Journal of Chromatography Library - Volume 3
LIQUID COLUMN CHROMATOGRAPHY A Survey of Modern Techniques and Applicatio...
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Journal of Chromatography Library - Volume 3
LIQUID COLUMN CHROMATOGRAPHY A Survey of Modern Techniques and Applications
JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G.H. Wagman and M.J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G. Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z. Deyl, K. Macek and J. Jan& Volume 4 Detectors in Gas Chromatography by J. SevEik
Journal of Chromatography Library - Volume 3
LIQUID COLUMN CHROMATOGRAPHY A Survey of Modem Techniques and Applications
Editors
Zden&kDeyl Physiological Institute, Czechoslovak Academy ofsciences, Prague
Karel Macek 3rd Medical Deparrmenr, Medical Faculty, Charles University, Prague
Jaroslav Ja nhk Knstitute of Instrumental Analysis, Czechoslovak Academy of Sciences, Brno
ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM - OXFORD - NEWYORK1975
ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 21 1, Amsterdam, The Netherlands AMERICAN ELSEVIER PUBLISHING COMPANY, INC. 52 Vanderbilt Avenue New York, New York 10017
Library of Congress Card Number: 73-89151 ISBN 0-444-41156-9 Copyright 0 1975 by Elsevier Scientific Publishing Company, Amsterdam
AU 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 written permission of the publisher, Elsevier Scientific Publishing Company, Jan van Galenstraat 335, Amsterdam Printed in The Netherlands
Contributors V. Betina, Department of Microbiology and Biochemistry, Faculty of Chemistry, Slovak Polytechnical University, Bratislava (Czechoslovakia) K. Capek, Laboratory of Monosaccharides, Prague Institute of Chemical Technology, Prague (Czechoslovakia) J. ChuriEek, Department of Analytical Chemistry, Technical University, Pardubice (Czechoslovakia) J. toupek, Institute for Macromolecular Chemistry, Prague (Czechoslovakia) J. Davidek, Department of Food Science and Analysis, Prague Institute of Chemical Technology, Prague (Czechoslovakia) Z. Deyl, Physiological Institute, Czechoslovak Academy of Sciences, Prague-KrE (Czechoslovakia) J. DrXata, Department of Biochemistry, Faculty of Pharmacy, Charles University, Hradec KrdovC (Czechoslovakia) J. GaspariZ, Department of Physical Chemistry, Faculty of Pharmacy, Charles University, Hradec Krilovk (Czechoslovakia) I. M. Hais, Department of Biochemistry, Faculty of Pharmacy, Charles University, Hradec KrdovB (Czechoslovakia) J. G. Heathcote, Department of Chemistry and Applied Chemistry, University of Salford, SaIford (Great Britain) S. Heiminek, Institute for Nuclear Research, k e i near Prague (Czechoslovakia) J . Jana'k, Institute of Instrumental Analysis, Czechoslovak Academy of Sciences, Brno (Czechoslovakia) M. Janda, Department of Organic Chemistry, Prague lnstitute of Chemical Technology, Prague (Czechoslovakia) P. Jandera, Department of Analytical Chemistry, Technical Univeisity, Pardubice (Czechoslovakia) M. Juhcova, Physiological Institute, Czechoslovak Academy of Sciences, Prague-KrE (Czechoslovakia) F. Julsi'k, Department of Inorganic Chemistry, Prague Institute of Chemical Technology, Prague (Czechoslovakia) I. Kluh, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) M. KrejEi, Institute of Instrumental Analysis, Czechoslovak Academy of Sciences, Bmo (Czechoslovakia) M. Kubin, Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) K. Macek, Third Medical Department, Medical Faculty, Charles University, Prague (Czechoslovakia) 0. Mike:, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) V
VI
CONTRIBUTORS
0. Motl, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) J. Novik, Institute of Instrumental Analysis, Czechoslovak Academy of Sciences, Brno (Czechoslovakia) Z. Pechan, Department of Biochemistry, University J. E. Purkynt, Brno (Czechoslovakia) J. Pokomy, Department of Food Science and Analysis, Prague Institute of Chemical Technology, Prague (Czechoslovakia) 2. Prochrizka, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) Z. Prusik, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) Z. Sestik, Institute of Plant Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) J. StanBk, Jr., Laboratory of Monosaccharides, Prague Institute of Chemical Technology, Prague (Czechoslovakia) I. Stibor, Department of Organic Chemistry, Prague Institute of Chemical Technology, Prague (Czechoslovakia) J. Turkovi, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) R. Vespalec, Institute of Instrumental Analysis, Czechoslovak Academy of Sciences, Bmo (Czechoslovakia) R. J. Washington, Department of Chemistry and Applied Chemistry, University of Salford, Salford (Great Britain) S. WiEar, Institute of Instrumental Analysis, Czechoslovak Academy of Sciences, Brno (Czechoslovakia) J. Zabranskf, Research Institute of Food Technology, Czechoslovak Agricultural Academy, Prague (Czechoslovakia) S. ZadraZil, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) Z. J. Zmrhal, Research Institute of Plant Production, Prague-Ruzynk (Czechoslovakia)
Contents Foreword . Preface .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XVII XIX
THEORETICAL ASPECTS OF LIQUID CHROMATOGRAPHY
.
Fundamental concepts (J Novik and J . Janik) . . . . lntroduction . . . . . . . . . . . . . . Principle of chromatography . . . . . . . . Chromatographic systems . . . . . . . . . . Chromatographic techniques . . . . . . . . Basic chromatographic quantities . . . . . . .
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Basic processes in chromatography (J . Novik. J . Janik and S . WiEar) . Flow of mobile phase through a packed column . . . . . . Diffusion of solute within the phases . . . . . . . . . Equilibration of solute between the phases . . . . . . .
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Physico-chemical basis of chromatographic retention in liquid-liquid and liquid-solid systems (J.Nov6k) . . . . . . . . . . . . . . . . . . . . . . . . Interaction of solute with the phases . . . . . . . . . . . . . . . . Thermodynamics of sorption equilibrium . . . . . . . . . . . . . .
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General description of the chromatographic process (J Novik. J . Janik and S . WiEar) . Solute mass balance in the chromatographic system . . . . . . . . . Concept of ideal linear chromatography . . . . . . . . . . . . . Concept of the theoretical plate . . . . . . . . . . . . . . . Dynamics of zone spreading . . . . . . . . . . . . . . . Chromatographic resolution . . . . . . . . . . . . . . . .
Gel permeation chromatography (M Kubfn) . . Introduction . . . . . . . . . . Principles of gel permeation chromatography Physical basis of the separation process . .
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3 3 4 5 7 8 11
11 15 17 25 25 31 33 35 40
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45 45 48
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57 57 57 59
. . . . . . . . . . . Fundamentals of ion-exchange chromatography (0. Mike;) Principles and terminology . . . . . . . . . . . . . . . . . . . . Characterization of ion exchangers . . . . . . . . . . . . . . . . . Reactions. affinity and selectivity in ion exchange . . . . . . . . . . . . . lon-exchange equilibria and kinetics . . . . . . . . . . . . . . . . Column operation and ion-exchange chromatography . . . . . . . . . . . . Ion exclusion. ion retardation. the ion-sieve process and partition chromatography on ion exchangers . . . . . . . . . . . . . . . . . . . . . . Ligand-exchange chromatography . . . . . . . . . . . . . . . . . Ion exchange in non-aqueous solutions . . . . . . . . . . . . . . .
83 85 85
Affinity chromatography (J . Turkovi) . Principles of affinity chromatography
89 89
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII
69 69 73 75 77 80
VIII
CONTENTS
Choice of bound affinant . . . . . . General aspects of the affinant-sorbent bond
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
92 94
TECHNIQUES OF LIQUID CHROMATOGRAPHY Instrumentation for liquid chromatography (M . KrejEf. Z . Pechan and Z . Deyl) . . . . . Classical instrumentation for liquid chromatography . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . Columns and accessories . . . . . . . . . . . . . . . . . . Column preparation and introduction of sample . . . . . . . . . . . Techniques of elution . . . . . . . . . . . . . . . . . . . Analysis of effluent . . . . . . . . . . . . . . . . . . . . Preparative and industrial liquid chromatography . . . . . . . . . . . How tolearn the technique . . . . . . . . . . . . . . . . . Techniques of high-efficiency liquid chromatography . . . . . . . . . . . Principal differences between classical and high-efficiency liquid chromatography . . The function of a liquid chromatograph . . . . . . . . . . . . . . Mobile phase reservoirs . . . . . . . . . . . . . . . . . . . . Gradient-forming devices . . . . . . . . . . . . . . . . . . Manipulation . . . . . . . . . . . . . . . . . . . . . . . Pumpingsystems . . . . . . . . . . . . . . . . . . . . . Pressure pulse-damping device . . . . . . . . . . . . . . . . . Sample introduction devices . . . . . . . . . . . . . . . . . Columns . . . . . . . . . . . . . . . . . . . . . . . . Thermostats . . . . . . . . . . . . . . . . . . . . . . . Detectors . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of different detectors . . . . . . . . . . . . . . . . Counter-current chromatography . . . . . . . . . . . . . . . . .
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.
Sorbents (J Janik. J . Coupek. M KrejEi. 0. Mike: and J . Turkovl) . Rational classification . . . . . . . . . . . . . . Sorbents for liquid-solid chromatography . . . . . . . Supports and stationary phases for liquid-liquid chromatography Column packings for gel chromatography . . . . . . . Ion-exchange materials . . . . . . . . . . . . . . Sorbents for affinity chromatography . . . . . . . .
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233 234 248 261 270
Operation of a modern liquid chromatograph (R . Vespalec and M KrejEf) Preparation of the apparatus . . . . . . . . . . . . Sorting of sorbents according to particle size . . . . . . . Determination of the activity of alumina by thin-layer chromatography Column preparation . . . . . . . . . . . . . . . . Sample preparation and application . . . . . . . . . . General comments . . . . . . . . . . . . . . . .
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283 283 285 290 291 295 297
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Mobile phases (0 Mike: and R Vespalec) . . . Mobile phases for liquid-liquid chromatography Mobile phases for liquid-solid chromatography . Mobile phases for ion-exchange chromatography Calculation of gradients . . . . . . .
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101 102 102 103 110 112 115 120 122 123 123 127 128 129 132 133 137 139 143 145 146 162 162
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169 170 174 182 187 202 215
PRACTICE OF LIQUID CHROMATOGRAPHY
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.
IX
CONTENTS Practice of gel chromatography (J . coupek. M . Kubh and Z . Deyl) . . . . . . . Choice of gel packing . . . . . . . . . . . . . . . . . . . . Choice of solvent and operating temperature . . . . . . . . . . . . Apparatus for gel chromatography . . . . . . . . . . . . . . . Special gel chromatographic techniques . . . . . . . . . . . . . . Evaluation of gel permeation chromatographic data . . . . . . . . . . Determination of molecular weights of naturally occurring macromolecular compounds by molecular sieve chromatography . . . . . . . . . . . . . .
. . . Practice of ion-exchange chromatography (0. Mike:) Introduction . . . . . . . . . . . . . . Choice of suitable ion exchangers . . . . . . . . Methods for the fractionation of ion exchangers . . . Decantation and cycling of ion exchangers . . . . . Buffering of ion exchangers . . . . . . . . . Deaeration of ion exchangers and filling of chromatographic Application of samples . . . . . . . . . . . Methods of elution . . . . . . . . . . . . Calculation of flow-rates . . . . . . . . . . Evaluation of fractions . . . . . . . . . . . Regeneration and storage of ion exchangers . . . . . Practice of affinity chromatography (J . Turkovi) . . . Preparation of the solid support with a bound affinant . Sorption conditions . . . . . . . . . . . Conditions for elution . . . . . . . . . . Preservation of solid sorbents with a bound affinant .
. . . . . . . . . . . . . . . . . . . . . . . . columns .
. . 301
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301 303 304 311 312
. . 317
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325 325 325 . 353 . 354 . 355 . 356 . 360 . 363 . 364 . 366 . 366
. . . . . . . . . . . 369 . . . . . . . . . . . 369 . . . . . . . . . . . 370 . . . . . . . . . . . 372 . . . . . . . . . . . 375
Analytical utilization of chromatograms (J . Novik. J . Janik and S . WiEar) Identification . . . . . . . . . . . . . . . . . Quantitation . . . . . . . . . . . . . . . . . .
. . . . . . . 377
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377 386
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Radiochromatographic techniques (I.M. Hais and J . Drxata) . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . Detectors . . . . . . . . . . . . . . . . . . . . . . . . Detection modes . . . . . . . . . . . . . . . . . . . . . .
. . 403
. . .
403 404 408
APPLICATIONS Hydrocarbons (J . ChuriEek) . . . . . . . . . . . Introduction and general techniques . . . . . . . Chromatography on adsorbents . . . . . . . . Chromatography on gels . . . . . . . . . . . Other methods of chromatography of hydrocarbons . .
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Alcohols and polyols (J . ChurPEek) . . . . . . . . . . . Introduction and general techniques . . . . . . . . . . High-speed liquid and gel permeation chromatography of free alcohols Chromatography of alcohols on ion exchangers . . . . . . . Chromatography of derivatives of alcohols and glycols . . . . . Separation of polyols and polymeric diols . . . . . . . .
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417
. . 417 . . 417
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421 423
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437 438
X
CONTENTS
Phenols (J . ChurPEek and J . Eoupek) . Introduction . . . . . . . Gel chromatography . . . . Adsorption chromatography . . Ion-exchange chromatography . .
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Ethers and peroxides (J ChurlEek)
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441 441 441 442 445
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0x0 compounds (J ChurPEek). . . . . . Introduction . . . . . . . . . . Aliphatic and cyclic aldehydes and ketones Quinones . . . . . . . . . . . Applications in lignin chemistry . . . .
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455 455 456 459 461
Carbohydrates (K. capek and J . Stansk. Jr.). . . . . . . . . . . . . . . . 465 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 466 General techniques . . . . . . . . . . . . . . . . . . . . . . 461 Liquid-solid chromatography . . . . . . . . . . . . . . . . . . 467 Liquid-liquid chromatography . . . . . . . . . . . . . . . . . 469 Gel chromatography . . . . . . . . . . . . . . . . . . . . 412 Ion-exchange chromatography . . . . . . . . . . . . . . . . . . 413 Automated detection methods . . . . . . . . . . . . . . . . . 415 Mono.. oligo- and deoxy saccharides . . . . . . . . . . . . . . . . . 483 Chromatography on charcoal-Celite . . . . . . . . . . . . . . . . 483 Chromatography on cellulose . . . . . . . . . . . . . . . . . . 486 Chromatography on ion-exchange resins . . . . . . . . . . . . . . . 481 Chromatography on molecular sieves . . . . . . . . . . . . . . . . 493 Amino sugars . . . . . . . . . . . . . . . . . . . . . . . 496 Free amino sugars . . . . . . . . . . . . . . . . . . . . . 496 Mutual separation of amino sugars and amino acids . . . . . . . . . . . . 499 Derivatives of amino sugars and chromatographic methods used in the synthesis of amino sugars . . . . . . . . . . . . . . . . . . . . . . . . . 500 Sugar derivatives . . . . . . . . . . . . . . . . . . . . . . . 501 Alditols . . . . . . . . . . . . . . . . . . . . . . . . 501 Glycosides . . . . . . . . . . . . . . . . . . . . . . . 504 Ethers and acetals . . . . . . . . . . . . . . . . . . . . . 506 Esters . . . . . . . . . . . . . . . . . . . . . . . . . 501 Sugar acids . . . . . . . . . . . . . . . . . . . . . . . 507 Sugar phosphates . . . . . . . . . . . . . . . . . . . . . 515
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Polysaccharides (K capek and J . Stangk. Jr.) Introduction . . . . . . . . . Ion-exchange chromatography . . . . Gel permeation chromatography . . .
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Polysaccharide-protein complexes(M. JuTicovPandZ. Deyl) . Glycosaminoglycans (mucopolysaccharides) . . . . . Glycoproteins and glycopeptides . . . . . . . .
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523 523 524 525
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529 529 538
Lower carboxylic acids (J Chur9Eek and P Jandera) . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . Generaltechniques . . . . . . . . . . . . . . . . . . . . . . Separation of carboxylic acids on the basis of molecular sorption. using aqueous and nonaqueous organic solvents . . . . . . . . . . . . . . . . . .
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543 543 543
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545
XI
CONTENTS
Ion-exchange chromatography of carboxylic acids in various aqueous acids or buffered solvent systems . . . . . . . . . . . . . . . . . . . . High-speed ion-exchange chromatography of carboxylic acids with anion exchangers of controlled surface porosity . . . . . . . . . . . . . . . . . Other separation techniques for carboxylic acids . . . . . . . . . . . Higher carboxylic acids (J . Pokomf) . . . . Introduction and general remarks . . . . Separation as fatty acid derivatives . . . Chromatography on adsorbents in general use Chromatography on specific adsorbents . . Gel and ion-exchange chromatography . .
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Steroids (2. Prochrizka) . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . General techniques . . . . . . . . . . . . . Introductory and theoretical considerations . . . . Sample preparation and application . . . . . . Liquid-solid chromatography . . . . . . . . Liquid-liquid chromatography . . . . . . Gel chromatography . . . . . . . . . . Ion-exchange chromatography . . . . . . . . Detection . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . Sterols . . . . . . . . . . . . . . . Androgens . . . . . . . . . . . . . . Estrogens . . . . . . . . . . . . . . . . Gestagens (progestins) . . . . . . . . . . Corticosteroids . . . . . . . . . . . . . Bile acids and other steroid acids . . . . . . . Steroidal glycosides . . . . . . . . . . . Steroidal insect hormones . . . . . . . . .
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565 567
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575 575 575 576 577 578
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. . 581 . . . . . 581 . . . . . 581 . . . 585
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588
. . . 593 . 593 . 594 . . 594 . . 595
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. 595 . . . 597 . . . 601 . . . 602
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603 604 604 . . 604 . . 605 . 613 614 . . . . . . . . . . 617 . . . . . . . . . . 618 . . . . . . . . 619
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Amines (Z . Deyl) . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . Aliphatic mono-. di- and polyamines . . . . . . Aromatic mines . . . . . . . . . . . . . Aromatic mines and aliphatic polyamines in mixtures .
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551
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Lipids (J . Pokomy) . . . . . . . . . . . . Introduction and general remarks . . . . . . . Separation of lipids into classes . . . . . . . Separation of glycerol esters and other neutral lipids . Separation of phospholipids and other polar lipids . .
Terpenes (0. Motl) . . . . Introduction . . . . . Hydrocarbons . . . . Ethers. epoxides and furans Esters . . . . . . . Aldehydes and ketones . Lactones . . . . . . Alcohols . . . . . . Acids . . . . . . .
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623 623 624 629 . . . . . . . . . . . . . . . . . 630 . . . . . . . . . . . . . . . . . . . 631 . . . . . . . . . . . . . . . . . . . 632 . . . . . . . . . . . . . . . . . 633 . . . . . . . . . . . . . . . . . 633
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637 637 637 643 . 645
CONTENTS
XI1
Tryptophan metabolites . . . . . . . . . Quaternary ammonium compounds and amino alcohols Biogenicamines . . . . . . . . . . . .
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645 649 650
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657 651 657 659 661
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665 666 668 675 688 692 691 704 105 708
Other non-heterocyclic nitrogen compounds (J . Chudzek) Introduction . . . . . . . . . . . . . . Nitro compounds . . . . . . . . . . . . Amides . . . . . . . . . . . . . . . Guanidine and urea derivatives . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
Amino acids (Z.J. Zmrhal. J.G.Heathcote and R.J . Washington) Analyticalchromatography . . . . . . . . . Ion-exchange chromatography . . . . . . . . Amino acid analyzers . . . . . . . . . . Packings for chromatographic columns . . . . . Preparation of eluents and reagents . . . . . . Chromatographic elution systems . . . . . . . Preparationofsample . . . . . . . . . . Calculation of the elution curve . . . . . . . Preparativechromatography . . . . . . . . .
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Amino acid derivatives (Z Deyl and M . JuficovB) . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . 2. 4.Dinitrophenyl (DNP) amino acid derivatives . . . . . . . 5-Dimethylaminonaphthalene-l-sulphonyl(Dns) aminoacids . . . . Hydantoins and substituted hydantoins . . . . . . . . . . Miscellaneous derivatives . . . . . . . . . . . . . . . .
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Peptides (I Kluh) . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . Methodsfor theseparationof peptides . . . . . . Analysis of the effluent from the chromatographic column Gel permeation chromatography . . . . . . . . Ion-exchange chromatography . . . . . . . . . Affinity chromatography . . . . . . . . . . Partition chromatography . . . . . . . . . .
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113 713 714 726 731 736
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741 742 742 744 749 756 168 110
Proteins (Z . Prusik) . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . General rules for the separation of proteins . . . . . . . . Gel permeation chromatography . . . . . . . . . . . Chromatography on glass with controlled pore size . . . . . . Ion-exchange chromatography . . . . . . . . . . . . Chromatography on hydroxyapatite and on calcium phosphate . Solubility chromatography . . . . . . . . . . . . . Technique of gel permeation chromatography in a detergent gradient Affinity chromatography . . . . . . . . . . . . . Detection of proteins in the effluent . . . . . . . . . .
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713 773 174 778 781
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188 189 198 199
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Enzymes (0. Mike:) . . . . . . . . . . . . . . . . Special requirements for the chromatography of enzymes . . . Techniques and automated analyses . . . . . . . . . Oxidoreductases . . . . . . . . . . . . . . . . .
. . . . . . . 182
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800 807 807 809 813
CONTENTS
XI11
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816 818 823 825 826
Low-molecular-weight constituents of nucleic acids. Nucleosides. nucleotides and their analogues ( S . Zadrdil) . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . General techniques in the separation of low-molecular-weight components of nucleic acids Automated procedures for the analysis of nucleic acid components . . . . . . . Individual types of nucleic acid constituents . . . . . . . . . . . . . .
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.
831 831 832 836 839
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859 859 862 873 878 880
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887 887 888 888 894
Transferases Hydrolases Lyases . . Isomerases Ligases .
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Nucleic acids ( S . ZadraZil) . . . . . . . . . . . . . Introduction and general techniques in nucleic acid separations Deoxyribonucleic acids . . . . . . . . . . . . . Ribonucleic acids . . . . . . . . . . . . . . Polynucleotides and large oligonucleotides . . . . . . Automated procedures andpolynucleotidesequenceanalysis . Alkaloids (K . Macek) . Introduction . . . Preparation of samples Techniques . . . Applications . . .
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. . . . Other heterocyclic compounds (J . Davidek. M . Janda and I . Stibor) Introduction . . . . . . . . . . . . . . . . . . . . Derivatives of ypyrone . . . . . . . . . . . . . . . . . Anthocyans . . . . . . . . . . . . . . . . . . . . Aflatoxins and mycotoxins . . . . . . . . . . . . . . . Other compounds containing heterocyclic oxygen . . . . . . . . . Porphyrins and related compounds . . . . . . . . . . . . . Indoles . . . . . . . . . . . . . . . . . . . . . Pyridine and related compounds . . . . . . . . . . . . . . Polynuclear aza-heterocyclics and complex mixtures of heterocyclic compounds
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895
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896 909 912 915 917 919 920 921
Organic sulphur compounds (J . ChuriEek) Introduction . . . . . . . . Sulphonic acids . . . . . . . Other sulphur compounds . . . . High-speed liquid chromatography .
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Organic phosphorus compounds (J . Zabranski) Application of column chromatography .
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939 939
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927 927 927 932 934
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Boron compounds ( S . Hefmbek) . . . . . General techniques . . . . . . . . Boranes and substituted boranes . . . . Carboranes . . . . . . . . . . Ligand derivatives of boranes and carboranes Metallocarboranes . . . . . . . .
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945 945 947 950 950 95 0
XIV
CONTENTS
.
Vitamins (J Davidek) . . . . . . . . Introduction . . . . . . . . . . Fat-soluble vitamins . . . . . . . . . Vitamin A group . . . . . . . . Calciferols . . . . . . . . . Tocopherols . . . . . . . . . Vitamin K group . . . . . . . . Water-soluble vitamins . . . . . . . Thiamine . . . . . . . . . . Riboflavinandotherflavins . . . . Nicotinic acid and its derivatives . . . Pyridoxinegroup . . . . . . . Biotin . . . . . . . . . . . Pantothenic acid and coenzyme A . Folic acid and other pteridine derivatives Corrinoids . . . . . . . . . L-Ascorbic and L-dehydroascorbic acids
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Antibiotics (V Betina) . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . Penicillins and cephalosporins . . . . . . . . Carbohydrate antibiotics . . . . . . . . . . Macrocyclic antibiotics . . . . . . . . . . . Tetracyclines and related antibiotics . . . . . . Nucleoside antibiotics including polyoxins . . . . Peptides and related antibiotics . . . . . . . Miscellaneous antibiotics . . . . . . . . . .
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979 979 . 980 985 994 . 996 . 999 .1000 1003
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. 1009 . 1009 . 1014
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. . .1033 . . 1033 . . 1033 . . . 1034 . . .1035
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Pesticides (J . Zabranskf and J ChurlEek) . . . . Introduction and general techniques . . . . . Chlorinated pesticides and their metabolites . . . Phosphorus pesticides . . . . . . . . . . Carbamate pesticides and their metabolites . . . Pyrethrins . . . . . . . . . . . . . Synthetic dyes (J . ChuriiEek and J . GaspariE) . Introduction . . . . . . . . . . . General techniques . . . . . . . . Chromatography on adsorbents . . . . Chromatography on hydrophilic gels . . .
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953 954 955 . 955 . 957 . 960 . 961 . 962 . 962 . 965 . 967 . 968 . 970 . 971 . 972 . 913 . 975
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Pigments of plastids and photosynthetic chromatophores (Z Sestlk) Introduction . . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . . . Chromatographic procedures . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . .
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Macromolecula substances and plastics (M . Kubin and J coupek) Introduction . . . . . . . . . . . . . . . . Vinyl polymers . . . . . . . . . . . . . . . Rubbers . . . . . . . . . . . . . . . . . Polyolefins . . . . . . . . . . . . . . . . Polycondensates . . . . . . . . . . . . . . .
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1018
. 1024 1029
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1051 1053 1056 1057 1062
xv
CONTENTS Copolymers . . . . Miscellaneous polymers . Oligomers . . . . .
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Cells and subcellular particles (M Juiicovi and Z . Deyl) Introduction . . . . . . . . . . . . Ribosomes . . . . . . . . . . . . Viruses . . . . . . . . . . . . . Bacteriophages . . . . . . . . . . . Blood cells . . . . . . . . . . . . Cells from the spleen . . . . . . . . . Bone marrow cells . . . . . . . . . .
1063 1064 1066
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Inorganic. coordination and organometallic compounds (F. Jursik) . . . . . . . . . 1087 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 1087 Simple inorganic compounds . . . . . . . . . . . . . . . . . . . 1088 . 1088 Cations . . . . . . . . . . . . . . . . . . . . . . . . 1096 Anions . . . . . . . . . . . . . . . . . . . . . . . Coordination and organometallic compounds . . . . . . . . . . . . . . 1099 . 1099 General survey . . . . . . . . . . . . . . . . . . . . . Geometrical isomers . . . . . . . . . . . . . . . . . . . . 1100 1101 Optical isomers and diastereoisomers . . . . . . . . . . . . . . . Relationship between chromatographic behaviour and configuration of optical isomers . 1103 . 1108 Fer rocenes . . . . . . . . . . . . . . . . . . . . . . . 1109 Metallocenes . . . . . . . . . . . . . . . . . . . . . .
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Isotopes and radioactive compounds (J . DGata and I.M. Hais) . Isotopic effects in liquid column chromatography . . . Separation of radioactive substances . . . . . . . Subject index
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List of compounds chromatographed
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Foreword Contemporary liquid column chromatography is a rapidly expanding analytical technique. In order to ensure its further growth, it is important that periodically all the information available should be summarized in a clear and easily understood manner. Such a compilation serves three purposes. For the novice, it represents the basis on which additional knowledge can be built and practical observations interpreted. For the practical analyst facing different problems, it often allows the possibility of checking whether his “new” problem has already been solved. Finally, for the “expert” - the analyst who is experienced in both the theory and practice of the technique and who may have participated in its development - it permits him to organize his thoughts, to examine the various modifications of the technique in their proper context, and t o realize those interrelationships which might have escaped his attention. In chromatography, the technique that is being discussed here, there are good examples of the influence of such compilations. In classical liquid column chromatography, Zechmeister’s book, first published in Europe in the mid-1 930s, represented the springboard for the rapid expansion of the technique within a few years and the English translation, published in the early 1940s, contributed significantly to the adaptation of the technique in the United States. The handbook by Stahl on thin-layer chromatography, first published in 1962, served a similar purpose in the expansion of this very practical technique. In gas chromatography, most of us who worked with the technique in its infancy obtained the theoretical basis from Keulemans’ book, while the books by Dal Nogare and Juvet, Purnell, and Littlewood, which were published virtually simultaneously in 1962, served as the foundation that enabled many thousands of analysts to increase their practical knowledge. Modern liquid column chromatography as we know it is only a few years-old and was clearly developed from the knowledge accumulated in gas chromatography. However, the technique itself is not young; in fact, it represents the oldest variant of chromatography, and many of those who recently started to apply the “new” technique in their research were not aware of the excellent results obtained by early workers and hence could not apply this old experience t o their present work. Also, by restricting their coverage of liquid column chromatography to only the latest results reported in the analytical journals, they may have overlooked the opportunity of examining the technique in its proper context in relationship to other liquid chromatographic techniques that are also usually carried out in columns. The aim of this book is precisely what was specified above as the proper purpose of a compilation that deals with a rapidly growing analytical technique: to represent the basis on which newcomers to the field can build their future work, to give a rich compendium of the various applications of liquid column chromatography, with adequate references to lead the reader t o additional information, and to show the interrelationships of the technique and the common theoretical basis of the individual variants. I believe the Editors have succeeded in achieving these objectives.
XVIII
FOREWORD
The essence of many research and review papers has been reported in this fine volume. To Liquid Column Chromatography,its Editors and its readers, my best wishes for success.
University of Houston, Houston, Texas (U.S.A.)
