New comprehensive biochemistry vol 08 separation methods

SEPARATION METHODS New Comprehensive Biochemistry Volume 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 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 Separation (Technology) I Deyl ZdenEk II Series QD415.N48 VOI 574.19’2s (574.19’2851 84-1502 [QD63.S4] ISBN 0-444-80527-3 Printed in the Netherlands V Contents Chapter Principles and theory of chromatography, by J Novak 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 2 22 25 27 27 Chapter 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 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 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 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 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 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 321 322 322 324 326 331 334 334 337 340 341 341 343 345 347 512 per unit of length are obtained at low absolute values of the voltage across the channel is an experimental advantage of EFFF The heat, generated due to high voltage values, is an interfering element that impairs separation characteristics of direct electrophoretic methods 7.4.4 Flow FFF Flow field-flow fractionation (FFFF) has been until now the most universally used subtechnique of FFF With this subtechnique, the flow of the solvent, perpendicular to the flow of the basic medium in the channel, is an external field The earliest works belonging to this class of FFFF were published by Lee and co-workers [51,52], and were called one-phase chromatography [51] or ultrafiltration-induced polarization chromatography [52] Their experimental arrangement consisted of a circular tube, 0.02 cm in diameter, made of semi-permeable material permitting penetration of the solvent through the walls, but preventing the penetration of a high-molecular mass solute They elaborated a basic theoretical model of the separation in this arrangement and demonstrated it in practice for the separation of blue-dextran and human plasma [51], bovine serum albumin and some polydextrans ~21 Giddings and co-workers [53,54] designed the FFFF channel in a classical manner, i.e., using two planparallel semi-permeable membranes, and a thin (0.38 mm) spacer into which the shape of the channel was cut They developed theoretical bases of FFFF and fractionated successfully a series of monodisperse spherical PS latex and a number of proteins They reached an excellent agreement between the theoretical assumptions of the retention and the experimental results As with other FFF subtechniques, the agreement between the theoretical and the experimental characteristics of dispersion was poorer The problem further requires a more extensive study In FFFF the perpendicular flow having the velocity U acts on all of the solutes uniformly For this reason the separation in FFFF is determined only by the differences in the values of the diffusion coefficient, D, or the friction coefficient, f The retention parameter, A, is then determined by the relationship [9] A = R*TV0/3rNqV,w2d (24) where V, is the volumetric perpendicular flow (inducing the field), is the viscosity of the medium, V o is the dead volume owing to the channel, and d is the effective Stokes' diameter The effect of relaxation on the retention and resolution in FFFF was studied in further detail in the subsequent work [55] A substantial improvement in the resolution of the fractionation of f2 virus was proved as long as the stop-flow technique was being applied after the injection into the channel within the relaxation time sufficient for the establishment of the quasi-equilibrium FFFF can be applied as a dialysation or ultrafiltration cell [56] to a continuous 513 separation For instance, two components, of which one is capable of permeating through a semi-permeable membrane, whereas the other is not, can be separated effectively by selecting the proper proportions among various experimental parameters, such as geometry and dimensions of the channel, the ratio of the flow along the channel, and across the channel through the pembranes creating the walls The operation of one such unit was demonstrated in practice for the isolation of low molecular mass ethylene blue from bovine serum albumin [56] High venality in the use of FFFF was shown by examples of effective separations of various solutes having particular characteristics Various viruses [57] and a number of proteins [58] were separated, purified and characterized from the viewpoint of diffusivity; colloid silica gel samples [59] with particle