158 The Basics of Chemical Instrumentation Chap. 5

electrophoresis are molded into flat slabs or poured into columns

made from glass or plastic tubing. The continuous phase is a

three dimensional cross-linked polymer having an essentially

random bond repetition pattern, but producing a constant

"pore" size. The pore size, which can be controlled within

certain limits, can provide a sieving effect which is conceptually

similar to the action of molecular sieves in GSC. For a given

net ionic charge, smaller molecules will be transported through

the gel faster than larger ones. The pore size is controlled by

varying the relative amount of polymer in making up the gel;

adding additional continuous phase polymer will decrease the

average pore size. In preparing polyacrylamide gel slabs, the

pore size is controlled by the quantity of cross-linking agent.

The disperse phase in an electrophoresis gel is a conductive

buffer solution which will dissolve the samples under study.

The principal types of gels used in electrophoresis are

made from starch, polyacrylamide, agarose, and a polyacrylamide—agarose combination. The earliest gel work was done

using hydrolyzed potato starch. Although most of the work

today is done using other gel media, starch gels are still used in

many laboratories because of their low cost, speed and relative

ease of preparation and operation. It is still one of the best

choices for the analysis of isoenzymes (separate species of

protein having the same catalytic activity). The gel is prepared

by heating the starch in a solution of the buffer until the

resulting colloidal suspension becomes transparent. The mixture

is then poured into a mold, in which the gel forms. The pore

size of a starch gel is variable only over a very narrow range, by

slightly changing the starch concentration. Too much starch
Electrophoresis 159

will produce a gel which is too stiff to be poured and molded,

while too little will give an excessively soft, "runnyM gel.

Another disadvantage of starch is due to its chemical structure,

which contains negatively charged side chains. These can

interact with protein molecules to hinder migration and also

cause an electro—osmotic effect similar to that observed with

paper and cellulose acetate.

Polyacrylamide gels are the most important and widely

used media in electrophoresis. They are prepared by crosslinking acrylamide (in an approximate concentration of 15

percent mass) with a co-monomer, N,N'—methylenebisacryl—

amide. Only the highest purity monomers can be used, since the

cross-linking reaction is very sensitive to the presence of

impurities. Other copolymers can be used to provide special

solubility properties. For example, gels can be prepared using

N,N'—bis—acrylylcystamine, producing a gel which can be easily

dissolved under specific conditions, allowing the sample to be

recovered for further analysis. In addition to the co—monomers,

an initiator and a catalyst are required. The common chemical

initiators are ammonium persulfate and potassium persulfate,

although light will initiate the reaction in the presence of

riboflavin. The most common catalyst is NjNjN'jN'—tetra—

methylethylenediamine (TEMED). The pore size of a

polyacrylamide gel is controlled by the ratio of this reagent with

the acrylamide monomer. The reaction scheme is shown in

Figure 5.4

The gel is either poured as a flat slab (typically 10 cm

square) or poured into glass or plastic tubes (7 to 10 cm long,

5 mm in diameter). A schematic diagram of the slab apparatus
160 The Basics of Chemical Instrumentation Chap. 5

Figure

5.4

Reaction

to

form

polyacrylamide

gel

medium.
Electrophoresis 161

is shown in Figure 5.5. In the case of the slab configuration, a

slot former (usually made from Teflon) is incorporated into the

gel to form sample cavities, which are then loaded with sample

in a buffer solution, usually containing sucrose or glycerol to

control density. It is possible to analyze many samples (along

with several known standards) under identical conditions using

slab gels, and the efficient heat dissipation of the thin medium

allows the use of high field strengths. The resolving power is

somewhat better than tube gels. The slab gel is more expensive

(since it requires more gel) and difficult to prepare than the tube

gel, however it has found a great deal of utility in RNA and

DNA separations.

Agarose gels are formed from a linear polymer of D—

galactose and 3,6—anhydrogalactose, with the structure being

maintained by hydrogen bonds. The average pore size, which is

controlled by the agarose concentration, is larger than either

starch or polyacrylamide, making them suitable for the analysis

of larger protein species (1.5*10 8 g/mol). The polymer is soluble

in boiling water, with the gel formation occurring upon cooling

to about 38° C. The apparatus needed for using agarose gels is

the same as for polyacrylamide and starch gels. The problem of

heat dissipation is more critical using agarose, since the viscosity

shows a strong temperature dependence. The main historical

disadvantage with agarose gels, however, is the presence of a

small number of ionized sulfate and carboxyl groups. This leads

to interaction with sample molecules and the presence of

electro-osmotic effects. Hydrolysis with an alkali can minimize

these difficulties.
sample

slots

162 The Basics of Chemical Instrumentation Chap, 5

Figure

5.5

An

apparatus

used

for

gel

slab

electrophoresi
Electrophoresis 163

The use of a polyacrylamide—agarose hybrid gel can

produce a gel of reasonable mechanical strength, yet with a pore

size which is larger than that in polyacrylamide. These

mixtures have been useful in nucleic acid work, and in

separations of very large proteins.

5.7 DETECTION

Once an electrophoretic separation has been completed, the

separated bands must be rendered visible. This will allow a

determination of sample purity (by observation of the number of

separated zones), qualitative analysis (possibly from a

comparison with standards), and recovery of the individual

bands for further analysis or study. The usual methods of

detection are zone staining, ultraviolet absorption,

radiochemical analysis, and biological assay.

5.7.1 Staining

The most widely used method of detection is zone staining,

in which the sample is reacted with a chemical which renders

the zone opaque (to ultraviolet or visible light) or fluorescent.

Stains which render a zone opaque in visible light are termed

chromogenic, since they generate a color in the band. Staining

reagents can be applied either before electrophoresis or after

completion. If the stain is applied before, the zone separation

can be observed as it progresses. It is usual to "fix” the

developed zones after electrophoresis, in order to stop further
164 The Basics of Chemical Instrumentation Chap. 5

zone diffusion. This is done by drying (the paper or cellulose

acetate media) or precipitating the sample (in gel media) using

trichloroethanoic add or ethanoic acid. Once the electrophoretogram is fixed, excess staining reagent is removed by

either soaking in a destaming solution, or by electrophoresis at a

right angle to the original electrophoretogram. Some of the

more common chromogenic stains are ninhydrin, Coomassie

brilliant blue, and Ponceau red. Some stains provide quantitative information from the intensity of the colored band. An

example is acridene orange, which is used for polynucleotides.