ALBERT ZLATKIS
In the last 10 years, the advances made in liquid column chromatography have been comparable with those of flat-bed techniques and gas chromatography several years ago. In principle, there are two reasons for this increasing interest in liquid column chromatography. Firstly, the present situation reflects the steadily increasing demands being made upon separation techniques, mainly in biochemistry, drug analysis, the analysis of environmental pollutants, etc. Secondly, the theoretical aspects of liquid column chromatography have been developed substantially, mainly as a result of the application of model situations in gas chromatography which resulted in the integration of the knowledge achieved in the techniques and instrumentation of the different individual chromatographc variants. A deeper understanding of the process of chromatographic separation, especially more complete knowledge about the factors that influence equilibria and the selectivity of separations, showed clearly how far from the optimum are the conditions used in classical liquid column chromatography. These advances resulted in liquid column chromatography becoming a more complete analytical procedure, both qualitatively and quantitatively, and it became a very rapid method, offering numerous ways of automation and a delicate approach to the separation of different substances. The success in this field is intimately connected with the application of new types of sorbents that allow rapid mass transfer and are suitable for many types of separations. In addition to their chemical properties, the importance of physical properties such as particle size and shape have been stressed during the recent development of the new techniques of separation. The high speed of mass transfer enables the separation time to be shortened substantially and flow-rates to be increased by using high pressures. A high column overpressure is, of course, inevitable when one uses small size particles, which, on the other hand, help the rapid separation to occur. The application of high pressures also necessitates increased investment costs. As the use of high pressures is one of the most striking differences compared with classical techniques, these modern procedures are referred to as high-pressure or, alternatively, high-speed or high-resolution chromatography. One of the most rapidly developing features of column chromatography lies in the detectors. Instead of slicing the column or using fraction collectors with subsequent laborious assays on each fraction, the column eluate is nowadays analyzed continuously in devices based on different physical and chemical principles. Sometimes these detectors are very specialized and can be applied only to a single type of compound. On the other hand, however, the sensitivity of these detectors does not attain the sensitivity that we expect in gas chromatography. As always, there are a few exceptions from this general rule. The level of sensitivity in liquid column chromatographic techniques is the result of the different nature of the mobile phase compared with gas chromatography. It is t o be expected that attempts will be made to increase the detector sensitivity in the near future. In spite of the advantages mentioned above, high-speed techniques may not ptovide a universal solution t o the many difficulties that one may meet in chromatographic separa-
XIX
xx
PREFACE
tions. In some fields they already dominate or will dominate in the near future, while in other areas classical techniques will be retained. It is not only the high costs involved in the new techniques, but it might well be the nature of the separation itself which may direct the chemist to make his own choice between the classical and modern techniques. We are currently witnessing a paradoxical situation. In many laboratories in which classical liquid column chromatographic methods are used, considerable delays have arisen that could have been avoided but for a poor knowledge of the theoretical principles, methods and apparatus involved in the modern version of liquid column chromatography. On the other hand, however, there are a number of laboratory workers who have approached the application of modern liquid column chromatography armed only with theoretical and instrumental experience related to gas chromatography. These workers usually have some gaps in the vast range of experience that has been accumulated in the years of developmelit of classical liquid column chromatography, and there seems to be an undesirable tendency to forget about the past and to re-discover facts already published a long time ago. Other publications on liquid column chromatography have also indicated this situation. We have tried to amalgamate the existing knowledge on classical and modern liquid column chromatography in the belief that it is the most useful way to stimulate the imagination of those who wish to use this method in the solution of practical problems. In general, we have tried t o maintain a balance when preparing the book. We have tried to include sufficient theory to allow an inexperienced worker to acquire an adequate level of understanding of chromatographic separations and possibly to adopt procedures of his own for a particular problem, while on the other hand we have tried to collect reliable separation procedures for individual types of compounds, w h c h are surveyed in the Applications part of the book. To some, it may seem that the lengths of the different chapters are disproportionate. We believe, however, that this reflects precisely the current status of liquid chromatographic techniques. While separations of hydrophilic substances such as nucleotides, amino acids, proteins, enzymes, etc. are so frequent that there is hardly a paper on enzymes, for instance, which would avoid the use of a liquid chromatographic separation step, with hydrophobic compounds there are far fewer papers but those which have appeared usually involve substantially new procedures. Again, the development is far from proportionate even when judged separately in these two areas. The system of listing individual chapters in the Applications section follows the general arrangement of organic chemistry, with additional chapters devoted to inorganic separations, radiochemical techniques, dyes and plastics, and is easily comparable with the system of listing references in the Bibliography Section of the Journal of Chromatography and in the Bibliography of Column Chromatography 1967-1970. In our opinion, this may help to bring everyone rapidly up-to-date without losing too much time in tedious literature searching. However, the rapid expansion of the literature will make parts of t h s book obsolete even before its appearance in print. It is recommended, therefore, that additional sources providing a fast information service should be followed, e.g., the Bibliography Section of the Journal of Chromatography. In discussing separations of individual types of compounds, we have tried to offer readers as much practical laboratory information as possible for the size of the book,
PREFACE
XXI
keeping in mind that it is desirable to have a book that can be used directly in the laboratory without the need to study the original literature. In order to be concise, we have included the maximum amount of information in figures and tables and have less frequently described individual methods directly in the text. The latter method was used in situations in which there are widely used separations available for a particular type or series of compounds. The extent to which we have succeeded in achieving the aims outlined above must be judged by the reader. We are, of course, aware that a book of this size could not be perfect and we would therefore appreciate any comments and criticisms in this respect. It is also a matter of courtesy to acknowledge the help given by collaborators who are neither authors of individual chapters nor Editors of the book. However, we feel that there should be something more than mere courtesy expressed in these acknowledgements: when preparing this manuscript, we realized that technical aid may frequently be more important than the undecipherable original manuscript of the authors, and apparently simple things such as attaching figures and tables to chapters and giving them proper numbers or listing references may easily turn Hercule Poirot’s job. We express our deepest gratitude to Miss J. Krausovi, M. CiprovP and H. MBlkovP of the Laboratory of Subcellular Structures, Physiological Institute of the Czechoslovak Academy of Sciences, Prague, to Dr. 2. Prochizka of the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague for translating a substantial part of this book, and, last but not least, to MIS. R. Diartovi, for drawing most of the figures.
Prague April, 19 74
Z. DEYL K. MACEK J. JANAK
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THEORETICAL ASPECTS OF LIQUID CHROMATOGRAPHY
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Chapter I
Fundamental concepts J . NOVAK and J. JANAK
CONTENTS Introduction ................................................................... Principle of chromatography ...................................................... Chromatographic systems ........................................................ Chromatographic techniques ...................................................... Basic chromatographic quantities ................................................... References ....................................................................
3 4 5
7 8 10
INTRODUCTION It is now about 70 years since Tswett (1903,1906) described his classical experiment on the separation of coloured pigments of leaves and termed the procedure “chromatography”. Although earlier experiments by Runge on capillary analysis and observations by Davy on changes in the composition of crude petroleum when in contact with rocks displaying adsorptive activity can now be defined as chromatographic experiments, it was only Tswett who understood and defined his method as a general means of separating substances (including colourless substances) and coined the term “chromatography”. This priority is now generally recognized. In Tswett’s original experiment, a light petroleum extract of the colouring substance of leaves was introduced on to a calcium carbonate column in a glass tube under continuous percolation of light petroleum. The result was the separation of the mixture into a series of green and yellow bands (Fig.l.l). This invention came at a time when experimental methods of scientific investigation were still concerned mainly with the mere description of phenomena, and the problems of industrial practice were not considered in detail, and the importance of Tswett’s work was not recognized and was revived only in the 1930s by Kuhn et aZ., in connection with investigations on models of natural substances. Of major significance in the further development of chromatographic methods was the work by Martin and Synge, the former being a Nobel prize winner; they used a liquid as the sorbent and showed the possibilities deriving from the linear sorption isotherm and copious selection in suitable liquids. This work was a precedent to the development of other chromatographc techniques, such as paper chromatography (Consden et a[.), countercurrent distribution (Craig), ionexchange chromatography (Mayer and Tompkins, Samuelson), electrophoresis (Haugaard and Kroner), thin-layer chromatography (Ismailov and Shraiber, Kirchner et QZ.), molecular sieve action and, later, gel permeation chromatography (Barrer, Porath and Flodin) and, eventually, gas chromatography (Claesson, James and Martin). References pi0
3
4
FUNDAMENTAL CONCEPTS LIGHT PETROLEUM [SOLVENT)
J
Fig. 1.1. Representation of Tswett’s classical chromatographic experiments.
The adventof gas chromatography resulted in major advances in the theory as well as in the instrumentation and methodology of chromatography. It is therefore not surprising that this technique served as the basis for the development of a consistent theory of chromatography, applicable to all or a large group of chromatographic techniques (Giddings). Recently, the application of the theoretical and methodological knowledge obtained in gas chromatography led to a renaissance of the classical Tswett chromatography in the field of column techniques. A significant contribution to the theory of the modern high-speed procedures of liquid chromatography in columns was made by Huber. It appears that liquid chromatography affords both a separation efficiency and time of separation that are comparable with those usual in gas chromatography.
PRINCIPLE OF CHROMATOGRAPHY
Chromatography can be characterized as a separation method based on the differential migration of solutes through a system of two phases, one of which is mobile. The mechanism proper that determines the regularities of the movement and spreading of the chromatographic zone can be considered as a continual convective disturbance of the equilibrium distribution of a solute between the phases and simultaneously compensating for the deficienciesby diffusion. As only those molecules of the substance chromatographed which occur in the mobile phase are subject to convective transport, the velocity of migration of the zone is proportional to the probability of the occurrence of the solute
CHROMATOGRAPHIC SYSTEMS
5
molecules in the mobile phase. It follows that although the chromatographic process involves inseparably the processes of convective transport, diffusion and sorption equilibration, it is an appropriate difference in the distribution constants that definitively determines the possibility of the differential migration of the chromatographc zones. However, if the mobile phase ceased to move, there would be n o migration at all. On the other hand, were it not for diffusion, the differences in distribution constants would lose their significance as there would be no possibility of restoring the disturbed equilibrium. Hence, convective transport, diffusion and differences in the distribution constants are inseparable factors in the chromatographic process, each representing a necessary, but not complete, condition for achieving a chromatographic separation. The above factors constitute, on the one hand, conditions for separation, but on the other hand, they imply a limitation in the separation efficiency. Geometrical irregularities of the chromatographic bed and the hydrodynamic properties of fluids lead to an uneven flow velocity of the mobile phase and to the formation of velocity profiles. Further, owing to a finite rate of equilibration (finite rate of lateral solute diffusion), the leading (sorption) part of the chromatographic zone during migration contains a certain excess of solute in the mobile phase, as compared with the equilibrium concentration, whereas the rear part of the zone shows a deficiency in the solute concentration in the mobile phase. Finally, longitudinal diffusion proceeds in both phases during chromatography. All of the above factors cause a spreading of the chromatographc zone. As will be shown later, chromatographic separation is feasible owing to the fact that while the spacing between two given zones increases linearly with increasing length of the migration path, the broadening of the zones increases with only the square root of the migration path. Hence, from the viewpoint of separation efficiency, the problem of zone spreading is as important as the problem of selectivity, i.e., the problem of differences in distribution constants. The above qualitative treatment indicates that a chromatographic separation will be the more effective the greater are the differences in the distribution constants of the solutes chrornatographed, the more uniform is the chromatographic bed and the flow of the mobile phase, the more rapid is the establishment of sorption equilibrium, and the slower is the longitudinal diffusion of the solute in both phases. The problems of flow, diffusion and sorption equilibrium are discussed in greater detail in the following sections.
CHROMATOGRAPHIC SYSTEMS Chromatographic systems can be classified according to (i) the state of aggregation of the phases; (ii) the physical arrangement of the phases; (iii) the mechanism underlying the distribution equilibrium. Hence one can combine gaseous, liquid and solid phases to form a total of five two-phase systems, i.e., solid-solid, liquid-solid, liquid-liquid, gas-solid and gas-liquid. Systems typical in liquid chromatography are liquid-solid and liquid-liquid. A solid-solid system is undoubtedly real, but it can hardly be of any use in chromatography as it is impossible in such a system to effect, under the usual chromatographic conditions, the relative References p . 1 0
6
FUNDAMENTAL CONCEPTS
movement of the phases without the interface being disturbed. Systems involving a gas as the mobile phase are typical in gas chromatography. The further discussion will be centred around liquid-liquid and liquid-solid systems. As for the physical arrangement of the phases, chromatographic systems can be divided into two large groups - columnar and planar systems - and columnar systems can further be classified with respect to the geometry of the chromatographic bed proper. Thus, we can speak about capillary columns, coated either with a film of a stationary liquid or with a layer of a solid adsorbent adhering to the inner wall, and packed columns, containing a grained solid material that functions either directly as the partitioning phase or merely as a support for a stationary liquid. Columnar systems are much easier to define than planar systems, represented by paper and thin-layer chromatography. In liquid-liquid systems, regardless of their arrangement, a very important point is the way in which the stationary liquid film is formed. Basically, there are two alternatives; in the first, the liquid film is being created during chromatography by adsorption of components of the solvent, while in the other a defined amount of stationary liquid insoluble in the mobile phase is deposited prior to carrying out the chromatographic run. The latter procedure is obviously preferable if it is desirable t o define the amount of the stationary liquid held up within the chromatographic bed. The possibility of defining the amount of the partitioning material is essential whenever relationships between retention data and the parameters of sorption equilibrium are to be expressed. Differential zone migration occurs by virtue of the differential distribution of solutes between the phases. This distribution can be due to various mechanisms, such as physical interactions of solute molecules with the solvent, formation of chemical bonds between the solute and the solvent, or merely a kind of hindered diffusion of solute molecules in the matrix o f the sorbent. Depending on the nature of the system, the solute-solvent interactions can take place on a two-dimensional or three-dimensional scale, i.e., on the surface or within the bulk phase. It is these two alternatives from w h c h the terms “adsorption” and “partition” chromatography, respectively, have been derived. However, if viewed on a molecular level, both alternatives can involve either of the above mechanisms; in other words, both ordinary physical interaction chromatography and ionexchange or ligandexchange chromatography and affinity chromatography can be effected as either a partition or an adsorption process. It is worth mentioning that electrophoresis displays phenomena similar to those of chromatography, but the differential zone migration in electrophoresis is based on a different mechanism and the above concepts of solute distribution between the phases are completely inapplicable. Therefore, this interesting separation method has not been included in this book. The distribution equilibrium in a solute-sorbent-mobile phase system is characterized by a sorption (distribution) isotherm, representing the relationship between the solute equilibrium concentrations in the coexisting phases. If the distribution isotherm is linear, the distribution constant is a constant independent of the solute concentrations. In this case, we speak about linear chromatography. If the distribution isotherm is curved, the distribution constant is a function of the solute concentrations in the system, which results in non-linear chromatography.
7
CHROMATOGRAPHIC TECHNIQUES
CHROMATOGRAPHIC TECHNIQUES Any chromatographic system allows elution or frontal chromatography to be performed. In systems with a solid adsorbent in the role of the sorbent proper, displacement chromatography can also be included. In the elution technique, a narrow band of the mixture to be chromatographed is introduced intermittently at a starting point (column inlet) on the chromatographic bed. Upon the action of the flowing mobile phase, the zone begins to migrate, the forward velocities of the individual components of the initial zone being inversely proportional to their distribution constants. The mobile phase is assumed to be virtually non-sorbed on the stationary phase in this technique. An elution chromatogram is shown in Fig. 1.2A. The distances of the peak maxima from the starting point are characteristic of the quality of the solute substances, whereas the peak areas are proportional to the total amounts of the substances in the chromatographc zones in elution chromatography. R
h I
I
j/q I I
I
I
I
I
I
I
‘Rl
‘112
‘R 3
* TIME
FRONTAL
I
I t R1
tR2
I fR3
t
TIME
DISPLACEMENT
1
1
I
I 1 I ‘RI
‘R’2 ‘R3
* TIME
Fig. 1.2. Chromatographic records: ( A ) elution chromatography; (9)frontal chromatography; (C) displacement chromatography. R = Response (see also Chapter 15).
References p.10
8
FUNDAMENTAL CONCEPTS
In frontal chromatography, the mixture to be analyzed enters the chromatographic bed continuously at the start, and hence the mobile phase is actually formed by the mixture analyzed. The resulting course of the concentrations of the solute components in the effluent is shown in Fig. 1.2B. The relative movement and the eventual spacing of the individual steps are controlled by the distribution coefficients in the same manner as quoted for the zones in elution chromatography. The height of the front is proportional to the concentration of the component in the mixture being introduced. In displacement chromatography, the procedure is similar to that in elution chromatography, but, instead of employing a non-sorbed eluent, the migration of zones is accomplished by displacement with a substance that is sorbed more strongly than any component of the mixture being separated. A displacement chromatogram is illustrated in Fig. 1.2C. 'Ihe analytical meaning of the position and the area of a chromatographic peak is the same as in elution chromatography. When employing solid adsorbents as chromatographic materials (liquid-solid and gas-solid systems), the displacement effects are operative, to a certain extent, also in elution and frontal chromatography. "he chromatographic zones can either be eluted from the chromatographic bed or the chromatographc run can be stopped before their elution. In the first instance, the volume of the eluent necessary t o elute the concentration maximum of the zone is a measure of retention. In the second instance, the distance travelled by the centre of the zone from the starting point serves as a criterion of retention. Planar techniques represent the second alternative. As the elution technique with the complete elution of the zones is of greatest concern in modern liquid chromatography, the discussion will be concentrated only on this version of chromatography.
BASIC CHROMATOGRAPHIC QUANTITIES When monitoring the composition of the effluent leaving the column outlet during the elution process, one obtains an elution chromatogram in either a differential (Fig. 1.3A) or an integral form (Fig. 1.3B). The ratio of the net retention time (tR - t,) of a solute component to the retention time (t,) of a non-sorbed substance is called the capacity ratio and is denoted by k :
where rR is the total retention time of the solute substance. Actually, the value of k represents the ratio in which a given amount of solute distributes itself between the stationary and the mobile phases in the given sorption system, i.e., k=
amount of solute in the stationary phase amount of solute in the mobile phase
It follows from eqn. 1.2 that (1.3)
where K is the distribution constant,
V, and
V, are the volumes of the stationary and the
9
BASIC CHROMATOGRAPHIC QUANTITIES DIFFERENTIAL A 1
INTEGRAL
Fig. 1.3. Schematic representation of a differential (elution) chromatogram (A) and the corresponding integral chromatogram (B).
mobile phase, respectively, in the column, and qs and qm are the cross-sections of the respective phases. The distribution constant is defined by
K = c”/cI
(1.4)
where C” and c’ are the solute equilibrium concentrations (number of moles per unit volume) in the stationary and in the mobile phase, respectively. The multiplication of tR and t, in eqn. 1.1 by the volume flow-rate of the mobile phase and combination with eqn. 1.3 gives
VR =
v,
(1 + k ) =
v,
+KV,
(1 5)
where VR is the overall retention volume and V, is equal to the dead retention volume. Hence, for a non-sorbed substance, k = 0, K = 0, tR = t, and VR = V,. Eqn. 1.5 can be rearranged (Martin and Synge, Phillips) to read as follows:
(1.6) where R is the so-called retardation factor. The quantity R is theoretically identical with V,/V,
= V,/(V,
References p . 1 0
+KV,)=R
10
FUNDAMENTAL CONCEPTS
the quantity R, enipioyerl in planar techniques (LeRosen):
RF =
distance of the solute zone from the start distance of the eluent front from the start
In practice, the equation R = RF/O
(1 -8)
holds (Giddings et al.), where B is a factor the value of which varies within the range 0.8-0.9. The extent of the separation of a pair of solutes is expressed empirically by a quantity called “resolution”; the calculation of resolution (R,) from the chromatogram is carried out by using the equation
Rs = 2 ( t ~ f~~ ~ ,/(At2 -I-At,)
(1 *9>
where rR2 and tR1are the retention times of the two components and At2 and At, are the time-widths of the respective zones. Resolution is dependent on the separation efficiency, expressed by the number of the theoretical plates, N : N = 16(tR/At)’
(1.10)
where At is again the time-width of the peak. The number of theoretical plates is given by the column length ( L ) and the height equivalent to a theoretical plate, H , the latter quantity being defined by
H = u2/L = LfN
(1.11)
where u is the standard length deviation of the chromatographic zone.
REFERENCES Barrer, R. M., J. SOC.Chem. Ind., London, 64 (1 945) 130. Claesson, S., Ark KemiMineral. Geol. A23, No. 1 (1946). Consden, R., Gordon, A. H.and Martin, A. J. P., Biochem. J., 38 (1944) 224. Craig,L.C.,J. Biol. Chem., 155 (1944) 519. Davy, D. T., Proc. Amer. Phil. SOC.,36 (1 897) 112. Giddings, J. C., Dynamics ofChromatography, Marcel Dekker, New York, 1965. Giddings, J. C., Steward, G. H. and Ruoff, A. L., J. Chromatogr., 3 (1960) 239. Haugaard, G. and Kroner, T. D., J. Amer. Chem. SOC.,70 (1948) 2135. Huber, J. F. K., in E. Kovits (Editor), Column Chromatography, Lausanne 1969. S a u e r l i d e r AG, Aarau, 1970,p. 24. Ismailov, N. A. and Shraiber, M. S., Farmacia, 3 (1938) 1; C.A., 34 (1940) 855. James, A. T. and Martin, A. J. P., Biochem. J., 50 (1952) 679. Kirchner, 1. G., Miller, J. M. and Keller, G. I.,Anal. Chem., 23 (1951)420. Kuhn, R., Winterstein, A.and Lederer,E., Hoppe-Seyler’sZ. Physiol. Chem., 197 (1931) 141. LeRosen, A. L.,J. Anrer. Chem. SOC.,67 (1945) 1683. Martin, A. J. P. and Synge, R. L. M., Biochem. J.,35 (1941) 1358. Mayer, S. W. and Tornpkins, E. R., J. Amer. Chem. SOC.,69 (1947) 2866. Phillips, C. S. G., Discuss. Faraday SOC.,7 (1949) 241. Porath, J. and Flodin, P., Nature (London), 183 (1959) 1657. Runge. F. F., Farbenchemie, Band Ill, Mittler u. Sohn, Berlin, 1850. Samuelson, O., Ion Exchange in Analytical Chemistry, Wiley, New York, 1963. Tswett, M. S., Proc. Warsaw SOC.Nut. Sci., Biol. Sect., 14, No. 6 (1903). Tswett, M. S., Ber. Deut. Bot. Ges., 24 (1906) 234,316 and 384.
Chapter 2
Basic processes in chromatography J. NOVAK, J . JANAK and S . WICAR
CONTENTh Flow of mobile phase through a packed column ........................................ Diffusion of solute within the phases ................................................ General rules of diffusion. ...................................................... Diffusionin liquids ............................................................ Equilibration of solute between the phases ............................................ Equilibrium in binary two-phase liquid systems.. .................................... Equilibrium in ternary two-phase liquid systems ..................................... Equilibrium in liquid-solid systems .............................................. Binary two-phase liquid-solid system. .......................................... Binary single-phase liquid system .............................................. Binary liquid mixture-solid adsorbent system .................................... References .....................................................................
11 15 15 17 17 17 19 20 21 21 21 23
FLOW OF MOBILE PHASE THROUGH A PACKED COLUMN There are basically two reasons for studying the hydrodynamics of the mobile phase in chromatographic systems. The first is the fact that the direct convective solute mass transport along the stream-lines between separate regions of the column is one of the most important solute transport mechanisms in the column. The second reason is of rather a technical nature and concerns the estimation of the pressure necessary to achieve the required flow-rate of the mobile phase through the column. The state of a moving liquid within a column is determined by a group of six quantities, f), i.e., by the values of u1@, t ) , namely, by three components of the velocity_*vectorIt@, u2 t ) and u3(?, t ) ,and by the density, p(r , t ) ,pressure p(7, t ) and temperature T(7,t ) , where r'represents the positional vector and f the time, respectively. When restricting the problem to the study of steady isothermal streaming of an incompressible liquid through a column, the number of determining quantities will be reduced to four: three components u z ( T )and u3(T),and the pressure,p(?). of the velocity vector, The above four independent variables are connected by four relationships:
6.
u,(q,
pdivu'=o p (u'grad)
(2.1)
u'= 3- gradp +I.(div grad 2
(2 .2) The scalar equation 2.1 expresses the fact that in any region within the column, the mass entering that region equals the mass leaving it. Hence, eqn. 2.1 is a hydrodynamic expression of the mass conservation principle. The vectorial equation 2.2, i.e., the Navier-Stokes equation, expresses the equality between the differentiation with respect to time of the References p.23
11
12
BASIC PROCESSES IN CHROMATOGRAPHY
momentum of an arbitrarily chosen volume of the streaming liquid (left-hand side) and the res$tant of the exterior forces acting on the above element (right-hand side). The force F is the re$ta$ of volume forces; in chromatography, these forces are represented by gravity, i.e. F = pg ,where g is the gravitational constant. While in classical liquid column chromatography gravity is usually the main driving force for the flow of the mobile phase, in modern high-speed liquid chromatography the role of gravity is negligible in comparison with the pressure forces represented by the second term of the right-hand side of eqn. 2.2. The last term of the equation stands for the resultant of the shear forces. Eqn. 2.2 is equivalent to three scalar equations corresponding to the individual components of the velocity vector, u l , u2 and 243. Eqns. 2.1 and 2.2 represent a non-linear system of partial equations of the second order, expressing merely the general regularities of streaming of liquids. In application to a specific problem, it is necessary to solve the respective boundary problem, i.e., to seek such solutions of the system that fit the given boundary conditions. These conditions also involve the geometry of the bed within which the movement of the liquid takes place. Such solutions are known only for some instances of geometrically simple and laminar flow (Schlichting); Hagen-Poisseuille streaming in tubes of generally non-circular crosssections serves as an example. Thus, the Poisseuille equation holds for steady isothermal streaming of an incompressible liquid through a tube of a circular cross-section: uz(d= [(az-rz)/41 Ap/&
(2.3)
provided that the volume forces are negligible. In eqn. 2.3, u, is the forward velocity of the streaming liquid along the tube axis at a distance r from the axis,a is the radius of the tube, Ap = p l - pzis the difference in the pressures at the inlet and the outlet of the tube, and p is the dynamic viscosity of the liquid. The need to describe analytically the complex structure of a packed column is one of the obstacles to applying eqns. 2.1 and 2.2 directly to the movement of the mobile phase in the chromatographic column. Therefore, the necessary relationships are searched for experimentally and the results are generalized by virtue of the methods of similarity theory. Eqn. 2.2 can be rewritten in a component form while omitting the gravitational term:
where i, k = 1 , 2 , 3 . Upon introducing the substitutions Xi = xi/l, Ui = ui/w and P = p/pw2, where 1 and w are undefined characteristic quantities, eqn. 2.4 will become a system containing the Reynolds number,Re = wZp/p, as a single parameter. This parameter is a dimensionless constant comprising the original constants of the system and the characteristic quantities. Hence, it follows from the similarity theory (Ehrenfest-Afanassjewa, Konakov) that the functions q.= q(P, X i ) for a given value of Re will be identical for geometrically similar systems, provided that the boundary conditions are similar. The Reynolds number, which characterizes the ratio of the effects of shear and inertial forces in the streaming liquid, is alone a determining criterion of isothermal steady streaming of an incompressible liquid. Under the predominant influence of shear forces,
FLOW OF MOBILE PHASE THROUGH A PACKED COLUMN
13
when Re < Reo, the streaming is of laminar (viscous) character and the stream-lines follow more or less the shapes of the bodies past which the liquid is streaming. At higher Re values (Re >Rel), the effect of inertial forces is predominant and one can speak about fully developed turbulent streaming, at which the velociiy of streaming acquires locally non-stationary values and only the mean values of the local velocities remain constant. The interval within Reo and Rel corresponds to a transitory region in which both types of flow occur. When choosing the mean velocity u , determined from the volume flow-rate, for the characteristic velocity w ,then the following general function for similar systems holds: where tl,t 2 ,etc., are geometrical simplexes derived from the boundary conditions. However, the actual shape of the function P has to be found experimentally. The first assumption for the applicability of the similarity theory to the hydrodynamic properties of packed columns is the geometrical similarity of the arrangement of the particles that constitute the packing. The simplest case of a packed bed formed by regularly arranged spheres of equal diameters can be considered first. The spheres can be arranged regularly in a plane to form either a rectangular lattice with the centres of the spheres representing the corners of a square with sides of length equal to the sphere diameter, d , or a triangular lattice in which the centres of the spheres form the apexes of an equilateral triangle with sides of length d. Spatial structures can now be formed from the above planar arrangements by gradually depositing the layers upon one another and shifting them appropriately. However, only those shifts will be permissible which enable mechanically stable structures to be formed. Geometrical considerations now lead to two limiting spatial structures that differ from each other by the degree of filling the space of the bed, Le., by the porosity, E , defined by the relationship E
= 1 - (v/v>
(2.6)
where v / V is the volume of the futed phase per unit volume of the bed. The loosest possible arrangement is represented by square nets deposited upon one another without any shift. In such a bed, each sphere touches six neighbouring spheres, and the entire bed is interwoven with a continuous network of channels. The porosity of such a structure is E = 1 - (n/6)= 0.476. The other extreme is the arrangement derived from the triangular net; the individual layers are positioned in such a way that the apexes of the triangles of a layer coincide with the centres of the triangles of the neighbouring layers. In this way, the most stable structure is formed, in which each sphere of the bed is in contact with eleven other spheres, and the porosity of such a structure has a value of about 0.26. It should be emphasized that the porosity of a bed composed of spheres of equal dimensions is independent of the size of the spheres, but depends on their arrangement. The porosity of randomly packed beds, composed of spheres of equal diameters, varies withm the range 0.35-0.40 and is therefore substantially higher than that of the expected most stable structure. A possible explanation of this discrepancy may be the breakdown of the bed structure into smaller agglomerates %th the minimum porosity and the formation of some bridges between the agglomerates (Giddings, 1965a). In this References p.23
14
BASIC PROCESSES IN CHROMATOGRAPHY
case, more than a quarter of the free volume of the bed would be in the spaces between the agglomerates. Another possibility is a combination of agglomerates of unequal structures and smaller porosities connected with bridges. The smallest volume unit for which it is meaningful to speak about the porosity defined by eqn. 2.6 is an elementary cube with edges of length d . The porosity of this element will be the same as that of the bed of any unconfmed size and of the same structure. However, real beds are always bound by the walls of the container, and the structure of the bed has to conform, at least at its outside, to the shape of the wall restricting the bed. Provided that the wall is a planar or cylindrical surface, as is normal in practice, the accommodation of the structure t o the container wall is always associated with a sharp increase in porosity in the immediate proximity of the wall. At the surface proper of the wall, the formally understood porosity is unity, as there are only point contacts between the spheres and the wall; at some distance from the wall (6 > 0.5) the porosity remains approximately constant. The course of porosity in the proximity of a wall was described by a semiempirical equation by Sonntag: E =
1 -A6(1-S)
(2.7)
which is applicable for the region where 6 < 0.5, where 6 is the distance from the wall expressed in multiples of the sphere diameter and A is an empirical constant with a value of 3.2 for loosely packed beds. A similar course of the bed porosity in the proximity of a wall was also found by Schwartz and Smith, who measured radial velocity profiles in a packed column. Hence, the porosity as determined experimentally will always be higher than that of the interior structure of the bed; the following equation holds approximately for cylindrical beds (Sonntag): E,,
= €0
+ 0.263 (1 - € 0 ) d/R
(2.8)
where E,, is the average porosity, e0 is the porosity of the interior structure and d/R is the ratio of the diameter of the sphere and the radius of the column. Substantially more complex are beds produced by loosely pouring spheres of different diameters or even particles of irregular shapes yet with the characteristic particle dimensions varying withn narrow limits. Although the equality of the porosities of two beds obviously does not necessarily imply that the structures of the beds are similar, the porosity is the only useful representation of the structure of the bed. It is now useful to consider again a column the packing of which is formed by regularly arranged spheres of the diameter d , the length of the bed and the porosity being L and E respectively. Further, assume that the ratio d/R (cj: eqn. 2.8) is sufficiently small and that it is possible to write em= f o = E. The geometry of the bed is characterized by the diameter of the spheres and by the porosity, and the length of the bed is irrelevant to the above problem. Let us now insert in eqn. 2.5 the diameter of the sphere, d, as the characteristic dimension 1 of the bed. Hence, the geometry of the bed will be described by two dimensionless ratios, t1 = d / L apd t2 = E , and eqn. 2.5 will become:
It can be seen that p in the variable P has been replaced by the pressure difference, Ap. A
DIFFUSION OF SOLUTE WITHIN THE PHASES
15
form of the function fdescribing the regions of both low and high values of Re was given by Ergun: -2 Ap
pu
.d_ . - c3
L 1--E
= 1 5 0 . 1 - ~+ 1.75 Re
< 10, eqn. 2.10 becomes the well known Kozeny-Carrnan --El2 .@ 2 = constant -
For Re/(l - E )
e3
L
(2.10) equation:
d2
(2.1 1)
Eqn. 2.1 1 was derived from the concept that the packed bed is a band of parallel capillaries; the proportionality (2.1 2) for small Re values has been known for a long time as the Darcy law. The applicability of relationship 2.9 was postulated only for beds formed by a random arrangement of spheres of equal diameters. The concept of the mean hydraulic diameter, dh, of the bed particles (Ergun and Oming): dh =
6qL (1
-
-E)
S
(2.13)
where q is the bed cross-section normal to the direction of flow of the liquid, L is the length of the bed and S is the total geometrical surface area of the particles constituting the bed, makes it possible to extend the applicability of eqns. 2.9-2.1 2 to beds composed of spheres of unlike diameters as well as particles of irregular shapes. However, the determination of the geometrical surface area of the particles with their own internal porosity is a separate problem.