diameters of 0.01-0.13 pm were fractionated and analyzed in this manner FFFF complements SFFF as far as size distribution analysis is concerned [lo] FFFF of water-soluble polyelectrolytes, sulphonated polystyrene and sodium salt of polyacrylic acid proved the applicability of this subtechnique even to the separation of macromolecules [60] A complication which must be solved is represented by a concentration dependence of the effective dimensions of polyelectrolyte solutions For this reason it may be difficult to interpret the fractograms obtained so that they may result in a distribution curve of molecular masses of the polymer studied 7.4.5 Steric FFF Steric field-flow fractionation (steric FFF) occupies, among other subtechniques of FFF, rather an exceptional position It utilizes the upper limit of the field strength applied, at which rather a modified retention mechanism occurs The particles are compressed permanently closer to the wall as the field strength applied increases At the instant when the mean distance of Brownian motion is less than the particle radius, (I, steric F F F takes place The name steric describes the fact that the mean layer thickness is controlled by steric exclusion of the particles from the layer adjacent to the wall Hence larger particles migrate into the stream lines of higher velocities of the solvent than smaller particles and are, consequently, transferred more rapidly To illustrate the mechanism of the separation by steric FFF, its principle is shown schematically in Fig 7.8 Giddings [61] treated theoretical aspects of steric FFF and its comparison with the mechanism of hydrodynamic chromatography He derived the limit relationship for A, + for the value of R,which is the real condition with respect to the character of steric FFF mentioned above R = 6A,(l - A,) (25) where A, = a / w Theoretically it is possible that any effective field - electrical, sedimentation, etc - may be applied to the steric FFF mode However, the gravitational field represents the most practical means of the utilization of the principle of steric FFF 514 FLOW CHANNEL Particle + \ Small particle Larqe particle Fig 7.8 Principle of steric FFF Reprinted from Ref 62, by courtesy of Marcel Dekker, Inc for fractionations of 1-100 pm particles Experimental evidence for the applicability of steric FFF was presented by Giddings and Myers [62], who carried out the fractionation of glass beads of 10-32 pm in diameter The column was composed of a 0.127 mm thick spacer clamped between two glass plates The actual channel (10 X 860 mm) was cut into this spacer Various types of the chromatographic spherical packing were fractionated and characterized from the viewpoint of dimensions in the subsequent work [63] In this case, a dependence of the retention ratio, R, on the flow-rate, which was not predicted by the theory, was observed By inclining the transversal axis of the channel and by injecting the sample into the upper part of the channel, particles under separation were carried and slid towards the lower part of the channel where collection ‘pockets’ were placed along the channel [64] The particles that were carried along the channel slid at the same time to lower parts of the channel and were trapped in the ‘pockets’ Smaller, slower particles were trapped a longer distance from the injection port, larger particles, moving more quickly were carried and trapped nearer to the injection port Continuous fractionation of particles could be obtained in this manner by selecting the channel design properly Caldwell and co-workers [65] explained the dependence of R on the flow-rate (631 by the existence of lift forces Steric FFF represents a further principal progress in the methodology of FFF, and permits a simultaneous extension of applications into the range of large-diameter particles (hundreds of microns) Its applications to fractionation of cells, microorganisms, etc., in biochemistry may be expected 7.4.6 Magnetic FFF Magnetic field-flow fractionation (MFFF) has been the youngest subtechnique of FFF So far the only work [66] dealing with MFFF defined elementary theoretical principles of the separation, and demonstrated in practice retentions of bovine serum albumin in the presence of nickel(I1) ions in a magnetic field of 400 G A coiled Teflon capillary with an inside diameter of 0.15 cm and length of 304 cm was used as a channel In the absence of nickel(I1) ions no retention was observed 515 For the value of X of spherical particles the relationship was derived X = ( r / w ) (k T / p , A H ) * (26) where r is particle radius, A H is the gradient of the magnetic field strength, and pp is the magnetic permeability Although the results obtained with the aid of MFFF are of preliminary significance only, the assumption formulated in the work cited above that this subtechnique of FFF is potentially a very promising method for the analysis, particularly of objects of biological character, is justified 7.4.7 Concentration FFF So far we have been treating FFF subtechniques making use of physical forces of a certain type to induce migration of molecules or particles of the solute Concentration field-flow fractionation (CFFF) has been the only subtechnique of FFF that makes use of a concentration gradient in order to induce effective chemical forces or chemical field [67] for the separation With this subtechnique it is a concentration gradient of a mixed solvent across the channel that is the effective field inducing a gradient of chemical potential with respect to the solute The chemical potential gradient is dp"/dx It follows from the theory [67] that the value of X is then - where ApO, = (dp"/dx) w is the total increment of the chemical potential across the channel If the ratio of the concentrations near both walls is a' = co/c,, then it holds for the retention ratio, R , R a'+1 = 6/ln a'[ a' - -1 In a' It was found by analyzing Eqn 28 that, for an effective fractionation, a' must be at least 10-100 Moreover, additional conditions necessary, with respect to the concentration gradient in the channel and to the total flux of the solute or the total concentration difference between the reservoirs of the mixture, were derived theoretically The design of the channel for CFFF again consists of two semi-permeable membranes forming the walls of the channel The reservoirs of the mixed solvent at various concentrations of active components are adjoined to these walls from the opposite sides The solubility of proteins in alcoholic solutions of various salts was studied On the basis of this study the conditions for the concentration gradient across the channel that are required for CFFF could be determined In addition, permeabilities of various membranes were studied in order to define the conditions of CFFF more 516 precisely The above study led to the finding that, for a successful utilization of CFFF, the design of the FFF channel must be modified substantially in order that all of the assumptions that are discussed in the work and that affect the success of the separation might be satisfied CFFF has been the most difficult subtechnique to realize,but there exists a prospect that, owing to its unique retention mechanism, the effort required for its practical realization and application will be made 7.5 Prospects of FFF In the preceding sections a survey of the results obtained in the development of the FFF methodology in the course of the fiteen years since its establishment is presented Except in rare instances, there has been no comparison of FFF subtechniques with other separation methods capable of solving similar analytical problems The methods that are comparable, as to the range of the applications to the solution of actual separation problems, are in most instances older Consequently, their methodology has been experimentally more developed and elaborated in detail elsewhere In spite of this it is possible to state in general that a number of efficient separations have already been obtained with the aid of FFF, surpassing other separation methods in many parameters Hence FFF can be considered to be a relatively young separation method, the improvement, utilization and expansion of which is to be expected Several recent works have suggested both theoretically and practically further possibilities that can be provided by FFF An increase in the retention and the capacity of the FFF channel, and an increase in the selectivity, can be obtained by modifying the surface of the channel wall on which the solute is accumulated with the aid of transversal barriers as shown by Giddings et al [68] These barriers form spaces in which the solvent does not move, and where the solute can permeate both in and out by diffusion only Consequently, the fractionation characteristics mentioned above are improved The channels established transversally could be used to trap even the second phase, and to combine thus the action of field strength and the partition between the phases Preliminary results were obtained in experiments with the fractionation of PS standards by the TFFF method using the channel with transversal