SEPARATION METHODS


New Comprehensive Biochemistry

Volume 8

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Urrechr

ELSEVIER
AMSTERDAM * NEW YORK * OXFORD


Separation Methods

Editor

Z. DEYL
Prague

1984

ELSEVIER
AMSTERDAM * NEW YORK * OXFORD

1984 Elsevier Science Publishers B.V.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise
without the prior permission of the copyright owner.

0

ISBN for the series: 0-444-80303-3
ISBN for the volume: 0-444-80527-3

This book has been registered with the Copyright Clearance Center. Inc. Consent is given for copying
pages for personal or internal use, or for the personal or internal use of specific clients. This consent is
given on the condition that the copier pay through the Center the per-page fee stated below for copying
beyond that permitted by the U.S. Copyright Law. The appropriate fee should be forwarded with a copy
of the front and back of the title page of the book lo the Copyright Clearance Center, Salem, MA 01970.
This consent does not extend to other kinds of copying, such as for general distribution, resale, advertising
and promotional purposes, or for creating new works. Special written permission must be obtained from
the publisher for such copying.
The per-page fee code for this book is 0-444-80527-3 : 84/$0 + .80.

Published by:
Elsevier Science Publishers B.V.
PO Box 211
lo00 AE Amsterdam
The Netherlands

Sole distributorsfor the U.S.A. and Canada:
Elsevier Science Publishing Co. Inc.

52 Vanderbilt Avenue
New York. NY 10017
USA

Library of Congress Cataloging In Publication Data
Main entry under title:
Separation methods.
(New comprehensive biochemistry; v. 8)
Includes index.
1. Separation (Technology) I. Deyl. ZdenEk.
II. Series.
QD415.N48 VOI. 8 574.19’2s (574.19’2851 84-1502 [QD63.S4]
ISBN 0-444-80527-3

Printed in the Netherlands


V

Contents
Chapter 1. Principles and theory of chromatography, by J. Novak

1

1.1 Basic terms
1.2 Classification of chromatographic systems and procedures
1.2.1 State of the aggregation of the coexisting phases
1.2.2 Physical arrangement of the system and the accomplishment of the chromatographic
experiment
1.2.3 Development of the chromatogram

1.2.3.1 Frontal chromatography
1.2.3.2 Elution chromatography
1.2.3.3 Displacement chromatography
1.2.4 Mechanism of the distribution of the solute compound between the phases
of the system
1.3 Development of chromatography - a review
1.4 Theoretical models of chromatography
1.5 Description of models of linear chromatography with an incompressible mobile phase
1.5.1 Linear non-ideal chromatography
1.5.2 Linear ideal chromatography
1.6 Simplified description of linear non-ideal chromatography
1.6.1 Retention equations
1.6.2 Spreading of the chromatographic zone
1.6.3 Concept of the theoretical plate
1.7 Mobile phase flow
1.8 Sorption equilibrium and the distribution constant
1.8.1 Problem of sorption equilibrium in a migrating chromatographic zone
1.8.2 Relations between the chromatographic distribution constant and the thermodynamic
properties of chromatographic system
1.8.3 Dependence of the standard differential molar Gibbs function of sorption and the
chromatographic distribution constant on temperature and pressure
1.9 Chromatographic resolution
1.I0 Development of theories of chromatography
References

1
2
2

22

25
27
27

Chapter 2. Principles and theory of electromigration processes, by J .
Vacik

29

2.1 Principles of electromigration methods
2.2 Transport processes and equilibria during electrophoretic separations

29
32

6
6
8
8
8
10
11
11
13
16
17
18
18
19



vi
2.2.1
2.2.2
2.2.3
2.2.4
2.2.5
2.2.6
2.2.7
2.2.8
References

Migration velocity
Mobility
Diffusion velocity
Velocity of convection
Hydrodynamic flow
Electro-osmotic flow
The velocity of the thermal flow
The distribution of the potential gradient

33
34
37
37
37
38
38
39
39


Chapter 3. Gas chromatography, by M. Noootny and D. Wiesler

41

3.1 Introduction
3.2 Modern instrumentation of gas chromatography
3.2.1 General considerations
3.2.2 Operating conditions
3.2.3 Multiple-column systems
3.2.4 Sampling systems
3.3 Chromatographic columns
3.3.1 Phase systems
3.3.2 Capillary columns
3.4 Detection methods
3.4.1 General considerations
3.4.2 Selective detectors
3.5 Solute identification techniques
3.5.1 Retention studies
3.5.2 Ancillary techniques
3.6 Metabolic profiles
3.7 Steric resolution
3.8 Derivatization methods
3.8.1 General aspects
3.8.2 Derivatization of alcohols and phenols
3.8.2.1 Silylation agents
3.8.2.2 Other derivatization agents
3.8.3 Derivatization of carboxylic acids
3.8.4 Derivatization of aldehydes and ketones
3.8.5 Derivatization of amines and amino acids

3.8.6 Derivatization for the separation of enantiomers
3.9 Sample preparation
3.10 Selected applications
3.10.1 Steroids
3.10.1.1 General
3.10.1.2 Steroid hormones in blood and tissue
3.10.1.3 Urinary steroids
3.10.1.4 Sterols
3.10.1.5 Bile acids
3.10.2 Lipoid substances
3.10.2.1 General
3.10.2.2 Intact lipids
3.10.2.3 Fatty acids
3.10.3 Acid metabolites
3.10.4 Carbohydrates