DIFFUSION OF SOLUTE WITHIN THE PHASES General rules of diffusion Diffusion is a direct consequence of the tendency of any system to reach a state of minimum potential energy and maximum randomness, as dictated by the second law of thermodynamics. In this respect, diffusion is one of the most universal phenomena of nature, characterized by spontaneous dilution and mixing of matter. Regarding the nature of chromatography, diffusion is a basic constituent of the chromatographic process. The diffusional transport is usually considered to be the result of a concentration gradient. As discussed by Giddings (1965b), this interpretation is essentially incorrect, despite being in accordance with observation. In fact, the only driving force in diffusion is the tendency of the system to attain a minimum free energy, i x . , a state of equilibrium. Diffusion is a random process in which the molecules present in regions of different concentrations undergo their diffusional transport independently of one another. As the number of molecules diffusing out of a given region is proportional to the number of References b.23
16
BASIC PROCESSES IN CHROMATOGRAPHY
molecules present in the region, there will always be a net solute flux towards the more dilute region, which gradually levels the concentration difference. One-directional isothermal diffusion under steady-state conditions of a substance (solute) through a stationary layer of another substance is expressed by Fick’s first law: (2.14)
where J i s the number of moles of solute passing in unit time through a unit area normal to the concentration gradient, D is the diffusion coefficient and ac/& is the concentration gradient. At low solute concentrations, the diffusion coefficient is virtually constant. A case in which the solute concentration can change with time‘in any region of the solution is described by Fick’s second law. The solute mass balance for a region of unit cross-section and of length dz leads to (2.1 5)
where &/at is the increase in solute concentration within the above region in unit time. It will be shown in Chapter 3 that eqn. 2.15 is of basic importance in the general description of the chromatographic process. A solutian of this equation for an extremely thin initial profile introduced at z = 0 (Crank) is
c=
no . exp (--z 2 /4Dt) (47rDt)% ~
(2.16)
where no is the number of moles of solute introduced per unit cross-section of the concentration profile (column). Eqns. 2.15 and 2.16 describe diffusion in stationary media. In chromatography, however, diffusion of the solute in flowing fluids usually occurs and, when employing fixed coordinates, this case can be described by (2.17)
where the term @c/az) accounts for the convective transport (along the coordinate z ) at a velocity u . Assuming that the same initial and boundary conditions as those mentioned in the solution of eqn. 2.15 apply, and provided that Dt < z’, the solute concentration profile can be described in this case by
no c=exp [ - (z - ut)’/4Dt] (47rDt)’h
(2.18)
Eqns. 2.16 and 2.18 represent Gaussian profdes, which are usually characterized by means of the standard deviation, u , through the use of Einstein’s equation: a’ = 2Dt
A more detailed discussion of the role of convective diWusion in chromatography is presented in Chapter 3.
(2.19)
EQUILIBRATION OF SOLUTE BETWEEN THE PHASES
17
Diffusion in liquids The rates of diffusion in liquids are lower by a factor of 104-105 than in gases. The great difference between liquid and gas diffusivity is very important in many respects when comparing gas and liquid chromatography, and it is necessary to be very cautious if the results obtained in GC are to be applied to the problems of LC. Typical diffusion coefficients in liquids are of the order 1O5 cm’lsec. One of the main causes of the difference between gas and liquid diffusivity is the difference in the lengths of the random steps travelled by the molecules in their thermal movement. W e with gases the length of these steps is approximately equal to the mean free path of the molecules, with liquids this length is similar to the molecular diameter. According to the random walk model (see Chapter 3), the rate of diffusional transport should be proportional to the second power of the step length. Further, as the molecules of a liquid are held together relatively strongly by intermolecular forces, a molecule must accumulate an appreciable amount of thermal energy in order to undergo a displacement step. Hence, a considerable activation energy is associated with diffusion in liquids, thus making the diffusion coefficient strongly dependent on temperature (Frenkel). On the other hand, owing to the relative incompressibility of liquids, diffusivity in liquids is only slightly dependent on pressure. Of numerous equations that have been suggested for calculating diffusion coefficients in liquids (Gambill), only that due to Wilke and Chang will be quoted here. This equation is: (2.20)
where M, is the molecular weight of the solvent, T is the temperature, ps is the dynamic viscosity coefficient (a function of temperature) of the solvent, 5 is the molar volume of solute and F, is an association factor ranging from about unity for nonpolar compounds to 2.6 for water. This equation proved successful when applied to liquids with molecules of small and medium size. The equation clearly shows the dependence of Dion the nature of both the solvent and the solute. It follows from the above discussion that there are significant differences in the diffusion coefficients as well as in their dependences on temperature, pressure and type of substance for liquids and gases. These differences may lead to very different situations when comparing the effects of the operatirig conditions on the performance of gas and liquid chromatographic systems.
EQUILIBRATION OF SOLUTE BETWEEN THE PHASES Equilibrium in binary two-phase liquid systems It follows from the definition of chromatography that a chromatographic system of any type must always involve two immiscible phases. The composition of the phases and the physical arrangement of the system are chosen with respect t o the given separation problem. Immiscibility is a problem mainly in liquid-liquid systems and will therefore be discussed with reference to the latter only. The phenomenon of immiscibility is closely related to the concept of non-ideal solutions with positive deviations from Raoult’s law. References p.23
18
BASIC PROCESSES IN CHROMATOGRAPHY
Increasing positive difference in the cohesive forces between like and unlike molecules will raise the tendency of both the molecules to escape from the mixture. When the above difference is sufficiently large, the liquids will show incomplete miscibility within some temperature range. Azeotropic solutions can be considered as intermediates between systems of complete and partial miscibility. A thermodynamic condition for two liquids to show incomplete miscibility is that the system composed of these liquids should have a minimum free energy when it is split into two phases. The solubility gap usually (but not always) narrows and, eventually decreases upon increasing the temperature. The above situation is shown schematically in Fig. 2.1, which represents a temperature-composition diagram of a binary system composed of substances M and S. The symbol cs represents the molar concentration (number of moles per unit volume) of component S,and the points on the line MmysS correspond, respectively, t o pure component M, the M-rich phase (just saturated with S), a two-phase system, the S-rich phase (just saturated withM) and pure component S. Phases m and s are called conjugated solutions. The curve, called binodd, separates the homogeneous (outer) and the heterogeneous (inner) areas. The point a! on the binodal curve is a critical mixing point corresponding to the temperature at which the entire system becomes homogeneous. Lines connecting the points of the composition of the coexisting phases are called tie lines or conjugation lines.
Fig. 2.1. Phase diagram of a binary two-phase liquid system.
Such a system is bivariant according to the Gibbs phase rule; if the temperature and pressure are fixed, the compositions of both conjugated phases are invariable provided that the phases are in equilibrium. A simple mass balance shows that the ratio of the number of moles of phases m and s, n,/n,, corresponding to pointy of the heterogeneous area, is given by n, In, = yTf/my (2.21) where y a n d Ey denote the distances between the respective points in the diagram.
EQUILIBRATION OF SOLUTE BETWEEN THE PHASES
19
Equilibrium in ternary two-phase liquid systems While the binary systems discussed above can be compared with any liquid-liquid chromatographc system without the solute component, a three,-component two-phase liquid system is the simplest representation of a solute zone in a liquid-liquid chromatographic system. Considering a system of three components of which only one pair displays incomplete miscibility, this pair represents the stationary and the mobile phase (components S and M)in the corresponding chromatographic system whde the third component, which is completely miscible with the other two, represents the solute (i). If it is imagined that component i is gradually added, at constant temperature and pressure, to the initially binary two-phase system of components S and M, the solute will distribute itself in certain proportions between the conjugated phases, thus changing both the composition and relative amount of the latter. The change in composition arises not only because the binary conjugated solutions become ternary solutions with an increase in the amount of the third component, but the presence of the third component will alter the equilibrium in such a way that the proportions of components S a n d M will also gradually be changed to different extents in both phases. This is in accordance with Gibb’s phase rule; as a ternary two-phase system is essentially trivariant, the concentration of only one component can be varied independently at a constant temperature and pressure. The effect of the third component in a ternary system such as that discussed above is similar to the effect of temperature in binary two-phase systems. As more solute is added to the ternary system, the compositions of the coexisting phases move closer to each other and, at a certain critical composition (point a), the system becomes homogeneous. Ternary systems can be represented most conveniently by means of triangular diagrams, and such a diagram is shown schematically in Fig. 2.2. The corners of the triangle represent pure components M, S and i. The concentration of i is plotted along the sides ZI a n d g and the concentrations of the other two components are plotted similarly (cf:,the dotted lines to point a). Hence, on any line drawn parallel to a side bound by two components, the concentration of the third component is constant. In Fig. 2.2, the side m c o r r e s p o n d s to a binary system such as that shown in Fig. 2.1. The I
Fig. 2.2. Phase diagram of a ternary two-phase liquid system.
References p.23
20
BASIC PROCESSES IN CHROMATOGRAPHY
binodal curve mDLT binds the heterogeneous area, while the remainder of the diagram and (above the curve) corresponds to a homogeneous system. The tie lines iiis , connect the points representing the compositions of the respective conjugated solutions. As with binary systems, any point on a given tie line represents a system composed of two phases of constant compositions. Provided that the concentrations are expressed in numbers of moles per unit volume, the ratio of the number of moles of the coexisting phases corresponding to point yl is given by
r n x
r n x
__-
nm,lns, = Y 1sllmlYl
(2.22)
It is apparent from the diagram that each pair of conjugated points on the binodal curve actually represents data for expressing the distribution constant of component i . The distribution constant is usually defined as the ratio of the solute equilibrium concentrations in the sorbent and mobile phases, which can be directly read off on the side Mi. For example, the distribution constant corresponding to the conjugated pair m, and sz is:
K = c!’/c! ‘2 5
(2.23)
In some instances, the tie lines may be parallel to the m s i d e or can even show an opposite slope, so that the distribution constant can be either zero or less than unity, respectively. The diagram indicates that the value of K can be appreciably dependent on the overall composition of the system, i.e., on the solute concentration. However, at very low solute concentrations in the system, the dependence between the concentrations of solute in the coexisting phases can be approximated as linear, thus resulting in a constant distribution constant, which is the situation of interest in chromatography. The situation discussed above concerned isothermal and isobaric conditions. While condensed systems depend only slightly on pressure, changes in temperature can lead to appreciable alterations in the equilibrium concentrations and, consequently, in the distribution constants.
Equilibrium in liquid-solid systems Owing to relatively strong intermolecular interactions in liquids, a molecule in the proximity of the liquid surface is pulled towards the interior owing to surface tension. +4san increase in the surface area is always associated with the transfer of molecules from the bulk phase to the surface, energy must be used in order to accomplish this increase. The energy necessary to increase the surface of a given amount of a liquid by 1 cm2 is equal to the surface free energy of unit surface area, which is identical with the surface tension of the liquid. The surfaces of solid substances also display a surface free energy, i e . , surface tension, but solids cannot reduce their surface area as the particles of solids do not undergo translational thermal movement. However, the free energy of the surfaces of solids is a most important factor in the adsorption of gases and liquids on solid adsorbents. The process of adsorption can be understood by discussing separately the following three systems: binary two-phase liquid-solid system; binary single-phase liquid system; and binary liquid mixture-solid adsorbent system.
EQUILIBRATION OF SOLUTE BETWEEN THE PHASES
21
Binary two-phase liquid-solid system A system is considered in which a liquid ( m ) is in intimate contact with an adsorbent (s). Both substances display certain intermolecular cohesion forces and thus certain surface tensions. Hence, at the interface, cohesion forces occur'from both sides so that the interfacial tension is lower than the surface tensions. In this event, the interfacial tension, om, can be expressed as us- = us - om, where us and am are the surface tensions of components s and m , respectively. In contrast to gas-solid systems, the formation of an adsorption layer of increased density is impossible with liquids, as they are incompressible.
Binary single-phase liquid system This system is simply a mixture of two miscible liquids. Strictly, such a system should be considered as a ternary two-phase system if the liquid is assumed to be in contact with air. As mentioned above, a pure liquid can attain a minimum free surface energy by reducing its surface area to a minimum. A mixture of two or more liquids has yet another possibility of reaching a state of minimum surface free energy. If the components have different surface free energies, the system will try to remove from the surface layer those molecules the presence of which requires a higher surface energy, while accumulating in the layer the molecules of the other components. Thus, there will be a higher concentration of the component of lower surface tension in the surface layer than in the bulk phase at equilibrium. Such a segregation can, of course, occur only in systems that involve a surface-active compound.
Binary liquid mixture-solid adsorbent system In this system, a difference in the solute concentration in the bulk liquid and at the liquid-solid interface can be achieved by virtue of both of the mechanisms described in the sections Binary two-phase liquid-solid system and Binary single-phase liquid system. It can be inferred from the previous discussion of the problem of adsorption that the presence of a solid adsorbent in a binary liquid mixture can lead to three different situations: in the first two, the chemical nature of the adsorbent can be such that either the solute or the solvent molecules will be preferred in interactions with the solid surface; the third possibility is that there is no preference for either compound. The larger the extent of the interactions, the greater is the effect of decreasing the interfacial tension by the presence of the respective component in the interfacial layer and, consequently, the greater is the enrichment of the layer by t h s component. If the adsorbent does not discriminate between the solute and solvent in the above respect, some enrichment of the interfacial layer can still occur as a result of the mechanism discussed in the section Binary single-phase liquid system, provided the component to be adsorbed is a surface-active one. In any event, the adsorbent provides a large interfacial area, thus making the total amount of the component accumulated in the adsorbed layer appreciably large. The above concepts are expressed quantitatively by the Gibbs adsorption isotherm, References p.23 .
22
BASIC PROCESSES IN CHROMATOGRAPHY
which can be written for dilute solutions in the form (2.24) where r is the so-called concentration excess of the solute component in the interfacial region, c' is the molar concentration of this component in the bulk liquid, R is the gas constant, and u is the interfacial tension. The dependence of the interfacial tension on c' can be described by the empirical equation (Szyszkowski) uo - u = ah(1
+ bc')
(2.25)
where uo is the interfacial tension in the adsorbent-pure solvent binary system and a and b are constants. The differentiation of u with respect to c' and substitution into eqn.
2.24 gives (2.26) Eqn. 2.26 actually represents the Langmuir adsorption isotherm, where a/RT= a' is the maximum attainable concentration of solute in the interfacial layer (c' % l/b). The quantity r can be expressed from the following solute mass balance. Let n be the number of moles of solute component in the initial solute-solvent mixture of volume V . After adding an adsorbent to the solution, the solute wdl distribute itself between the bulk and interfacial phases. At equilibrium, the situation can be described by
n = Vc = c'( V - V,)
+ Q''
(2.27)
where c is the solute concentration in the initial solution, c' and c'' are the solute concentrations in the bulk phase and in the interfacial adsorption layer of the liquid-solid system, respectively, and V, is the volume of the interfacial layer. I? is defined as the difference between the number of moles of solute in the initial solution and in the bulk phase after adding a weight amount w, of the adsorbent, related td'l g of the adsorbent. Thus, dividing both sides of eqn. 2.27 by ws and rearranging gives
r = ( v/w,) (C - c') = ( v,/w,)
(c"
- c')
(2.28)
As V,c"/w, can be expressed as n"/w,, i.e., the number of moles of the adsorbed solute per unit weight of the adsorbent, the quantity r can be expressed by
r = (n"/w,)
-
(2.29)
( v,/w,)c'
On combining eqns. 2.29 and 2.26, we obtain -n"= a * WS
.bc' + -V- ca 1
+ bc'
w,
'
(2.30)
The ratio V,/w, in eqn. 2.30 expresses the volume of the interfacial adsorption layer per gram of the adsorbent. This volume is small, and if, in addition, c' is also small, the term can be neglected. Hence, at very low solute concentrations (c' < l/b), eqn. 2.30 can be reduced to
n"/ws = a* bc'
(2.31)
23
REFERENCES
Eqn. 2.31 is very suitable for expressing the distribution constant. As c' = n'/Vm, where n' is the number of moles of solute in a volume V, of the solvent (mobile phase), we can write
(2.32) where the subscript a in K, differentiates between the adsorption distribution constant and that defined for liquid-liquid systems ( K ) .
REFERENCES Carman, P. C., Trans. Inst. Cbem. Eng., 15 ( 1 937) 150. Crank, J., TbeMatbematics of Diffusion, Oxford Univ. Press, New York, 1956. Darcy, H., Les Font.7ines Publiques de la Ville de Dgon, Pans, 1856. Ehrenfest-Afanassjewa, T.,Mutb. Ann., 77 (1916) 259. Ergun, S., Cbem. Eng. Progr.,48 (1952) 89. Ergun, S a n d Orning, A. A.,Ind. Eng. Cbem., 41 (1949) 179. Frenkel, J., Kinetic Theory of Liquid, Clarendon Press, Oxford, 1946, Ch. IV. Cambill, W. R., Cbern. Eng. (London), June 30 (1958) 113. Giddings, J. C., Dynamics ofCbromatograpby, Marcel Dekker, New York, 1965a, p.200. Giddings, J. C., Dynamics of Chromatography, Marcel Dekker, New York, 1965b, p.228. Konakov, P. K.,Izv. Akad. NaukSSSR, Otd. Tekb. Nauk, (1949) 240. Kozeny, J., Sitzungsber. Akad. wiss. Wien, Math.-Naturwiss. Kl., Abt. 2B, 136 (1927) 271. Navier, M., Mem. Acad. Sci. Inst. Fr., 6 (1 827) 389. Schlichting, H.,Boundary Layer Theory, McCraw-Hill, New York, 1955, Ch. V. Schwartz, C. E. and Smith, J. M.,Ind. Wg. Chem.,45 (1953) 1209. Sonntag, G., Cbem.-Ing.-Tech., 32 (1960) 317. Stokes, G. G., Trans. Cambridge Phil. Soc., 8 (1845) 287. 'Szyszkowski, B.,Z. Phys. Cbem. (Leipzig!, 64 (1908) 385. Wilke,C. R. and Chang, P., AICbEJ., 1 (1955) 746.
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Chapter 3
General description of the chromatographic process J. NOVAK, J . JANAK and S. WICAR
CONTENTS
.. . . . .. . . . . . . . . . .. . . .. .. . . . . .. . . . . . . . .. . . . . . . . . . .. . .. . . . . .. . .. . . . .. . . . . . .. . . . . . . . . . . .. .. . . . . . . .. . . .. .. .. .. .. .. . . . . . .. . ... . . . . .
Solute mass balance in the chromatographic system . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Equations of the chromatographic zone . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 29 Concept of ideal linear chromatography . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Concept of the theoretical plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 33 Dynamics of zone spreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Non-uniformityofflow ....................................................... 36 Longitudinal diffusion in the mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Longitudinal diffusion in the stationary phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Non-equilibrium in the sorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 One-site adsorption kinetics (adsorption chromatography) . . . . . . . . . . . . . . . . . . . . . 37 Diffusion-controlled kinetics (par tition chromatography) . . . . . . . . . . . . . . . . . . 37 Non-equilibrium in the interparticle mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Non-equilibrium in the intraparticle mobile phase . . . . . . . . . . . . . . . . .. . . . 38 Combination of plate height contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Chromatographic resolution . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 40 Extra-column zone broadening . . . . . . . . . . . . . . . . . . . . ... . . . . . 42 References .................................................................... 43
..
..
SOLUTE MASS BALANCE IN THE CHROMATOGRAPHIC SYSTEM The transport of the solute mass in a chromatographic column takes place by two basic mechanisms - molecular diffusion and convection. The intensity, of both of these transport phenomena can be described by means of the vectors of diffusional and respectively), convective flow (subscripts Dj and
5,
&(T, r) = - Dj grad cj (r+,t )
The magnitudes of the above vectors indicate the velocity of the mass flux through a unit area and the direction of the vectors is identical with that of the mass flux at any point within the column. In eqns. 3.1 and 3.2, Dj is the diffusion coefficient of the j t h component of the given phase, u'is the forward velocity of the phase at a point of the position vector 7,and cj is the concentration of the jth component at the point of the position vector r'and at a time t in the given phase. Let us choose a volume element V enclosed by a continuous surface S in the interparticle space of the column through which the mobile phase flows. Further, let (.I& and (.I&.& References p.43
2s
GENERAL DESCRIPTION OF THE CHROMATOGRAPHIC PROCESS
26
be the components of the vectors, as defined by eqns. 3.1 and 3.2, normal to a surface element dS (see Fig. 3.1). The integral
s""bi',
+ (J+J
(3.3)
S
represents the difference between the rates of the supply and outflow of the mass of the jth component of the mobile phase within the element enclosed by surface S . It follows from the mass conservation principle that the value of the integral 3.3 must be equal to the rate of accumulation of mass of the component within the element, provided that no chemical reactions take place within the element. Hence,
Fig. 3.1. Representation of the solute mass fluxes in the interparticle space of the column.
According to the Gauss theorem, the surface integral on the right-hand side of eqn. 3.4 can be expressed by a volume integral over the volume closed by the surface S :
As the choice of the volume element in the mobile phase was arbitrary, eqn. 3.5 also holds true in the differential form:
(3.6)
The same treatment for a volume element of the stationary phase leads to a mass balance equation for the jth component in the stationary phase:
where c;.' is the concentration of component j in the stationary phase; the convective flow within the stationary phase can also be brought about by the diffusion fluxes. The system of eqns. 3.6 and 3.7 holds for any component in both phases, but the
SOLUTE MASS BALANCE IN THE CHROMATOGRAPHIC SYSTEM
27
solute component is of most concern in the context of the present discussion. At low solute concentrations in the column, the changes in the carrier liquid and stationary phase concentrations can be neglected and the convection in the stationary phase can be omitted. Hence, if the solute concentrations in the mobile and stationary phases are denoted simply by c’ and c’: respectively, the system of eqns. 3.6 and 3.7 becomes a system of two equations : at
= div (-
D’grad c’ + u’c’)
(3.6a)
(3.7a) provided that the solute components in both phases behave independently of each other at low concentrations. The complicated geometry of a packed column does not permit the boundary problem to be formulated analytically, thus hampering the solution of eqns. 3.6a and 3.7a for a packed column. The only means of obtaining approximate analytical solutions is the replacement of the local concentrations c‘(( t ) and c”(Z t ) and the local flow velocity by mean concentrations < c’(z, t ) > and < c”(z, t ) > and a constant vector , respectively. The averaging is carried out over the cross-sections normal to the column axis. This substitution will reduce significantly the problems that arise from the complicated pattern of the packed column, but it is necessary to employ a number of empirical coefficients and the results will be only approximate. Let us imagine a column of a circular cross-section cp and length L. The entire volume of the column is homogeneously filled either with a solid adsorbent or with a liquid stationary phase deposited on a solid support. The column void volume through which the mobile phase flows is E @ and the velocity of the latter within the void space is u = v/etp, where v is the volume flow-rate of the mobile phase. Hence, the vector < > is assumed to be always in a direction parallel to the column axis and of a magnitude equal to u. The volume occupied within the column by the packing is (1 - e&L. If the packing is a solid support coated with a stationary liquid, the volume of the latter within the column is q ( l - e)(PL,where 9 is the volume fraction of the stationary liquid of the entire column-packing volume. The discussion below will concern this type of packing. In the column idealized as shown above, if we consider a volume element bound within two sections normal to the axis and situated at distances z I and z 2 from the column inlet, the volume V of this element is V = cp ( z 2 - z,). The concept of the mean concentrations and the mean velocity of flow eliminates the problem of radial concentration gradients and radial components of the vector , and hence there will be no mass flux through the cylindrical surface enclosing the element. With exact models also there is zero mass flux through the column wall owing to the impermeability of the wall, but in the case discussed here, the above fact is part of the boundary conditions. When substituting mean values consistently into eqns. 3.1 and 3.2 in place of the local values and neglecting the longitudinal diffusion of solute in the stationary phase, as it proceeds on the very small cross-section q(l - e ) ~the , difference between the intake and outflow of solute mass in the mobile phase (see Fig. 3.2), as expressed for the above
z(3
References p.43
28
GENERAL DESCRIPTION OF THE CHROMATOGRAPHIC PROCESS
specified element, will acquire an especially simple form (cf, integral 3.3 of the exact model):
=e
J (-D'grad)dS S
Fig. 3.2. Solute mass balance in an idealized column.
On the other hand, the difference is equal to the rate of increase of the solute mass in the element of the volume q(z2 - zl),ie., in both phases of the element, which is given by the integrals
"M
VS
where V, = E V and Vs = q( 1 - E)K The formal difference, as compared with the exact equation 3.4, is already apparent here. Combining eqns. 3.8 and 3.9, employing the Gaussian theorem, and with regard to the arbitrary choice of the volume element, the mass balance for the column is also valid when expressed in a differential form: (3.10)
Eqn. 3.10 describes precisely the properties of the idealized column, but the behaviour of a real column of the same mean concentrations and mobile phase volume flow-rate, as with the idealized column, is characterized only approximately. For instance, there is no theoretically substantiated relationship between real and apparent diffusional flow in a packed column, < D grad c(< t ) > and D grad < c(z, t ) >, or between real and apparent convective flow, ~
y ) = (k/n)” exp [- h (v
-
y)*]
(5.7)
The information on the resolving power of the column is now contained in the parameter h (which may or may not depend on the elution volume); its relation to the variance u2 of a single-component peak centred at y is
(5.8)
h = 112 u2
Methods proposed for solving eqn. 5.6 are discussed in more detail in Chapter 3; in the present context, it is of interest that the resolution factor, h , is usually an increasing function of elution volume (May and Knight; h i t el al.; Tung, 1966b; Tung et al., 1966), although sometimes a minimum is observed (Hendrickson; Tung and Runyon; Yau et al., 1970).