Stains which produce a fluorescent product are usually

applied before electrophoresis. The electrophoretogram is

scanned using an ultraviolet lamp of appropriate wavelength.

The most common fluorescent stain is dansyl chloride, which is

also commonly used as a derivatizing reagent in HPLC.

Acridene orange, which is used for the quantitation of

polynudeotides, can also be used as a fluorescent stain.

5.7.2 Ultraviolet Absorption

It may be possible to detect the separated zones on gels

from their absorption of ultraviolet light, without the use of

staining compounds. Most proteins have chromophoric groups

(the carboxylic carbonyl, for example) which show absorption

maxima between 230 and 280 nm using a deuterium lamp as the

excitation source. Scanning with uv radiation using a recording

densitometer provides a quantitative record of the zone profile

along the gd. Polyacrylamide gds present problems because of

the presence of unreacted monomer in the gel which will also
Electrophoresis 165

absorb in the uv region. This will cause a high background

level. Agarose gels are usually dried to produce a thin film in

order to decrease light scattering.

5.7.3 Radiochemical Methods

Radiochemical detection methods are based upon

prelabeling the sample with a radiotracer, the level of which is

measured by liquid scintillation counting or autoradiography.

The most common tracer isotopes are 125I

( a gamma emitter),

32P (a high energy beta—emitter), 14 C, 35 S (both medium energy

beta emitters) and 3 H (a low energy beta emitter). These

tracers can be incorporated into the sample somewhere in the

preparation sequence, especially using alkylation. The addition

of radioactive labels will very rarely change the biological

activity of the material, and will not change the ionic charge.

To use liquid scintillation counting, the gel or paper is

dried and the zones are cut apart using a scalpel or special

sectioning instrument. Samples separated on paper or cellulose

acetate can be dissolved directly in a suitable scintillation

mixture, but the samples on gels must be solubilized. Some

samples can be removed using a buffer solution, while others

require an organic reagent such as hyamine hydroxide or

piperidine. The gel structure can be disrupted using ultrasound

in a suitable liquid (formamide for agarose, 30 percent hydrogen

peroxide for polyacrylamide). Alternatively, pre—solubilized

gels (as we discussed earlier) may be used.

Although a detailed discussion of scintillation counters is

beyond the scope of our treatment of electrophoresis, a brief
166 The Basics of Chemical Instrumentation Chap. 5

overview is in order. The radioactive label atoms listed above

will decay and in the process release beta or gamma particles,

which have the potential of electronically exciting absorber

molecules. When certain absorber molecules "de-excite,'* they

release a photon which can be detected and amplified using a

photomultiplier tube.

Because most of the radiotracers used in electrophoresis

have low penetration, the sample is dissolved directly into a

solution (called a cocktail) containing absorber molecules in an

appropriate solvent. The solvent is usually an aromatic

hydrocarbon such as toluene or xylene, since these molecules are

readily excited by the radiation, and transfer this energy to the

scintillator. The scintillator is an organic molecule which

accepts the energy from the solvent, and re-emits it as a photon

in the 300 to 400 nm range. This is the photon that is detected

and amplified by the counter. Sometimes a secondary

scintillator is added to the cocktail, mainly for very high

sensitivity work. The secondary scintillator absorbs the photon

emitted from the (primary) scintillator, and re—emits a photon

at a longer wavelength, near 400 to 500 nm. This is

advantageous since the photomultiplier tube is more sensitive in

this region. Some typical scintillators are shown in Table 5.1.

The samples are dissolved on an appropriate cocktail and

placed in special sealed glass vials. An automatic conveyor

system will bring the samples above the counter, where they will

be held for a predetermined integration time. Quantitative data

can then be obtained by comparison with suitable calibration

standards.
Electrophoresis 167

Conpoimd

p—terpnenvl

1 .4—diphenvloxazole

(PPO)

1 .4—diphenvloxadiazole

(PPD)

secondary scintillator:

Table I

Scintillator Compounds:

Relative

Structure Scintillation

Efficiency*

'relative to PPO

1 .4—di-2-i 5-Phenvloxazole)

(POPOP)
168 The Basics of Chemical Instrumentation Chap. 5

Autoradiography is a simple technique especially

applicable to soft, large pore agarose gels which cannot easily be

sliced for scintillation counting. The gel slab is first dried under

vacuum, and placed in a cassette containing a sheet of

fine-grained x-ray film, which is backed by a fluorescent

intensifying screen. The cassette is then stored at low

temperature (below 0°C) for a few days. The tracer species

cause the appearance of dark bands on the film corresponding to

the labeled zones on the slab.

Biological assay methods are more complex and often

much more time consuming than other detection methods we

have discussed, but have nonetheless been of great value. The

technique is based on observing the biological or biochemical

reactions of the separated materials after electrophoresis. The

analyses are very sensitive, and detection is selective for specific

proteins. The biochemical activity observation is usually

enzymatic action (if the species is catalytic) or antigenic (or

immunological) action. Usually the gel slab is cut into sections

and the activity of each section is recorded. When the

enzymatic or antigenic action will produce a visible change (such

as a precipitation or color change), the reaction can be done in

situ, directly on the intact slab. One can also cut a trough

parallel to the lane in which the sample migration has occurred.

The antibody or antisera is then placed in this trough.
Electrophoresis 169

SUGGESTED READING

Introductory Treatments

M. Melvin, Electrophoresis, John Wiley and Sons (on behaif of

ACOL), Chichester, 1987.

R. Stock and B. F. Rice, Chromatographic Methods, 3rd Ed.,

Chapter 3, Chapman and Hall Science Paperbacks, London, 1978.

E. Heftmann, Ed., Chromatography: A Laboratory Handbook of

Chromatographic and Electrophoretic Methods, Van Nostrand

Reinhold, New York, 1975.

Detailed Treatments

A. T. Andrews, Electrophoresis: Theory, Techniques, and

Biochemical and Clinical Applications , Clarendon Press, Oxford,

1986.

Basic Biochemistry

M. Yudkin and R. Offord, Biochemistry, Houghton Mifflin Co.,

Atlanta, 1975.

L. Stryer, Biochemistry . 2nd. Ed., W. H. Freeman Co., San

Francisco, 1975.

M. Malm, Fundamentals of Organic and Biochemistry , Van

Nostrand, New York, 1980.