41
45
45
47
49
53
62
62
68
72
72
75
79
79

80
83
87
89
89
90
90
93
95
99
100
103
104

108
108
108
108
111
114
115
116
116
117
118
121
124


vii

3.10.5 Biological amines
3.10.6 Prostaglandins
3.10.7 Amino acids and peptides
References

Chapter 4. Liquid column chromatography (4.1-4.7)

125
128
129
135

149

Chapter 4.1. Types of liquid chromatography, by S.H. Hansen, P.
Helboe and U. Lund
1.51
4.1.1 Introduction
4.1.2 Adsorption
4.1.3 Partition
4.1.4 Bonded phases
4.1.5 Ion exchange
4.1.6 Size exclusion
4.1.7 Affinity
References

Chapter 4.2. Instrumentation, by S.H. Hansen, P. Helboe and U.
Lund

151

151
151
152
152
152
153
153

15.5

4.2.1 Introduction
4.2.2 The column
4.2.3 Injection devices
4.2.4 Solvent delivery systems
4.2.5 Detectors
4.2.6 Technical optimisation of the LC system
4.2.7 Conclusion
References

155
156
157
157
158
158
158
159

Chapter 4.3. Detection, by S.H. Hansen, P. Helboe and U.Lund


161

4.3.1 Introduction
4.3.2 Detectors
4.3.2.1 The ultraviolet detectors
4.3.2.2 The fluorescence detector
4.3.2.3 The electrochemical detector
4.3.2.4 The refractive index detector
4.3.2.5 The radioactivity detector
4.3.2.6 liquid chromatography-mass spectrometry
4.3.3 Detection enhancement
References

161
162
162
163
163
164
164
164
164
166

Chapter 4.4. Absorption and partition chromatography, by S.H.
Hansen, P . Helboe and U. Lund

167

4.4.1 Phase systems

4.4.1.1 General aspects

167
167


viii
Adsorption chromatography
Liquid-liquid partition chromatography
Bonded phase chromatography
Dynamically coated phases
4.4.2 Derivatization
4.4.3 Experimental techniques
4.4.3.1 General aspects
4.4.3.2 Sample pre-treatment
4.4.3.3 Choice of the chromatographic system
4.4.3.4 Quantitative analysis
4.4.3.5 Identification
4.4.3.6 Preparative liquid chromatography
4.4.4 Applications
References
4.4.1.2
4.4.1.3
4.4.1.4
4.4.1.5

Chapter 4.5. Ion exchange chromatography, by 0. Mikes’
4.5.1 Ion exchange in biochemistry
4.5.1.1 Classic methods
4.5.1.2 Modem trends

4.5.2 Ion exchangers
4.5.2.1 Classification and fundamental properties of ion exchangers
4.5.2.2 Materials for batch processes and packings for low-pressure liquid column chro-

ma tography
4.5.2.3 Packings for medium- and high-pressure liquid chromatography
4.5.2.4 Packings for ampholyte displacement and chromatofocusing
4.5.3 Mobile phase systems
4.5.3.1 Aqueous solutions and organic solvents
4.5.3.2 Volatile and complex-forming buffers (special additives)
4.5.3.3 Amphoteric buffers for ampholyte displacement chromatography and chromatofo-

cusing
4.5.4 Experimental techniques
4.5.4.1 Principles of chromatographic separation procedures
4.5.4.2 Choice of a suitable ion exchanger
4.5.4.3 Preliminary operations. equilibration (buffering) of ion exchangers, and filling or

packing of chromatographic columns
4.5.4.4 Application of samples and methods of elution
4.5.4.5 Evaluation of fractions
4.5.4.6 Regeneration and storage of ion exchangers
4.5.5 Areas of application
4.5.5.1 Biochemically important bases and acids
4.5.5.2 Saccharides and their derivatives
4.5.5.3 Amino acids and lower peptides
4.5.5.4 Proteins and their high molecular weight fragments
4.5.5.5 Enzymes
4.5.5.6 Nucleic acids and their constituents
4.5.5.7 Other biochemically important substances


References

168
171
174
183
185
185
185
186
187
187
188
189
201
201

205
205
205
206
208
208
211
215
219
220
220
223

225
226
226
229
230
232
234
231
238
238
238
243
243
248
256
258
259


ix

Chapter 4.6. Gel chromatography, by D. Berek and K. Macinka

2 71

4.6.1 Introduction
4.6.2 General concepts and principles of theory
4.6.2.1 Mechanism of ideal gel chromatography
4.6.2.2 Real gel chromatography
4.6.2.3 Resolution power and calibration in gel chromatography