Calibration of GPC column systems The hopes that GPC with well characterized porous packings could be developed into an absolute method for determining molecular weights and their distribution (Beau et al.) have not been substantiated, and calibration of GPC columns with narrow fractions of known molecular weight is necessary. This calibration is carried out simply by injecting a series of well defined fractions. The logarithms of their molecular weights are then plotted against the elution volumes measured at the peak maximum and the best line is drawn through the points either by hand or by means of some standard statistical procedure. It has been ascertained for a wide variety of polymers on various packings and with different solvents that a linear relationship of the form
logM=A
-
B
v,
(5.9)
is approximately valid. The unique properties and advantages of a linear semi-logarithmic calibration curve have been discussed by Bly (1969). It is usually necessary to combine several columns (capable of separating polymers of different maximum molecular weights) in series in order to obtain good linearity of the calibration curve. The effect of some operational variables on the reliability of calibration was studied by several workers. Boni et al. (1 968a), Lambert, Meyerhoff (1965a) and Rudin exanlined the effect of sample size on retention volume and discussed methods of minimizing it. It is recommended that the calibration should be carried out at the same values of the operational variables (flow-rate, temperature) as used in the subsequent analyses of the given polymer. In this manner, it is possible to obtain molecular weights and distributions of good accuracy from GPC data, and the calibration curve remains unchanged for a long time, although some deterioration of column efficiency has been reported (Hazel1 et al.). References p . 6 6
64
GEL PERMEATION CHROMATOGRAPHY
The necessity to have, for each analyzed polymer, a series of narrow fractions of known molecular weights for calibration is a serious disadvantage. Attempts to construct calibration curves on the basis of polymers with broad molecular-weight distributions have been reported by Cantow et al., Frank et al., Purdon and Mate and Weiss and CohnGinsberg, but the results seem to be less accurate than those from narrow fractions. Many workers have therefore proposed methods for the correlation of calibration curves for different polymers or polymer-solvent systems. The solution of this problem would permit calibrations to be made with polystyrene or polypropylene oxide fractions, which are commercially available, and then the calibration curve thus obtained to be used for other polymers of interest. Thus, Moore and Hendrickson proposed the plot of extended
Id
I8
20
22
24
26
28
ELUTION VOLUME (COUNTS 1
Fig. 5.3. Universal calibration in GPC (Grubisic e t a l . ) expressed as the dependence of the hydrodynamic volume parameter, [ p ] M ,on elution volume for several polymers and copolymers with different structures. 0 , Polystyrene; 0 , “comb-type” branched polystyrene; +, “star-shaped’’ branched polystyrene; X, poly(methy1 methacrylate); 0 , poly(viny1 chloride); “ladder-type” polyphenylsiloxane; 0,polybutadiene; a,graft styrene-methyl methacrylate copolymer; A, heterograft (block) copolymer polystyrene-poly(methy1 methacrylate)-polystyrene. Results obtained on four Styragel columns in tetrahydrofurane (1 ml/rnin) at room temperature.
.,
PHYSICAL BASIS OF THE SEPARATION PROCESS
65
chain-length versus retention volume as the basis of such a universal calibration method, but it soon became apparent that significant differences between individual polymers persist (Salovey and Hellman). Meyerhoff (1965b) used the product of the effective hydrodynamic radius and the square root of the molecular weight as a universal parameter, but the suggestion of Benoit et al. and Grubisic et al. that the effective hydrodynamic volume is a decisive factor in the elution behaviour of different polymers on GPC columns was soon widely accepted. This universal calibration parameter has a sound physical basis and is easy to apply, as the hydrodynamic volume of a polymer at a given temperature in a certain solvent is proportional to the product [q] ' M ,and the intrinsic viscosity, [q], can easily be measured. Fig. 5.3 shows the excellent agreement of calibration curves plotted as log ( [ V I M )versus V, for several very different polymers, including linear and branched polystyrenes, two grafted styrene-methyl methacrylate copolymers with different structures, polybutadiene, poly(viny1 chloride), poly(methy1 methacrylate) and stiff-chain ladder polyphenylsiloxane. These results have been confirmed by other workers (Boni et al., 1968b; Dawluns and Hemming; Le Page et al. ; Rohn; Wild and Guliana). Some conflicting evidence has also been published (see Crouzet et al.; Meyerhoff, 1965a,b; Swenson e t al.). Recently, Rudin and Hoegy attributed part of this discrepancy to the fact that the hydrodynamic volume of the solvated polymer a t infinite dilution is implicitly introduced into the universal calibration parameter, [q]M , whereas the calibration is performed at finite concentrations of the solute, and they proposed a modified procedure in order to remove the apparent discrepancies. If it is desirable to correct the chromatogram for imperfect resolution prior to converting it into the molecular-weight distribution, the columns must also be calibrated with respect to the resolution factor, h (see eqn. 5.7); this calibration is carried out either by the already mentioned reversed-flow technique (Tung, 1966b; Tung et al.), by a method using the leading edge of chromatographic peaks (Tung and Runyon) or by means of the additivity of variance method proposed by Hendrickson (see also May and Knight). Balke and Hamielec also proposed a method that obviated the use of the tedious and timeconsuming reversed-flow technique. A method that is particularly attractive for its simplicity has been proposed by Kendrick: he assumed the validity of the calibration curve according to eqn. 5.9 and further assumed that the molecular-weight distribution of all fractions can be adequately approximated by the log-normal distribution (this latter approximation is certainly valid for the narrow fractions' that are normally employed for calibration purposes). Under these conditions, an analytical solution of eqn. 5.6 exists (Tung, 1966a) and the following relationship can serve for determining the spreading factor, h , from the chromatogram of a fraction and from its known polydispersity: (5.10)
where the left-hand side is the height of the peak divided by the total area of the chromatogram, fl= 2 In (a,,,/G,,)and B is the slope of the calibration curve, defined by eqn. 5.9; fi, and M , are the weight- and number-average molecular weights of the fraction, respectively. References p.66
66
GEL PERMEATION CHROMATOGRAPHY
REFERENCES Ackers, W., Biochemistry, 3 (1964) 723. Algelt, K. H. and Moore, J. C., in M. J. R. Cantow (Editor), Polymer Fractionation, Academic Press, New York, London, 1 9 6 7 , ~123. . Balke,S.T.and Hamielec, A. E.,J. Appl. Polym. Sci.,l3 (1969) 1381. Beau, R., Le Page, M. and De Vries, A. J., Appl. Polym. Symp., 8 (1969) 137. Benoit, H., Grubisic, Z., Rempp, P., Decker, D. and Zilliox, J.,J. Chim. Phys., 63 (1966) 1507. Biesenberger, J . A. and Ouano, A.,J. Appl. Polym. Sci., 14 (1970) 471. Billmeyer, Jr., F . W., Johnson, G. W. and Kelley, R. N., J. Chromatogr., 34 (1968) 316. Billmeyer, Jr., F. W. and Kelley, R. N., J. Chromatogr., 34 (1968) 322. Bly, D. D.,J. Polym. Sci., PLvtC, 21 (1968) 13. Bly, D. D., Anal. Chem., 41 (1969) 477. Boni, K. A., Sliemers, F. A. and Stickney, P. B.,J. Polym. Sci., Part A-2,6 (19684 567. Boni, K. A., Sliemers, F. A. and Stickney, P. B.,J. Polym. Sci., Part A-2,6 (1968b) 579. Brewer, P. J., Nature (London), 188 (1960) 934. Cantow, M. J . R. and Johnson, J. F., J. Polym. ScL, Part A-I, 5 (1967) 2835. Cantow, M. J. R., Porter, R. S. and Johnson, J. F.,J. Polym. Sci., Part A-I, 5 (1967) 1391. Carmichael, J. B.,J. Polym. Sci., Part A - 2 , 6 (1968) 517. Casassa, E. I;., J . Polym. Sci., Part B, 5 (1967) 773. Casassa, E. F., J. Phys. Chem., 75 (1971) 3929. Casassa, E. F. and Tagami, Y.,Macromolecules, 2 (1969) 14. t o u p e k , J . a n d H e i t z , W.,Makromol. Chem., 112(1968)286. Crouzet, P., Martens, A. and Mangin, P.,J. Chromatogr. Sci., 9 (1971) 525. Dawkins, J. V. and Hemming, M., Makromol. Chem., 155 ( 1972) 7 5 . Determann, H.,Angew. Chem., 76 (1969) 635. DiMarcio, E. A. and Guttman, C. M., J. Polym. Sbi., Part B, 7(1969) 267, DiMarcio, E. A. and Guttman, C. M.,Macromolecules, 3 (1970) 131. Flodin, P.,J. Chromatogr., 5 (1961) 103. Frank, F. C., Ward, I. M. and Williams, T.,J. Polym. Sci., Part A-2,6 (1968) 1357. Gelotte, B., in A. T. James and L. J. Morris (Editors), New Biochemical Separations, Van Nostrand, Princeton, N. J., 1962, p.93. Giddmgs, J . C., Dynamics of Chromatography. Part 1 : Principles and Theory, Marcel Dekker, New York, 1965. Giddings, J. C., Anal. Chem., 39 (1 967) 1027. Giddings, J . C.,Anal. Chem.,40 (1968) 2143. Giddings, J. C. and Eyring, H.,J. Phys. Chem., 59 (1955) 416. Giddings, J . C., Kucera, E., Russel1,C. P. and Myers, M. N., J. Phys. Chem., 7 2 (1968) 4397. Giddings, 3. C. and Mallik, K. L., Anal. Chem., 38 (1966) 997. Grubisic, Z., Rempp, P. and Benoit, H.,J. Polym. Sci., Part B, 5 (1967) 753. Guttman, C. M. and DiMarcio, E. A.,Macromolecules. 3 (1970) 681. Haller, W. J.,J. Chromatogr., 32 (1968) 676. Hazell, J . E., Prince, L. A. and Stapelfeldt, H. E.,J. Polym. Sci., Part C, 21 (1968) 43. Hendrickson, J . G . , J. Polym. Sci., Part A-2, 6 (1968) 1903. Hermans,J. J.,J.Polym. Sci., PartA-2,6 (1968) 1217. Kelley, R. N. and Billmeyer, Jr., F. W., Anal. Chem., 41 (1969) 874. KeUey, R. N. and Billmeyer, Jr., F. W., Anal. Chem., 42 (1970a) 399. Kelley, R . N. and Billmeyer, Ji-., F. W., Separ. Sci., 5 (1970b) 291. Kendrick, T. C . , J. Polym. Sci., Part A-2,7 (1969) 297. K u b h , M., Collect. Czech. Chem. Commun., 30 (1965) 1104 and 2900. Lambert, A.,Polymer, 10 (1969) 213. Laurent, T. C. and ffillander, J.,J. Chromatogr., 14 (1964) 31 7. Le Page, M., Beau, R. and De Vries, A. J., J. Polym. Sci., Part C, 21 (1968) 119.
REFERENCES
67
Lindqvist, B. and Storgbds, T., Nature (London), 175 (1955) 511. McQuarrie, D. A., J. Chem. Phys., 38 (1963) 437. May, Jr., J. A. and Knight, G. W., J. Chromatogr.,55 (1971) 111. Meyerhoff, G., Ber. Bunsenges. Phys. Chem., 69 (1965a) 866. Meyerhoff, G., Makromol. Chem., 86 (1965b) 282. Moore, J . C., J. Polym. Sci.. Part A , 2 (1964) 835. Moore, J . C. and Hendrickson, J. G., J. Polym. Sci., Part C, 8 (1965) 233. Morris, J . C. 0. R. and Morris, P., Separation Methods in Biochemistry, Wiley-Interscience, New York, 1963. Ogston, A. C., Trans. Faraday Soc., 54 (1958) 1754. Ouano, A. and Biesenberger, J . A., J. Appl. Polym. ScL, 14 (1970) 483. Porath, J.,J. Pure Appl. Chem., 6 (1963) 233. Porath, J . and Ilodin,P.,Nature (London), 183 (1959) 1657. Pouchlq, J . , Collect. Czech. Chem. Cornmun., 28 (1963) 1804. Pouchly, J.,J. Chem. Phys., 52 (1970) 2567. Purdon, Jr., J. R. and Mate, R. D.,J. Polym. Sci., Part A - I , 6 (1968) 243. Rohn, C. L.,J. Polym. Sci.,Part A-2,5 ( 1 967) 547. Rudin, A.,J.Polym.ki., P a r t A - 1 , 9 ( 1 9 7 1 ) 2 5 8 7 . Rudin, A.and Hoegy, H. L. W . , J . Polym. Sci., Part A - I , 10 (1972) 217. Salovey, R. and Hellman, M. Y ., J. Polym. Sci., Part A-2,5 ( I 967) 333. Smit, J . A. M., Hoogervorst, C. J . P and Staverman, A. J . , J. Appl. Polym. Sci., 15 ( 197 1 ) 1479. Smith, W. B . and Kollmansberger, A., J. Phys. Chem., 69 (1965) 4157. Swenson, H. A., Kaustinen, H. M. and Almin, K.-E., J. Pofym. Sci., Part B , 9 (1971) 261. Synge, R. L. M., Inst. Int. Chim. Solva.v, Cons. Chim. (Rapp. Discuss.), 9 (1953) 163. Tung, L. H., J. Appl. Polym. Sci,10 (1966a) 315. Tung, L. H., J. Appl. Polym. S c i , 10 (1966b) 1271. Tung, L. H., Moore, J . C. and Knight, G. W., J. App2. Polym. Sci., 10 (1966) 1261. Tung, L. H . and Runyon, J . R., J. Appl. Polym. Sci., 13 (1969) 2397. Vaughan, M. I:.,Nature(London), 188 (1960) 5 5 . Verhoff, F. M. and Sylvester, N. D., J. Macromol. Sci.,Chem., 4 (1970) 979. Vink, H., J. Chromatogr., 5 2 ( 1 970) 205. Weiss, A. R. and Cohn-Ginsberg, E.,J. Polym. Sci., Part A-2, 8 (1970) 148. Wild, L. and Guliana, R., J. Polym. Sci., Part A-2,5 (1967) 1087. Yau, W. W. and Malone, C. P.,J. Polym. Sci., Part B , 5 (1967) 663. Yau, W. W. and Malone, C. P., Polym. Prepr., Amer. Chem, Soc., Div. Polym. Chem., 1 2 (1 971) 797. Yau, W.W., Malone, C. P. and Fleming, S . W., J. Polym. Sci.,Part B , 6 (1968) 803. Yau, W. W., Malone, C. P. and Suchan, J . L., Separ. Sci., 5 (1970) 259.
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chapter 6
Fundamentals of ion-exchange chromatography 0. MIKEB CONTENTS Principles and terminology ....................................................... Characterization of ion exchangers ................................................. Reactions, affinity and selectivity in ion exchange ..................................... Ionexchange equilibria and kinetics ................................................ Column operation and ion-exchange chromatography ................................... Ion exclusion, ion retardation, the ionsieve process and partition chromatography on ion exchangers ................................................................ Ligandexchange chromatography .................................................. Ion exchange in non-aqueous solutions .............................................. References ...................................................................
69 73 75 77 80
83
85 85 86
PRINCIPLES AND TERMINOLOGY Ion exchangers can be defined as polyvalent materials that are insoluble in water, contain bound ionogenic groups and are capable of dissociating and exchanging ions in solutiorl. Sometimes the shortened term ionex is used instead of ion exchanger. In spite of the fact that there are natural ion exchangers, most ion exchangers have been prepared synthetically. Natural and synthetic ion exchangers may consist of inorganic or organic materials and are usually solid substances, but liquid ion exchangers are used in special circumstances. Ion exchangers and their properties have been described in thousands of papers and tens of monographs, and many of these articles also deal with chromatographic aspects. The most important publications in this field since 1960 are those by Dorfner (1963a, b), Helfferich( 1962a), Hering, Inczedy, Marinsky, Osborn, Paterspn, Reuter, Saldadze et al. and Samuelson. Griesbach (now Reuter) is producing a comprehensive German work in numerous volumes. There are specialized chapters on ion exchange in monographs on chromatographic methods by Flaschka and Barnard, Genge, Kunin, Mikes’, Morris and Morris, and Walton (1 967). Systematic reviews have been published every other year, the last three being by Walton (1968, 1970, 1972). In a typical ion exchanger (see the schematic representation in Fig. 6.1), there are two main components: a porous matrix (or network) and electrically charged, covalently bound functional ionogenic groups. Ion exchangers can be divided into four main groups, depending on the composition of the matrix: (1) inorganic exchangers, based on aluminium silicates and other suitable minerals; (2) synthetic resins of many types; (3) ion-exchange cellulose; (4) ion-exchange polydextran. References p . 8 6
69
70
FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY C
A
Fig. 6.1. Schematic representation of resinous ion-exchange particles. A = Anion exchanger; C = cation exchanger. The lines illustrate the polymer chains and cross-linking of the matrix, and the charges in circles the functional ionogenic groups. Counter-ions have been omitted.
There are five principal classes of functional groups present in ion exchangers, and hence exchangers can be classified on this basis as follows: (1) cation exchangers; ( 2 ) anion exchangers; (3) amphoteric and dipolar ion exchangers; (4) chelating ion exchangers; (5) selective (or specific) ion exchangers. Cation exchangers are high-molecular-weight polyvalent insoluble anions (polyvalent acids), the ionogenic groups of which are saturated with individual soluble cations. These are able to dissociate when the exchanger comes into contact with an aqueous solution, and thus may be exchanged, e.g. : COONa COONa
-
COO-Na’ COO-Na+
CaCI2-
Ca2+
+
2 NaCl
The shortened term catex is also used instead of cation exchanger. An anion exchanger (anex) is a high-molecular-weight polyvalent insoluble cation (polyvalent base), which is able to exchange electrostatically bound soluble anions, e.g. :
Amphoteric ion exchangers are polyvalent insoluble “zwitter-ions” (polyvalent “inner salts”), which dissociate in aqueous solution without the release of ions into the solution. However, they are then able to bind dissociated salts from the solution, cations to anionic groups and anions to cationic groups, e.g. :
71
PRINCIPLES A N D TERMINOLOGY COO' Na' &R3CI-
*
$$O0-
+
NaCl
NR3
This binding represents a dynamic equilibrium. After washing with a large amount of water, the original internally neutralized form of the exchanger is regenerated and the salt is released. It is difficult to prepare these exchangers with exact equivalence of ionogenic groups. Dipolar ion exchangers can be prepared by binding amino acids (e.g., arginine via amino group) to the matrix. These dipolar ion exchangers are used advantageously for the separation of biopolymers. Chelating ion exchangers are resins prepared by incorporating complex-forming groups, e.g., iminodiacetic acid. These ion exchangers retain only specific ionic groups (e.g., heavy metals or alkaline earths) and are therefore more selective than cation exchangers. An example is given below, in which M2+represents, for example, Cuz+,NiZ+,ZnZ+,Co2+or
uop:
Selective (or specific) ion exchangers are synthesized experimentally for a specific purpose. They contain functional groups that are able to retain only one type of ion or a very limited number of types. For example, Skogseid synthesized a resin with trinitrophenyliminodinitrophenyl groups. These groups specifically bind potassium ions:
This type of ion binding cannot be considered as a true ion exchange, but is more closely related to potassium precipitation by hexanitrodiphenylamine (dipicrylamine). The principle of selective ion exchange resembles that of affinity chromatography. The homogeneity of functional groups is very important in ion-exchange chromatography. Exchangers can be classified as monofunctional if they contain only one type of ionogenic or chelating group; these are sometimes called homoionic exchangers, and are most suitable for chromatography. Cheaper polyfunctional exchangers are used only for certain technical purposes. The presence of different kinds of ionogenic (or chelating) groups leads to a loss of resolution in the chromatography. The terminology of ions should now be explained. Let us consider a homoionic exchanger, in which there is one type of charged functional group covalently bound to the matrix (cf Fig. 6.2). In every case, dissociable counter-ions with an opposite charge are bound to it by electrostatic forces and thus form part of the ion exchanger. Counterions are mentioned in the literature and in commercial pamphlets when the ionic form of an ion exchanger is illustrated. Co-ions play an important role in the mechanism of ionexchange chromatography and are ions with the same type of charge as the functional group, but they are soluble and capable of forming salts, acids, bases or water with counter-ions. The co-ions compete with the functional group in attracting counter-ions, References p.86
72
FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY CI -
CI-
clCI -
CI-
Na+
CI-
-2’
car
CI -
Na’
CINa b- CI-
CI
CI-
CINa’
Na+
CI-
CI -
-
Na+
Fig. 6.2. Scheme of cation exchange. Left: bead of cation exchanger in the Na+ form (the negative circles represent carboxylic functional groups, Na’ the counter-ions in the ion-exchanger, Ca2+the counter-ions in solution and Cl- the co-ions); right: bead after exchange. The process can be expressed by the equation 2(R-COO-Na+)
+ CaCl, * (R-COO-),CaZ+ + 2 NaCl
where R = resin, Caz+are the counter-ions in the ionexchanger, Na+ the counter-ions in solution and
c1- the co-ions.
thereby facilitating the exchange of the desired counter-ion. Examples to illustrate this terminology by Helfferich (1962a) are as follows: o p e of exchanger
Cation Cation Anion Anion
Functional POUP
-coo- $0;
-y(CH3)3 -N(CH,
Counter-ion in ion exchanger (ionic form of exchanger)
Co-ion in solution
Counter-ion in solution [to be exchanged)
N a+ H+ C1OH-
CIc1N a+ N a+
Caz+
K+ OHCH, COO-
The first of these four examples is represented schematically in Fig. 6.2. The complete change of counter-ions in an ion exchanger is called cycling, and sometimes the term “cycle” is used instead of “ionic form”. Cycling at the end of an ionexchange operation to produce the ion exchanger in its original form is called regeneration.
73
CHARACTERIZATION OF ION EXCHANGERS
CHARACTERIZATION OF ION EXCHANGERS Information describing the properties of individual ion exchangers is necessary before they can be used for chromatography. It is usually presented in the form of tables (cf: Chapter 13). First the type of exchanger must be known. Both cation and anion exchangers are classified according to the nature of the active groups, as shown below: Ion exchanger
5Pe
Usual functional group
Cation exchanger
Strongly acidic Medium acidic Weakly acidic Strongly basic Medium basic
Sulphonic Phosphonic Carboxylic Quaternary ammonium Mixture of tertiary and quaternary ammonium groups Amines, polyamines
Anion exchanger
Weakly basic
The ionic form of a commercial ion exchanger is usually indicated by the manufacturer. Cation exchangers are produced in the acidic form, designated H'(or H*),or in the salt from @a+, L,i+ and others). Anion exchangers are produced as the free base, designated O H (or B*), or as salts (usually Cl-). The type of matrix (lattice) of an ion exchanger must be considered carefully. Polystyrene or polyacrylic types find a wide application, while phenolic types are, in general, not suitable for chromatography, because phenolic R-OH groups dissociate in alkaline media and thus form an additional cation exchanger group, R-0- , with different properties from those of the main functional group. For the chromatography of many biopolymers, polystyrene or other aromatic matrices are not suitable because of their denaturating effect. Polyacrylic types are better, but a cellulose or a polydextran matrix is usually the best. The degree of cross-linking of the matrix is very important in chromatography, and defines the average porosity of exchangers. The symbol X, accepted as a measure of the degree of cross-linking, represents the percentage of divinylbenzene in the styrene polymerizationmixtureused to prepare this type ofresin(cf: Chapter9, Ion-exchange materials). The process ofcross-linkingis easily controlled and therefore it is possible to produce a resin withaporositysuitable foragivenpurpose,and in commercial resins it varies from X1 to 16, X2 to X9 being most often used. The more cross-links present, the less an exchanger swells. The functional groups of individual exchangers have the same electric charge and therefore they have the tendency to extend the network to a maximum, and this process is accompanied by the hydration of functional groups and bound ions. The strength of the repulsing force is influenced by the type of functional group, ionic strength, pH and the nature of any bound ions, but it is strictly limited by the cross-linking. Swelling is reversible and can be considered as a state of balance between the tension of the elastic network and the osmotic pressure of the inside solution, arising from the presence of counter-ions. Volume changes in swollen resins, due to changes in the composition of the surrounding solution, some times disturb the chromatographic operation, and these difficulties occur more often with resins with a low degree of cross-linking. However, the lower cross-linked resins change ions more rapidly, but they are less selective. As a result References p.86
74
FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY
of variations in the cross-linking reactions, Pepper et al. recommended the use of water regain values (W.R.) to characterize ion exchangers instead of the X values. The water regain is defined as the maximum weight in grams of water taken up to 1 g of completely dry ion exchanger. The relationshp between the X and W.R. values for strongly acidic cation exchangers is shown below: X
w.R.
X
W.R.
2 4 6 8
3.45 1.92 1.36 1.04
10 15 20 25
0.83 0.59 0.48 0.38
The cross-linking and porosity are equivalent terms used for ion-exchange resins, but this terminology is not valid in the case of macroreticular resins (cf, Chapter 9). The cross-linking defines here the composition of the matrix only; very large pores of macroreticular resins are best expressed by the maximal molecular weight of substances penetrating the beads. The capacity of an ion exchanger is a measure of the total amount of ions the resin is able to bind and is usually expressed as milliequivalents (mequiv.) per gram of dry resin (in the Hi or C1- form) or as milliequivalents per millilitre of fully swollen wet resin (in the H'or C1- form) packed in the bed. T h s value, which is a measure of all charged groups present, is not usually achieved in practice, but is lower for various reasons, e.g., operating under non-equilibrium conditions. Factors that influence the available capacity are concentration and ionic strength of the eluent, pH, temperature, the accessibility of functional groups and the nature of the counter-ions. The dependence of the ion-exchange capacity on pH is illustrated by the titration curves in Fig. 6.3. It can be seen that for strongly acidic and strongly basic exchangers, the capacity is virtually independent of pH and they can therefore be used over a wide pH range. However, the capacity of weakly acidic cation exchangers and of weakly basic anion exchangers is strongly dependent on pH, so the use of weakly acidic exchangers is therefore limited in media of low pH and weakly basic anion exchangers are not very efficient in alkaline media. A
C 1
0
5
10 HC I (rnequiv /g)
0
10
5 NaOH(mequiv/g)
Fig. 6.3. Titration curves of ion exchangers in a dilute solution of neutral salts. A = anion exchangers; C = cation exchangers; a = strongly basic anion exchanger or strongly acidic cation exchanger;b = medium basic or acidic exchanger; c = weakly basic anion exchanger (mine-type) or weakly acidic (carboxylic) cation exchanger.
75
REACTIONS, AFFINITY A N D SELECTIVITY
Particle size and particle form are important characteristics of ion exchangers. The particle size determines how quickly equilibrium is established and hence influences the sharpness of a chromatographic separation: the smaller the particle size, the sharper is the separation. If the particles are too small, the flow resistance of the chromatographic column is increased and higher pressures are required for elution and care must therefore be taken when choosing a suitable grain size. The particle form is also an important factor. Ion exchangers are delivered either in the form of grains prepared by grinding the resin gel (these have irregular shapes) or as uniform beads (spheres), prepared by polymerization in an emulsion. Generally, the latter are better for chromatography, because they do not pack the column so tightly and consequently there is a lower flow resistance. The bead form possesses better mechanical properties and hence the losses caused by friction are lower. The particle size is usually expressed in terms of the size range of dry copolymer beads before any ionic groups are attached, and is measured by standard mesh screens. Sometimes the particle size is expressed as the wet mesh range after maximal swelling". The wet mesh size depends, of course, on the many factors mentioned in the preceding paragraphs. Millimetres (mm) and microns (pm) are also used to measure grain size. For the conversion table for U S . standard mesh screens, see Table 1 1.1 (p.286). For fine chromatographic separations, it is important that the particle size should be as uniform as possible. The narrower the variation in grain size, the sharper is the separation obtained.
REACTIONS, AFFINITY AND SELECTIVITY IN ION EXCHANGE Exchange reactions of simple ions are best described in terms of an ionic redistribution between the ion-exchange gel and the aqueous phase. These reactions are always stoichiometric because the electroneutrality of the resin must be maintained. As no covalent bonds are formed or broken during this process, there is little heat evolution or absorption accompanying ion exchange. The only exceptions are neutralization reactions involving a cation exchanger in the H+ form or an anion exchanger in the O H form, in which the formation of low-dissociated water is the source of heat. Ion exchange is, in general, a reversible process and therefore an equilibrium is obtained, e.g. : 2(R-SO;Na+)
+ CaZ++ (R-SO;)?
CaZ++ 2 Na'
where R = resin. This equilibrium depends not only on the relative affinities of ions for the exchanger, but also on the relative ionic concentrations. Therefore, ions with a low affinity for the exchanger can regenerate it and replace ions of a greater affinity, if the former are present at a higher concentration. In practice, this is made use of in the regeneration of water softeners: CaZ+and Mg2+are present in natural hard water at relatively low concentrations. Because they have a higher affinity for the cation exchanger (in the Na' form), they exchange with Na' . A large amount of water can be treated in this way, to produce water that contains only monovalent ions which do not precipitate *American manufacturers use U.S. standard mesh screens as their standard, and British manufacturers B.S.S. standard mesh screens.
'References p.86
76
FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY
soap. When the ion exchanger is exhausted, a small amount of concentrated sodium chloride solution quickly regenerates the ion exchanger to the original Na' form. The affinity of ions for an ion exchanger is sometimes called the "ion-exchange potential" and in dilute aqueous solutions it increases with the size of the ionic charge. Polyvalent ions are more strongly bound than monovalent ions, the affinity being proportional to the charge. For ions of the same charge, the exchange potentials are inversely proportional to the radius of the hydrated ions. Because the radii of many cations are inversely proportional to their atomic weights, the affinities of these cations can be arranged in order of atomic weights. The exchange potentials of cations are similar to or identical with the so-called lyotropic series. The affinity of anions is governed by similar rules. In addition, the exchange potentials increase with the ability of anions to polarize. Examples (cf. Kunin and Myers, and Nachod) of typical affinity sequences are given below. Composite affinity sequence for cations: (CH3)4w < Li' < Na' < NW4 < K' < Rb' < Cs' < T1' < Ag';Mg2+< CaZ+< Sr" < BaZ+;Fez+ < Co2+< NiZ+< Cuz+< Zn"; A13' < Sc3+< Lu3+< Yb" < Tm3+ < Er3+< Ho3+< Y3' < Dy" < Tb" < Gd3' < Eu3+< Sm3+< Pm3+ < Nd3' < Pr3+ < Ce3+< La3+.The position of €Ion ?strongly acidic cation exchangers is nearly the same as that of L f , and on weakly acidic (carboxylic) cation exchangers it is about the same as that of Ba". Composite affinity sequence for anions: fluoride < acetate < formate < chloride < bromide < chromate < molybdate < phosphate < arsenate CH2 CO-NNH
CH=CH*
I COOH
I
CHeCH2
CH-
-
I
I
CH2-CH-CH2-
CO-NH ‘CH2 CO-NH’
I
-CH-CHP-CH-CH~-CH-
I coo H
I COOH
CH2-
Some ion-exchange resins are prepared by condensation reactions. Heteroionic cation exchangers can be prepared by condensing formaldehyde with phenolsulphonic acid:
Weakly basic resins can be synthesized by condensation of ethylene dichloride with polyamines or of epichlorohydrin with polyethyleneimine: CH2-
C ,H2-
-NU-CHz-
CH-CH*-N\
I
OH
CHz-CH
NH -
-N H -
I OH
207
ION-EXCHANGE MATERIALS
An extensive survey of various methods of preparing synthetic organic ion exchangers was given by Helfferich.