171

APPENDIX I

CALIBRATION

I.l INTRODUCTION

In the preceding chapters, we have discussed some of the most

important and valuable instrumental techniques available to

perform chemical analysis. These techniques can provide both

qualitative and quantitative information. Qualitative

information includes, for example, the number of components in

a natural gas mixture from a wellhead or the identity of the

proteins present in an industrial fermentation broth prior to

extraction. To obtain quantitative information (for example,

how much, what concentration, what level of purity?), one must

usually use some sort of standardization or calibration on the

instrumental response. This is in contrast to absolute methods,

which do not depend directly on the comparison of instrumental

response of a sample with that of a standard.
172 The Basics of Chemical Instrumentation App. I

Chemical analysis always seeks to answer some question or

series of questions. We referred to such questions above with

our hypothetical natural gas mixture and fermentation broth.

The personnel who require the "answers" from a chemical

analysis are typically plant engineers or managers who may have

little or no background in analytical chemistry. It can

sometimes be difficult for the analyst to communicate the

difference between "quant" and "qual." For example, upon

providing a plant manager with a qualitative statement such as "the trouble with extraction 51 is an excess alcohol content" the

next question will invariably be, "well, how much excess is

there?" The difference between these two levels of information

lies in calibration. Calibration can be a simple process, or it can

be a long, involved time-consuming task requiring the utmost

skill on the part of the chemists or technicians. It is therefore

important to thoroughly understand the questions which have

been posed for chemical analysis to answer.

1.2 ABSOLUTE VERSUS COMPARATIVE METHODS

There are very few analytical methods which can be considered

truly absolute. Even the methods which are usually thought of

as being absolute (such as gravimetry, gasometry, coulometry,

and titrimetry) use instrumentation which is "standardized" in

some way. In the United States, the National Institute of

Standards and Technology (NIST, formerly the National Bureau

of Standards) under the Department of Commerce is legally

charged with the responsibility of standardizing basic weights
Calibration 173

and measures. For example, even the most precise and sensitive

analytical balances built for use in gravimetry must be

standardized using weights traceable to a primary standard

weight set available for reference at NIST.

Analytical methods which are not absolute are called

comparative methods. These methods require calibration of the

instrumental response against the response of known standards

under identical experimental conditions. It cannot be

emphasized too strongly that without some type of calibration,

no quantitative information whatsoever can be obtained from

comparative methods. The standards which are employed

should be prepared to match the character of the sample as

closely as possible. This is done to minimize possible interfering

effects of other substances. This process is called matrix

matching, and should be practiced as far as is practical with all

the calibration methods we discuss. Of course, exact matching

of the standard and sample matrix may often be impossible,

especially in the case of very complex sample mixtures. In these

cases approximate matrix matching may be adequate. We must

remember that practicality, expedience and economics also play

a role in "real world" analytical problems.

The methods used for calibration range from simple, fast

comparisons to detailed and costly designed studies. In keeping

with the overall philosophy of this volume, the simplest, fastest,

safest and most economical approach that will provide the

desired information at the required level of accuracy is the

approach which is advocated. Our discussion will begin with the

simplest methods, and graduate to the more involved

procedures. Treatment of the most sophisticated research—
174 The Basics of Chemical Instrumentation App. I

oriented methods, which often require complex nonlinear

analysis software, are beyond the scope of both this book and

most of the problems encountered in chemical analysis.

All quantitative information obtained using chemical

instrumentation is based on the observation of a change in

response of some detection device with an increasing or decreasing concentration of sample. This change in response

may be an increase in absorbance indicated by an ultraviolet

detector in HPLC, or the decrease in current sensed by an

electron capture detector in GC. Many instrumental responses

are linear with sample concentration (Figure A1.1) even if only

over a small range of concentrations. This linear relationship

provides a level of predictability in the response of the detector.

It is not absolutely necessary that the relationship be a straight

Line, but it is necessary that (1) a relationship actually exists,

and (2) the relationship and its associated uncertainty must be

confidently predictable.

1.3 SINGLE POINT CALIBRATION

The simplest (and least certain) form of instrument calibration

is the single point method. While fast and easy, it can only be

justified under a strictly defined set of conditions. In applying

the method, we assume that the response is in fact a straight

line, passing through the origin (Figure A1.2), and that there

are no systematic errors in the method. We must also establish

that the method gives a zero blank response, that is, the

response is in fact zero when no analyte (the substance being
Calibration 175

Figure A1.1 A plot showing a linear relationship between the measured

ultraviolet absorbance and solute concentration.

analyzed for) is present. That these criteria are indeed met

must have been established previously by a more extensive series

of analyses. Only then can a single point calibration be used

with any certainty for routine work. The measurement of a
176 The Basics of Chemical Instrumentation App. I

Figure A1.2 A plot illustrating a single point calibration.

standard sample of known concentration then allows the

calculation of the unknown concentration by a simple ratio:

CS (RU/RS ) = Cu (Al.l)
Calibration 177

where Ru and Rs are the responses of the unknown and

standard, respectively, and Cs and Cu are their associated

concentrations. The concentration of the standard is, of course,

chosen to be higher than that of the unknown. This serves to

bracket the unknown between a known concentration and a

previously established zero point response at the origin.

This calibration method is subject to a number of

important disadvantages. Mistakes in procedure cannot be

detected if only one standard is used, since one must rely on the

"known” value as being correct. There is no way to average or smooth the indeterminate or random instrumental error which is

always present in any method. In addition, it may be difficult

to know of or correct for the presence of interfering chemical

species in the sample.

The single point calibration can be improved somewhat by

always running a blank solution. This will allow the analyst to

correct for the possible presence of impurities, or slight

differences in analytical conditions between the time the

standard and the unknown are run. Naturally, when an

analytical method is known to produce a finite, nonzero blank

value, this measurement will be absolutely necessary. The value

of the response to the blank is then subtracted from that of the

standard and the unknown.

L4 TWO POINT CALIBRATION

The two point (plus a blank) method offers a refinement which

does not significantly add to the complexity or cost of most
178 The Basics of Chemical Instrumentation App. I

analyses. Two standard samples are measured, and the response

of a blank solution is subtracted. The concentrations of the two

standards are chosen to bracket the expected value of the

unknown concentration. It is often useful to make the high

concentration standard either near the limit of linearity of the

method, or near the highest expected sample concentration.

The lower concentration standard should be near the minimum

detectable level of the sample using the particular method. This

will provide the calibration with a wide operating range.

Naturally, our assumption of a straight line behavior with

concentration must have been previously established. The

unknown concentration can then be determined graphically or by computer.