4.6.2.4 Processing experimental data
4.6.3 Equipment and working procedures in gel chromatography
4.6.3.1 Scheme of a gel chromatograph
4.6.3.2 Transport of mobile phase
4.6.3.3 Sample preparation and application
4.6.3.4 Separation columns
4.6.3.5 Operational variables
4.6.3.6 Detection
4.6.3.7 Measurement of effluent volume
4.6.3.8 Auxiliary equipment
4.6.3.9 High speed separations
4.6.3.10 Preparative separations
4.6.3.11 Special working procedures
4.6.4 Materials for gel chromatography
4.6.4.1 Column filling materials - gels
4.6.4.2 Mobile phases - eluents
4.6.4.3 Reference materials - standards
4.6.5 Areas of applications
4.6.5.1 Proteins and peptides
4.6.5.2 Nucleic acids and nucleotides
4.6.5.3 Nucleoproteins
4.6.5.4 Saccharides
4.6.5.5 Other biological materials and biologically active substances
4.6.5.6 Applications in clinical biochemistry
References

271
272
272
274

275
277
280
281
282
283
284
286
287
289
289
290
290
291
294
294
301
303
304
306
310
312
313
314
314
316

Chapter 4.7. Bioaffinity chromatography, by J. Turkova

321


4.7.1 Introduction
4.7.2 General considerations on the preparation of bioaffinity adsorbents and their use in sorption
and desorption
4.7.2.1 Required characteristics of solid matrix support
4.7.2.2 Choice of affinity ligands for attachment
4.7.2.3 Affinant-solid support bonding
4.7.2.4 Sorption and elution conditions
4.7.3 Solid matrix support and the most common methods of coupling
4.7.3.1 Survey of the most common solid supports
4.7.3.2 Survey of the most common coupling procedures
4.7.3.3 Blocking of unreacted groups
4.7.4 Experimental techniques
4.7.4.1 Classic bioaffinity chromatography
4.7.4.2 High-performance liquid bioaffinity chromatography (HPLAC) of proteins
4.7.4.3 Automatic time-based instrument for preparative application
4.7.4.4 Extracorporeal removal of substances in vivo

321
322
322
324
326
331
334
334
337
340
341
341

343
345
347


X

4.7.5 Areas of application
4.7.5.1 Enzymes, their subunits and inhibitors
4.7.5.2 Antibodies and antigens
4.7.5.3 Lectins, glycoproteins and saccharides
4.7.5.4 Receptors, binding and transfer proteins
4.7.5.5 Nucleic acids and nucleotides
4.7.5.6 Viruses, cells and their components
4.7.5.7 Specific peptides
4.7.5.8 Others
References

348
353
353
354
354
354
355
355
355
356

Chapter 5. Flat bed techniques, by J . Sherma and B. Fried


363

General introduction
5.2 Thin-layer chromatography
5.2.1 Introduction and history
5.2.1.1 Introduction
5.2.1.2 History
5.2.2 Sorbents, layer preparation and precoated plates
5.2.2.1 Sorbents
5.2.2.2 Layer preparation
5.2.2.3 Precoated layers
5.2.3 Sample preparation, derivatization and solvent systems
5.2.3.1 Sample preparation
5.2.3.2 Derivatization
5.2.3.3 Solvent systems
5.2.4 Development modes and chambers
5.2.4.1 Development modes
5.2.4.2 Chambers
5.2.5 Detection
5.2.5.1 General
5.2.5.2 Methods of detection
5.2.5.3 Detection reagents
5.2.6 Identification
5.2.7 In situ densitometry
5.2.8 Applications
References (Part A)
5.3 Paper chromatography
5.3.1 History and introduction
5.3.2 Chromatography papers

5.3.3 Sample preparation and application
5.3.4 Mobile phase (solvent) systems
5.3.5 Development methods
5.3.5.1 Descending development
5.3.5.2 Ascending development
5.3.5.3 Horizontal and radial development
5.3.5.4 Multiple development
5.3.5.5 Two-dimensional development
5.3.5.6 Miscellaneous techniques
5.3.6 Drying of the chromatogram
5.3.7 Detection of zones
5.3.8 Qualitative identification of zones
5.1

363
364
364
364
365
366
366
369
369
371
371
373
373
374
374
375

378
378
378
379
380
382
388
388
392
392
393
395
396
398
398
400

40 1
402
402
402
402
403
404


5.3.9 Quantitative PC
5.3.10 Applications

References (Part B)


404
405
410

Chapter 6. Electromigration techniques, by Z. Deyl and J. Hofejii

415

6.1 Introduction
6.2 Zone electrophoresis
6.2.1 Paper electrophoresis
6.2.1.1 Equipment for low and lligh voltage paper electrophoresis
6.2.1.2 Two-dimensional separations
6.2.1.3 Cellulose and cellulose acetate membranes
6.2.1.4 Ion exchange papers
6.2.1.5 Ultramicroelectrophoretic methods
6.2.1.6 Electrophoresis in non-aqueous buffers
6.2.2 Thin-layer electrophoresis
6.2.3 Electrophoresis in fused salts
6.3 Moving boundary electrophoresis
6.4 Electrophoresis in gel media
6.4.1 Starch gel electrophoresis
6.4.2 Polyacrylamide gel electrophoresis
6.4.2.1 Disc electrophoresis - general considerations and solutions
6.4.2.2 Rod shaped gel system
6.4.2.3 Slab gel system
6.4.2.4 Gradient gel electrophoresis
6.4.2.5 SDS-polyacrylamide gel electrophoresis
6.4.2.6 Two-dimensional polyacrylamide gel electrophoresis and the lsodalt system