Cellulose ion exchangers Cellulose fibres were found to be excellent carriers for ionogenic functional groups for the ion-exchange chromatography of proteins, nucleic acids and their high-molecularweight fragments. Their fibrous nature makes the functional groups accessible to macromolecules from the solution and their hydrophilic properties do not cause undesirable denaturing hydrophobic interactions that occur with other ion exchangers containing aromatic matrices. Native cellulose is a polycondensate of anhydroglucose monomers and is composed of thousands of cellobiose units:
I
0t i
Ch20 t i
The cellulose chains associate in fibres to form “crystalline” areas (micelles). The free hydroxyl groups in positions 2 and 6 (and sometimes also in position 3 ) can be easily substituted. Guthrie introduced ionogenic groups into cellulose, but these derivatives were not used in chromatography until Peterson and Sober, Porath, Sober et al. and Sober and Peterson described methods by which cellulose ion exchangers can easily be prepared in the laboratory and showed the importance of these materials in the chromatography of biopolymers. Cellulose swollen in alkali reacts with chlorine derivatives, which can be illustrated by the synthesis of carboxymethylcellulose from cellulose and chloroacetic acid:
I
OH
I
0-CH2-COOH
These ion exchangers are now commercially available in various forms (cf , Table 13.6, p. 346/347).
Ion-exchange denva tives of polydextran Ion-exchange derivatives of polydextran have favourable properties for the chromatography of biopolymers similar t o those mentioned for cellulose derivatives. Dextran is formed from sugar solutions by Leuconostoc mesenteroides and, after copolymerization with epichlorohydrin, cross-links are formed and the product represents a suitable carrier References 11.227
208
SORBFNTS
for ionogenic groups (Porath and Lindner). The structure of cross-linked polydextran is
-CH2
I . I
0
I
CHp
I I CH2 I
CHOH
I
OH
0
- & o & y p o ~ > o b -0
0-CH,
0-
OH
OH
OH
The free hydroxyl groups are available for substitution in the same way as with cellulose. In alkaline media, they react with suitable halogen derivatives t o produce ion exchangers. These ion exchangers are commercially available as SephadexFThey are produced with two porosities corresponding to Sephadex G-25 and GdO (cJ,Table 13.7, p. 348/349) and contain all necessary ionogenic groups. With respect to the porosity of the matrix, ionexchange chromatography of biopolymers on these derivatives can be influenced by the molecular sieving process. Functional groups of ion exchangers A survey of the most important functional groups in cation exchangers is given in Table 9.14 and in anion exchangers in Table 9.15. In ion-exchange resins, the ionogenic groups are often attached to aromatic nuclei of the matrix: -Cti2-CH-CH-
-CHz-CH
$ so;
-CH2-
c)
CH2
c H + -2 -
CHZ
I
so;
N (CH,), I
II
m
209
ION-EXCHANGE MATERIALS
(I = cation exchanger; sulphonated polystyrene; 11 = anion exchanger; chloromethylated polystyrene treated with a tertiary amine; 111 = cation exchanger; sulphonated phenolic resin). The functional groups can also be incorporated into the lattice in the form of aliphatic monomers. In ion-exchange derivatives of cellulose and polydextran, the functional groups are bound through the oxygen aioms of the hydroxyl groups. TABLE 9.14 FUNCTIONAL GROUPS IN ORGANIC CATION EXCHANGERS ~
~
Group
Formula
Type
Carriers
Sulphonic
-so;
Strongly acidic
Sulphomethyl Sulphoethyl (SE)
-CH, -SO; -C,H, -SO;
Strongly acidic Strongly acidic
Sulphopropyl (SP) Phosphonic
-C, H, -SO;
Strongly acidic
Aromatic nuclei in polystyrene or phenolic resins Aromatic nuclei in phenolic resins Hydroxyl groups in cellulose or polydextran; aromatic nuclei in a phenolic lattice Hydroxyl groups in polydextran
-Po:-
Moderately acidic
Carboxyl
-coo-
Weakly acidic
Carboxyniethyl (CM)
-CH, -COO-
Weakly acidic
(P)
Aromatic nuclei in resins; hydroxyl groups in cellulose or polydextran Polyacrylic resins or polyacrylic gels Hydroxyl groups in cellulose or polydextran; phenolic groups in resins
TABLE 9.1 5 FUNCTIONAL GROUPS IN ORGANIC ANION EXCHANGERS ~~
~~
Group
Formula
Trimethylaminomethyl Hydroxyethyldimethylaminomethyl Triethylaminoethyl (TEAE) Quaternary aminoethyl (QAE) Guanidinoethyl (GE)
-CH,h(CH3)3
Methylpyridinium Diethylaminoethyl (DEAE)
Carriers Strongly basic (type I) Strongly basic (type 11)
Aromatic nuclei in resins Aromatic nuclei in resins
- c , H , ~ ~H( c~ ,) ,
Strongly basic
-C,H, fi(C2H,),CH,CH(OH)CH,
Strongly basic
Hydroxyl groups in cellulose Hydroxyl groups in polydextran
-c, H, N H C ( ~ ~ H , )
Strongly basic
Hydroxyl groups in cellulose
'NH, -C,H,fiCH, -C,H~&H(C, H A
Strongly basic Intermediate basic
Polystyrene lattice Hydroxyl groups in cellulose and polydextran
-CH, fi(CH, ),C, H,Otl
II
Continued on p. 21 0 References p.22 7
210
SORBENTS
TABLE 9.1 5 (continued) Group
Formula
Type
Carriers
Mixed amino (ECTEOLA)
Undefined
Hydroxyl groups in cellulose
Mixed amino
I
Aminoethyl iAE) Polyethyleneimine (PEI) Alkyldmino
-c, H,AH,
Intermediate basic (mixture of weakly, moderately and strongly basic groups) Intermediate basic (mixture of tertiary and quaternary ammonium groups) Intermediate basic
p-Aminobenzyl (PAB)
-~H(cH,), -&YcH,),c, H,OH
-IC, H, I ~ H , ) ~ cH,,
f i ~ ~
Intermediate basic
AHR,
Weakly basic
-CH,C, H, h H 3
Weakly basic
Epoxy pol yamine lattice
Hydroxyl groups in cellulose Hydroxyl groups in cellulose Aromatic nuclei in polystyrene or phenolic polyamine resin Hydroxyl groups in cellulose
TABLE 9.16 FUNCTIONAL GROUPS IN SPECIAL RESINS Group Trimethylammoniummethyl and carboxyl
p - Manine Bis(carboxymethy1)iminomethyl Hydroquinoyl
Formula
l -coo>c ~ H - ( c H , -cH,~(cH,
=
-CHzN
)3
Type
Carriers
Amphoteric resin
Aromatic nuclei in polystyrene and polyacrylic resin Hydroxyl groups in polydextran Aromatic nuclei in resins
Dipolar ion I ~ C O O - exchanger Chelating resin CH,COOH
,
‘CH,COOH -c6
H 3 ‘2
Electron-exchange resins (redox resins)
Resin for resolution of enantiomers
Vinyl group of vinylhydroquinonedivinylbenzene copolymer Carbonyl group of carboxylic resin
Special exchangers
In addition to cation and anion exchangers, special types of ion exchangers have been developed (Table 9.16). Amphoteric resins are prepared by saturating a strongly basic anion exchanger with
21 1
ION-EXCHANGE MATERIALS
acrylic acid and polymerizing the latter (Retardation IIA8 is manufactured in this way from Dowex 1 ) . Another type is prepared by saturating a strongly acidic cation exchanger with vinylpyridine followed by polymerization. In both instances the linear polymer formed from the additive nionorners is trapped in the network of. the original resin and cannot diffuse out (Hale et al., Hatch et d.).Owing to the marked shape of the polyelectrolyte, these types of exchangers are also called “snake cage resins”. The introduced ionogenic groups with opposite charge are oriented to the vicinity of the functional groups originally present and are capable of interacting with them. These ion exchangers must be distinguished from mixed bed resins. The newest form of amphoteric ion exchangers are dipolar ion exchangers (Porath and Fornstedt, Porath and Fryklund). These exchangers are prepared by covalent binding of dipolar monomers (e. &, amino acids) to a suitable carrier (e. g., polydextran). They are used advantageously for the separation of biopolymers. Mixed bed resins are mixtures of beads of common cation exchangers in the H‘ form and anion exchangers in the OH- form and are used for the demineralization of water and aqueous solutions in a single column (a single bed). After being exhausted by desalting, the individual exchangers must be separated for regeneration, which is carried out separately, then the ion exchangers are mixed again. Chelating ion exchangers of the iminodiacetic acid type have been prepared (Pepper and Hale) from chloromethylated cross-linked polystyrene: -CH -CH2I
-CH
I
-CH2-
-CH-
CH2-
I
This type of resin is manufactured under the name Chelex 100. Other chelate ion exchangers have been reviewed by Nickless and Marshall and by Hering. Conventional ion exchangers exchange ions with the surrounding solution. Cassidy, and Updegraff and Cassidy prepared electron-exchange resins (redoxites), which are polymers containing groups capable of reversibly exchanging electrons with molecules in the surrounding solution. The redox resins can be prepared by polycondensation of hydroquinone with phenol and formaldehyde or by copolymerization of vinylhydroquinone with divinylbenzene:
-CH-CH2-
These resins were proposed as insoluble reducing and oxidizing agents. In spite of t\e References p.22 7
.
212
SORBENTS
large number of possibilities for utilizing them, they have not been used extensively in practice. Many unsuccessful attempts have been made to copolymerize optically active monomen into various resins in order to prepare materials that are capable of separating optical isomers from racemic mixtures. Grubhofer and Schleith prepared a carbonyl chloride derivative from a monofunctional carboxylic resin by treatment with thionyl chloride. This resin, containing --COCl groups, then underwent a reaction with quinine via esterification of its secondary alcohol group so that the asymmetric carbon atom'in the quinine molecule was retained :
(R = resin). This resin was used successfully for the column separation of racemic mandelic acid.
Form of ion exchangers Ion exchangers are usually solid, but liquid ion exchangers have been developed for special purposes. Solid exchangers can be prepared in the form of membranes, tubes and fibres for various ion-exchange applications, especially for continuous processes and electrophoresis. For column chromatography, the granular form of ion exchangers is the most usual. Ion exchangers are supplied commercially for these purposes as ground grains or, more usually, as spherical beads. The difference between them can be seen in Fig. 9.10. Grains prepared by grinding the dried gel have an irregular shape. In comparison with beads, they offer greater losses caused by friction, less mechanical resistance and greater flow resistance of the column. The bead form of ion exchangers has a regular spherical shape and is obtained by polymerization in suspension. The liquid monomer mixture is stirred with water in such a way that stable drops of reacting mixture are formed in the form of regular spheroids, whch are converted into solid spheres of the same size by polymerization (Groggins). The functional groups are introduced into the spheres by subsequent treatment, which does not destroy their form. The washed products are termed ion-exchange beads. Regular small beads can also be prepared by dropping the reaction mixture from capillary tubes of suitable dimensions. The bead form of ion exchangers, with its regular dimensions, is important in modem column chromatography, because the lower flow resistance of the beads in columns enables the particle size to be diminished and the greater mechanical stability permits the use of higher pressures, which results in a substantial increase in the speed of the chromatographic process. A special porous form of ion exchanger with large pores has been developed that
ION-EXCHANGE MATERIALS
213
Fig. 9.10. Microphotographs of granular ion exchangers. (A) Grains prepared by grinding of the gel; (B) beads prepared by submersion polycondesation in emulsion,
contains two types of cross-linkages (cfiSamuelson). Initial cross-linking is obtained in the normal way by copolymerization of styrene with small amounts of divinylbenzene in the bead. Additional cross-linking is achieved by Friedel-Crafts catalyzed methylene bridging in the swollen state. Dowex 21K is an example of this type of resin. It has volume changes and a capacity comparable with those of X8 resin but otherwise it has many of the desirable properties of X4 resin, especially a higher permeability and a faster equilibration rate. A special modern form of ion exchanger are the so-called macroreticular resins (Kun and Kunin; Kunin er al., 1962a, b). The beads (Fig. 9.1 1) are aggregates consisting of tiny granules (with diameters of a few hundred h g s t r o m s ) with large pores between them (diameter up to 1000 They are prepared by suspension copolymerization in the presence of a good solvent for monomers (styrene and divinylbenzene), which is simultaneously a poor swelling agent for the polymer. Macroreticular resins have a large internal surface area (several tens of m2/g). These ion exchangers allow the penetration of large molecules and are also able to operate in non-aqueous solutions. This type of resin was introduced commercially under the name Amberlyst. For high-speed analytical chromatography, pellicular ion-exchange resins have been developed (cfiKirkland, 1971). This type of ion-exchange bead consists of an inert solid core that displays no ion-exchange properties, covered with a thin shell of ion-exchange resin (Fig. 9.1 1). Diffusion into the very thin ion-exchange fdm lasts only a few seconds and equilibrium is reached very quickly in comparison with the usual beads. Therefore, this type of ion exchanger exhibits a much higher chromatographic efficiency than conven-
a).
References p.22 7
214
SORBENTS
Fig. 9.1 1 . Schematic representation of the inner structure of three types of ion-exchange beads. (1) Microreticular resins are composed of a gel containing micropores defined by the X value. (2) Macroreticular resin beads contain macropores (several hundred Angstroms wide) with a large inner surface area. The structural part is composed of a highly cross-linked gel with narrow micropores. (3) Pellicular resins (porous layer beads) contain a solid inert core surrounded by a shell (ca. 1 fim thick film) composed of cross-linked microporous ion-exchange gel.
tional ion-exchange resins. Kirkland (1969, 1970) published the following relative data: cation exchange of 5’-uridine monophosphoric acid 3.5 and anion exchange of 2-aminobenzimidazole 8 theoretical plates per second; conventional gel ion exchangers often give results of less than 0.1-0.5 theoretical plates per second. The carrier velocity has high values, e.g., 2-3 cm/sec in columns of 2-3 mm I.D. The non-porous impervious spherical silica support particles (3-40 pm) are uniformly coated with a thin ( I pm) porous crust of adsorbent and these types of beads are generally called controlled surface porosity ion exchangers*. The strongly acidic cation-exchange shell consists of a very stable fluoropolymer containing sulphonic acid groups with a capacity of 3.5 pequiv./g of Zn2+. The strongly basic anion exchanger has a capacity of 12 pequiv./g and contains tetraalkylamonium groups. These porous layer beads of ion exchangers are suitable for use in high-speed chromatography. The dry packing technique can be used for preparing the columns, because these ion exchangers undergo no detectable changes due to swelling when the pH and/or ionic strength or buffer composition are vaned. The dimensions of the columns used vary, with ratios of diameter to length from 1: 10 to very high values, and stainless-steel columns (straight or coiled) are used with lengths up to 3 m and 1.D.s of several milli*E. I. du Pont de Nemours & Co. (Wilmington, Del., U.S.A.) uses the trade-name Zipax for controlled surface porosity supports.
SORBENTS FOR AFFINITY CHROMATOGRAPHY
215
metres (Horvith and Lipsky). The mechanical stability of the beads allows a column input pressure of up to 5000 p.s.i. to be used. The separation is accelerated by increasing the temperature. The disadvantage of this type of ion exchanger is the low capacity. Owing to the lower retention of substances, the ionic strength usually has to be reduced in comparison with the resolution of similar samples using conventional ion exchangers. The possibility of increasing the efficiency of high-speed chromatography when only a thin sorbing porous layer will be active on the surface of the beads has been considered theoretically by various workers (e.g., Bohemen and Purnell, Golay, Knox, Knox and McLaren, Parrish, Weiss). The desirability of using porous layer beads in ion-exchange chromatography has been demonstrated by Horvith et al. Other information and examples, in addition to the literature cited, have been given by Burtis ef al., Gere, Schmit, Uziel and others. For some continuous or counter-current processes with ion exchange, especially in industry and in nuclear chemistry, liquid ion exchangers are used (Hogfeldt). The ion exchange can occur not only between the crystal or gel and the solution of an electrolyte, but in principle also between two non-miscible phases. One phase is an aqueous electrolyte solution and the other can be a solution of a base or acid in an organic solvent, e.g., longchain amines (Moore, 1960), or a special liquid compound. Substances used as liquid ion exchangers must contain an ion-exchanging group and a large non-polar part that is insoluble in aqueous phase. Di(2-ethylhexyl)phosphoric acid is an example of a weakly acidic liquid ion exchanger, and dinonylnaphthalenesulphonic acid is a strongly acidic liquid exchanger. Liquid ion exchangers are also used in analytical chemistry (Coleman et al.) and can be applied in the impregnation of inert supports for column processes (Cerrai).
SORBENTS FOR AFFINITY CHROMATOGRAPHY In their review article, Cuatrecasas and Anfinsen (1971a) mention the properties which a sorbent for affinity chromatography should possess. Firstly, it should have a minimal non-specific adsorption. When preparing an insoluble affinant, it is important that this should be on the carrier only in the form of covalently bound molecules. The molecules of the affinant that are not bound covalently must be washed out, which is difficult with supports that strongly adsorb the affinant molecules. Similarly, when substances that form a specific and reversible complex with the bound affinant are isolated, it is important that, as far as possible, only their retention should take place on the column of insoluble affinant and only in the form of a specific complex with the bound affinant. This is one of the main reasons why carriers that contain ionogenic groups, such as the copolymer of ethylene with maleic anhydride, which set carboxyl groups free after the affinant had been attached, have never been as widely applied as the neutral agarose in affinity chromatography. For a smooth c o m e of affinity chromatography, good flowing properties are important, i.e., the eluent should penetrate the support column at a sufficient rate even when the affinant is bound on to it. The support must possess a sufficient number of chemical groups which can be activated or modified in such a manner as to be able to bind affinants. The capacity of a specific adsorbent prepared by the attachReferences p.227
216
SORBBNTS
ment of the affinant to the solid support is dependent on the number of these groups present. The activation or modification should take place under conditions that do not change the structure of the support. No less important are the chemical and mechanical stabilities of the carrier under the conditions of the affinant binding, but also at various pHs, temperatures and ionic strengths, in the presence of denaturating agents, etc., which are necessary for good sorption and elution of the isolated substance. The possibility of repeated use of a specific adsorbent depends on these stabilities. Should the isolated substance be sorbed on the bound affinant, the support must be sufficiently porous to provide for sufficient freedom of formation of the specific complex. An example is the sorption of P-galactosidase on t o sorbents prepared by binding the inhibitor of p-aminophenyl-Pi>-thiogalactopyranoside through a hydrocarbon chain both to the polydextran gel Sepharose and to the polyacrylamide gel Bio-Gel P-300 (Steers et aL). The contents of the bound inhibitor were virtually identical in the two instances, but the isolation of the 0-galactosidase by affinity chromatography was successful only when Sepharose was used. In spite of a high concentration of inhibitor (50 prnole/ml), the enzyme was not retained on Bio-Gel P-300. This might be explained by an excessively large volume of the tetramer of 0-galactosidase (molecular weight 540,000; Craven et al.), which could not enter into the Bio-Gel pores. On the other hand, when nuclease of molecular weight 17,000 (Cuatrecasas, 1970a) was isolated from Staphylococci, Bio-Gel P-300 appeared to be a suitable support. A high porosity of the solid support is further indispensable for the isolation of substances with a relatively weak affinity for the bound affinant (dissociation constant > lo-'). The concentration of the bound affinant, freely accessible to the isolated substance, should be very high in this instance, in order to achieve a strong interaction which would retain physically the isolated substances migrating down the column. When choosing gels, their specific surface area should also be taken into consideration in addition to the pore size. In Table 9.17 are given the amounts of chymotrypsin bound to 1 ml of hydroxyalkyl methacrylate gels of various pore sizes (given by the value of exclusion molecular weights), and various specific surface areas (Turkova et al., 1973). From Table 9.17 it is evident that the amount of bound chymoTABLE 9.1 7 AMOUNTS OF CHYMOTRYPSIN BOUND TO HYDROXYALKYL METHACRYLATE GELS (SPHERON) AS A FUNCTION OF THE MAGNITUDE OF THEIR SPECIFIC SURFACE AREAS Spheron
1 o5 103 700 500 300 200 100
Molecular-weight exclusion limit
10' 1 O6 700,000 500,000 300,000 200,000 100,000
Specific surface area (mZiml)
Amount of bound chymo trypsin
(mgiml)
Relative pro teolytic activity (%)
0.96 5.9 3.6 23 19.5 0.6 0.2
0.73 7.8 6.7 17.1 17.7 6.9 2.6
44 49 37 44 53 38
-
217
SORBENTS FOR AFFINITY CHROMATOGRAPHY
trypsin is directly dependent on the magnitude of the specific surface area, which is at a maximum in Spheron 300 and 500. The relative proteolytic activity is also given in this table.
Cellulose and its derivatives The binding of affinants of a predoininantly proteinic nature on to cellulose and its derivatives was discussed in a review by Silman and Katchalski, the binding of enzymes by Crook et al., and the binding of nucleotides, polynucleotides and nucleic acids by Gilham. These reviews mention very varied methods of binding. In view of the present limited use of cellulose in affinity chromatography, we shall briefly mention some of them here. The most commonly used method of binding substances with a free amino group on to cellulose (CEL) is the Curtius azide method, used for the first time by Micheel and Ewers and today applied mostly in the modification o f Hornby et al.: CEL-OH
+ CI-CH~-COOH
NaOH
Prote8n-NHz PH8 *
CEL-O-CH2-CO-NH-urotein
C H OH C E L - 0 - C W - C O O H e
CEL-O-CH>-COOCHII
-I
H~N-NHz
NUN02
CEL-O-CH2
CO-N3
HCI
CEL-O-CH2-CO-NNH-NNH2
After the preparation of carboxymethylcellulose azide by Curtius rearrangement, an isocyanate is formed on to which the amino group of the affinant is bound. Affinants with basic amino groups can be further coupled to the carboxyl groups of carboxymethylcellulose in the presence of carbodiimide (Weliky et al.): CEL-O-CH2-COOH+
R-NH2
+ H20
bCEL-O-CH2-CO-NH-R N ,N'-dicycl ohexylcarbodi irnide
Kay and Lilly worked out the triazine method of protein binding. 2-Amino4,6-dichloros-triazine is bound t o the hydroxyl group of cellulose and reacts rurther with the amino group of the protein:
c1
NHI
orotein
Jagendorf et al. developed a method of protein binding based on the acylation of the hydroxyl group in cellulose with bromoacetyl bromide and subsequent alkylation of the amino group o f the protein: CEL-OH
+ Br-CO-CH2-Br
-.
CEL-0-CO-CH2-Br
Protein-NHZ =-CE L-0-CO-CH2 - ~ H-
protein
The first attachment of an affinant on to cellulose was carried out by means of diazonium References p.227
218
SORBENTS
groups (Campbell er al.):
HO
The affinants are bound by their aromatic residues, in the case of proteins mainly by tyrosine and histidine, but also non-specifically and more slowly by their amino groups (Gundlach et al., Tabachnik and Sobotka). Nucleic acids are bound t o aminoethylcellulose mostly by using periodate oxidation (Gilham). Cellulose and its derivatives are produced by a number of firms*. In addition to Whatman (Maidstone, Great Britain), Serva (Heidelberg, G.F.R.) lists both current cellulose derivatives and bromoacetylcellulose (BA-cellulose) and p-aminobenzylcellulose (PAB-cellulose). Bio-Rad Labs. (Richmond, Calif., U .S .A.) supply p-aminobenzylcellulose under the trade-name Cellex PAB and aminoethylcellulose under the name Cellex AE. MilesSeravac and Miles-Yeda (Maidenhead, Great Britain) produce a hydrazide derivative of CM-cellulose (Enzite-CMC-hydrazide), bromoacetylcellulose (BAC) and rn-aminobenzyloxymethylcellulose (ABMC). In addition to the supports for the binding of affinants, they also supply insoluble affinants, such as CM-cellulose-bound cysteine, and further the enzymes trypsin, chymotrypsin, papain, protease from Srreptomyces griseus , bromelain, ficin, peroxidase, RNase, amylase and cytochrome c, DEAE-cellulose-bound leucineaminopeptidase, alcohol dehydrogenase (YADH), glucoso-oxidase and urease under the names consisting of the name of the enzyme with the prefix Enzite. Cellulose-bound trypsin is also produced by E. Merck (Darmstadt, G.F.R.). Although cellulose was used as a carrier mainly during the initial period of the development of affinity chromatography, it is still used. Some workers (Dean and Lowe; Lowe and Dean) still consider cellulose to be the most suitable support for nicotinamideadenine dinucleotide (NAD) and they isolated on it the NAD-binding dehydrogenase. Similarly, Fritz and his co-workers (Fink et al. ; Fritz et al., 1970; Tschesche er a/.) still make use of cellulose-bound proteases for the isolation of their inhibitors from most various sources, and Sat0 et al. used halogenoacetylcellulose for the isolation of aminoacylase.
*Only firms known to the authors are mentioned in t h e text. Therefore, the list is necessarily incomplete and it should in no case be considered as implying recommendation of any particular firm or product.
219
SORBENTS FOR AFFINITY CHROMATOGRAPHY
Copolymer of ethylene and maleic anhydride The linkage of affinants to a copolymer of ethylene and maleic anhydride (EMA) was discussed in a review by Goldstein. The method of binding enzymes t o this support was worked out by Levin et al. in 1964. The protein is bound to anhydro groups of the polymer by its amino groups: -CHz-
CH --CH -CH2-CH2-CH-CH
o=c
I
I c=o
o=c
-CH,-CH,
I
t NHz-protein-NHZ
/ c=o
-
‘ 0 ’
‘0’
prbtein
I
NH
o=c’
coo1
-CH2-CH-CH
1
-CH2-CHp--CH
coo- cooI
I
-CL---11H2
-C+,--
When the affinant is bound, carboxyl groups are set free (either after the binding with proteins or hydrolysis in aqueous medium), which give the support a polyanionic character. The copolymer of ethylene and maleic anhydride is produced by Monsanto (St. Louis, Mo., U.S.A.). The British firm Miles-Yeda binds the enzyme trypsin, chymotrypsin, papain and subtilopeptidases A and B on to this polymer and supplies them under the names Enzite-EMAXenzyme name). These preparations are characterized by a high content (about 60%) of the bound enzyme. The properties of enzymes bound to EMA cariiers have been intensively investigated (Goldstein and Katchalski, Silman and Katchalski). This support with bound proteases was utilized mainly by Fritz, Werle and co-workers for the preparation of a series of inhibitors of proteolytic enzymes (Fritz et al., 1967, 1968, 1972; Hochstrasser et al., 1972a, b) and with bound inhibitors for the isolation of proteases (Fritz et al., 1969).
Agarose and its derivatives Agarose is a polydextran carrier which is presently most used in affinity chromatography. As demonstrated by Cuatrecasas and Anfinsen (1971a), agarose fulfils virtually all the requirements of an ideal carrier. The most utilized method of affinant binding on Sepharose activated by cyanogen bromide was developed by Porath et al. and AxBn et al. The affinants are bound by means of primary aliphatic or aromatic amino groups. AxBn and Ernback proposed a two-step activation scheme. In the first step, a labile intermediate is formed under the effect of halogenocyanates, which is then converted in the second step into an inert carbamate and a reactive imidocarbonate. The amino group of the affinant is then bound to the latter in a weakly alkaline medium with the formation of a stable covalent bond. On the basis of a study of model reactions with methyl 4,6-O-benzylidene-cu-~-glucopyranoside (Ahrgren et d.), t h s course seems improbable and at present the prevailing view is that the main part References p.22 7
220
SORBENTS
of the affinant binding t o the support takes place via isourea derivatives. The degree of agarose activation, measured on the basis of the capacity of binding of small peptides (AxBn and Ernback), is directly proportional to the pH. Activation takes place at pH 1 1. The whole procedure for the activation of Sepharose with cyanogen bromide (AxCn er al., Cuatrecasas, Porath et al.) is described in detail below.