slits [68] The results proved, in principle, the applicability of this system Subramanian [69]published an interesting work, describing the separation method, making use of a perpendicular field across a part of the channel that establishes concentration distribution across the channel without the flow, and later on, separation with the aid of the flow without the field action By its character, this method can be classified as a category of FFF However, it differs from FFF in some basic aspects The field acts in a short part of the channel only, and the fluid does not move at that point As the fluid moves, the separation itself is performed owing to hydrodynamic phenomena only; the field being no longer in action By selecting properly the experimental conditions, i.e., intensity and the time of the field 517 action, channel length and the solvent flow-rate, a high efficiency of fractionations can be reached within a relatively short time, as shown by a theoretical analysis of the problem [69] Except TFFF, all of the other subtechniques of FFF involved the establishment of a parabolic velocity profile If the profile is not parabolic and is asymmetric with regard to the longitudinal central axis of the channel, and can be described by a general function of a polynomial type, both theoretical retention and zone spreading will correspond to this distribution of velocities across the channel Theoretical analysis of this general problem was performed by Martin and Giddings [70] Retention could be controlled by intentional variations in the shape of the velocity profile inside the channel JanEa and Giddings [71] showed a prospective possibility of utilizing non-Newtonian behaviour of some liquids They used the flexible three-parameter equation of Ellis, describing non-Newtonian phenomena, to perform a theoretical analysis and derived the dependence of R on X for different conditions of the non-Newtonian flow This phenomenon could be utilized actively to increase the selectivity of various FFF separations, particularly with the solutes, retained more strongly, i.e., with low values of A The utilization of variations in the shape of the velocity profile in a single separation run was also suggested [71], i.e., programming of the values that are decisive for the range of nowNewtonian behaviour of the liquid applied An extension of FFF to the separation of nonspherical particles and the influences of the wall effect have been studied both theoretically and experimentally by Gajdos and Brenner [721 High-resolution polymer separations have been achieved with a TFFF channel of a new construction [73] Retention and zone spreading equations in SFFF have been verified [74] Recent studies of electroretention of proteins [75,76] have resulted in more detailed understanding of previously observed retention anomalies in EFFF Several papers have demonstrated many possibilities of application of the FFF subtechniques in various fields of chemistry, biology and technology [77-791 The papers published so far have illustrated well the fact that FFF methodology has a wide range of possibilities that have not been used as yet In contrast to the first decade of its existence a greater number of research laboratories have started to investigate this methodology and its practical applications [80-81] It can therefore be expected that the elementary knowledge of the theoretical, experimental and application fields of FFF will be extended and elaborated in the years to come Acknowledgements I am indebted to Dr J Jan& for stimulating discussions 518 References Giddings, J.C (1966) Sep Sci 1, 123 Giddings, J.C 157th National Meeting, American Chemical Society, Minneapolis, April 13-18, 1969, Anal 003, ACS, Washington, 1969 Giddings, J.C., Myers, M.N., Lin, G.C and Martin, M (1977) J Chromatogr 142, 23 Giddings, J.C., Fisher, S.R and Myers, M.N (1978) Am Lab 10, 15 Giddings, J.C Proc 6th Discuss Conf Macromol., IUPAC, Prague, July 1978 Giddings, J.C (1979) Pure Appl Chem 51, 1459 Grushka, E., Caldwell, K.D., Myers, M.N and Giddings, J.C (1974) In Separation and Purification Methods, Vol 2, p 127 (Perry, E.S., Van Oss, C.J and Grushka, E., Eds.) Marcel Dekker, New York Giddings, J.C (1976) J Chromatogr 125, Giddings, J.C., Myers, M.N., Yang, F.J.F and Smith, L.K (1976) In Colloid and Interface Science (Kerker M., Ed.) Vol 4, Academic Press, New York 10 Giddings, J.C., Myers, M.N and Moellmer, J.F (1978) J Chromatogr 149, 501 11 Giddings, J.C., Myers, M.N., Caldwell, K.D and Fisher, S.R (1980) In Methods of Biochemical Analysis p 79, (Click, D Ed.) 