6.4.3 Agarose gel electrophoresis
6.4.4 Composite gel (acrylamide-agarose) electrophoresis
6.5 lmmunoelectrophoretic procedures
6.5.1 Apparatus and equipment
6.5.2 Crossed immunoelectrophoresis
6.5.3 Fused rocket immunoelectrophoresis
6.5.4 Rocket electrophoresis
6.5.5 Crossed line immunoelectrophoresis
6.5.6 Tandem crossed immunoelectrophoresis
6.6 Isoelectric focusing
6.6.1 Carrier ampholytes
6.6.2 lsoelectric focusing in polyacrylamide gel
6.6.3 Thin-layer isoelectric focusing
6.6.4 Density gradient isoelectric focusing
6.6.5 Free solution isoelectric focusing
6.6.6 Two-dimensional procedures involving isoelectric focusing
6.6.7 Transient state isoelectric focusing
6.7 Isotachophoresis
6.7.1 Apparatus for isotachophoresis
6.7.2 Detection in isotachophoretic separations
6.7.3 Buffer systems for isotachophoretic separations of serum proteins
6.8 Affinity electrophoresis
6.9 General detection procedures
6.9.1 Detection by ultraviolet absorbance
6.9.2 Detection by fluorescence measurement

415
415
415
416

418
422
422
423
424
425
425
426
427
427
428
428
431
433
435
436
439
443
445

446
446
448
45 1
45 1
453
454
454
455
456

457
45 7
45 8
458
459
460
461
462
463
464
467
468
468


xii
Detection by staining
6.9.3.1 Silver based staining of polypeptides
6.9.4 Scanning of electrophoretograms
6.9.5 Detection by radioactivity counting
6.9.5.1 Autoradiography and fluorography
6.9.5.2 Spark chamber detection
6.9.5.3 Direct counting
6.9.5.4 Elution or solubilization of radioactive material
6.9.5.5 Counting after combustion
6.9.5.6 Disruption of gel structure
6.10 Preparative procedures
6.10.1 Electrophoresis in columns
6.10.2 Preparative agar gel electrophoresis
6.10.3 Preparative electrophoresis in polyacrylamide gel

6.10.4 Preparative isoelectric focusing
6.10.4.1 Preparative isoelectric focusing in a density gradient
6.10.5 Preparative flat bed isoelectric focusing
6.10.5.1 Continuous flow isoelectric focusing
6.10.6 Preparative isotachophoresis
6.10.7 Continuous flow through electrophoresis
6.11 Drying of polyacrylamide gels
References

469
472
473
47 3
473
474
475
4 15
415
47 5
416
476
477
478
481
48 1
483
483
484
487
489

489

Chapter 7. Field-flow fractionation, by J. JanCa

497

6.9.3

References

497
498
500
500
501
502
502
505
506
508
510
512
513
514
515
516
518

Subject index


52 1

7.1 Introduction
7.2 Principle of FFF
7.3 Theoretical backgrounds of FFF
7.3.1 Retention
7.3.2 Zone spreading
7.3.3 Relaxation
7.3.4 Optimization of FFF
7.4 FFF Subtechniques
7.4.1 Thermal FFF
7.4.2 Sedimentation FFF
7.4.3 Electrical FFF
7.4.4 Flow FFF
7.4.5 Steric FFF
7.4.6 Magnetic FFF
7.4.7 Concentration FFF
7.5 Prospects of FFF


Deyl (ed.) Separation Methods
Elsevier Science Publishers B.V.

1

0 1984

CHAPTER 1

Principles and theory

of chromatography
JOSEF NOVAK
Institute of Analytical Chemistry, Czechoslovak Academy of Sciences,
611 42 Brno, Czechoslovakia

1.1 Basic terms
It is useful to begin the chapter on the theory of chromatographic separation
methods with a definition of chromatography. However, several such definitions can
be formulated according to various classification aspects. For the sake of accuracy a
phenomenological definition, a molecular kinetic definition and various working
definitions can be introduced. According to the first definition chromatography is
understood as aphenomenon of differential migration of solute compounds in a
system of two phases, of which one is stationary and the other mobile. According to
the molecular kinetic definition, chromatography is taken as a continuous process of
convective upsetting and diffusional reestablishment of equilibrium between the
concentrations of the solute compound in the stationary and in the mobile phase of
the chromatographic system. This process results in a differential migration of the
solute compounds. According to the working definitions chromatography is a certain
method (specifically a separation and analytical method and various methods of
physicochemical measurements). From the point of view of the theory of chromatography we are particularly interested in the chromatographic process.
Whereas the realization of a chromatographic experiment is often surprisingly
simple - a number of important chromatographic processes proceed spontaneously
- the mechanism of the chromatographic process is relatively complex. A prerequisite of the proper understanding of the mechanism of chromatography is the
concept of dynamic equilibrium between the concentrations of a solute in a system
of two coexisting phases; more accurately, equilibrium between the concentrations
of the solute should be understood as a result of the identity of its chemical
potentials in the individual phases of the system. Even when assuming that such a
system is stationary and in equilibrium, molecules of the solute permanently pass
from one phase to the other, remaining for a certain time in one or other phase after
each transition. As the process is random at this level, the individual time intervals