1.5 MULTI-POINT CALIBRATION CURVE

A logical extension of one and two point calibrations is the

determination of a calibration curve using a set of known

standards having concentrations varying over the range of

interest for a particular analysis. While this is usually done by

preparing a set of standard solutions of increasing

concentrations, an in situ approach is also possible. The in situ

method is performed by sequentially adding increasing amounts

of the standard to a single solution, taking into account the

change in solution volume that this process causes. A blank

sample should always be run and included in the calibration

curve. The blank reading should not, in general be subtracted
Calibration 179

from the rest of the standards, since it is subject to the same

instrumental errors. Two examples of calibration plots are shown in

Figure A1.3, one being a straight line and the other curved.

Although both straight line and curved plots are usable, the

linear plot is to be preferred. The main reason for this is that

the curved plot requires more standards in order to adequately

describe the response, especially in the region of curvature.

Statistical analysis software packages which perform linear

regression are readily available for use on nearly all personal

computers. These programs are capable of fitting the standard

plots to equations (often called mathematical models) which

account for the pattern or structure of the response. While the

most familiar functional form of the fitted equation is a straight

line (Y = mC + b, where Y is the instrument response, C the

concentration, m the slope of the line and b the intercept), other

forms can be calculated. Higher order linear polynomial models

(such as quadratic and cubic), exponential, logarithmic, and

power models are easily calculated with standard software

packages available commercially, usually at a modest cost.

An important advantage of the calibration curve modeled

to an equation is the ability to generate statistical measures of

error, tests of coefficient significance, residual magnitude and

pattern, analysis of variation (anova) tables, and probability

plots. While we will not treat the calculation or interpretation

of these parameters in this discussion, they are very important

since they allow the analyst to take full advantage of the

available data. Mistake recognition and outlier identification
180 The Basics of Chemical Instrumentation App. I

are made much easier using these statistics, a feature not shared

with the simpler calibration methods discussed earlier. In

addition, a much higher degree of precision can be obtained
Calibration 181

using a calibration curve. The accuracy, as usual, will depend

on the accuracy of the individual standards.

1.6 STANDARD ADDITION METHODS

We have discussed the effects of the sample matrix, and how

matrix matching is desirable to prevent interferences from other

chemical species present in the sample. When matrix matching

is difficult or impossible, standard addition methods are

especially useful. The method involves measuring the

instrumental response of the sample solution, followed by the

addition to this solution of a known quantity of the analyte.

The change in the instrument response is related to the amount

of the standard added.

The method is applicable only when a known straight line

relationship has been established between the instrument

response and the analyte concentration. If a determinate or systematic error is present in the measurement technique, it

must be constant over the whole range of analyte concentration

of interest. This means, in effect, that a single blank value must

apply to all analyte concentrations, even though a blank sample

may not be available. The sample cannot contain any chemicals

which can be mistaken for the analyte, since this will be

perceived as being built into the determinate error. The change

in volume of the solution upon the addition of the standard must

either be negligible or accounted for computationally.

While in some well defined and studied circumstances it

may be possible to use a single standard addition, most of the
182 The Basics of Chemical Instrumentation App. I

time it is far better to use multiple standards. Single standard

addition suffers from some of the same disadvantages as single

point methods which we discussed earlier. There is no built-in

error check to guard against mistakes in technique, and no way

to assess or average over the experimental errors. Using

multiple standard addition, one can apply the mathematical

modeling software described earlier. This will provide the

analyst with a more reliable error analysis. The number of

standards used will have to be decided, based upon consideration

of two primary factors. The first factor is the extent of linearity

of the measurement. It is undesirable to use measurements

outside of this region. The second factor is the required

precision of the results.

While the standard addition method is generally rapid and

addresses the sample matrix problem, it suffers from some

disadvantages. Often, larger quantities of sample may be

required as compared to other methods. The most serious

shortcoming from a conceptual point of view is that it is an

extrapolation method, rather than in interpolation method (as

in the case of the calibration curve earlier). This makes the

method inherently less precise than using a calibration curve.

1.7 INTERNAL STANDARD METHODS

One of the most useful methods for the calibration of

chromatographic analyses is the internal standard method. The

method is especially valuable when it is not possible to exactly

control the quantity of sample analyzed. An example would be
Calibration 183

the GC analysis of a hydrocarbon mixture using syringe

injection. Sample evaporation from the syringe can change

component concentrations and ruin analyses. Another

application would be when using instrumental methods having

parameters which are difficult to control, such as the gas flow

rates in some types of atomic spectroscopic techniques. In

general, the precision of the internal standard method is very

high as compared to the calibration curve methods we have

discussed earlier.

To implement the method, a standard solution is prepared

which contains not only a known quantity of the analyte, but

also a known quantity of another compound, called the internal

standard. Since this compound must be chosen carefully, we

will discuss the important characteristics more fully later on. The internal standard compound is also added to the unknown

solution containing the analyte. The instrument response of the

standard solution is then measured, and the response factor, R12,

determined according to:

(Si/S 2 ) (Ca/CO = R12 (A1.2)

where Si and S2 are the instrument responses of the analyte and

internal standard, respectively, and Ci and C 2 are the

corresponding concentrations. The instrument responses can be,

for example, chromatographic peak area counts (as in

chromatography) or absorbance units (as with an ultraviolet

spectrophotometric detector). The next step is to measure the

instrument response of the sample solution, which contains the
184 The Basics of Chemical Instrumentation App. I

unknown analyte concentration and the known internal standard

concentration. The unknown concentration is then determined

from:

(S1/S2) (C2/R12) = Ci (sample solution) (A 1 . 3 )

where, as before, the subscript 1 refers to the analyte and 2

refers to the internal standard. It is possible to use a series of

standards, all prepared with a different concentration of the

analyte but the same concentration of the internal standard.

One then plots a calibration curve of the response ratio, S 1/S2,

versus the analyte concentration Ci for each solution. The

resulting plot should be a straight line having the origin as its

intercept and the response factor R12 as its slope. The unknown

solution is prepared in the same way as the standards, having

the known quantity of internal standard added. The

concentration can then be determined graphically by

interpolation of the S 1/S2 ratio, or by using an equation fitted to

the curve.