Coupling of affinants on agarose Washed and decanted Sepharose is suspended in distilled water (1 : 1). The suspension is placed in a well ventilated hood, a pH meter electrode pair is immersed in the suspension, and finely divided cyanogen bromide (50-300 mg per millilitre of packed Sepharose) is added gradually with constant stirring. The suspension is maintained at pH 11 by addition of sodium hydroxide solution. The concentration of the sodium hydroxide solution used depends on the amounts of Sepharose and cyanogen bromide present; for 5-10 ml of packed Sepharose and 1-3 g of added cyanogen bromide, Cuatrecasas recommends the use of 2 M sodium hydroxide, and for 100-200 ml of packed Sepharose and 20-30 g of cyanogen bromide an 8 M solution is suitable. The temperature should not exceed 20°C; if cooling is necessary, ice may be added. The reaction is completed in 8-1 2 min. After a rapid transfer of the suspension on a biichner funnel under constant suction, the activated Sepharose is washed with cooled buffer solution of the same composition as will be used for the subsequent coupling of the affinant. The buffer volume should be 10-1 5 times that of the Sepharose to be activated. The washed Sepharose is suspended as rapidly as possible in an equal volume of the affinant solution. According to Cuatrecasas, the washing, addition of affinant solution and mixing should not take more than 90 sec. Even at a low temperature, the activated Sepharose is unstable. The conditions used for the linking of the affinant, such as pH, buffer composition and temperature, are dependent on the nature of the bound affinant. The binding reaction proceeds most satisfactorily at pH 8-10, but if the nature of the affinant requires a lower pH, the binding of a sufficient amount of affinant can usually be carried out successfully even at that pH if the amount of cyanogen bromide is increased during activation and the amount of affinant during the binding. The active groups that remain after the binding of the affinant are eliminated by blocking them with 1 M ethanolamine. A detailed procedure of the binding of affinant to cyanogen bromide-activated Sepharose is given below. Cyanogen bromide-activated Sepharose 4 B is produced commercially by freeze-drying with the addition of dextran and lactose, which must be washed out before use. The manufacturer (Pharmacia) gives the following procedure for coupling the affinant with cyanogen bromide-activated Sepharose. The required amount of gel is allowed to swell in 1 0-3 M hydrochloric acid and the solution is then used for washing the gel for 1 5 min. The volume of 1 g of the freeze-dried gel when swollen is approximately 3.5 ml. It is recommended that 200 ml of the solution per gram of dry gel should be used for the washing, in several batches. Immediately after washing, the solution of the affinant to be coupled is added. The optimum conditions for coupling the affinant, i.e., pH, buffer composition and temperature, are dependent to an appreciable extent on its character. Generally, the coupling reaction takes place most effectively at pH 8-10, but if the nature of the coupled affinant requires it, lower pH values can also be used. The affinant, especially if it is of a proteinic character, is dissolved in a buffer of high ionic strength (about 0.5) in
SORBENTS I O R AFFINITY CHROMATOGRAPHY
22 1
order to prevent non-specific adsorption. The higher ionic strength then facilitates subsequent washing. Carbonate or borate buffers with sodium chloride added can be used. The amount of the affinant coupled depends on the proportion of the affinant in the reaction mixture and the volume of gel, the pH of the reaction, the nature of the coupled affinant (number of reactive groups, etc.), and also on the reaction time and temperature. For example, when chymotrypsin is coupled with 2 ml of cyanogen bromide-activated Sepharose at pH 8, only 5 mg were coupled when 10 mg of protein were present, at 20 mg of protein ca. 8 mg were coupled, and at 30 mg the amount coupled was ca. 10 mg. At room temperature (20-25"C), the coupling is usually completed after 2 h, while at lower temperatures it is recommended that the mixture should be allowed to stand overnight. During the coupling, the reaction mixture must be stirred. Stirring with a magnetic stirrer is not recommended, as it may cause mechanical destruction of the gel. When the coupling is completed, the gel with the coupled affinant is transferred on to a sintered-glass filter and washed with the buffer used during the coupling. In order to eliminate the remaining active groups, the manufacturer (Pharmacia) recommends blocking them with 1 M ethanolm i n e at pH 8 for 2 h. The final product should then be washed four or five times alternately with buffer solutions of high and low pH. For example, acetate buffer (0.1 M , pH 4) and borate buffer (0.1 M , pH 8.5) can be used, each being 1 M i n sodium chloride. As already stated, all non-covalently bound substances should be eliminated during the washing. In addition to the binding of affinant on to agarose, described above, the triazine method of binding affinants on agarose is also used. It was originally developed by Kay and Lilly for binding of affinants to cellulose (see p. 217), using 2-amino-4,6-dichloro-striazine. Affinity chromatography of some substances takes place only if the affinant is sufficiently distant from the surface of the solid support (Steers er af.),which can be achieved by inserting between them a sufficiently long bridge. For this purpose, aliphatic diamines are usually used, for example ethylenediamine, which can be bound directly to cyanogen bromide-activated Sepharose in the conventional manner. In order t o eliminate the undesirable formation of additional cross-linkages owing to the reaction of both terminal amino groups, a large excess of diamine should be employed (Cuatrecasas, 1970a). A detailed procedure for the preparation of w-aminoalkyl derivatives of agarose is given below. Aliphatic diamino compounds, for example ethylenediamine, can be coupled directly on to cyanogen bromide-activated Sepharose. In order to avoid the undesirable formation of additional cross-linkages owing to the reaction of both terminal amino groups, a large excess of the diamine is used. Into a suspension of Sepharose 4B in water (1 :I ) , 250 mg of cyanogen bromide per millilitre of packed Sepharose are added and the reaction is carried out as described in the procedure for the activation of Sepharose with cyanogen bromide. An equal volume of cold distilled water containing 2 mmole of ethylenediamine per millilitre of packed Sepharose is added to the washed and collected activated Sepharose, which has been adjusted with 6 M hydrochloric acid to pH 10. After 16 h of reaction at 4"C, the gel is washed with a large volume of distilled water. Thus Sepharose derivatives can be obtained containing about 12 pmole of aminoethyl group per millilitre of packed Sepharose. By use of various diamino compounds of the general formula NH2(CH2XNH2,various w-aminoalkyl derivatives can be prepared. References p.227
222
SORBENTS
Affinants that contain a free carboxyl group may be bound to aminoethyl-agarose by water-soluble carbodiirnides (Cuatrecasas, 1970a), as mentioned in the procedure given below for the preparation of estradiolSepharose. A 300-mg amount of 3-0-succinyl-[3 Hlestradiol dissolved in 400 ml of dimethylformamide is added t.0 40 ml of packed aminoethyl-Sepharose 4B. The dimethylformamide is needed in order to solubilize the estradiol, and it is not necessary for affinants that are soluble in water. The suspension is maintained at pH 4.7 with 1 N hydrochloric acid. Then 500 mg (2.6 mmole) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, dissolved in 3 ml of water, are added to the suspension over 5 min and the reaction is allowed to proceed at room temperature for 20 h. Substituted Sepharose, after being transferred into the column, is washed with 50%aqueous dimethylformamide until the eluate is no longer radioactive. It is recommended that the derivative should be washed with ca. 10 1 of the washmg liquid over 3-5 days. Using this procedure, about 0.5 pmole of estradiol may be bound per millilitre of packed Sepharose. Of other Sepharose derivatives, Cuatrecasas (1 970a) prepared bromoacetamidoethyl-Sepharose by reaction of 0-bromoacetyl-Nhydroxysuccinimide with aminoethyl-Sepharose, succinylaminoethyl-Sepharose by reaction with succinic anhydride, diazonium derivatives from p-aminobenzamidoethylSepharose, and also tyrosyl-Sepharose and sulfhydryl-Sepharose. He bound affinants with free carboxyl groups to sulfhydryl-Sepharose by thiol-ester bonds using water-soluble carbodiimides. The preparation of various derivatives of Sepharose 4B was reviewed by Cuatrecasas (1970b). As regards the stability of agarose, it is stable in the pH 4-9 range and temperatures below 0°C or above 40°C are not recommended. Sepharose is resistant to high salt concentrations, urea (7 M) and guanidine hydrochloride (6 M) (Cuatrecasas, 1970a). It is stable even when exposed at room temperature to 0.1 M sodium hydroxide solution and 1 M hydrochloric acid for a short time (2-3 h). For tHe affinity chromatography of weakly water-soluble substances, 50% dimethylformamide or 50% ethylene glycol can also be used. Lyophilization can be carried out only after addition of protective substances, for example dextran, glucose and serum albumin. The main manufacturers of agarose are Pharmacia (Uppsala, Sweden), who produce agarose under the name Sepharose, and Bio-Rad Labs., who produce agarose under the name Bio-Gel A. Both preparations are produced in several varieties that differ in their pore sizes and the molecular-weight exclusion limits depending on them. For facilitating the binding of affinants, Pharmacia produce lyophilized cyanogen bromide-activated Sepharose. They also produce the ready-made insoluble affinant Con A-Sepharose, representing concanavalin A bound to Sepharose, which is suitable for the isolation of polysaccharides or glucoproteins containing the glycosyl residue as the terminal group (Aspberg and Porath, Edelman et al., Lloyd, Yariv e t al.). Miles-Seravac produce a series of insoluble affinants based on agarose, for example agarose-bound enzymes such as trypsin, chymotrypsin, papain and protease from Streptomyces griseus. Their commercial names are Enzite-agarose-(name of enzyme). For the isolation of chymotrypsin, MilesSeravac produce agarose-bound e-aminocaproyl-D-tryptophan methyl ester (Cuatrecasas et al.), for the isolation of papain agarose-bound tetrapeptide Cly-Gly-Tyr (0-benzy1)Arg (Blumberg et al.), for the isolation of ribonuclease agarose-5'(4'-aminophenyl)uridine2'13')-phosphate (Wilchek and Gorecki), for the isolation of L-tryptophan-binding proteins
223
SORBENTS FOR AFFINITY CHROMATOGRAPHY
agarosea-tryptophan and agarosea-tryptophan (Sprossler and Lingens), for L-tyrosinebinding proteins agarose-L-tyrosine (Chan and Takahashi), and for thyroxine-binding proteins agarose-thyroxine (Pensky and Marshall). Concanavalin A bound to agarose is produced under the name Glycosylex A. Among immunoadsorbents, Miles-Seravac produce for the isolation of antibodies agarose-bound antigens,' for example bovine serum albumin, human and goat immunoglobulins, and insoluble haptens, for example agarosebound dinitrophenyl, xsanilic acid, gibberellic acid and 3indolylacetic acid. For the isolation of antigen, they produce agarose-btjund antibodies which are formed against bovine albumin, growth hormone, glucagon, human IgG, dinitrophenol, gibberellic acid and 3-indolylacetic acid. It is expected that the number of commercially produced affinants bound t o agarose and other supports will increase substantially in the future, which is indicated by the present rapid increase in the numDer of publications that describe the affinity chromatography of a wide range of substances on supports prepared from agarose. The number of such papers is already so large that it is impossible to review them within the scope of this chapter: in 1968, only two papers describing the use of affinity chromatography on agarose appeared, in 1969 the number of such papers was ten, in 1970 twenty, in 1971 forty-six, in 1972 sixty-seven and in 1973 seventy. A review of most of the papers utilizing Sepharose for affinity chromatography is offered by Pharmacia in a leaflet on cyanogen bromide-activated Sepharose 4B used for the immobilization of biopolymers.
Polyacrylamide supports and their derivatives Polyacrylamide gels are composed of a hydrocarbon skeleton on to which carboxamide groups are bound: -C H 2-CH -C H -C H -C H 2-CH -
I
CO-NH2
I
CH?NH2
I
CO-NH2
On reaction with a suitable compound, they can be converted into solid carriers suitable for the binding of a series of affinants (Inman and Dintzis). Their aminoethyl derivatives may be prepared by using a large excess of ethylenediamine at 90"C, and hydrazide derivatives by using excess of hydrazine at 50°C. Aminoethyl derivatives of Folyacrylamide gels can be converted into their p-aminobenzamidoethyl derivatives by reaction with p-nitrobenzoylazide in the presence of N,N-dimethylformamide, triethylamine and sodium thiosulphate. After activation with nitrous acid, the hydrazide derivative can bind affinants with its amino groups: -CH-CH2-
-CH-CH2-
-CH-CH2-
HN02 I 1 CO-N H -N H 2 -CO-N3
P r o t e i n -NH2
1
WCO-NH-protein
Polyacrylamide gels containing residues of aromatic acids, when diazotized with References p.227
224
SORBENTS
nitrous acid, bind affinants mainly through their aromatic residues:
'CH~- protein
The same gels, when activated with thiophosgene, bind affinants by means of their free amino groups :
'
-CH-CHzCO -NH
CIC--5
I
protein-NH2
-2L CO-NH
'
-CH-CH2 CO-NH
a
NH-
S
II
C -NH-protein
The procedures for the binding of proteins on to all three derivatives of polyacrylamide gels are given below.
Coupling of proteins with commercially produced polyacrylamide derivatives (Enzacryls) Coupling of affinants on polyacrylamide gels containing aromatic amino acid residues (Enzacryl A A ) after activation with nitrous acid To a suspension of 100 mg of Enzacryl AA in 5 ml of 2 M hydrochloric acid, cooled t o O'C, 4 ml of an ice-cold 2% solution of sodium nitrite are added and the mixture is stirred magnetically for 15 min. The diazo-Enzacryl formed is then washed four times with the buffer in which the affinant will undergo coupling (for example, a phosphate buffer of pH 7.5). After centrifugation and decantation, the affinant is added, for example an enzyme (2.5 mg) in a suitable buffer (0.5 ml). The coupling is allowed to proceed with magnetic stirring for 48 h. The reaction is terminated by addition of an ice-cold solution of phenol (0.01%) in sodium acetate (10%). After a further 15 min, the Enzacryl with the coupled affinant is first washed with a dilute buffer, then with the same buffer made 0.5 M in sodium chloride. T h s washing should be carried out very carefully. The manufacturer (Koch-Light) recommends carrying out the whole experiment first with nondiazotized Enzacryl, in order to determine the best conditions for washing out all of the adsorbed material. Coupling of affinants on polyacrylamide gels containing aromatic amino acid residues (Enzacryl A A ) after activation with thiophosgene To a suspension of 100 mg of Enzacryl AA in 1 ml of phosphate buffer (3.5 M, pH 6.8-7.0), well stirred with a magnetic stirrer, 0.2 ml of a 10%thiophosgene solution in chloroform is added. After vigorous stirring for 20 min, a further 0.2 ml of the thiophosgene solution is added, and after additional stirring for 20 min the NCS-Enzacryl is
SORBENTS FOR AFFINITY CHROMATOGRAPHY
225
washed once with acetone, twice with 0.5 M sodium hydrogen carbonate solution and twice with a buffer suitable for coupling (for example, a borate buffer of pH > 8.5). After centrifugation and decantation, 0.5 ml of an affinant solution (for example, 2.5 mg of enzyme) is added and the coupling is carried out as described inathe preceding section. Activation of the hydrazine derivative of polyacrylamide gel (Enzacryl AH) with nitrous acid and subsequent coupling is carried out in the same manner as described for EnzdCryl AA. Coupling of proteins on polyauylamide gels by using glutaraldehyde Weston and Avrameas developed a method of direct binding of affinants on to polyacrylamide gels using glutaraldehyde, which, if present in excess, reacts with one of its two aldehydic groups, which is bound to the free amido group present in the polyacrylamide gel. The remaining free active group then reacts with the amino group of the affinant added during the subsequent binding reaction. Thus a firm bond is formed between the support and the affinant. Bio-Gel P-300 is allowed to swell in water and is washed twice with a four-fold volume of 0.1 M phosphate buffer of pH 6.9. Then 19.4 ml of gel (1 g of dry beads per 45 ml) are mixed with glutaraldehyde solution (4.8 ml; 25%, v/v) and incubated at 37°C for 17 h. The gel is washed and centrifuged four times with a four-fold volume of 0.1 M phosphate buffer of pH 6.9, then three times with 0.1 M phosphate buffer of pH 7.7. The coupling of the protein is carried out after mixing of 3 ml of gel in 13.5 ml of a buffer of pH 7.7 with 0.3 ml of enzyme solution (20 mg/ml) at 4°C for 18 h on a shaker. After the reaction, the gel is centrifuged and washed. Using this method, 70 mg of acid phosphatase could be coupled per gram of dry gel. Polyacrylamide gels are stable in the pH range 1-10 and they support well all generally employed eluents. They do not contain charged groups, and so ion exchange with the chromatographed substances is minimal. They are biologically inert and, as they are synthetic polymers, they are not attacked by microorganisms. As the gel particles adhere strongly to clean glass surfaces, lnman and Dintzis recommend the use of siliconized glass or polyethylene laboratory vessels. The main producer of polyacrylamide gels is Bio-Rad Labs., who produce them under the commercial name Bio-Gel P by copolymerization of acrylamide and N,N’-methylenebisacrylamide. Bio-Gel P of various pore sizes range from Bio-Gel P-2 with a molecularweight exclusion limit of 1800 up to Bio-Gel P-300 with a molecular-weight exclusion limit of 400,000. All brands are of 50-100,100-200,200-400 and 400 mesh size. In addition to these gels, Bio-Rad Labs. produce ion-exchanging derivatives of the gels, for example the weakly acidic cation exchanger Bio-Gel CM, and also intermediates for affinity chromatography, such as the aminoethyl and hydrazide derivatives of Bio-Gel P-2 and P-60. For the linking of affinants, mainly enzymes, Koch-Light (Colnbrook, Great Britain), produce Enzacryls. Enzacryl AH is a hydrazide derivative of polyacrylamide gels, and Enzacryl AA is a polyacrylamide gel containing aromatic acid residues. Enzacryl polythiolactone, polythiol and polyacetate are currently being introduced. Although Cuatrecasas (1970a) demonstrated the suitability of acrylamide gels for the isolation of staphylococcal nuclease by affinity chromatography and Truffa-Bachi and References p. 22 7
226
SORBENTS
Wofsy isolated specific cells on Bio-Gel P-6with bound hapten, the utilization of these gels in affinity chromatography is still limited.
Hydroxyalkyl methacrylate gels Hydrophilic hydroxyalkyl methacrylate gels are prepared by polymerization of a suspension of hydroxyalkyl esters of methacrylic acid and alkylene dimethylacrylate (toupek et al., 1972a, b) by varying the ratio of the concentrations of monomer and inert components. The number of reactive groups, porosity, and the specific surface area of the gel may be changed within broad limits. The gel has the following structure: ?H3 -C-CH2-C
I
co I 0 I
7%
CH3 y
3
C-
co
co
co
O-CCHz-CHzOCl
O-CH2-CCH20H
0
I
1
I
I
I
I
0
0
I
I
co I
c H3
I
CH2
7%
-C-CH:,
I
I
CHZ
7H2
I
I
C __ CH2-
-CHz-
CH3 y
- C-
3
I
CHz p C - C H 2 -
I
1
co
co
I
0-CH2-
CH20H
1
CO
I I
CCH3
O--CH,--CH2OH
Hydroxyl groups of the gel possess analogous properties to those of agarose. After cyanogen bromide activation, they bind the affinants in the same manner as Sepharose by their amino groups (Turkovi et al., 1972, 1973). The gel activation and the affinant binding are virtually identical with those described in the procedures for the binding on agarose. The gels form regular beads with excellent chemical and physical stabilities. They stand chromatography well under pressure, and do not change their structures after heating for 8 h in 1 N sodium glycolate solution at 150°C or after boiling for 24 h in 20% hydrochloric acid. They are biologically inert and, as acrylamide gels, are not attacked by microorganisms. Their production is being started by Lachema (Brno, Czechoslovakia) under the name Spheron. Spherons of various pore sizes and exclusion molecular weights are manufactured from Spheron 100, with a molecular-weight exclusion limit of 100,000,up to Spheron l o 5 ,with an exclusion molecular weight of l o 8 .The production of cyanogen bromideactivated dried Spheron is already also under consideration. Spherons have been used successfully for a series of affinity chromatographic procedures, of which only the isolation of the chymotrypsin inhibitor from potatoes on Spheron 300 (with bound chyrnotrypsin) and the chromatography of chymotrypsin on Spheron 300 with bound trypsin-inhibitor has been published up to the present time (Turkovi et al., 1972).
REFERENCES
227
Glass and its derivatives Workers at Corning (Corning, N.Y., U.S.A.) demonstrated that glass, when treated with y-aminopropyltriethoxysilane, becomes a suitable support for a series of affinants (Line et al.; Messing; Weetall, 1969a, b, 1970, 1971; Weetall and Baum; Weetall and Hersh, 1969,1970; Weibel and Bright; Weibel et al.). Affinants can be bound on to the amino groups of the glass derivatives by their carboxyl group using soluble carbodiimides, and by their amino groups after activation with thiophosgene, and by aromatic residues with the azo-bond to arylamine derivatives. Glass derivatives have outstanding stability and are not attacked by microorganisms. However, in some instances they cause undesirable unspecific adsorption (Cuatrecasas and Anfinsen, 197 1b). Corning produces a series of glass supports of various bead and pore sizes, which are, however, not yet commercially available. In affinity chromatography, only Weibel et al. have used a glass support as a specific adsorbent prepared by diazo-coupling of nicotinamide-adenine dinucleotide, which proved an effective coenzyme for the apoenzyme of alcohol dehydrogenase from yeast.
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Coupek, J., Kfivlikovd, M. and PokornL, S., IUPAC International Symposium on Macromolecules, Helsinki, 1 9 7 2 ~J.; Polym. Sci., Part C. in press. Coupek, J.. Turkovd, J . and-KFiv&ova’, M., 2nd Discussion Conference on Macromolecules, Prague, 19728, Abstr. No. D9/3 b. Craven. G . R., Steers, Jr., E. and Anfinsen, C. B., J . Biol. Chem., 240 (1965) 2468. Crook, E. M., Brocklehurst, K. and Wharton, C. W., Methods Enzymol., 19 (1970) 963. Cuatrecasas, P., J. B i d . Chem., 245 (1970a) 3059. Cuatrecasas, P., Nature (London), 228 (1970b) 1327. Cuatrecasas, P. and Anfinsen, C . B., Methods Enzymol., 22 (1971a) 345. Cuatrecasas, P. and Anfinsen, C. B., Annu. Rev. Biochem., 40 ( 1 97 1 b) 259. Cuatrecasas, P., Wilchek, M. and Anfinsen, C. B., Proc. Nut. Acad. Sci. U S . , 61 (1968) 636. Dean, P. D. G. and Lowe, C. R., Biochem. J., 127 (1972) 1 1 P . Determann, H., Angew. Chem., 76 (1 964) 635. Determann, H., Kriever, M. a n d Wieland. T . , Makromol. Chem., 114 (1968a) 256. Determann, H. and Lambert, K . , J. Chromatogr.. 56 (1971) 140. Determann, H., Liiben, G. and Wieland, T.,Makromol. Chem., 73 (1964) 168. Determann, H., Rehner, H. a n d Wieland, T., Makromol. Chem., 114 (1968b) 263. Deuel, H. and Neukom, H., Advan. Chem. Ser., 1 I ( 1 954) 5 1. Deuel, H., Solms, J . and Anyas-Weisz, L., Helv. Chim. A c f a , 33 (1950) 2171. De Vries, A. J., Le Page, M., Beau, R. and Guillemin, C. L., 3rd International Gel Permeation Chromatography Seminar, Geneva, 1966, Waters Ass., Framingharn, Mass., 1966. Edelman, G . M., Rutishauser, U. and Millette, C. F., Proc. Nut. Acad. Sci. U.S., 68 (1971) 2153. Eriksson, K. E., Pctterson, B. A. and Steenberg, B.,Sv. Papperstidn., 71 (1968) 695. Fawcett, J . S . and Morris, C. J . 0. R., Sepur. Sci., 1 (1966) 9 . Fink, E., Jaumann, E., Fritz, H., Ingrisch, A. and Werle, E., Hoppe-Seyler’s 2. Physiol. Chem., 352 (1971) 1591. Friedman, L., J. Amer. Chem. Soc., 52 (1930) 1311. Frisque, A. J. and Bernet, K., US.Pat., 3,644,305. Fritz, H., Brey, B. and Biress, L., Hoppe-Seyler’s 2. Physiol. Chem., 353 (1972) 19. Fritz, H., Brey, B., Schmal, A. and Werle, E., Hoppe-Seyler’s 2. Physiol. Chem., 350 (1969) 617. Fritz, H., Gebhardt, M., Mester, R., Illchmann, K. and Hochstrasser, K., Hoppe-Seyler’s Z. Physiol. Chem., 351 (1970) 571. Fritz, H., Hochstrasser, K., Werle, E., Brey, E. and Gebhardt, B. M., Z. Anal. Chem., 243 (1968) 452. Fritz, H., Schult, H., Hutzel, M., Wiederman, M. and Werle, E., Hoppe-Seyler’s 2. Physiol. Chem., 348 (1967) 308. (;ere, D. R., in J. .I. Kirkland (Editor), Modern Practice of Liquid Chromatography, Wiley-Interscience, New York, 1971, p. 417. Ghetie, V. and Schell, H. D., Rev. Roum. Biochim., 4 (1967) 179. Giddings, J. C. and Mallik, K. L., Anal. Chem., 38 (1966) 997. Gilham, P. T.,Methods Enzymol., 21 (1971) 191. Golay, M. J. E., in R. P. W. Scott (Editor), Gas Chromatography 1960, Butterworths, London, 1960, p. 139. Goldstein, L.,Methods Enzymol., 19 (1970) 935. Goldstein, L. and Katchalski, E., Z. Anal. Chem., 243 (1968) 375. Greber, G. and Hausmann, P., Angew. Chem., Int. Ed. Engl., 7 (1968) 394. Groggins, P. H., Unit Processes in Organic Synthesis, McGraw-Hill, New York, 3rd ed., 1947, p. 803. Grubhofer, N. and Schleith, L., Naturwissenschaften, 40 (1 953) 508; Hoppe-Seyler’s 2. Physiol. Chem., 296 ( 1 954) 262. Gundlach, G., Kohne, C . and Turba, F.,Biochem. 2.. 336 (1962) 215. Guthrie, J. D., Ind. Eng. Chem., 44 (1952) 2187. Hadeball, W. and Seide, Plast. Kaut., 16 (1969) 418. Halisz, I., Engelhardt, H., Asshduer, J. and Karger, B.. L.,Anal. Chem., 4 2 (1970) 1460. Hala’sz, I . and Holdinghausen, F., in H. G. Struppe (Editor), Gas Chromatographie 1968, Akademie Verlap, Berlin, 1968,.p. 31 1.
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Chapter I 0
Mobile phases 0 . MIKES and R . VESPALEC
CONTENTS Mobile phases for liquid-liquid chromatography . . ................ Equilibrium between the mobile and stationary phases .............................. Selectivity of the liquid-liquid systems and solubility coefficients ..................... Programming of the mobile phase .............................................. Programming of the solvent flow ............................................ Gradientelution ......................................................... Temperature programming ................................................. Mobile phases for liquid-solid chromatography ...................................... Properties of the mobile phase .............................. Effect of the physical properties of the mobile phase on column efficiency . . . . . . . . . . . . . . Strength of the mobile phase .................................................. Elution strength of the binary mobile phase ................................... Demixing effects .................................... ................. Selectivity of the mobile phase ............................ ................. Mobile phases for ionexchange chromatography . . ...................... Aqueoussolutions ......................................................... Importance of ionic strength and pH of the mobile phase ......................... Choice of the mobile phase ................................................ Increasing concentrations of acids and bases ................................... Mobile phases for chromatography on buffered columns ........................... Special additives t o buffers ................................................. Complex-forming mobile phases and phases that alter the solutes . . . . . . . . . . . . . . . . . . . Mixed and non-aqueous phases ................................................ Temperature of mobile phases ................................................. Calculation of gradients ......................................................... General aspects ............................................................ Classification of gradients .................................................... Gradient formation ......................................................... Calculation of concentration gradients .......................................... “Exponential” gradients .................................................. “Proportional” gradients .................................................. “Disproportional” gradients ............................................... pl-l gradients ................................ Theory of gradient elution .................................................... References ........ .................................