of the Occurrence of the solute molecules in a given phase are also random and,


2
hence, very different. The mean time intervals of the occurrence of all solute
molecules in each phase during a certain time are, however, constant under given
conditions, and their ratio represents a basic factor of chromatographic retention.
Thus, the ratio at which a given amount of the solute at equilibrium is distributed
between the phases of the system is not determined by a static presence of the solute
molecules in these phases but rather by the probability of their occurrence in the
phases of the system. When, under these conditions, one phase moves with respect to
the other, the solute molecules move together with the moving phase during their
occurtence in that particular phase, but remain stagnant when in the stationary
phase. Due to the statistical fluctuation some molecules of a given solute migrate a
shorter or longer distance during a certain time interval than that corresponding to
the mean time intervals of the occurrence of the molecules of this solute in the
phases. This results, together with the longitudinal diffusion, in a spreading of the
migrating zone of the solute. However, due to its statistical nature, this spreading
increases only as the square root of the mean migration distance, so that, in the case
of differential migration of zones of different solutes, the zones can be separated.
This assumption of the mechanism of the chromatographic process will be formulated quantitatively in subsequent paragraphs of this chapter.

I .2 Classification of chromatographic systems and procedures
1.2.1 State of the aggregation of the coexisting phases

The traditional definition of the phases in a chromatographic system is often rather
problematic. Whereas the term mobile phase is usually clear, specification of the
chromatographic stationary phase is not always unambiguous. For instance, the
whole content of the chromatographic column is sometimes considered as the
stationary phase, but sometimes only those components of the packing that are

functioning as sorbents of the solute compound are termed in this way. In the
former case, the concept of chromatographic stationary phase apparently differs
from the classical physical concept of the phase. Whereas in the physical conception
the phase is a homogeneous part of the system, the chromatographic stationary
phase may contain even more physical phases. In the latter case, the inert support of
the sorbent is not considered to be the stationary phase, in spite of the fact that it
represents a rather substantial physical phase of the system. However, when an
active adsorbent plays the role of the sorbent support, it must then be considered as
the chromatographic stationary phase. A problem then arises, viz. what part of the
used adsorbent is really active with respect to the solute compound in the given
system. Naturally, in a given chromatographic packing, chromatographic stationary
phases cannot be unambiguously identified with physical phases. The above indeterminacies should be considered when classifying chromatographic systems according to the state of the aggregation of the phases; a summary of typical chromatographic systems according to this classification is presented in Table 1.1.


3
TABLE 1.1
Chromatographic systems
Stationary
phase

Mobile phase
Liquid

Gas

Solid compound
Solid compound + liquid
Liquid

LSC

LSLC
LLC

GSC
GSLC
GLC

LSC, liquid-solid chromatography; GSC, gas-solid chromatography; LSLC, liquid-solid-liquid chromatography; GSLC, gas-solid-liquid chromatography; LLC, liquid-liquid chromatography; GLC, gas-liquid
chromatography.

1.2.2 Physical arrangement of the system and the accomplishment of the chromatographic experiment

According to the physical arrangement chromatographic systems can be divided into
planar and column ones. The planar arrangements are represented by systems of
paper and thin layer chromatography. When further dividing the planar systems
according to their physical arrangement we come to systems in the equilibration
chamber and to the so-called sandwich systems. According to development procedures (flow of the mobile phase in the planar bed) the systems can be further
classified as ascendent, horizontal, descendent and, occasionally, centrifugal; in
orthogonal beds the development may proceed in one or more directions. When,
during the development of the chromatogram, the composition of the mobile phase
remains constant the development is termed isocratic, on the other hand, when the
composition of the mobile phase varies, we speak of gradient development.
A more exact classification of column systems according to the physical arrangement leads to various types of packed and capillary columns. In column chromatography the use of several columns that can be suitably switched over, so that
chromatographic fractions eluted from one column can be further chromatographed
on other columns, is somewhat analogous to two-dimensional development in planar
beds. In column chromatography the separation may proceed isocratically or with a
programmed gradient of composition of the mobile phase, isothermally or with
programmed changes of column temperature, and isobarically or with programmed
changes of mobile phase pressure at the column inlet. The programming of the
composition of the mobile phase is important practically only in liquid chromatography, whereas temperature and pressure programming is used primarily in gas

chromatography.
In planar chromatographic systems the solute compounds are usually not eluted
from the chromatographic bed but rather detected directly in it, whereas in modern
column chromatography the solute compounds are gradually eluted with the mobile
phase and detected in the effluent at the column outlqt.