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46,1917 41 Yang, F.J.F Myers, M.N and Giddings, J.C (1974) Anal Chem 46, 1924 42 Giddings, J.C., Caldwell, K.D., Mwllmer J.F., Dickinson, T.H., Myers, M.N and Martin, M (1979) Anal Chem 51 30 43 Yang, F.J., Myers, M.N and Giddings, J.C (1977) J Colloid Interface Sci 60, 574 44 Giddings, J.C., Yang, F.J.F and Myers, M.N (1975) Sep Sci 10, 133 519 45 Yau, W.W and Kirkland, J.J., 13th Int Symp on Chromatography, Cannes, June 30-July 4, 1980, p 1, P-4 46 Kirkland, J.J., Yau, W.W., Doerner, W.A and Grant, J.W (1980) Anal Chem 52, 1944 47 Caldwell, K.D., Kesner, L.F., Myers, M.N and Giddings, J.C (1972) Science 176, 296 48 Kesner, L.F., Caldwell K.D., Myers, M.N and Giddings, J.C (1976) Anal Chem 48, 1834 49 Subramanian, R.S., Jayaraj, K and Krishnamurthy, S (1978) Sep Sci Technol 13 273 50 Giddings, J.C., Lin, G.Ch and Myers, M.N (1976) Sep Sci., 11, 553 51 Lee, H.L., Reis, J.F.G., Dohner, J and Lightfoot, E.N (1974) AIChE J 20, 776 52 Lee, H.L and Lightfoot, E.N (1976) Sep Sci 11,417 53 Giddings J.C., Yang, F.J and Myers, M.N (1976) Science 193, 1244 54 Giddings, J.C., Yang, F.J and Myers, M.N (1976) Anal Chem 48, 1126 55 Yang, F.J., Myers, M.N and Giddings, J.C (1977) Anal Chem 49, 659 56 Giddings, J.C., Yang, F.J and Myers, M.N (1977) Sep Sci 12, 499 57 Giddings J.C., Yang, F.J and Myers, M.N (1977) J Virol 21 131 58 Giddings, J.C., Yang, F.J and Myers, M.N (1977) Anal Biochem 81, 395 59 Giddings, J.C., Lin G.C and Myers, M.N (1978) J Colloid Interface Sci 65, 67 60 Giddings, J.C., Lin, G.C and Myers, M.N (1978) J Liq Chromatogr 1, 61 Giddings, J.C (1978) Sep Sci Technol 13, 241 62 Giddings, J.C and Myers, M.N (1978) Sep Sci Technol 13, 637 63 Giddings, J.C., Myers, M.N., Caldwell, K.D and Pav, J.W (1979) J Chromatogr 185, 261 64 Myers, M.N and Giddings, J.C (1979) Powder Technol 23, 15 65 Caldwell K.D., Nguyen, T.T., Myers, M.N and Giddings, J.C (1979) Sep Sci Technol 14, 935 66 Vickrey, T.M and Garcia-Ramirez, J.A (1980) Sep Sci Technol 15, 1297 67 Giddings, J.C., Yang, F.J and Myers, M.N (1977) Sep Sci 12, 381 68 Giddings, J.C., Smith, L.K and Myers, M.N (1978) Sep Sci Technol 13, 367 69 Subramanian R.S (1978) J Colloid Interface Sci 63, 49 70 Martin, M and Giddings, J.C (1981) J Phys Chem 85, 727 71 JanEa, J and Giddings J.C (1981) Sep Sci Technol 16, 805 72 Gajdos, L.J and Brenner, H.(1978) Sep Sci Technol 13, 215 73 Giddings, J.C., Martin, M and Myers, M.N (1981) J Polym Sci Polym Phys Ed 19, 815 74 Karaiskakis, G., Myers, M.N., Caldwell, K.D and Giddings, J.C (1981) Anal Chem 53, 1314 75 Chiang, AS., Kmiotek, E.H., Langan, S.M., Noble, P.T., Reis, J.F.G and Lightfoot, E.N (1979) Sep Sci Technol 14, 453 76 Shah, A.B., Reis, J.F.G Lightfoot, E.N and Moore, R.E (1979) Sep Sci Technol 14, 475 77 Myers, M.N Graff, K.A and Giddings, J.C (1980) Nuclear Technol 51, 147 78 Giddings, J.C., Graff, K.A., Myers, M.N and Caldwell, K.D (1980) Sep Sci Technol 15, 615 79 Caldwell, K.D., Nguyen, T.T., Giddings, J.C and Mazzone, H.M (1980) J Virol Methods 1, 241 80 Inagaki, H and Tanaka, T (1980) Anal Chem 52, 201 81 Martin, M and Reynaud, R (1980) Anal Chem 52, 2293 This Page Intentionally Left Blank 52 Subject index Accelerated swelling 230 Acetyltrimethylammonium bromide (CTMA) 184 Acidic metabolites 121 Acrylamide-agarose gels 445 Adsorption 151, 168 397 Affinant, solid support bonding 326 Affinity 153 chromatography, applications 348-355 automated 345 classical 341 high performance 343 electrophoresis 464 ligands, choice 324 criteria 324 group specific 325 Agar gel electrophoresis, preparative 477 Agarose gel(s) 443 Alumina for TLC 367 Amberlyst 213 Amino acids 129 Ampholyte(s) 32, 455 displacement 219, 225, 229 Amphoteric buffers 208, 225 Amphoteric ion exchangers 208, 209 Anaerobic column chromatography 224 Analytical grade resins 213 Ancillary techniques in G C 80 Anexes 209 Argentation chromatography 227 Artifacts in G C 105 Backings for TLC 369 Balanced slurry method 286 Bidimensional columns 294 Bifunctional reagents in affinity chromatography 340 Bile acids 115 Bioaffinity see also Affinity elution 228 sorbents, requirements 322 specificity 325 Biogenic amines 125 Bleeding in G C 46, 49 Blocking of unreacted groups in affinity chromatography 340 Bonded phase(s) in LC 152 174 Bonded phase materials 171 Boronic acids as derivatization agents 94, 98 p-Bromophenacyl bromide 97 Buffered partition systems in PC 397 [err-Butyldimethylsilyl ethers (TBDMS) 92 fen-Butylpentafluorophenylmethylchlorosilane 93 Capacitance detectors 288 Capacity