233
234 234 239 245 245 246 247 248 249 252 255 257 258 261 261 261 263 264 264 267 267 269 269 270 270 270 271 273 273 275 276 277 277
234
MOBILE PHASES
MOBILE PHASES FOR LIQUID-LIQUID CHROMATOGRAPHY Equilibrium between the mobile and stationary phases In liquid-liquid chromatography, the fundamental process of distribution is based on dissolution. Hence, this technique may be looked upon as a repeated distribution of samples between two mutually immiscible liquids. The high efficiency of the distribution process may be achieved by a frequent repetition of the elementary distribution process effected by the movement of at least one liquid phase. From these basic characteristics, several conclusions ensue directly: (1) The stationary arid the mobile liquids should be chosen so that they are mutually sufficiently poorly soluble, but sufficiently good solvents for all the components of the sample. (2) The dissolution isotherm is usually a straight line or can be a t least approximated to a straight line better than and in a broader interval than in adsorption. Therefore, symmetrical elution zones can be easily obtained. (3) As in gas-liquid chromatography, it is usually desirable to suppress maximally the adsorption of the sample on the carrier used. (4) If the mobile phase and the sample are poorly soluble in the stationary phase employed, the controlling mechanism may consist in the adsorption of the sample on the surface of the stationary liquid. In this case, the adsorbing surface is energetically substantially more homogeneous than the surface of the commonly used adsorbents. (5) Two completely immiscible and mutually insoluble liquids do not exist. Therefore, the experimental conditions should be chosen so that the passage of the mobile phase through the column does not result in washing out of the stationary phase and hence a change in the column properties. The prerequisite for a chromatographically utilizable sample retention in a liquidliquid system is the achievement of a measurable concentration of solute 2 in the mobile phase m and the stationary phase s. The magnitude of the elementary partition step is characterized by the experimental distribution coefficient, K , .which is equal to the ratio of the solute concentrations in the stationary and the mobile phases: (10.1)
where n: is the number of moles of solute dissolved under the equilibrium state in phase i , the volume of which is K. For very dilute solutions, which are of interest in chromatography, n\ = x i . ni,where x i is the molar fraction of the solute in phase i , of which ni moles are present in the column. Then, (10.2) where v/ is the molar volume of the pure phase i. At a given pressure and temperature, the requirement for the equilibrium of the component in the mobile and the stationary phases is the equality of chemical potentials: P W ,
=P X T
( 10.3)
23 5
LIQUID-LIQUID CHROMATOGRAPHY
From this basic condition and from the relationship for the chemical potential of the solution component, Locke and Martire deduced the theory of retention for LLC. The chemical potential of a solution component at any chosen pressure P and temperature T, p2(T, P),can be expressed by using the standard chemical potential of the component at the given temperature and standard pressure P*, I*'' (T, P*),the molar volume of the pure solute , the molar fraction of the solute x i , and the activity coefficient at the given pressure and temperature y2(P, 7):
ui
+
p2(T, P) = p$>'(T, P*) (P - P*)u;
+ R T 1nx:y2(T, P)
( 1 0.4)
Substituting into eqn. 10.3, it follows that (10.5) If the state of the pure liquid is chosen as the standard state of the solute for dissolution in a chosen phase at a chosen pressure and temperature, i e . , if 1im y; = 1 , then x;-1
&"(T,
P*) = p:jS(T, P*)
(10.6)
The activity coefficient of the component at a chosen pressure P can be expressed, when the activity coefficient at standard pressure P* is used, by the equation
R Tln y2(T, P) = RTln y2(T, P*)+ (7 - u ! ) (P- P*)
(1 0.7)
where v2 is the partial molar volume of the component 2 in solution. Combining eqns. 10.5, 10.6 and 10.7, and taking into consideration eqn. 10.2, a relationship for K is obtained. IfP* = 1 atm and P(the pressure at which equilibrium is attained in the column) is taken as being equal to the average pressure i", then (1 0.8) The solutions of the solute 2 in both the pure stationary and mobile liquids, of molar volumes v$ and ,:v respectively, can be considered to be infinitely diluted during are replaced chromatographic conditions. Therefore, in eqn. 10.8, the symbols y:! and by yY1" and y?"'". Av, is the difference of partial molar volumes of the solute in the mobile and the stationary phase. If the specific retention volume, Vg,is defined by the relationship
5= VN/ws = KIP,
(1 0.9)
where V,,, is the corrected retention volume, ws is the weight of the stationary phase in the column and p, is its density, then (10.1 0) where Miis the molecular weight of the phase i and p,(7') is the density of the mobile phase at temperature T. As Av, is small and the mean pressure on the column is usually several tens of atmospheres, the magnitude of the last term in eqns. 10.8 and 10.10 can be References p.277
236
MOBILE PHASES
neglected in current analytical practice. Then, approximately (10.1 1) and (1 0.1 2) From eqns. 10.8 and 10.10-1 0.12, distribution constants and retention volumes can be predicted for systems in which the activity coefficients of solutes have been determined either by calculation from the theory of solutions or by independent measurements. In contrast, activity coefficients can be derived from chromatographic measurements. For the relative retention, a , of two solutes A and B, which is a measure of selectivity of their separation, it follows from eqns. 10.10 and 10.12 that ( 10.1 3)
Hence the resolution of two components in LLC is given only by the difference in their activities (deviations from ideality) in the chosen phases. Therefore, the selectivity of the separation of a chosen pair of substances can be regulated by the choice of both the stationary and the mobile phase. As the activity coefficients are temperature dependent, temperature can also be utilized for the control of selectivity. Deviations from ideal behaviour of the solute in solutions may be positive or negative and be of different magnitudes. In solutions, where only weak interactions of molecules occur due mainly to dispersion forces, the values of activity coefficients may be expressed in values close to unity. Strong intermolecular interactions lead to negative deviations characterized by small values of -yy, which, during complex formation, may be even of the order of In contrast, if the solute--solvent interactions are weaker than the solute-solute or solvent-solvent interactions, then the deviations are positive and the activity coefficients may be high. In analytical practice, it is usually required that 1 < K B 100. The capacity factor, k’,which affects the resolution distinctly (cf. eqn. 10.28), is connected with the term K by the relationship
k‘=K.v, ( 10.14) Vm As usually V,/ V, < 1, values of K < 1 as a rule lead to a low resolving power even when the relative retention of the separated components is good. It therefore follows that the stationary phase should be chosen so that the activity coefficients of the separated components are lower in that phase than in the mobile phase, so that it is a better solvent for the sample. If the activity coefficient according t o Pierotti et of. (1956, 1959) is expressed as a function of the structure of the solute and the solvent, then in LLC the activity coefficients of solutes that form homologous series may be described for both phases by the first two terms only. Then, approximately In
= kl
+ kznz
(1 0.15)
LIQUID-LIQUID CHROMATOGRAPHY
237
where n 2 is approximately equal to the number of carbon atoms in the solute molecule and k l and k 2 are the differences in the empirical constants of the first two members of the relationship of Pierotti et al. for the stationary and the mobile phase. Hence, for each homologous series, a linear relationship between the number of carbons and the logarithm of the specific retention volume may be expected. The effect of the molecular weights of the stationary and the mobile phases on retention, following from eqn. 10.12, is demonstrated for the case of the stationary phase. Liquids with as low a molecular weight as possible are usually chosen as the mobile phase. However, any liquid can be used as the stationary phase. Often high-molecular-weight compounds are used, as in gas chromatography, and sometimes polymeric compounds. Therefore, it can be stated that the relationshipM,/M, < 1 applies generally. Let us consider two columns in which two chemically similar liquids, X and Y, of molecular weights M, and M y and densities px and py , are used. From the theory of Flory and Huggins, it follows that (1 0.16) For the deduction, it is assumed that p x = p y = ps and that the molecular weight of each of the two stationary phases is higher than the molecular weight of the sample. Hence, with an increase in the molecular weight of the stationary phase (of the same type), the retention decreases, and the decrease is greatest for M , / M y < 20 (Fig. 10.1). Similarly, for the relative retention, Q = (%),I( Vg)B, of the separated substances A and B on chemically similar stationary phases X and Y , (10.17) From eqns. 10.16 and 10.17, it is evident that if the molar volumes of the pure solutes,
u l and v i , are equal, then by changing the molecular weight of the stationary phase only
the retention values can be affected. The relative retentions of substances A and B, which represent a measure of the separation selectivity, do not change. If u i > v i , then the relative retention decreases with increasing molecular weight of the stationary phase. In the opposite case, the selectivity increases with increase in M,. The higher the difference in the molar volumes of substances A and B, the lower are the retention volumes and a better resolution is obtained on increasing the molecular weight of the stationary phase. The effect of the molecular weight of the mobile phase is the reverse. In order to achieve short analysis times, it is therefore advisable t o choose a mobile phase with a low molecular weight and a stationary phase with a high molecular weight. The temperature dependence of the retention voluhe is expressed by the equation (10.18) where ah,is the partial molar energy of the transfer of the solute from the mobile to the stationary phase, which is equal to the difference in the partial energies of mixing of the solute with the mobile and the stationary phases, and % is the temperature coefficient of References p.277
MOBILE PHASES
Fig. 10.1. Change in the retention of the solute with change in molecular weight of the stationary phase, calculated according to eqn. 10.16. M x = 2 . lo4 ;p , v: = 100,200 and 350.
the mobile phase dilatation. In typical cases, R
dln V
usually has a value of about d(l/T) 3 kcal/mole, which is of the same order as those in gas chromatography. It therefore follows that in order to achieve good reproducibility of retention measurements in LLC, the temperature of the column should be thermostatted to ca. kO.1"C. It was found also experimentally that in LLC the elution volume changes distinctly with temperature. Schmit et al. found that on increasing the temperature by 30"C, the elution volume decreases by about 50%. The effect of pressure on the specific retention volume is very small. At medium pressures in the column, i.e., units to tens of atmospheres, the simplified equation 10.12 can be employed. The general relationship of eqn. 10.10 can be used even for very accurate measurements up to mean pressures of 102-103 atm, because the difference in the partial molar volumes of the solute in the mobile and the stationary phases, AV, is very small. The effect of pressure on Aij should be considered only for pressures higher than lo3 atm. Hence, it may be said that for current analytical practice, the simple relationship of eqn. 10.12 should suffice in most instances. Only for very accurate measurements, carried out at mean pressures in the column, i.e., about 100 atm and higher, should a more accurate relationship be used.
LIQUID-LIQUID CHROMATOGRAPHY
239
Selectivity of the liquid-liquid systems and solubility coefficients The most important criterion for the choice of the partition system in liquid-liquid chromatography is its selectivity. Selectivity is a property characteristic of a given system of two phases with respect to the chosen solutes. Therefore, i t is possible to discuss only the effect of a single phase on the system selectivity in an actual system. In addition, each component of the chosen liquid pair, especially if both are of low molecular weight, can be used, in principle, as either the stationary or the mobile phase. When selectivity is calculated on the basis of the relative retention of the separated components, it is irrelevant which of the liquids was considered as the stationary or mobile phase. In practice, the system of phases is most often chosen empirically. The main reasons for this are the inadequacy of the theory of solutions for giving an effective and sufficiently precise prediction of partition coefficients in any given system, a lack of data necessary for the prediction (molar volumes, critical temperatures and solubility parameters) and, to a certain extent, also the difficult calculations encountered. Another route based on the theory of solutions is the determination of partition coefficients from the characteristic parameters of the sample and both liquid phases, which was proposed by Huber et al. In this case, a procedure can be used based on the theory of regular solutions, according to Hildebrand and Scott (1962, 1964), or on the method of structural increments (Dealetal.; Derr et al.; Huggins; Martire; Pierotti et a/., 1956, 1959; and others). The sum of the empirical partition coefficients may also be useful when choosing a system of phases. In general, it can be stated that one of the conditions necessary for the achievement of adequate selectivity in an LLC system are the differences in the properties of the phases that are usually indicated as differences in polarity. Therefore, before discussing selectivity, a description of these properties should be given. The so-called polarity of a liquid phase is, in fact, the result of intermolecular interactions in the liquid. These intermolecular interactions are divided into two basic types: unspecific, which are caused by dispersion forces, and specific, which are caused by other interactions. The different types and intensities of these interactions are evident not only from the different behaviour of liquids as solvents, but also from their boiling points and latent heats of evaporation. The stronger are the intermolecular interactions, the more effective the given liquid usually is as a solvent for polar solutes and thus it also has a higher heat of evaporation. Its magnitude, relative t o unit volume, was used by Hildebrand and Scott (1962, 1964) for the quantitative characterization of the magnitude of intermolecular forces. They introduced as one of the fundamental concepts of the theory of regular solutions the so-called solubility parameter, 6 , defined as the square root of the ratio of the molar heat of evaporation, AE'. and the molar volume, V: (10.19) The solubility parameters ofliquids used as mobile phases range between 5.00 for perfluoroalkanes and 21 for water. In addition to Hildebrand and Scott's data, the solubility parameters of various compounds are listed in papers by Burrel, Crowley et al., Gardon, Lieberman, Morrison and Freiser, and Polak. The critical properties of liquids, necessary for calculations, were summarized by Kudchadker et al. References p.277
TABLE 10.1 SELECTED SOLVENT CHARACTERISTICS Solvent
Molecular weight
Density
Wml)
.\ Viscosity (cp, 20°C)
Surface area of the molecule,
2
6***
'd
605
6,
0
SA* Fluoroalkanes§§ CFCI, -CF, IsooctaneOOO Diisopropyl ether n-Pentane (33, -CF, n-Hexane n-Heptane Diethyl ether Triethylamine n-Decane Cy clopentane Cyclohexane nPropy1 chloride Isopropyl chloride Tetrachloromethane Diethyl sulphide Ethyl acetate Propylamine Ethyl bromide rn-Xylene Toluene chloroform Tetrahydrofuran Methyl acetate Benzene Perchloroethylene Ethyl methyl ketone Acetone Dichloromethane Chlorobenzene
-
-0.25 170.92 114.23 102.18 72.15 187.38 86.18 100.21 74.12 101.19 142.29 70.13 84.17 78.54 78.54 153.84 90.19 88.10 59.11 108.99 106.16 94.12 119.39 72.10 74.08 78.12 236.74 7 2.1 0 58.08 84.94 112.56
1.455 0.691 0.7 24 0.626 1.579 0.660 0.684 0.7 14 0.7 25 0.730 0.751 0.779 0.892 0.859 1.595 0.837 0.901 0.7 19 1.456 0.864 0.866 1.489 0.888 0.934 0.879 2.091 0.805 0.792 1.326 1.107
0.50 0.37 0.23
7.6 5.1 5.9
0.01 0.28 0.00
0.33 0.41 0.28
4.5
0.01 0.01 0.38
0.92 0.47 1.oo 0.35 0.33 0.97 0.45 0.45
10.3 5.2 6 .O 3.5 3.5 5.O 5.0 5.7
0.04
3.4 7.6 6.8 5.0 5 .O 4.8 6 .O
0.37 0.26 0.29 0.40 0.45 0.60 0.32
0.62 0.59 0.5 7 0.37 0.65
0.32 0.44 0.80
4.6 4.2 4.1 6.8
fih'
(A1,03)**
0.05 0.04
0.30 0.29 0.18 0.38 0.58
0.5 1 0.56 0.42 0.30
6.0 6.2 7 .O 7 .O 7.1 7.1 7.3 7.4 7.4 7.5 7.8 8.1 8.2 8.3 8.4 8.6 8.6 8.6 8.7 8.8 8.8 8.9 9.1 9.1 9.2 9.2 9.3 9.3 9.4 9.6 9.6
6.0 5.9 7.0 6.9 7.1 6.8 7.3 7.4 6.7 7.5
0 1.5 0
0 0 2 3.5
0 0 0 0 0 0 0 0 0 0
8.1 8.2 7.3
0 0 3
0 0 0
0 0 0
8.6 8.2 7.0 7.3 7.8 8.8 8.9 8.1 7.6 6.8 9.2 9.3
0 2 3 4 3 0 0 3 4 4.5 0 0
0.5 0.5 2 6.5 0 0.5 0.5 0.5 3 2 0.5
o
=
6.8 6.4 9.2
5 5.5 2
2.5 0.5
o
m
0.5
0
0.5
0 1.5 0 0 2 0
1.0 0 0 0.5 0 0.5
0.5
0
0 0 0.5
0 0 0 OT
o O
o
g G
w
%2 30
2
s
tu
u u
Anisole 1,2-Dichloroethane Methyl benzoate Dioxane Amyl alcohol Methyl iodide Bromobenzene Carbon disulphide n-Propanol F'yridine Benzonitrile Ni tromethane Nitrobenzene Ethanol Phenol Dimethylformamide Acetonitrile Methylene iodide Acetic acid Dimethyl sulphoxide Methanol Ethanolamine Ethylene glycol Formamide Water Amyl chloride Isobutyl methyl ketone 1-nitropropane Aniline Diethylamine
108.14 98.96 166.18 88.10 88.16 141.94 157.03 76.14 60.09 79.10 103.12 61.04 123.12 46.07 94.12 73.10 41.05 267.87 60.05 78.13 32.04 61.08 67.02 45.04 18.02 106.60 100.16 89.09 93.13 73.14
0.995 1.256 1.157 1.035 0.814 2.279 1.499 1.263 0.803 0.982 1.010 1.130 1.200 0.7 89 1.07 1 0.944 0.783 3.226 1.049 1.101 0.791 1.018 1.109 1.134 0.998 0.883 0.801 1.022 1.022 0.711
1.32 0.79
4.8
1.54 4.1 0.50 1.19 0.37 2.3 0.93 1.24 0.67 2.03 1.20 12.7
6.0 8.0
0.49 0.56 0.61
3.7 8.O 5.8
0.15 0.82 0.7 1
3.8
0.64
8.O
0.88
0.37
10.0
0.65
1.26 2.2 0.60
8.O 4.3 8.0
1 .o 0.6 0.95
8.0
1.11
4.2 5.3 4.5 6.7 7.5
0.26 0.43
19.9
1.00 0.43
4.4 0.38
9.I 9.7 9.8 9.8 9.8 9.9 9.9 10.0 10.2 10.4 10.7 11.0 11.1 11.2 11.4 11.5 11.8 11.9 12.4 12.8
12.9 13.5 14.7 17.9 21
9.1 8.2 9.2 7.8
2.5 4 2.5 4
2 0
9.3 9.3 10.0 7.2 9.O 9.2 7.3 9.5 6.8 9.5 7.9 6.5 11.3 7.O 8.4 6.2 8.3 8.0 8.3 6.3
2 1.5 0 2.5 4 3.5 8 4 4.0
0.5 0.5 0.5 4 5
8 1
2.5 0.5
0 0
7.5 5
5 7.5
0 7.5
1
3
1.5
1 0.5
0 0 0
0 0 0 4 0 0 0 0
0.53
0.62 0.63
*The surface area of the molecule as derived by Snyder from chromatographic measurements, expressed in relative units. For transformation to A=, the values given should be multiplied by 8.5. **Elution strength in liquid-solid systems for elution from alumina. ***Solubility parameters calculated from boiling points. 5 Approximate values. 5 8 Average values for various compounds. 8 8 8 2,2,4-Trimethylpentane. ?Some papers give higher values.
242
MOBILE PHASES
When liquids are ordered according to their increasing solubility parameters, a series is obtained which is very similar to the sequence of some eluotropic series. Hence, the solubility parameter 6 can also be considered t o be a measure of the elution power of a solvent in liquid-liquid systems. In spite of this, when this series is compared with the actual elution behaviour, in some instances (for example, according to Macek and Prochizka), appreciable discrepancies are found. This is probably due to the effect of specific interactions of various types in these liquids, which, however, need not display their effects to the same extent during the dissolution of samples of various types. It follows that the magnitude of 6 , characterizing the magnitude of intermolecular forces, may not always be sufficiently reliable. Keller et al. have shown that, assuming that individual contributions to the solubility parameter can be added to give a total, resultant, solubility parameter, 6 may be divided into four contributions: the contribution of dispersion forces, 6,; the contribution of the orientation of molecules, 6,; the contribution given by the ability of accepting protons, 6;, and the contribution due to the ability of donating protons, 6,. Each of these contributions, with the exception of S, is in fact a measure of another type of “polarity”, and the percentage fraction of each is different for different substances. It therefore follows that a single precise and generally applicable eluotropic series cannot exist. When an eluotropic series is composed for an actual case, in addition to the dispersion contribution, those contributions to the total polarity which might apply in the considered case must be taken into account. However, the lack of necessary numerical data is a practical problem. A review of selected characteristics that are important for the selection of mobile phases is given in Table 10.1. This table shows that the,re is no direct relationship between eo, characterizing the elution power in liquidsolid systems, and the solubility parameter, 6. The reasons for this were discussed in detail by Keller and Snyder. From the thermodynamic point of view, the problem gf selectivity in LLC for similar (and hence poorly separable) solutes was investigated by Martire and Locke. Assuming that In y2 = In yih
+ In yazth
(10.20)
they demonstrated that even for relative retention the following equation can be written: In
(Y
= (In
+ (In a)ath
(10.21)
The component (In is given by athermal, configurational contributions of activity coefficients, ylth, following from the different sizes of the solute and solvent molecules. It only reflects the effects of the arrangement and therefore it can be considered to be a purely entropic term. The thermal component (In is determined by thermal contributions of activity coefficients, rib, following from the intermolecular forces between the solute and solvent molecules. According to Martire, it is given by both enthalpic and entropic contributions to non-ideality. Using Flory-Huggins’ relationships, it can be shown for athermal contributions to the relative retention that (1 0.22)
LIQUID-LIQUID CHROMATOGRAPHY
243
From eqn. 10.22, it follows that the greater is the difference between the molar volumes of the pure stationary and mobile phases, vf and ,:v and those of the two solutes A and B, u g , the greater is also the athermal term. The contribution of the athermal term to the relative retention leads to the result that even compounds +at undergo absolutely identical energetic interactions with both phases (for example, isomeric solutes) and that differ sufficiently only in their molar volumes (densities), can be separated successfully by LLC. The solute with the lower molecular weight dissolves preferentially in phases with a larger molar volume, because a larger increase in entropy takes place during dissolution. For the thermal contribution to the relative retention, it can be deduced that
vi,
(1 0.23)
where u i is the molar volume of component A, approximately equal to v:; 6, and 6, are Hildebrand and Scott's (1962) solubility parameters for the mobile and the stationary phase; and and are the critical temperatures of substances A and B. The thermal contribution increases with increase in the difference between the intermolecular forces in the mobile and stationary phases, which always exist if the miscibility of the liquids is limited. For a given pair of solvents, the thermal contribution increases with the magnitude of the T i - T: * and hence a requirement for separation, based on energy terms, is a product
vi
T:
difference between the critical temperatures of the solutes A and B. A substance with a higher critical temperature is dissolved preferentially in the phase with a higher coefficient of solubility. If this solvent is the stationary phase, the substance with a higher critical temperature has a greater retention, while in the opposite case it is eluted preferentially. In view of the approximations that have not been discussed for the sake of brevity, the relationship for the thermal contribution is not applicable (a) to systems in which the solvent-solvent interactions are stronger than solute-solvent interactions, (b) to the separation of substances that differ appreciably in their behaviour and (c) to systems in which adsorption phenomena in interphases occur. Every system of solvents can therefore be characterized by the terms - - (u; ";)and (6: - 8 2 ) . If the stationary phase is less polar than the eluent (which is common in separations of non-polar or weakly polar solutes), then the elution sequence is in accordance with the increase in the molecular weights. If the eluent molecules are smaller than the molecules of the stationary phase, as is usual, then the contribution of the thermal term to the total retention is negative. In addition, 6; > 6: and, because T decreases with increasing molecular weight, the thermal contribution term is positive and larger than the athernial term. During analyses of polar solutes, the stationary phase must be more polar than the mobile phase. If, in this instance, the stationary. phase has a higher molecular weight, both contributions are positive. It therefore follows that for separation in a liquid-liquid system, the value of the thermal term is usually decisive. The athermal (configurational) term, the absolute contribution of which is usually smaller, may either enhance or adversely affect the separation. As the contribution of both terms during the separation of strongly polar solutes is positive, it is evident that LLC has potentially a higher selectivity for the separation of polar substances. The contribution of the thermal References p.277
244
MOBILE PHASES
0.4
Fig. 10.2. Contribution of the thermal to the relative retention, term, (In LY, for various values of f(r*)= ( ~ ~ - T ~ calculated ) / T ~ ~ according to eqn. 10.22.
1 1 700(---) v; v,”
Fig. 10.3. Contribution of the athermal term, (In of ( u i - v b ) , calculated according to eqn. 10.23.
to the relative retention, a, for various values
LIQUID-LIQUID CHROMATOGRAPHY
24 5
and athermal terms to the relative retentions is illustrated by Figs. 10.2 and 10.3. As already mentioned, a requirement for selectivity in LLC is the immiscibility of both chromatographic phases. From the theory of solutions, it follows that the higher is the heat of mixing of two liquids, AHM,the less miscible they are. For the mobile and the stationary phases, the corresponding relationship assumes the form:
AHM = ( X m V i
+ X,v,")@; -
(1 0.24)
where x, and x , are ~ the molar fractions of the stationary and mobile phases, respectively, and @, and @m are their corresponding volume fractions in the resulting solution. Hence, the miscibility is the lower the larger are the molar volumes and the greater is the difference between their solubility parameters. In view of the magnitude of the solubility parameters, the phase should be chosen so that 16, - 6, I > 4 and the solubility parameters of all separated components are between the values 6, and 6,. If, however, 6, and 6, differ too widely, adsorption could take place in the liquid-liquid interphase. The immiscibility of the liquids m and s may be enhanced by the choice of a stationary liquid with a high molecular weight. On the other hand, it is advisable to avoid the use of mobile phases of high molecular weight, because they have an excessively high viscosity, which is disadvantageous from the point of view of column efficiency (see p.249). The requirements for the choice of a suitable carrier for a given type of stationary phase are discussed in Chapter 9. Programming of the mobile phase When analyzing samples containing components of various types, capacity factors of the components very often exceed the optimum range 1 < ki < 10, so that the choice of constant conditions does not lead to a satisfactory separation. In a system that permits a good resolution of the most easily eluted components the last components are usually eluted over excessively long intervals and their zones are extremely broadened, which impairs detection or makes it impossible. Under the conditions suitable for elution of the slowest components, kl values for the first components are muchless than unity, so that even at a sufficient column effectivity and suitable values of the relative retentions, the separation in this regon is not effective. The so-called effect of small k ' applies here. The problem can be solved either by changing the column efficiency or the k,! value during the analysis; the latter method is more effective and is therefore more often used. Programming of the solvent flow
The column efficiency can be controlled by changing the linear flow-rate of the mobile phase in a continuous or discontinuous manner. On de&easing the flow-rate at the beginning of the analysis, the column efficiency may be increased. An increase in the flow-rate at the end of the analysis, accompanied by a decrease in column efficiency, can be utilized only if the last components are sufficiently well separated. The programming of the flow is suitable only when the lowest values are not much less than unity, and the highest values do not exceed a value of about 10. The condition for this is that the column efficiency should change sufficiently with the flow-rate, i.e., the constant C i n eqn. 10.29 References p.277
246
MOBILE PHASES
should have a sufficiently high value. For systems characterized by a very small dependence of the efficiency on the mobile phase flow-rate, the technique of flow programming is not very effective. From the practical point of view, this technique is very advantageous because of its simplicity and low demand on apparatus. Chemical properties and the physical state of the column do not change during the flow programme, which is important from the point of view of rapid repeatability of the analyses. As the value of k‘ does not change during the flow programming, the elution volumes remain constant.
Gradient elution The most commonly used technique of programming in liquid chromatography is the regulated change of the mobile phase composition, i.e., the so-called gradient elution. Its most important effect is the control of capacity factors, which are regulated so that their values for all eluted components vary within the limits 1 < k’ < 10. The effect of changes in the viscosity of the mobile phase on the column efficiency, caused by the change in composition, is usually negligible. Concentration gradients may be either discontinuous, created by one or several stepwise concentration changes of two or more components, or continuous of various types, Snyder and Saunders have shown that the most suitable is a continuous logarithmic concentration gradient, which gives the most effective separations with respect to time. In this method of separation, the value of k’ for each eluted substance during its passage through the column is close to the optimum. The composition, and hence also the elution power of the mobile phase can be changed within a broad range, which per&ts the effective control of the magnitude of the capacity factor over an exceptionally wide range. However, the successful application of this method assumes the fulfilment of several conditions. Firstly, a suitable stationary phase must be chosen, or the programme should be adapted t o a particular stationary phase. In systems with a liquid stationary phase, the danger of increased washing out arises during the changes in the mobile phase polarity. In some types of chemically bound phases, chemical reactions with the components of the mobile phase may take place under certain conditions, even if these are generally characterized by a much hgher stability. Adsorbents show the highest resistance towards the effect of the mobile phase and therefore they are the most suitable for use with concentration gradients. Therefore, in classical adsorption liquid chromatography or in thin-layer chromatography, where the preparation of the chromatographic bed is rapid and cheap, the use of simple gradients has found wide application. In high-efficiency liquid chromatography, complicated devices (see Chapter 8) are necessary for the generation of concentration gradients at high pressures. During gradient elution, the capacity factors change in time as a function of the chosen form of the gradient. If reproducibility of elution volumes and quantitative data is to be achieved, which would be comparable with the results obtained during work at a constant composition of the mobile phase, it is necessary that the form of the concentration gradient and its course with time should be reproducible as precisely as possible. After each analysis, complete regeneration of the column used is essential. This regeneration requires the
LIQUID-LIQUID CHROMATOGRAPHY
247
complete washing out of all components of the mobile phase that passed through the column during the programme and the re-attainment of the equilibrium state that existed in the column before the sample injection. In liquid-solid systems, regeneration is often time consuming, and it is advantageous to increase the mobile phase flow-rate or to change the column temperature. If the column is not completely regenerated, reproducibility of the measurements cannot be achieved. The gradient elution adversely affects the conditions of the functioning of detectors that are sensitive t o the composition of the mobile phase used, especially in binary detectors (refractometers, etc.). In spite of these disadvantages, the gradient technique remains the most effective means for the analysis of complex mixtures that contain substances of different chemical types. The development of the necessary instrumentation and the investigations devoted to the theory and practice of gradient elution indicate that this technique will find wide utilization in modern liquid column chromatography.
Temperature programming Relatively little attention has so far been paid to the possibility of temperature programming, which in liquid-liquid systems follows from the dependence of activity coefficients on temperature, and in liquid-solid systems from the values of heats of adsorption. During temperature programming, both the capacity factors and the column efficiency change. According to Schmit et al., an increase in temperature of 60°C decreases the' HETP by half. The capacity factor usually decreases with increasing temperature; in some instances the increase was observed only in systems with a multicomponent mobile phase. In liquid-liquid systems, this technique meets with difficulties caused by the increased solubility of the stationary phase in the mobile phase at elevated temperatures. If the temperature increases over the whole analytical column simultaneously, provision cannot be made that the mobile phase present in the column should be in equilibrium with the stationary phase at every moment and at each point in the column, even if the temperature of the pre-column changes at the same rate. A partial solution of this problem consists in heating the precolumn to a temperature slightly higher than that of the column (Kirkland, 1971b). However, the temperature programme of the pre-column should be chosen carefully with respect to the column diameter and the flow-rate. If the mobile phase is a single compound, the situation is similar to that in temperature programming in gas chromatography. Lebedeva et al. have shown that in this way the retention can be regulated in liquid-solid systems even when the heats of adsorption of the solutes are of the order of kilocalories per mole. The higher the heat of adsorption, the more distinct is the effect of temperature. If a temperature programme is applied to a system with a binary or a more complex phase, then the increase in temperature also leads to changes in equilibrium between the mobile and the stationary phases, which result in changes in the composition of the mobile phase during the programme. This effect was made use of by Scott and Lawrence in the programme of a temperature-regulated gradient elution characterized by a stable, instantaneous equilibrium between the stationary and mobile phases along the whole References p.277
248
MOBILE PHASES
column. The resulting effect is comparable with the influence of the concentration gradient prepared in the conventional manner, but with the advantage of a better resolution of the eluted components. Its disadvantage is that a relatively lengthy regeneration of the column and pre-column is required, which, however, can be shortened by using exchangeable pre-columns.
MOBILE PHASES FOR LIQUID-SOLID CHROMATOGRAPHY Properties of the mobile phase When comparing gas and liquid chromatography, it is evident that almost all of the basic differences between them, both theoretical and practical, follow from the different properties and the behaviour of the mobile phase in the chromatographic system. They appear in all basic aspects: in the thermodynamics of the chromatographic process, in its dynamics, in the operating technique and in the instrumentation. The physical basis of these differences is the different intermolecular interactions in the liquid and in the gas, which is directly evident, for example, from the very great differences in their physical properties (density, compressibility, viscosity, etc.) and in the diffusion coefficients. In a chromatographic system with a liquid mobile phase, and also in other liquid systems, intermolecular forces are the cause of the dissolution of the sample components in the mobile phase. They lead to their molecular dispersion, which is a prerequisite of the chromatographic process. Energetically important interactions of the mobile phase molecules with those of the stationary phase distinctly affect and modify the interaction of the sample with the stationary phase. Together with the mobile phasesolute interactions, they often distinctly regulate thkresulting selectivity of the chromatographic system used, so that in liquid chromatography, the mobile phase cannot be regarded as an inert component and its presence cannot be neglected or its effects underestimated. As the effect of the mobile phase in liquid chromatography is more complex than in gas chromatography, the criteria for its choice must also be more varied. It Aould also be kept in mind that the choice is not only dependent on other parameters (separation selectivity, equipment, operating technique, etc.) but often also exerts a reverse influence. If the basic requirement for achieving the chromatographic process is fulfilled, i.e., the sample is sufficiently soluble in the chosen mobile phase, it is mportant that this mobile phase should also fulfil some other requirements, as follows: (1) In combination with the stationary phase, it should ensure, at least for the most important components of a given sample, a sufficient selectivity of separation, which is essential for good resolution. For the capacity factors of single samples, k,!, 1 d kl!< 10 should usually apply. (2) It should enable such experimental conditions or operating procedure to be chosen as would prevent changes in the column properties. If conditions (1) and ( 2 ) cannot be fulfilled simultaneously, another mobile or stationary phase should be selected. (3) It should satisfy the requirements for work with the most suitable or at least a satisfactory type of detector.