As mentioned earlier, the choice of the internal standard is

very important. The standard should, of course, not already be

present in the sample. It should be similar to the analyte in

terms of physical properties such as vapor pressure, polarity,

density, and viscosity. Close attention must be paid to any

physical property winch may affect the detector response. The

sample and standard should give a similar magnitude of response

to the detector being used, without causing interference with the

other’s signal. For example, in GC work the two compounds
Calibration 185

should produce baseline resolved peaks having integrator area

counts within 20 or 30 percent of each other. The standard

substance must be miscible with the sample solution, and must

not cause a chemical reaction. The standard and the analyte

should be easily separable using standard methods such as chromatography, or should give spectroscopic signals which are

easily resolvable. The concentration of the internal standard

should be close to that of the analyte, to minimize error in the

response factor. While it may not always be possible to design a

calibration to fit all of these criteria, one can usually obtain a

nearly optimum measurement by using an internal standard

which is a homologue of the analyte.

The high level of precision of the method is the result of

making both standard and analyte measurements on the same

solution. In addition, the measurements are done either at the

same time or at times very close to one another. The advantage

of high precision that this method provides is offset somewhat

by the rigorous, time-consuming work required.

1.8 STANDARD REFERENCE MATERIALS

Throughout our discussion of calibration, we have made many

references to the process of comparing the instrumental response

of an analyte of unknown concentration with the response of a

standard under identical conditions. This standard is of a

known concentration, and is very often prepared in the

laboratory for the particular analysis at hand. There are many

cases, however, in which the preparation of a standard
186 The Basics of Chemical Instrumentation App. I

"in—house" is not adequate or desirable. A few examples might

be analyses for regulatory agency submission, analyses which

will figure in legal proceedings, and analyses used in the

generation of archival data compilations. In these instances,

materials of reference fill a vital role in chemical analysis.

The National Institute of Standards and Technology

(NIST) is the primary source of materials of reference in the

United States. There are actually three types of available

materials: reference materials (RM’s), certified reference

materials (CRM’s), and standard reference materials (SRM’s).

A reference material is a "material or substance one or more

properties of which are sufficiently well established to be used

for the calibration of an apparatus, the assessment of a

measurement method, or for assigning values to materials." A

certified reference material is a "reference material one or more

of whose property values are certified by a technically valid

procedure accompanied by or traceable to a certificate or other

documentation which is issued by a certifying body." Standard

reference materials are certified reference materials that are

issued by NIST.

The materials of reference defined above are developed for

three primary purposes. The continuing development of

accurate methods of analysis (reference methods) is one vital

goal. Another is the calibration of measurement systems used in

the exchange of goods (custody transfer) and in quality

assurance. The third important purpose is to assure the long

term integrity of a wide range of measurement programs. The

instrument response values of the NIST standard reference

materials are obtained by one of three possible measurement
Calibration 187

schemes. The first is the use of a previously validated reference

method. The second is through the results of two or more

independent, reliable measurement methods, and the third is

through measurements done in a network of cooperating,

competent and knowledgeable laboratories. In all cases, the

measurements are obtained through statistically—designed

experiments which are mathematically—planned in advance.

The application of an SRM can be most clearly illustrated

by an example. A municipality may wish to set a standard level

of several alcohols which may be added to gasoline for the

purpose of decreasing carbon monoxide emissions from

automobiles. The municipal petroleum inspector will sample the

fuel from the supplier’s tank, and submit the samples to the

laboratory for analysis. Since legal or judicial action may hinge

upon the results from such an analysis, it is vital to eliminate as much procedural uncertainty as possible. In such a case, the

laboratory will probably perform the analysis according to a

standard guideline (possibly one prescribed by the American

Society for Testing and Materials), and calibrate the method

using a standard alcohol in gasoline reference material obtained

from NIST. The use of the standard reference material removes

some of the uncertainty associated with the calibration process,

since the standard is a uniformly and confidently accepted

material. This is further reinforced by the fact that most other

laboratories performing analyses of alcohol in gasoline will also

use the same standard reference material. An additional benefit

is that the SRM provides a baseline of calibration comparability

among all of the laboratories engaged in this testing, regardless

of location or affiliation.
188 The Basics of Chemical Instrumentation App. I

SUGGESTED READING

Introductory Treatments

B. W. Woodget and D. Cooper, Samples and Standards , John

Wiley and Sons (on behalf of ACOL), Chichester, 1987.

D. McCormick and A. Roach, Measurement, Statistics and

Computation, John Wiley and Sons (on behalf of ACOL),

Chichester, 1987.

J. R. Taylor, An Introduction to Error Analysis , University Science

Books, Mill Valley, 1982.

Detailed Treatments

R. L. Anderson, Practical Statistics for Analytical Chemists, Van

Nostrand Reinhold, New York, 1987.

J. C. Miller and J. N. Miller, Statistics for Analytical Chemists, John Wiley and Sons (Halsted Press), New York, 1988.

B. Ostle and R. W. Mensing, Statistics in Research , 3rd Ed., The

Iowa State University Press, Ames, 1975.

Standard Reference Materials

The Role of Standard Reference Materials in Measurement Systems, National Bureau of Standards Monograph 148, 54 pp., January,

1975.

Standard Reference Material Catalog, 1988—89, National Bureau of

Standards Special Publication 260, January, 1988.
189

GLOSSARY

Accuracy A measure of how close a measured quantity is to the

actual or true value of that quantity. In general, the true value

is unknown, and measurements are approximations of the true

value. Systematic errors will decrease the accuracy of a

measurement.

Adsorption Isotherm A graphical and mathematical description

of the equilibrium distribution of a solute between two phases,

one being a solid surface and the other being a gaseous head

space above the solid. The most well known models are the

Freundlich and Langmuir isotherms.

Aerogel The dispersion of a gas or relatively small solvent

molecule in a solid or liquid structure. It may be considered the

opposite of an aerosol. The styrene-divinyl benzene polymeric

packings used in size exclusion chromatography columns are

loosely called aerogels.

Aliquot A part of a whole which is representative of the whole,

such as a sample of a material for analysis or a volume of a

reagent used in performing a test.

Beer—Lambert Law The simple relationship between the

concentration of a substance in solution which absorbs
190 The Basics of Chemical Instrumentation

electromagnetic radiation, and the extent of the absorption,

expressed as: A = e C/

where A is the observed absorbance of the solution, C is its

concentration, e is the molar extinction coefficient and i is the

measurement cell path length. The extinction coefficient is also

referred to as the specific absorption and as the Bunsen

coefficient. This relation is subject to many assumptions and

conditions which, when satisfied, allow the determination of

solute concentration from spectrophotometric measurements.

Buffer Solution A (usually) aqueous solution consisting of a

weak acid and its salt (called the conjugate base) or a weak base

with its salt (called the conjugate acid). The add or base

concentration will determine the pH of the buffer solution.

Buffer solutions are useful because they resist changes in pH

upon the addition of acid or base.