in affinity chromatography 328 Capillary G C 57, 68 Capsule autosampling in G C 55 Carbodiimide-promoted coupling 339 Carbohydrates 124 Carrier gas, choice 47 purity 48 Catexes 209 Cellulose acetate 422 Cellulose for TLC 367 Cellulose ion exchangers 213 Centrifugally accelerated electrophoresis 421 Charring reagents 380 Chelating ion exchangers 209 Chiral derivatizing agents 103 Chlorodimethoxymethylsilane 93 2-Chloroethoxyamine 100 N-Chloromethyl succinimide 97 Chlorotrimethylsilane 90 Christiansen effect-based refractometers 288 Chromatofocusing 206-209, 225, 229 Chromatographic grade resins 213 Chromatographic systems, A choice in LC 187 classification 2, Classification of phases for biological purposes in G C 63 Coating in situ (LCC) 171 Column deactivation techniques 69 electrophoresis 476 for GPC 284 for HPLC 156 selectivity in G C 64 switching 294 Complex-forming buffers 223 Composite gels 445 Concentration field flow fractionation 515 Conductivity 39 detectors 288 522 Constrictive effects 38 Continuous flow-through electrophoresis 487 Continuous gel chromatography 292 Copper complexes 224 Counterions 180, 209,223 Coupling capacity 338 Coupling procedures in affinity chromatography 337 Crossed electrophoresis 421 Crossed immunoelectrophoresis 448 Crossed line electrophoresis 453 Cyanogen bromide activated supports 337 Cyclodextrin complexation 224 Deaeration of aerogels 230 DEAHP - cellulose 215 DEAHP-starch 215 Decantation 230 Decolorizing ion exchangers 209 Decrease of mobile phase flow 273 Deflexion refractometers 288 Degasing 282 Demands upon an electrophoretic system 33 Densitometry in situ 382 Derivatization, for TLC 373 methods in GC 89 reactions 185, 186 Detection, at two different wavelengths 236 by autoradiography (in electrophoresis) 473 by dipping 378 by fluorescence in electrophoresis 468 by fluorography in electrophoresis 473 by radioactivity counting in electrophoresis 473 by spraying 378 by staining (in electrophoresis) 378 469, 471 by UV (in electrophoresis) 468 enhancement 164 in GC 92 in GPC 287 in PC 403 methods, destructive 234 non-destructive 234 reagents used in TLC 379 Detector@) electrochemical for LCC 163 electron capture 76 fluorescence, for LCC 163 for GC properties 74 universal 74 for LCC, general 162 survey of properties 165 photoionization 77 radioactivity 164 refractive index 164 selective 75 thermionic 75 ultraviolet, for LCC 162 Determination, of enzymes 237 of the molar mass 305 of the size of macromolecules 305 Development, methods in PC 398 of the chromatogram 1,3-Dichlorotetrafluoroacetone 102 Differential chromatography 292 Differential refractometer 288 Diffusion coefficient in gases 47 Diffusion velocity 37 Dimethylthiophosphinic chloride 94 Dipolar ion exchangers 209 Direct radioactivity counting 475 Disc electrophoresis 32, 428 Displacement chromatography , 21 Distribution coefficient 18, 272 Dithiocarbamates 100 Donnan effects 274 Double column GC system 51 Drying of paper chromatograms 402 Dynamically coated phases in HPLC 183 ECTHAM-cellulose 215 Eddy diffusion 211 Effluent volume measurement 289 Electrical field flow fractionation 510 Electric double layer 33 Electrochemical detectors 288 Electromigration methods, classification 30,31 Electroosmotic flow 33, 38 Eluent/solvent mixture choice 171 Eluotropic, series 169, 373 strength of solvents 169 Elution, chromatography 4, 211 conditions (in affinity chromatography) 331 of radioactive material 475 Enantiomers, derivatization for separation 103 labelling 89 Enzymic reactions on TLC plates 380 Epoxide containing supports (in affinity chromatography) 339 Equilibration of ion exchangers 230 Ethyldimethylsilylimidazole 92 Falling needle injector 56 Fatty acids 118 523 Field flow fractionation, optimization 502 principle 498 retention 500 zone spreading 501 theory 500 Filters in GPC 282 Flame ionization detector 73 Flame photometric detectors 75 Flow, in field flow fractionation 512 programming 49 rate, in GC 48 in ion exchange chromatography 211 Fluorescamine 235 Fluorimetric detection 234, 288 Focusing methods 32 Fraction collectors 289 Fractions, evaluation 234 Frontal analysis 211 Frontal chromatography Fused rocket immunoelectrophoresis 451 Fused salts electrophoresis 425 Fused silica columns 69 GC/FTIR coupling 81 GC, history 42 GC/HPLC combination 107 GC/MS combination 48, 81 GC/MS, computerization 82 GC, operating conditions 47 requirements upon design 46 specific requirements in biohemistry 47 Gel(s), disruption 475 for preparative electrophoresis 479 general demands 294 permeation