4

1.2.3 Development of the chromatogram
1.2.3.1 Frontal chromatography
A continuous supply of the analyzed material, or of its mixture with a non-sorbed

mobile phase, into the column or into the planar bed results first in frontal
chromatography and then in the saturation of the sorbent with all the components of
the supplied material. After the development of the chromatogram, and during
continuing supply of the mixture, the front of the least sorbed component is washed
out first, followed by a mixture of the first component and the more strongly sorbed
component etc., and, finally, after all the components of the mixture break through,
a mixture identical in composition to that of the mixture supplied flows out of the
column. By interrupting the supply of the analyzed mixture to the previously
saturated column, and connecting the supply of the mobile phase alone, the opposite
(desorption) frontal chromatogram arises. Initially, the mixture of all the components flows out of the column. After the least sorbed component has been eluted the
mixture deprived of this component flows out of the column. After the further, more
strongly sorbed component is eluted the mixture deprived of the first and second
components flows out of the column. Finally, the most strongly sorbed component is
washed out and only the supplied mobile phase leaves the column. Both versions of
development of the frontal chromatogram are schematically and in an idealized form
illustrated in Fig. 1.1.
I .2.3.2 Elution chromatography

Elution chromatography is simpler, and, with respect to the separation of an
analyzed mixture, more effective. With this alternative a dose of the analyzed
1

STARTINGTHECONTINUOUSIMROWCllON
OF MIXTURE0fOC)MPOVNDS1.2.3 AND MP

I1

BREAK THROUGH OF THE FRONT
OFCOMPOUND 1
SATURATIONOF THE COLUMN WITH
ALL THE COMPOUNDS

111

IV

V

STARTINGTHE INTRODUCTION OF PURE
MOBILE PHASE
ELUTIONOFALL THE COMPOUNDS

3+MP

_ _ - - - - - - - - - - - -- - - 1
_
GRAPHICAL RECORD OF THE SORPTION AND DE
SORPTION STAGES OF A FRONTAL CHROMATOGRAM


Fig. 1.1.

wA
ELUTED ZONES

1 + 2 * 3 MP


5
I INTRODUCTION C f A CHARGE OF
MIXTURE OF COMPOUNDS 1.2 AND 3
11. DEVELOPMENT OF CHROMATOGRAM

OF THE ZONES OF
COMPOUNDS 1.2 AND 3

1

111. ELUTION

-

FLOW OF MP
f

L
GRAPHICAL RECORD OF AN ELUTION CHROMATOGRAM

L


1tMP

ELUTED ZONES

Fig. 1.2.

material is supplied to the column inlet or to the planar bed and is then washed with
a non-sorbed mobile phase through the column. The development and differential
migration of elution zones of individual components of the mixture thus take place.
When the supply of the mobile phase continues the individual zones are gradually
washed out of the column; the zone of the most weakly sorbed component is washed
out first, followed by the zone of a more strongly sorbed component etc., and,
finally, after the elution of the zone of the most strongly sorbed component, only the
supplied mobile phase flows out of the column. A schematic illustration of the
elution chromatography is presented in Fig. 1.2.
1.2.3.3 Displacement chromatography
When the stationary phase functions as an adsorbent and a compound that is
adsorbed more strongly than any other component of the analyzed mixture serves as
the mobile phase, the procedure otherwise similar to that used with elution chromatography is termed displacement chromatography. With this alternative the most
weakly adsorbed component is displaced by the more strongly adsorbed component,
this latter is then displaced by the more strongly adsorbed component, etc., resulting
in a situation when the most strongly adsorbed component of the analyzed mixture
is displaced by the supplied displacement agent. After the chromatogram has been
developed, the zones of all the components migrate closely next to each other and,
when the supply of the displacement agent continues, they leave the column in the
order of increasing adsorption ability. In the case of elution chromatography (and in
frontal chromatography when the mixture of the analyzed material is supplied
together with the mobile phase) the eluted fractions are in fact mixtures of the solute
compounds with the mobile phase, whereas in the case of displacement chromatography the individual zones are more or less the solute compounds alone. A scheme of

displacement development is illustrated in Fig. 1.3.


INTRODUCTION OFA CHARGE OF
MIXTUREOFCOMWUNDS 1.2AND3

7

1

D E M L O M N T OF CHROMATOGRAM
DISPLACEMENT OF ZONES OF
COMPOUNDS 1,2 AND 3

-

FLOWOFMP

GRAPHICAL RECORD OFA DISPLACEMENT
CHROMATOGRAM

DISPLACED ZONES

Fig. 1.3.

1.2.4 Mechanism of the distribution of the solute compound between the phases of the
system

The mechanisms of sorption and/or the interaction of the solute with the mobile
phase can be summarized as follows: u , physical dissolution in the phase; b, physical

adsorption on the surface of the phase; c, chemical reaction in the bulk phase or on
its surface (acido-basic equilibrium, formation of coordination complexes or chelates,
association of ionic pairs, exchange of ions, precipitation); d , steric exclusion
(molecular sieving effect, gel permeation); e , bioaffinity association.
The cases presented in paragraphs 1.2.1-1.2.4 can be mutually combined. The
number of all possible combinations naturally exceeds the number of the real
combinations, however, the number of real chromatographic systems and procedures
is still very large. From the practical point of view, the alternatives of elution
chromatography are most important. Therefore, with the exception of general
problems, only elution chromatography will be discussed in this chapter.

1.3 Development of chromatography

-

a review

The oldest intentional chromatographic experiments were performed as frontal
chromatography in a liquid-solid system and date from the beginning of the 19th
century [l].Elution chromatography (liquid-solid) was discovered at the beginning
of the 20th century [2], but developed rapidly only after the discovery and theoretical
explanation of liquid-liquid elution chromatography [ 31 in the forties and particularly after the discovery of elution gas chromatography [4-61 in the fifties. The
pioneers in chromatography are noted in Table 1.2. A detailed description of the
development of chromatography can be found in reviews by Ettre [7,8] and Zechmeister [9].