Certified Reference Material A reference material one or more

of whose property values are certified by a technically valid

procedure, accompanied by or traceable to a certificate or other

documentation which is issued by a certifying body.

Chemical Potential A thermodynamic quantity defined as the

free energy per mole of a substance. It may be viewed as a

thermodynamic driving force, since matter will spontaneously

flow from a region of high chemical potential to one of low

chemical potential.
Glossary 191

Chirality The observation that a molecule is not super—

imposable upon its mirror image. Such a molecule is called

chiral, while one which is superposable upon its mirror image is

called achiral. The word is derived from the Greek "cheir,"

meaning hand, implying handedness.

Chxomophore A molecular group or radical which absorbs

energy in the ultraviolet or visible region of the electromagnetic

spectrum. Common chromophoric groups are the carbonyl

(^C=0), alkeneic and aromatic groups. They are

characterized by a delocalized system of electrons in 7r orbitals.

Colloid A two phase system consisting of a solvent and a

dispersed, gross phase of relatively larger molecules (as

compared with the solvent). Colloid dispersions show the

expected bulk properties of a solution, but do not show

colligative property behavior. Two types of colloidal dispersions

are possible, gels and sols.

Cross-Linking The formation of bonds in a more or less regular

fashion along the backbone of a polymeric molecule.

Cross-linking decreases molecular freedom and produces a

stronger, harder, and less soluble polymer.

Cryogen A cryogen or cryogenic fluid is usually defined as a

liquid which has a boiling point below —150° C at atmospheric

pressure. Another (broader) definition considers any fluid

having a critical temperature below room temperature to be a
192 The Basics of Chemical Instrumentation

cryogenic fluid. They are used extensively in the laboratory to

produce low temperatures. The most commonly used cryogen is

liquid nitrogen, which boils at 77 K at atmospheric pressure, and

is relatively inexpensive.

Density A fundamental thermophysical property that describes

the amount of a material which occupies a given volume at a

given temperature and pressure (state). The density of a fluid is

described in three dimension by the P—V—T surface, specific

volume being the reciprocal of density.

Diastereomers A class of stereoisomers in which the molecules

are not mirror images of one another. This includes ds/trans

isomers. Diastereomers have different physical properties (such

as vapor pressures and melting points), although the chemical

properties (reactivity, etc.) will be similar. As an example, one

can consider the following:

Cl H Cl Cl

\ / \ /

c=c c=c

/ \ / \

H Cl H H

Diffusivity A transport property which describes the tendency

of molecules of a given compound to disperse into molecules of

another compound or itself. Kinetic theory and

thermodynamics hold that if a gas mixture is of nonuniform

concentration, diffusion will take place until the concentration is
Glossary 193

uniform and no concentration gradients exist. The diffusivity is

both temperature and density dependent.

Dipole Moment A property of a polar molecule, evaluated by

multiplying the magnitude of the charges on the atoms (in

electrostatic units) of the molecule by the distance separating

the charges. A polar molecule is one in which the center of

negative charge does not coincide with the center of positive

charge.

Electronegativity All atoms which have fewer than eight

electrons in their outer electron shell (principal quantum level)

have low energy vacancies capable of accommodating an electron

from outside the atom, as attracted by the nucleus of the atom.

Electronegativity thus describes the relative tendency of the

atom to become negatively charged.

Elution The gradual passage of the components of a sample

mixture through a chromatographic column as the mixture is

carried by the mobile phase through the stationary phase.

Enantiomers A class of stereoisomers in which molecules are

not superposable upon their mirror images. They are sometimes

called optical antipodes. Enantiomers have the potential of

showing optical activity, in which the plane of polarized light

can be rotated by passing through a solution of such a

compound. Enantiomers have identical physical properties.
194 The Basics of Chemical Instrumentation

Entrainer A mobile phase modifier used for supercritical fluid

chromatography or supercritical fluid extraction. This term is

more popular in the engineering community.

Explosion—Proof (as in explosion—proof laboratory or instrument) This term refers to designs in accordance with

Class A, Group b of the U.S. National Electrical Code. This

subsection details the requirements of fixtures, cabinets,

switches, and procedures which are designed to minimize the

possibility of an explosion being caused by spark sources. It

should be noted that the term is not a scientific term, but a

specification of a body of code.

Fluorescence One of two principal types of chemical

luminescence in which an electron in a ground electronic and

vibrational level is excited (by a photon of appropriate energy)

to an excited vibrational level of an excited electronic state,

resulting in the reemission of the energy. The photon which is

reemitted is at a longer wavelength than the exciting photon due

to the dissipation of some of the excitation photon energy in the

excited vibrational levels. This provides the basis of one of the

most sensitive methods of spectroscopic analysis, due to the

absence of "noise" from the excitation source which is at the

higher frequency.

Fraction Collector A device which collects the liquid eluting

from a liquid chromatography column. The instrument

functions by placing a series of vials one by one in position to

receive the eluent for a specified time interval. It is most
Glossary 195

commonly used in exclusion chromatography of biological

samples.

Frit A disk or plug of sintered glass or metal, having a

controlled pore size, used for filtering or as a carrier for samples

in chromatography, and as a restrictor for the column exit in

supercritical fluid chromatography.

Gaussian A curve having a bell shape which is described

mathematically by:

(—(x—u) 2 /2<j 2

) y =

e

(Js/Tk

where y is the ordinate, x is the abcissa, u is the mean value

and a is the standard deviation. It is also called the normal

distribution. The shape is an idealization of the geometry of

chromatographic peaks, never really attainable in practice.

Gel A two—component colloidal system consisting of a

continuous phase and a disperse phase, in a semisolid form. The

disperse phase is usually a solvent species, while the continuous

phase is a network such as a polymer. Gels are usually

transparent, but may become opalescent.

Heterocycle An organic ring compound in which the ring is

composed of atoms of more than one element. An example
196 The Basics of Chemical Instrumentation

would be pyridine, which is analogous to benzene with one of the

ring carbons replaced by a nitrogen.

Homologous Series A series of chemical compounds in which

each member differs from the next by the addition of a constant

number of atoms. The series of ethane, propane, butane,

pentane, and hexane is an homologous series, since each member

differs by the addition of a methylene group, -CHr-. Each

member of the series is called a homolog.

Hydrolysis A reaction in which water reacts with a chemical

species to form a product, usually involving ionization of the

water before the reaction. An example is the conversion of a

starch into glucose by water in the presence of a suitable

catalyst.