chromatography, applications 304 calibration 275 data processing 277 mobile phase transport 282 parasitic effects 274 peak capacity 276 preparative 290 real 274 resolution 275 side effects 274 techniques 280 theory 272 pores 272 survey 296-299 Gibbs function 22 Glass-to-metal seals 47 Gradient(s) elution 233 gel electrophoresis 435 in electrophoresis 32 Heptafluorobutyric acid anhydride 100 Heteroionic ion exchangers 209 Hexamethyldisilazane 90 High performance silica gel (for TLC) 370 High speed separations in GPC 290 High voltage paper electrophoresis 416 Homoionic ion exchangers 209 Horizontal development in PC 401 HPLC, packings 215 sample filters 105 Hydrodynamic flow 37 Hydrogen as carrier gas 48 Identification in TLC 380 Immobilized stationary phases in GC 70 immunoaffinity chromatography 345 applications 347 lmmunoelectrophoresis, instrumentation 446 Impregnated layers 368 Infrared spectrophotometer 288 Instrumentation for LCC 155 Interferometers 288 Ion exchange 152 chromatography, applications 238 automated 249 classical 205 modern trends 206 mechanism, dynamic 181 papers in electrophoresis 422 TLC 368 Ion exchangers, characterization 212 choice 229 classification 208 for HPLC survey 216 functional groups 214 preliminary operations 230 regeneration 237 storage 237 Ion exclusion 226 Ion-pair chromatography 171, 184 Ion retardation 227 Ion sieving 227 Ionic strength, effect in affinity chromatography 333 in ion exchange 221 Isocratic elution 233 Isodalt system 439 Isoelectric focusing 32 continuous flow 483 density gradient 457 free solution 458 in polyacrylamide gel 456 preparative 481 flat bed 457,483 transient state 459 two dimensional 458 lsoelectric points of proteins 247 Isoeluotropic solvents 182 Isoporous ion exchangers 209 Isotachophoresis 31, 460 buffer systems 463, 465 detection 462 instrumentation 461 preparative 484 buffers 485 Kinetics of ion exchange 210 KMT 215 Kovac's index 79 Laurell's electrophoresis 448 LCC, classic systems 155 LC/MS coupling 164 Leading electrolyte 31 Lectins, in affinity electrophoresis 466 Ligand chromatography 227 Light scattering detectors 288 Linear, non-ideal chromatography 11 Lipids 116 Liquid crytalline phases 65 Liquid-liquid partition 171 Liquid-solid partition 168 Loop injection valves 157 Loss of stationary phase 171 Low pressure ion exchange chromatography, materials 211 Macroreticular resins 213, 218 Magnetic field effects in electrophoresis 421 Magnetic field flow fractionation 514 Mark-Houwink equation 306 Medium pressure chromatography 206 packings 215 Metabolic profiles in GC 83 N-Methyl-bis-trifluoroacetamide94 Methylchloroformate 101 N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) 91 Microporous ion exchangers 209 Migration velocity 33 Mixed bed resins 209 Mixed phase approach 67 Mobile phase(s) for GPC 301 Mobile phase systems for ion exchange chromatography 220 Mobility 34 Models of linear chromatography Modifiers 169 in RPC 178, 179 Molecular weight estimation by electrophoresis 436 440, 441 Molecular weight standards for electrophoresis 438 Moving boundary electrophoresis 426 MS as detector 74 Multidimensional chromatography 294 Multiple column systems in GC 49 Multiple development in PC 402 Multi-point bonding 328 Nernst film 210 Ninhydrin colorimetry 234 Non-aqueous buffers in electrophoresis 424 Non-aqueous systems in PC 397 Non-specific adsorption in affinity chromatography 323, 328 Normal (straight) phase chromatography 171,227 Number of theoretical plates 16, 62 Oleophilic ion exchangers 209 On-column injectors for capillary GC 60 One-to-one bonding 329 Operation variables in GPC 286 Optimal column dimensions in HPLC 157 Optimization of LCC 158 Organic solvents in ion exchange 220, 222 Overlayering 232 Packing, of columns for GPC 285 of ion exchangers 231 Paper chromatography, applications 405-410 history 392 Paper electrophoresis 415 apparatus 416 Papers for chromatography 393 Partition 151 chromatography on ion exchangers 227 Pellicular ion exchangers 209, 217 Pentafluoropropionic acid anhydride 100 Peptides 129 Perfluoroacylimidazole 100 Permethylation 102 Pharmalyte 225 Phase systems, in GC 62 in LCC 167 Phenyldiazomethane 96 Phosphomolybdic acid 380 525 Photometric detection 287 o-Phthaialdehyde detection 234 PI-Sepharose 225 Pneumatic column switching 52 Polyacrylamide gel(s), drying 489 Polyacrylamide gel electrophoresis 428 preparative 478 Polyamide for TLC 368 Polybuffer ion exchangers 219 225 Polymer reference materials, natural (calibration) 302 synthetic (calibration) 303 Pore geometry 274 Porous glass 