TABLE 1.2
Pioneers in chromatography
Stationary
phase:


Solid sorbent

Liquid sorbent

Mobile phase:

Liquid (LSC)

Gas (GSC)

Liquid (LLC)

Gas (GLC)

Elution
development

M.S.Tswett (1906);
R. Kuhn, A. Winterstein and E. Lederer

E. Cremer (1951);
J. Jan& and
M.Rusek (1953);
H.W. Patton,
J.S. Lewis and
W.I. Kaye (1955)

A.J.P. Martin and
R.L.M. Synge (1941)


A.T. James and

(1931)

A.J.P. Martin
(1952);
N.H. Ray (1954):
B.W. Bradford,
D. Harvey and
D.E. Chalkley
(1955)

Frontal
development

D.T. Day (1897);
A. Tiselius (1943);
S. Claesson (1949)

C.S.G. Phdlips

Displacement
development

A. Tiselius (1943);
S. Claesson (1949)

N.C. Turner (1943);
C. Claesson ( 1946);

N.M. Turkel'taub
(1950); C.S.G.
Phillips (1953)

(1953)

C.S.G. Phillips
(1952)

C.S.G. Phillips
(1954)


8

1.4 Theoretical models of chromatography
When describing the chromatographic process in terms of mathematics it is necessary to define a suitable (sufficiently realistic and yet mathematically tractable)
model of chromatography. From the point of view of theoretical considerations the
following models are of interest [lo].
a. Model of ‘ideal chromatography’, assuming a piston flow of the mobile phase,
infinitely rapid setting of equilibrium between the concentrations of the solute in the
coexisting phases, and zero lonptudinal diffusion of the solute.
b. Model of ‘non-ideal chromatography’, considering the actual velocity profile of
the mobile phase flow, finite rate of of equilibration between the concentrations of
the solute in the coexisting phases, and the actual longitudinal diffusion of the
solute.
c. Model of ‘linear chromatography’, using a linear sorption isotherm for calculations.
d. Model of ‘non-linear chromatography’, using a non-linear sorption isotherm for
calculations.
In t h s way four combined models of chromatography may be postulated: A,

ideal linear; B, non-ideal linear; C, ideal non-linear; and D, non-ideal non-linear.
Whereas the models B and D are real, the models A and C are apparently
hypothetical. In spite of this even the latter two models are very useful from the
theoretical point of view.

1.5 Description of models of linear chromatography with an
incompressible mobile phase
1.5.1 Linear non-ideal chromatography

The mass balance of a solute in the infinitesimal volume of a chromatographic bed
(column), delineated by two parallel sections of identical area A, drawn perpendicular to the direction of the mobile phase flow at distances z a‘iid z + d z from the
beginning of the bed leads to the equation:

where c , and
~ cis are the mean (over the cross-section) concentrations (mass/volume)

of the solute in the mobile and stationary phases, $M and cpS are the fractions of the
area A occupied by the mobile and the stationary phase, DM and Ds are the
diffusion coefficients of the solute in the mobile and stationary phases, u is the mean
forward velocity of the mobile phase, averaged over the cross-section i.e.. u = F/@M,
where F is the volumetric rate of the mobile phase, t is time and z is the longitudinal


9
distance from the beginning of the bed in the direction of the mobile phase flow. It
follows from the right side of equation 1 that the given mass balance includes the
convective transport of the solute in the mobile phase and the diffusional transport
of the solute in the mobile and stationary phases of the system. For + M and +s it
holds that:
+S/+M


=

where A, and A M the are absolute parts of the area A, occupied by the stationary
and mobile phases.
In the case of liquid-solid chromatography or gas-solid chromatography the value
+ M represents total porosity of the bed E , so that +s = 1 - E and +S/+M
= (1 - E ) / E .
Equation 1 has two unknown quantities, clM and cis, so that one additional
independent equation is necessary for the solution. Such an equation can be derived
on the basis of the concept of solute mass transfer between the phases of the system.
The volume element of the bed Adz is also considered here. The interphase transfer
of the solute is then given by the flow J(M e S) through the total area of the phase
interface in the volume element Adz, and the actual direction and density of this
flow are determined by the actual sense and degree of the deviation from equilibrium between the concentrations clMand cis. The difference between the actual
solute concentration in phase 1 and such a concentration in this phase, which would
be in equilibrium with the solute concentration in phase 2, is the driving force of the
solute transfer, e.g., from phase 1 to phase 2. The solute flow through a unit area of
the phase interface is given by the relation E[c,, - (cls/K)], where 5 is the mass
transfer coefficient and K is the distribution constant defined as the equilibrium
ratio of cls and cIM,i.e.,
= ( c ~ S / c ~ M )eq

(2)

If K is the area of the phase interface in unit bed volume, then for the flow J(M F? S)
it holds that

Changes in the concentration of the solute in the stationary phase occur due to
transfer of the solute across the phase interface and longitudinal diffusion ,in the

stationary phase. Thus, it may be written

(

A 'cis dz = [ K ciM- :)Adz
s at

a2cis
+ AsDs--dz
aZ

and after dividing by the volume Adz the equation

(4)


10

is obtained. Equation 5 is the second equation required for the solution of the
problem.
Let us now define the initial and boundary conditions for the case of column
elution chromatography. At the beginning no solute is present in the column, i.e.
at t = 0 and 0 < z < 00, ciM= c , =
~0

(6)

The solute is applied to the column in the form of a concentration pulse of the
concentration c ~ and~ duration
. ~ S t , so that

at t > St and z = 0,cIM= 0
at 0 < t < St and z = 0, cIM= c , ~ , ~ .