Interatomic Distance The average distance between molecules

in a given state of matter at a given temperature and pressure.

It can also be thought of as the mean free path (of a fluid

phase), the average distance traveled by a molecule before it

encounters and collides with another molecule.

Isochoric A process or measurement which is done with the

volume of the system held at a constant value. It follows that if

the volume is constant, the density will also be constant.

Isomer A compound which has the same chemical formula as another compound, but which differs from the other compound

in the way the atoms are arranged (that is, the two compounds
Glossary 197

differ in an aspect of molecular geometry). The word is derived

from the Greek "isos," meaning equal, and "meros," meaning

part.

Isothermal A process which is performed at a constant

temperature. An isotherm is a response or measurement which

is obtained from a technique maintaining the system at a

constant temperature. It also refers to the measured or fitted

lines on a P—V—T surface or a phase diagram, in which the

represented response is at constant temperature.

Isotope One of two or more forms of an element having the

same atomic number (and position on the periodic chart) but a

different relative atomic mass. Some isotopes of important

common elements are radioactive, and are useful in chemical

analysis as tracers.

Labile A system or compound which is unstable and is easily

converted to a more stable system or compound by a slight

disturbance, such as a temperature increase. Such a system or

compound is said to be thermally labile.

Linearity (of a chromatographic detector) The range of sample

concentrations over which the response factor remains constant

within a specified range, usually plus or minus 20 percent. The

response factor is a ratio of signal to sample size. The linearity

is often expressed in orders of magnitude (or decades) of

concentration.
198 The Basics of Chemical Instrumentation

Mass Spectrum A histogram of ion mass (actually, the ion mass

divided by the elementary electronic charge) versus ion

abundance produced by a mass spectrometer. In the mass

spectrometer, the sample is decomposed into constituent and

reconstructed ions (either by an electron beam or by chemical

means) which are then separated according to mass.

Modifier A fluid added to the primary mobile phase of a

supercritical fluid chromatograph or supercritical fluid

extraction system in order to change the polarity, and therefore

the solvent strength, of the fluid. The most common modifier is

methanol, added to carbon dioxide (the most common mobile

phase) in amounts ranging from 1 to 3 percent.

Moiety Although usually defined as one part of approximately

two equal parts, a moiety, as the term is used in chemistry,

refers to a functional group on a molecule, such as the carboxyl

group or moiety.

Permanent Gas An archaic although commonly used term

which refers to the gases which are difficult to liquefy, and

which were previously though impossible to liquefy. These gases

include the noble gases, and hydrogen, nitrogen and oxygen.

Pi Orbital (or pi bond) A theoretical construction describing

the chemical bond produced from the overlap of two p—type

orbitals. The 7r-orbital or bond consists of an electron cloud of

two parts, one lying above the plane of the atoms and one lying
Glossary 199

below. The electron cloud is actually a mathematical

description of the probability of finding an electron in this

particular volume of space around the molecule.

Polydispersivity A characteristic of polymeric materials in

which the molecules occur with a range of molecular weights,

rather than a single compound of fixed molecular weight. Such

polymer systems are said to be polydisperse.

Precision A measure of the reproducibility of an experimental

measurement, due to the effects of random errors on the

measurement technique. In general, systematic errors will not

be noticed in the precision of a series of measurements.

Preparative-Scale Chromatography The use of chromatographic hardware to achieve separation of relatively large

quantities of chemical species, for other than analytical

purposes. The most common types of preparative scale

chromatography are done using packed column GC, and HPLC.

Relatively little is done using SFC, and no such work is done

using capillary column GC. Electrophoresis can also be done on

the preparative scale.

Quadrupole Mass Filter An electrostatic (rather than magnetic)

mass separation device consisting of four rods arranged

symmetrically, across which are applied voltages having a dc

(direct current) and rf (radio frequency) component. The

electrostatic field that results is used to induce controllable

trajectories on the ion paths.
200 The Basics of Chemical Instrumentation

Racemic Modification A mixture consisting of equal parts of a

given enantiomeric pair. A solution such as this is optically

inactive, and will not rotate the plane of polarized light. The

mixture is designated by the prefix ± placed before the chemical

name.

Ranque—Hilsche Tube See vortex tube.

Reference Material A material or substance one or more

properties of which are sufficiently well established to be used

for calibration of an apparatus, the assessment of a measurement

method, or for assigning values to materials.

Refractive Index The ratio of the velocity of light in a vacuum

to the velocity of light in a particular medium such as a solid or

fluid. The refractive index is dependent upon the wavelength of

the light, and the temperature and density of the medium. It is

often used in chemical analysis as the basis for qualitative

identification (using an Abbe refractometer, for example) or

solute detection (using a differential refractometer in HPLC).

Resin A synthetic resin is a man-made high polymer made

from a chemical reaction between two or more substances,

usually with heat and catalyst. A natural resin is most often a

vegetable—derived mixture of carboxylic acids, essential oils and

terpenes usually occurring in the bark of many tress and shrubs.

Response Factor The ratio of detector signal output (in units of

integrated area) to the size of the sample.
Glossary 201

Retention Time The time required for a solute to elute from a

chromatographic column. It is related to the retention volume,

which is the volume of mobile phase required to elute a solute

from a chromatographic column.

Rotary Evaporator A device which rotates a flask (which may

be fluted to induce the continuous mixing of its contents) while

a vacuum or heat or both is applied to the flask. The rotation

serves the dual purpose of preventing bumping or cavitation,

and producing a thin layer of solvent on the inside surface of the

flask (as it rotates) to hasten evaporation.

Septum A self—sealing flexible gasket that isolates the inside of

a chromatographic injector from the outside environment, and

through which a syringe is pierced in order to deliver sample to

the chromatograph. They are available in different sizes,

materials, and temperature ratings. The word is derived form

the Latin "saeptum," meaning wall or enclosure.

Septum Bleed Extraneous impurity material released form a

septum on a chromatographic injector, eventually entering the

detector and causing interference. The impurities may include

compounds from previously injected samples, material from the

septum itself, or impurities introduced by the operator careless

handling a septum during replacement.

Sintered Glass/Metal A solid structure formed by the

agglomeration of ceramic (or vitreous) or metallic material at a

temperature below the melting point. The energetic driving
202 The Basics of Chemical Instrumentation

force behind the formation of sintered materials is the reduction

of the surface area, and the associated reduction of free energy.

Sol A two component colloidal system consisting of relatively

large molecules dispersed in a continuous phase solvent.