218 Porous silica 218 Post column derivatization in LCC 164 Potential gradient distribution 39 Precoated plates, 366, 369 Precolumn derivatization in LCC 164 Precolumns, in GC 57, 59 in HPLC 157 Precolumn sampling techniques 60 Preconcentration in GC 104 Pre-cycling 230 Preimpregnation in TLC 378 Preparative electrophoresis 476 instrumentation 480 Profiling in GC 83 Propyldimethylsilylimidazole 92 Prostaglandins 128 Protolytic equilibria 35 Pulse elution 234 Pulse packing of ion exchangers 232 Pumps for GPC 282 Purification methods before GC 104 Quantitation, in LCC 187 in PC 404 Radial development in PC 401 Radioactivity counting after combustion 475 Reactor grade resins 213 Reagent removal in GC 52 Recycling 291 Redox ion exchangers 209 Reflection refractometers 288 Relations between structure and retention in GC 80 Relaxation in field flow fractionation 502 Reproducibility of results 176 Resolution 24 of optical isomers 224 Response(s), delay in detectors 287 distortion in detectors 287 enhancement 73 Restriction of diffusion 273 Retardation 38 Retention, equations 11 in GC 79 Reversed phase, chromatography 171, 227 systems in PC 397 TLC 370 R F, definition 364 Rocket electrophoresis 451 Rod gels 431 slicing 442 R,, definition 381 Sample application, in ion exchange chromatography 232 in PC 395 Sample, destructive detectors in GC 73 loop 233 for GPC 284 preparation for GPC 283 for PC 395 for TLC 371 pre-treatment in HPLC 186 Sampling devices in LCC 157 Sampling in GC 53 Sandwich chambers for TLC 376 Saturation column in HPLC 157 Scanning of electrophoretograms 473 SDS-polyacrylamide gel 436 Sedimentation field flow fractionation 508 Selective ion exchangers 209 Selectivity, influencing by bonded phases 176 in ion pair chromatography 173 Sephadex thin layers 368 Silica gel for TLC 366 Silicone based chiral phases 88 Silver stain in electrophoresis 444,472 Size exclusion (seealso gel permeation) 152 Slab gel system 433 Solid sampling in GC 55 automation 55 Solute property detectors 287 Solvent, delivery in LCC 157 evaporation technique 171 removal in GC 54, 57, 60 systems for TLC 373 in PC 396 Sorbents for TLC 366 Sorption, conditions in affinity chromatography 331 equilibrium 18 Spark chamber 474 Special additives in ion exchange chromatography 223, 224 Specific ion exchangers 209 Spheron 218 Splitless methods in GC 58 Split-stream technique 236 Splitting injectors 58 Spreading of the chromatographic zone 13 Starch gel 427 Stationary phases for GC, survey 66 Stepwise elution 233 Stereoselectivity in GC 87 Steric field flow fractionation 513 Steroid hormones 108 Sterols 114 Stop-and-go gel chromatography 293 Straight systems in PC 397 Supercritical fluid chromatography 292 Supports, for affinity chromatography, survey 334 for bonded phase chromatography 175 Surface silylation 68 Surface wettability 69 Syringe injection of sample 54 ascending development 374 chambers 374, 375 descending development 374 development modes 374 history 364 layer preparation 366, 369 multiple development 374 overrun development 375 programmed multiple development 375 radial 374 two dimensional 374 Tortuosity 38 Transport of compounds during electrophoresis 33 Trideuteroborane 102 Trifluoroacetic acid anhydride 100 Trifluoroacetimidazole 93 N-Trimethylsilylacetamide(MSA) 91 Trimethylsilyldiazomethane96 Trimethylsilylimidale (TSIM) 91 TRI-SIL 90 Turbidimeters 288 Two dimensional development in PC 402 Two dimensional electrophoresis 418, 439, 443 444 Temperature, gradient 39 in the mobile phase 222 programming 48 reproducibility 49 Terminating electrolyte 31 Ternary solvent mixtures 171 THAM-cellulose 215 Theoretical plate(s) 16 62 in ion exchange chromatography 210 Theories of chromatography, survey 26 Thermal diffusion coefficient 39 Thermal field flow fractionation 506 Thermal flow 38 Thermostats 289 Thin layer, electrophoresis 425 gel chromatography 293 Three-way valve 233 TLC, applications 384-388 Ultramicroelectrophoresis 423 Underlayering 232 Unusual metabolites, searching 85 Urinary steroids 111 UV detection in TLC 378 Vacancy gel chromatography 292 Vapor phase osmometry 288 Velocity gradient 36 Viscosity detectors 288 Volatile buffers 223 Water as detecting reagent 380 Xerogel 212 Zone electrophoresis 415 .. .SEPARATION METHODS New Comprehensive Biochemistry Volume General Editors A NEUBERGER London L.L.M van DEENEN Urrechr ELSEVIER AMSTERDAM * NEW YORK * OXFORD Separation Methods Editor... 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 Separation. .. 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