(7)

If the terms for the longitudinal diffusion of the solute in the stationary phase are
neglected in equations 1 and 5 , the following solution exists for the system of
equations 1, 5 , 6 and 7 [11,12]

where c ~ is the
~ actual
.
~ solute concentration in the mobile phase in the section
z = L (at the end of the column), t , is the elution time of peak’s maximum, u, is
standard deviation of the time record of the elution peak and m , is the total solute
mass in the elution zone.
For t R and uf in equation 8 it further holds

where K is distribution constant defined by equation 2, k is the so-called capacity
ratio defined as the equilibrium ratio of solute masses in the stationary and mobile
phases, i.e., k = (miS/miM)eq,and L is the length of the column. Solution 8, together
with equations 9 and 10, holds sufficiently accurately only in the case that 6r e t R
and uI e .1, Relation 9 represents the basic equation of chromatographic retention.
I . 5.2 Linear ideal chromatography
As already mentioned in paragraph 1.4, the concept of ideal linear chromatography

is based on the model [13] which should have the following properties: (i) infinitely
fast setting of equilibrium between the solute concentrations in the mobile and
stationary phases; (ii) zero longitudinal diffusion of the solute in both phases; (iii)



11
absolutely linear sorption isotherm; and (iv) piston flow of the mobile phase. In spite
of the fact that this model is not real it is interesting as it provides for a fairly
accurate description of chromatographic retention. Naturally, it does not yield any
information about zone spreading, as the spreading factors have not been considered
at all. The initial concentration profile of the solute would, under the conditions of
linear ideal chromatography, proceed through the column without any change of its
shape at such a rate at which the center of a broadening elution zone proceeds under
the conditions of non-ideal chromatography (a more rigorous treatment [14] of the
model of linear non-ideal chromatography shows that the retention time is not fully
independent of spreading factors).
When the terms representing the longitudinal diffusion of the solute in the mobile
phase in equation 1 is neglected and equation 5 is substituted by the following
equation
-ac,S

at

- K - aciM
at

the relation

is obtained, representing in principle the mathematical definition of linear ideal
chromatography. The solution of equation 12 leads to the fundamental retention
equation 9. According to the theorems about the properties of partial differentiations, and with respect to equation 12, it may be written

and under the assumption that ciM is invariant (which is one of the premises of ideal
linear chromatography) it holds

d t = [ ( l + k ) / u ] dz,['dt=-L

l+k

L

dzand

U

tR= L(l

+ k ) / u , where again k = K+s/+M

I . 6 Simplified description of linear non-ideal chromatography
I.6.I Retention equations
An exact solution of a completely general model of non-ideal linear chromatography
has not yet been found. Therefore, approximate methods [15,16]which would make


12

it possible to characterize this model on the basis of analysis of individual components of the mechanism of the chromatographic process were sought; Such an
approach leads very simply to the basic equation of chromatographic retention and
provides for the description of the individual spreading factors in terms of the
physical features of the system. When limited only to the aspects of chromatographic
retention this approach corresponds in general to LeRosen's concept of chromatography [17].
The migration rate of the center of the elution zone with respect to the rate of the
mobile phase is determined by the mean probability of the Occurrence of the solute
molecules in the mobile phase, hence


where t l M / ( f l M + t l s ) is the mean fraction of the total time spent by the solute
molecules in the chromatographic bed (column), for which the solute molecules
occur in the mobile phase, miM/(mIM + m,s) is the mean fraction of the total mass
of the solute component within the chromatographic zone, which is present in the
mobile-phase part of the zone, u , is the mean forward velocity of the center of the
elution zone and R is the so-called retardation factor (with certain reservations [18]
identical with R , used to express retention in systems of planar chromatography).
As t , , + t , , = f , , t , s / t , M + mIs/mlM = k and u , = L / t , , the relation t , = L ( l + k ) / u
is immediately obtained. For the ratio L/u it holds L/u = t , , where t , is the
so-called dead retention time (retention time of a non-sorbed compound).
Equation 9 can thus be written in the form

+

t , = tM(l k )

(15)

By multiplying this equation by the volumetric flow rate of the mobile phase the
relation
VR= VM(1 + k )

(16)

is obtained, where V , is the retention volume of the solute compb'und and V , is the
dead retention volume, i.e., the retention volume of a non-sorbed compound. As
k = K ~ # J ~ / C=#KJA, s / A , , and in a uniform bed (packing of the column) A J A , =
VJV,, equation 16 may be rewritten as
VR = V ,


+ KV,

(17)

where Vs is the volume of the sorbent in the column, and V , is generally identical
with the geometrical void volume of the column. For the quantity R it apparently
holds