Solute A compound that is dissolved in a solvent. The name

solute implies that the dissolved material is present as a minor

constituent, or is relatively dilute in the solution, such as (for

example) in the Henry’s law region or below.

Solvent Strength In HPLC, the solvent strength is the ability of

the solvent or solvent system to provide a desired capacity

factor, k' . Stronger solvents provide smaller capacity factors,

while weaker solvents provide larger values. A solvent of

appropriate strength for a separation provides the necessary

capacity factor to achieve separation.

Standard Reference Material A certified reference material

issued by the United States National Institute of Standards and

Technology (formerly the National Bureau of Standards).

Stereoisomers A class of isomers which differ from one another

only by the way the atoms of the molecules are arranged in

space. In other respects, the isomers are identical; the chemical

formulae are the same, and the same atoms are bonded to one

another in the same order.
Glossary 203

Steric An effect due to molecular geometry in which atoms on a

molecule cause or prevent a reaction or a molecular movement

due to what is imagined to be physical interference by the atoms

in close proximity to one another. "Steric strain" in a molecule

may prevent certain geometric conformations of structure. A

large, bulky group of atoms situated near a reactive site on a

molecule may prevent a reaction at that site due to "steric

hindrance."

Stokes’s Law A mathematical relationship which describes the

frictional force experienced by a spherical, charged body in a

viscous solution as the body moves under the influence of an

electric field.

Stokes-Einstein Equation A mathematical relationship derived

from hdyrodynamic theory which relates the binary interaction

diffusion coefficient of a spherically-shaped solute species with

the solvent viscosity, temperature and the size of the solute. It

can be expressed as:

D12

RT

67r 77p r a

where D 12 is the binary interaction diffusion coefficient, is the

solvent viscosity, T is the thermodynamic temperature, R is the

gas constant, and r a is the radius of the solute molecule.
204 The Basics of Chemical Instrumentation

Theoretical Plate An imaginary section or volume element of a

distillation column or chromatographic column in which solute

partitioning between phases occurs, and in which equilibrium is

established rapidly before the solute moves on to the next

"plate."

Thermal Conductivity A transport property which describes the

tendency of matter to transfer heat through a temperature

gradient which is set up across its volume. Thermal conduction

will occur until the temperature gradient is zero. The thermal

conductivity of most materials is both temperature and pressure

dependent. The primary thermal transport mechanism in solids

is conduction, while in fluids, both conduction and convection

play a role.

Transport Property A thermophysical property that describes

the movement of some physical quantity, such as heat, mass,

electrical charge or momentum, from one part of a system

(ensemble of molecules) to another. Examples of transport

properties are viscosity, thermal conductivity, and electrical

conductance.

Ultrasound Radiation just above the region audible to the

human ear. Normally, ultrasound that is used in scientific

applications is in the ‘35 kHz region. Ultrasound is used in

degassing HPLC solvents, and is the basis of a detector for gas

chromatography. There are many industrial applications of

ultrasound as well, such as in cleaning, soldering, plating, and in
Glossary 205

the acceleration of chemical reactions, especially those occurring

through heterogeneous catalytic mechanisms.

Vapor Pressure The pressure exerted by the evaporated vapor

or gas that forms above any condensed phase which is held in a

closed system. Condensed phases include both liquids and

solids.

Viscosity Viscosity is a transport property of fluids that

describes the resistance of a fluid to flow. It is the shearing

stress per unit area of a fluid undergoing shear (or flow).

Void Volume The total empty volume of a chromatographic

system. The void volume establishes the de facto minimum

retention time, since it represents the time required for an

unretained solute to elute from the column.

Vortex Tube A refrigeration device which produces a cold air

stream by producing a vortex from a flow of compressed air

confined in a pipe. The vortex is forced to decrease in radius at

constant angular velocity. Thermodynamics requires that the

air stream must lose energy during this process, which produces

the cooling effect. A stream of heated air is also produced,

which removes the energy given up by the cooling air.

Xerogel A gel—like structure consisting of a solution of linear

polymers which has been cross-linked to restrict movement.

Alternatively, the movement may be restricted by the presence

of intermolecular interactions such as hydrogen bonds.

NiST-i 1 4A U.S. DEPARTMENT OF COMMERCE

(REV. 3-89) NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY

i. PUBLICATION OR REPORT NUMBER

NISTIR 89-3933

BIBLIOGRAPHIC DATA SHEET

2. PERFORMING ORGANIZATION REPORT NUMBER 1

3. PUBLICATION DATE

February 1990

4. TITLE AND SUBTITLE

Basics of Chemical Instrumentation

5. AUTHOR(S)

Thomas J . Bruno

6. PERFORMING ORGANIZATION (IF JOINT OR OTHER THAN NIST, SEE INSTRUCTIONS)

U.S. DEPARTMENT OF COMMERCE

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY

GAiTHCRSBURQ,-*w>-2oa99- Boulder , Colorado 80303

7. CONTRACT/GRANT NUMBER

8. TYPE OF REPORT AND PERIOD COVERED

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (STREET, CITY, STATE, ZIP)

10,

SUPPLEMENTARY NOTES

DOCUMENT DESCRIBES A COMPUTER PROGRAM; SF-185, FIPS SOFTWARE SUMMARY, IS ATTACHED.

11.

ABSTRACT (A 200-WORD OR LESS FACTUAL SUMMARY OF MOST SIGNIFICANT INFORMATION. IF DOCUMENT INCLUOES A SIGNIFICANT BIBLIOGRAPHY OR

LITERATURE SURVEY, MENTION IT HERE.)

This report is intended to provide an introduction to chemical instrumentation to workers

whose training lies outside the area of experimental chemistry. It is especially geared

toward scientific managers in government and industrial laboratories who must interact on

a daily basis with chemical professionals, and who often have highly sophisticated analytical

chemistry laboratories under their jurisdiction. This first report deals with instrumental

separation methods. After an introduction to the nature of mixtures, the topics of gas

chromatography, high performance liquid chromatography, supercritical fluid chromatography,

and electrophoresis are discussed. These sections are followed by an appendix covering

calibration methods. A glossary is provided in which terms not covered in the body of the

report are defined.

12.

KEY WORDS (6 TO 12 ENTRIES; ALPHABETICAL ORDER; CAPITAUZE ONLY PROPER NAMES; AND SEPARATE KEY WORDS BY SEMICOLONS)

chemical instrumentation; chromatography; electrophoresis, gas chromatography, liquid

chromatography, separation methods; supercritical fluid chromatography.

13.

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