LIQUID-SOLID CHROMATOGRAPHY
249
(4) It should possess suitable properties that permit the maximum efficiency of the chromatographic bed and the shortest time of analysis to be achieved (low viscosity, high diffusion coefficients of the sample components). (5) It should permit the recovery of the separated components. (6) It should be inert to all of the materials of construction of the chromatograph with which it comes into contact during the measurements. (7) It should be safe and economically acceptable, or be capable of regeneration after use. Ofcourse, all of these requirements do not always apply to the same extent. The first two requirements, even if only on broad lines and according to general principles, should be considered in relation to the operating techniques used, and the basic types of chromatographic systems or the type of sample serve as criteria. Points (3), (6) and (7) do not require further discussion. The recovery of the separated components from the eluate is usually effected by the difference between their boiling points and those of the mobile phase. The effect of the physical properties of the mobile phase on the efficiency of the chromatographic bed will differ according to the shape of the bed (thin layer or a column) and, to a certain extent, also according to the method chosen. Point (4) applies equally to both column adsorption chromatography (LSC)and partition chromatography (LLC) because, from the point of view of the dynamics of the chromatographic process, the role of the mobile phase is the same in both instances. Therefore, the effect of the physical properties of the mobile phase on the efficiency of the chromatographic column will be discussed before the analysis of the role of the mobile phase in LSC and LLC.
Effect of the physical properties of the mobile phase on column efficiency
On the supposition that a system with an optimum, or at least an acceptable, selectivity has been found for a given analysis, mainly two criteria are important for estimating the efficiency of the analysis: the resolution of individual components and the time necessary for the analysis. Both parameters are affected to a considerable extent by the experimental arrangement (column length, pressure drop, linear flow-rate of the mobile phase) and the dynamics of the transport of the sample through the column. Assuming that the total time of analysis is equal to the elution time of the last detected zone, t z , then L tz = to( 1 + k;) =- (1 U
+ k;)
(1 0.25)
For an efficient column and a sufficiently large capacity factor, k;, and for a linear distribution isotherm, the elution time of the latter part of the last zone is negligible with respect to the total time of analysis. For given experimental conditions, the column length, L , and the capacity factor, k i , are constants. The elution time of the unretained component, t o ,is indirectly proportional to the linear flow-rate of the mobile phase in the column, u (cmlsec). From the equation for the column permeability, KO, the mobile References p . 2 77
250
MOBILE PHASES
phase flow-rate in the column can be expressed as a function of the pressure drop, AP,the mobile phase viscosity, 7, the column length, L , and its total porosity,$ (1 0.26) Substituting this expression into eqn. 10.25, it follows that tz = KO & . AP (l
+ki)
(1 0.27)
It is evident that tz is directly proportional to 7 , if the other terms are constant. The viscosity of the mobile phase can in practice be changed either by a change in the composition of the mobile phase or by an increase in temperature. However, an increase in temperature is generally accompanied by a change, usually a decrease, in the capacity factor. The change in the composition can usually be carried out so that kk remains constant if other.conditions do not change. The resolution, R,, of two components A and B can be expressed by the relationship (10.28)
where tA and t g are uncorrected elution times, wA and wg are the peak widths at the baseline expressed as time, a is' the relative retention, k; is the capacity factor of the later eluted component B and N is the number of theoretical plates of the column used. Terms (a) and ( b ) are unambiguously determined by the chosGn chromatographic system at a given temperature and are independent of the physical properties of the mobile phase. The effect of the physical properties of the mobile phase on the resolution becomes evident only in term (c), which characterizes the efficiency of the column used. Consideration of the effect of the physical properties of the mobile phase on the resolution can therefore be replaced by their effect on the efficiency. The effect of the properties of the mobile phase on the column efficiency is complex because it is connected with the dependence of the plate height on the linear flow-rate of the mobile phase. For the sake of clarity, a simple equation for the theoretical plate height may serve as the starting point:
H=A+Cu
(10.29)
Thls equation is most often used to describe the results in high-speed liquid chromatography. The mass transfer resistance coefficient, C,can be divided into the contribution of the resistance to the mass transfer in the stationary phase, C,, in the mobile phase fraction contained in the interparticulate space, C' ,and in the immobile fraction of the mobile phase contained within the packing particles, CA :
c=c,+c,+c;
(10.30)
LIQUID-SOLID CHROMATOGRAPHY
25 1
The contribution of the mobile phase can be expressed in the form (10.31)
c;
d2 = cp(@’, k’)2%
Dm
where w is a constant the magnitude of which is given by the non-uniformity of the mobile phase flow through the bed, dp is the average particle diameter, Dm is the diffusion coefficient of the solute in the mobile phase, cp(@‘, k‘) is a term dependent on the mobile phase fraction occupying the space within the particles, a’,and k’ is the capacity factor of the solute. For our purposes, the contribution of the stationary phase may be considered to be constant. The term A = 2Adp is also independent of the physical properties of the mobile phase because the constant h characterizes the bed geometry. After substituting in eqn. 10.29, it follows that ( 1 0.32)
From eqn. 10.32, it follows that at a given flow-rate, the contribution to the plate height of the mass transfer resistance in the mobile phase is indirectly proportional to the diffusion coefficient, so that with increasing O m ,the column efficiency, and hence the resolution, also increase. Substituting eqn. 10.26 for u in eqn. 10.32, it becomes evident that the column efficiency is also affected by q:
ap
( 1 0.33)
As the viscosity of the mobile phase also affects the magnitude of the diffusion coefficients (a more viscous liquid lowers the diffusion rate and decreases the diffusion coefficient value), Om is not constant when is changed in eqn. 10.33. The effect of the viscosity on the efficiency in liquid-solid systems was investigated experimentally by Snyder (1967, 1969). He found that when the viscosity of the mobile phase is doubled, the analysis time is also doubled when the pressure drop remains constant. For a constant time of analysis (constant u), however, the column efficiency decreases. It can be deduced that by increasing the viscosity 2%-fold, the plate height is approximately doubled. The effect of the viscosity on the separation efficiency in liquidliquid systems was described by Kirkland (197 la). Reversed-phase systems were studied from this point of view by Schmit et al., who found that when the temperature is increased from 20°C to 80°C, the column efficiency approximately doubles. From the point of view of the rate of analysis and the resolution, it is therefore preferable if mobile phases with as low a viscosity as possible are chosen, or if the work is performed at elevated temperatures. References p.277
252
MOBILE PHASES
As a series of liquids of very low viscosity (about 0.2-0.3 cP) exists, the viscosity of the mobile phase can be kept below 0.4-0.6 cP even when more viscous liquids are added. Only when aqueous solutions or phases that contain a large proportion of water are used can viscosities below 1 CPnot be achieved.
Strength of the mobile phase In the conventional arrangement of high-efficiency adsorption column chromatography, the adsorbent is wetted with the mobile phase. If the molecules of the transported sample A are to be retained by the surface, then a certain number of solvent molecules must first be expelled from the adsorbed layer. On the other hand, the surface set free by sample desorption is immediately occupied by molecules of the mobile phase M. In the zone moving through the column, a process is taking place that is characterized by the equilibrium equation
A,+nM,*A,+nM,
(1 0.34)
The subscripts m and s indicate molecules in the mobile and stationary phase, respectively. For the sake of simplicity and clarity, it is useful to assume that the mobile phase is composed only of a single type of molecules, that the injected sample is dissolved in the mobile phase, and that the interaction of the sample with the mobile phase does not lead to the so-called secondary solvent effects (hydrogen bonds, complex formation, etc.). The coefficient n in eqn. 10.34 is equal to the ratio of the effective adsorption cross-sections of the sample molecule and the mobile phase, and is therefore not a whole number. The net energy of adsorption during this process, AE, can be expressed as
AE=E~+nE,-Ek-nE~
(10.35)
where E:, E y , E t ,E: are the molar free energies of the interaction of sample molecules with the mobile phase molecules in the adsorbed layer and in the solution. Under the above conditions, to a first approximation the liquid phase energy terms E k , nE; can usually be ignored. Snyder (1964b) also corroborated experimentally that their contribution in typical adsorption systems is less than 10% of the contribution of the corresponding adsorption terms. This finding can be easily explained. If no specific interactions are involved in the solution with the mobile phase (in the case of nonpolar or weakly polar solvents), the intermolecular interactions are caused by non-specific Van der Wads forces. The magnitude of these dispersion forces per molecule is proportional to its cross-section. From eqn. 10.34, it follows that the surface area of the sample molecule is equal to n times the area of the mobile phase molecule. In solution, the sample molecule is surrounded by mobile phase molecules and, a t low sample concentrations and the covering of the adsorbent surface usual in chromatography, mutual interaction of the sample molecules does not come into consideration in the adsorbed layer either. Contact of the solvent molecules with the sample molecules is therefore disturbed only at the site of contact of the sample molecule with the adsorbent surface. Therefore, with complete solvation of the adsorbed sample molecules, a maximum of 50% of the solvation energy, represented by the term E:, can be released after desorption. Similar conditions also exist during the
Ll QUl D -SOL1D CH ROM A TOG RAP11Y
253
desorption of the mobile phase molecules, which causes the contribution of the dissolving members to the total energy change to decrease further. In addition, the terms that characterize adsorption interactions are usually much higher, especially the term E,". In the opposite case, the value of the distribution coefficient is so low with respect to the relationship AE=RTlnK
(1 0.36)
that a satisfactory separation cannot be achieved, so that the total energy effect of the adsorption process is approximately
AE=EP - n E y
(1 0.37)
where the terms E: and EY represent the molar free adsorption energies of the sample and the mobile phase, respectively, on the pure adsorbent surface. It is evident that the more strongly the mobile phase molecules are bound by the surface, the lower is the value of AE for a given sample during elution. Therefore, more strongly adsorbed mobile phases decrease the retention and act as more effective, stronger elution agents. Hence, the adsorption value of the mobile phase molecules is a measure of their elution strength. On the basis of the above, with respect to the magnitude of the adsorbing surface area and its activity, 8, and assuming the validity of Langmuir's isotherm for the adsorption of the mobile phase and sample adsorption, Snyder (1968) derived a very useful relationship for the prediction of the adsorption distribution coefficient, K , of the component A: log K = log V,
+ p(EA - SAeo)
( 10.3 8)
The magnitude o f K gives the ratio of the sample concentrations in the stationary and mobile phases. Numerically, it is also equal to the corrected retention volume in millilitres and referred to 1 g of adsorbent. The volume of the adsorbed phase, V,, is equal to the product of the specific adsorbent surface area, S (m'/g), and the thickness of the adsorption layer. To a first approximation, when it can be considered as being equal to the thickness of a monomolecular layer of adsorbed water,
5 = 0.000,35
S
(10.39)
Under this supposition, V, is a constant characteristic of a given adsorbent and independent of the nature of the sample. However, in general, the above supposition is not completely valid because the layer thickness depends on the shape and arrangement of the molecules. SAis the area of the adsorbent surface occupied by the sample molecule (adsorption cross-section). EA is proportional t o the energy of adsorption of the sample molecule from pentane solution*. The symbol e o , ie., the strength of the mobile phase, characterizes the magnitude of the interaction of the mobile phase molecules with the surface. According to Snyder (1968), its numerical value is proportional to the interaction energy of the mobile phase *Snyder (1968) used a different approach, so that with his original symbols eqn. 10.38 has the following form: log KO = log Va
References p.277
+ a(SD - As€')
254
MOBILE PHASES
with the pure adsorbent surface less the interaction of the pentane molecule with the adsorbent surface relative to unit area. For pentane as the mobile phase, E' = 0. From the mobile phase elution strength, it follows that the numerical value of E' is dependent on the adsorbent used. Experimentally, it was found that the result determined for elution from alumina can be applied directly to other adsorbents of the oxide type. For silica gel, magnesium oxide and Florisil, the following relationships can be used: Eo(siOz) = 0.77 ~'(A1~0~)
(10.40a)
e'(Florisi1) = 0.53 e0(A1203)
(10.40b)
e'(Mg0) = 0.58 ~'(AlzO3)
(10.40~)
The scattering of the calculated values, k0.04 unit, differs only slightly from the experimental results and it can therefore be considered that a single eluotropic series can be proposed for all adsorbents of the oxide type. During chromatography on non-polar adsorbents, polar interactions that are important in adsorbents of the oxide type cannot apply. Therefore, the polarity and polarizability of the molecules virtually does not affect the elution strength, and the magnitude of the nonspecific Van der Waals forces is the only factor. The elution strength in these instances increases approximately in proportion with the increase in the molecular size and the sequence of the elution strength is generally reversed in comparison with polar adsorbents, as was shown, for example, by Schorn, Williams et d. and others (compare Tables 10.1 and 10.2). The effect of the elution strength of the mobile phase on the distribution coefficient during the elution of a sample from an adsorbent follows from eqn. 10.38. For the ratio of the distribution coefficients of the given solute during elution with the mobile phases 1 and 2, the equation K log--] =psA(€; - €?) (10.41)
KZ
applies. TABLE 10.2 ELUOTROPIC SERIES FOR ELUTION FROM CHARCOAL ACCORDING TO SCHORN The elution strength increases from the fust member (water) to the last (benzene).
Solvent
Surface area of the molecule (A2)
Water Methanol Ethanol Acetone Propanol Diethyl ether Butanol Ethyl acetate ti-Hexane Benzene
12.7 24.6 32.3 35.7 40.0 47.6 47.6 48.4 57.8 51.0
LIQUID-SOLID CHROMATOGRAPHY
255
Without changing the surface area or the type of adsorbent, the sample retention can be changed merely by changing the mobile phase elution strength, The retention change is the greater the more active is the adsorbent used and the greater the area occupied by the sample. The greater these values, the smaller is the change in the elution strength necessary for the required shift of K . On the other hand, a given pair of m6bile phases affects the retention of solutes that differ in the cross-section of the molecule in different ways. Eqn. 10.41 represents the theoretical basis for the gradient elution technique in LSC.
Elution strength of the binaiy mobile phase The description and quantitative evaluation of the function of a multicomponent .mobile phase in a chromatographic system is complicated. For the sake of simplicity, only two-component systems will be described here, as they represent a special case of more complex phases. As two-component mobile phases permit a wide control of the selectivity and the elution strength, their use in practice is also more advantageous than more complex phases. The more complicated the mobile phase used, the more difficult is a preliminary estimation of its function in a chromatographic system, its preparation and the maintenance of constant properties during the measurement. The choice of a simpler mobile phase is also preferred from the point of view of the interpretation of the separation processes. A mixed mobile phase can be considered as a simple mobile phase with the addition of a sample. Therefore, in a similar manner to adsorption of the sample from a simple mobile phase, a dynamic equilibrium takes place when a binary mobile phase is introduced. This is described by the equation
M, + N , + M , + N ,
(10.42)
where N represents the more strongly sorbed component. For the formulation of the relationship for the elution strength of a binary mobile phase, Snyder (1 968) made the following assumptions: (1) No secondary effects of the mobile phase are observed in the system. ( 2 ) During the passage of the mobile phase through the adsorbent bed, no change in composition takes place which might cause the formation of a front of the stronger component of the solvent mixture, also indicated by demixing of the mobile phase. This condition is fulfilled only in the elution method after the passage of a volume of mobile phase that permits the front of the more strongly sorbed component t o leave the column. If the mobile phase is introduced on to a dry adsorbent during development (dry column technique), or if a continuous concentration gradient is applied, this condition is never fulfilled. The formation of a front can be neglected, as a rule, only if a mobile phase is employed in which the concentrations of components art comparable and the elution strengths not too different, and if the most rapidly eluted sample component has a retention such that it is eluted only after the front of the more strongly sorbed component. (3) The molecules of the mobile phase components are of equal size (n = 1). (4) Adsorption of both components on the surface of the stationary phase is described by Langmuir’s equation. References p.2 77
256
MOBILE PHASES
( 5 ) In the description of the competing adsorption equilibrium, solvation terms are neglected (see eqn. 10.35). The interaction of molecules in the adsorbed layer is also negligible. , obtained the follo.wing For the elution strength of a binary mobile phase, c $ ~ he relationship: (10.43)
where E$ and $ are the elution strengths of the pure components on a given adsorbent of activity fl> 5" is the area occupied by a molecule N of the mobile phase and xN is the molar fraction of the stronger sorbed component. The elution strength of the binary mobile phase, and hence of every more complex mobile phase, depends not only on its composition and the elution strengths of the individual components and their molecular size, bur also on the activity of the adsorbent used. Therefore, an eluotropic series that consists of simple and binary mobile phases, which would be the same for different adsorbents of the same type and also for the same adsor-
Fig. 10.4. Dependence of the elution strength of a binary mobile phase on its composition, for elution from silica gel. Specific surface area cu. 300 m 2/g, deactivation with 2% of water; p = 0.71. 1 = Hexane-carbon tetiachloride; 2 = hexane-chloroform; 3 = hexane-diethylamine; 4 = hexaneethanol. The curves were calculated from eqn. 10.43. In the region of the validity of the equation, the calculated values differ from the experimental values by 0 i 0.02 to 0.03 e o units.
LIQUID-SOLI D CHROMATOGRAPHY
257
bents with different activities, cannot be devised. It is important that the elution strength of a binary mobile phase changes most rapidly with the content of the stronger component in the region of low concentrations (Fig. 10.4). The component present in very low concentrations was termed a moderator by Mags. Eqn. 10.43 can also be used for the estimation of the elution strength of a multicomponent system if it is chosen so that the elution strengths of the two strongest components are much higher than the strengths of the remaining components. In such a case, the adsorbent surface is covered only by molecules of the strongest components, and the difference 1 - xN should be replaced in eqn. 10.43 by the molar fraction of the strongest component. A detailed analysis of the elution strength of the mobile phase and of the conditions of the validity and applicability of the above relationships was given by Snyder (1 968). Quantitative data characterizing the mobile phases are summarized in Table 10.1. Other magnitudes (adsorbent activities, p; areas of the surface occupied by the molecules of vaqous samples, SA;volumes of adsorbed layers, V,) necessary for calculations according t o eqns. 10.38,10.41 and 10.43 were also given by Snyder (1968). Demixing ejrects
The basis for the deduction of the sample retention and the relationship for the elution strength of a binary mobile phase consisted in competing adsorption of the molecules on the adsorbing surface, characterized at each point of the column by dynamic equilibrium between the molecules in the mobile and the adsorbed phases. From the point of view of this process, it is irrelevant whether the component added t o the mobile phase acts as the sample or another mobile phase component; only the method of its introduction into the column is of importance. If the component is introduced into the column as a discrete pulse, it is considered t o be a sample. As a result. of its passage through the column, its original shape, usually rectangular, is gradually changed until a t the column outlet an elution zone of the conventional shape is obtained. The maximum concentration of the zone, cnlaX., is always lower than the originally introduced concentration, co. If the added component of original concentration co is introduced into the column continuously, the technique is called continuous or frontal injection, which leads t o the formation of a frontal chromatographic zone. The maximum equilibrium concentration in the frontal zone is equal to the injected concentration, co. Its formation, in relation t o the shape of the adsorption isotherm, was described qualitatively for the first time by De Vault. He demonstrated that if the adsorption system is characterized by a convex isotherm (isotherm of type I), then the frontal zone has a sharp front and a diffuse rear. Systems with a convex isotherm (type 111) lead t o frontal zones with a diffuse front and a steep rear (for types of isotherms, see Brunauer, BrunaueTet al. or Young and Crowell). As a consequence of the added component being caught on the surface, its concentration decreases to zero in the first fractions of the injected sample. Its elimination by adsorption leads to a situation where the frontal zone formed moves through the column a t a lower rate than the pure mobile phase. The stronger is the added component adsorbed and the lower its concentration, c o , the more slowly its front moves through the column. It is evident that the frontal zone is formed in all instances, regardless of whether the References p.277
258
MOBILE PHASES
component acts as the sample or an additional component of the mobile phase. The position and the shape of the zone are determined by the shape of the adsorption isotherm, concentration co , amount of adsorbent and the flow-rate. For binary liquid systems, these processes were treated quantitatively, for equilibrium and non-equilibrium courses of the adsorption process, by Glueckauf (1945; 1947a, b, c; 1949). If several components dre injected simultaneously with a simple mobile phase, the number of fronts formed equals the number of components present. However, it can be demonstrated that the zones are also formed during the changes in concentration of the mobile phase components. The amount of the corresponding component in the stationary phase must always be changed so that it is in equilibrium with its content in the mobile phase. If the concentration increases, an adsorption front is formed, and if it decreases, a desorption front appears. As the attainment of equilibrium always takes a certain time, it appears that in this process, during the control of the mobile phase composition at the column outlet, more strongly adsorbed components, i.e., those with a greater elution strength, added at the column inlet were demixed in the column. The formation of frontal chromatographic zones in the mobile phase, the so-called ' demixing, is, therefore, a general phenomenon caused by the attainment of adsorption distribution equilibria in the chromatographic system under dynamic conditions, during changes in the composition of the frontally injected mobile phase. It always takes place regardless of the bed shape (column, thin-layer) and whether and how the bed was previously wetted. Therefore, a description and quantitative evaluation of the demixing effect should be undertaken from these points of view. When passing from a single to a binary mobile phase, the shape and the course of the zone formed, and also the volume of the phase necessary for its elution from the column, can generally be described by using known procedures. Certainly, for the interpretation or the prediction of the retention data, the described effects represent an undesirable and complicating factor, especially in work with changing gradients. As is shown for polyzonal thin-layer chromatography, sometimes they can be utilized with advantage. In other instances a volume of mobile phase must be allowed to pass through the column such that all zones formed during the change in composition are eluted. Only then can the column be considered to be conditioned and able to give equilibrium data reproducibly.
Selectivity of the mobile phase The basic equation 10.38 for the magnitude of the retention in liquid-solid systems was deduced on the assumption that the contribution of the liquid phase terms to the total energy change of the process (see eqn. 10.35) is negligible. This assumption is usually satisfactory for weak and not too strong mobile phases. When such phases are used, usually samples in which the components are chemically related or very similar in their adsorption behaviour and dissolving properties are analyzed. From the point of view of molecular interactions, this means that the interactions in the mobile phase are weak and non-specific (see the section Strength of the mobile phase). The interactions of the ,nolecules of single sample components with the surface of the stationary phase are substantially of the same type and do not differ in principle from the type and magnitude of
LIQUID-SOLID CHROMATOGRAPHY
259
interactions of the surface with the mobile phase molecules. Large differences lead to excessively high distribution coefficients, which are unsuitable from the chromatographc point of view. In strong mobile phases (with respect to polar sorbents of the silica gel type), both assumptions are, however, only rough approximations. From the definition of the concept of elution strength, it follows directly that in the case of strong mobile phases during the interaction with the surface of the adsorbent, interactions of other types (usually indicated as polar or specific) are involved in addition to dispersion intermolecular interactions. The molecules of the sample components must also undergo specific interactions during contact with the adsorbent. However, these interactions are even stronger, and if thls were not so, elution of all components would occur in the dead volume. However, if the values of the distribution coefficients are t o be maintained within the range 1 < K < 100, it is necessary, with respect to eqn. 10.36, for AE to remain within the same range of values as in the work with weak mobile phases. Only in a few instances can it be assumed, according to eqn. 10.37, that the difference in the adsorption interactions of the sample and mobile phase molecules will fulfil t h s condition. Therefore, it follows from eqn. 10.35 that the liquid phase terms must play an important role in the total energy balance. Larger differences in interactions in an adsorbed state must be compensated by larger differences in interactions in the mobile phase. T h s , however, is possible only if specific interactions between solvent molecules or between molecules of solute and solvent will, even in solution (in the mobile phase), occur at least in some instances. Then, of course, the basic conditions from which eqn. 10.38 was deduced are no longer valid and therefore it becomes at most a rough approximation. The magnitude of specific interactions in the mobile phase, indicated in the literature as secondary solvent effects, should be amended by introducing a correction term, A. Then log K = log
V , + xE A - SAe0) + A
(1 0.44)
For a given adsorbent, A is a function of the solute and solvent compositions. It is evident that the secondary solvent effects complicate the quantitative interpretation of the results and the prediction of the retention value according to eqn. 10.38. On the other hand, they permit the improvement of the separation selectivity or the achievement of the separation of components that are inseparable in the usual systems (Le., in systems without secondary effects). From the secondary effects of the mobile phase, the second important mobile phase characteristic follows, Le., selectivity. The magnitude of the relative adsorption and the migration rate of the sample through the bed are regulated by the choice of the elution strength of the solvent and the values of the distribution coefficients are optimized. The elution strength of the mobile phase can be considered to be independent on the sample type. However, the differences in the relative migration rates of the components of a given sample can be changed by controlling the selectivity of the mobile phase. It is evident that the selectivity of a certain mobile phase can be discussed only in connection with the given solutes. During the practical solution of an analytical problem, first a suitable elution strength of the mobile phase should be found for a given sample and a certain adsorbent, in order to optimize the distribution coefficients. Then, if the resolution of some sample components is insufficient, even after some simple adjustments of experimental conditions References p.277
260
MOBILE PHASES
(column length, flow-rate, column temperature, etc.), the required improvements can often be achieved by changing the selectivity of the mobile phase by changing its composition. However, the elution strength must remain constant even if the composition is changed. The so called equi-eluotropic series according t o Neher can serve as a guide (Fig. 10.5). For. each mixture of two components in this nomogram, the elution strength increases from left to right. The composition of the binary mobile phases of identical
1 3 5 1 0 I
I
1
1 1 1 1 1 1 ,
1
2
30
I
I
3 I
1
50
20
I
I
5
10
1
I
I
"
:
100 b
I f 100 I
I
1
2
3
4
5
I
I
I
1
#
I
1
1
1
I
I
l
l
~
2p
10 1
c
I
CHCI,
Diethyl ether Butyl acetate
10
20
I
I
50 I
1
I
2 I
I
I I I I
I
3 1
100
I 1 I
,
lp
5 I
:I
,
Ethyl acetate t - y - ? l o ; Acetone
b
I 1 1 1 1
1
6
,,I
,
Fig. 10.5. Equieluotropic series according to Neher. The numbers correspond to the content (%, V/V> of the stronger component in a binary mobile phase. The composition of mobile phases with equal elution strengths can be read from the intersection points of the lines parallel to the Y (coordinate) axis (in the direction of the arrows). HC = saturated hydrocarbons and cyclic hydrocarbons.
26 1
ION-EXCHANGE CHROMATOGRAPHY
elution strength can be read from the intersection points in a vertical line. The values obtained serve for orientation only. If preliminary considerations are inconclusive as to which solvent could affect the selectivity of the resulting mobile phase satisfactorily, the consecutive testing of single members of the equi-eluotropic series may prove time consuming. Insufficient attention has been devoted to the study of mobile phase selectivity in liquid-solid systems and therefore no general rules, relationships or numerical data can be given that would permit the choice of a mobile phase of suitable selectivity.
MOBILE PHASES FOR ION-EXCHANGE CHROMATOGRAPHY Aqueous solutions Most ion-exchange processes and ion-exchange chromatography are carried out in aqueous solutions, which are the best solvents for these purposes. Electrolytic dissociation in water immediately splits the dissolved substance that is to be sorbed into ions and the swelling of ion exchangers in water enables the ions so formed quickly to penetrate t o functional groups, which in water are also in a dissociated or'partly dissociated form. All these circumstances are in favour of rapid exchange in comparision with non-aqueous or mixed solutions.
Importance of ionic strength and pH of the mobile phase There are two main properties that must be considered carefully in ion exchange in aqueous solutions: pH and ionic strength. Very acidic or very basic solutions regenerate cation and anion exchangers to their H" or OH-(base) form or they convert the anion and cation exchanger completely into the corresponding salt forms. Any sorption of other ions under these conditions is not possible or is very limited. Therefore, in most instances ion-exchange chromatography is effective only in a certain range of pH values, which can be derived from titration curves of ion exchangers and are summarized in Table 10.3 for TABLE 10.3 USEFUL RANGE OF pH VALUES OF MOBILE PHASES FOR ION-EXCHANGE CHROMATOGRAPHY Type of ion exchanger
pH range for maximal capacity of ion exchanger*
Strongly acidic cation exchanger Moderately acidic cation exchanger Weakly acidic cation exchanger Strongly basic anion exchanger Moderately basic anion exchanger Weakly basic anion exchanger
2-12 4-12 5-12 12-2 8-2 6-2
*This pH range is limited by the stability of ion exchangers. The producer of Sephadex (Pharmacia, Uppsala, Sweden) recommends the following pH ranges: SPSephadex, pH 2-10; CM-Sephadex, pH 6-10; QAESephadex, pH 10-2; DEAESephadex, pH 9-2.
References p.277
262
MOBILE PHASES
various exchangers. Within these limits, the ion exchangers display a good capacity for sorption of corresponding ions. The ionic strength, p , is a measure of the intensity of the electrical field due to the ions in the solution. It is defined as half the sum of the terms obtained by multiplying the concentration,q (or molality), of each ionic species in the solution by the square of its valence, zi:
The ionic strength of mobile phases strongly influences the available capacity of exchangers. A high ionic strength in general decreases the sorption of sorbed ions (or zwitter-ions) also at nonextreme values of pH. Therefore, at the beginning of the chromatography the mobile phase must have a sufficiently low ionic strength. When operating with buffered ion exchangers (see below), the starting value for many applications is 1.1 = 0.05 or 0.1 and the final value is about 1.1 = 0.5; it does not usually exceed 1.1 = 2, at which value regeneraTABLE 10.4 CHOICE OF THE MOBILE PHASE FOR ION-EXCHANGE CHROMATOGRAPHY USING CATION EXCHANGERS Matrix and mobile phase
Form of ion exchanger H+
Mixed form (Hf t another cation)
Buffered columns
Composition of the matrix
Stable resins
Resins
Resins, cellulose, polydextran
Usual aqueous mobile phases
(1) Aqueous solution of a suitable acid (2) Sequence of aqueous solutions of the same acid with increasing concentration (3) Sequence of aqueous solutions of various acids with increasing acidity and concentration
(1) Aqueous solution of a weak or medium base (2) Sequence of solutions of bases with increasing basicity
(1) Anionic buffer
Mixed mobile phases
Solutions of polar organic solvents in water or dilute acids
Special mobile phases
with constant pH and low ionic strength (2) Sequence of anionic buffers with constant pH and increasing ionic strength (3) Sequence of buffers with increasing basicity and increasing ionic strength Admixture of small amount of organic solvent influences the relative sorption of solutes
Addition of organic solvents to mobile phase in instances when the desorbed base is not soluble in the aqueous phase Solutions of substances capable of forming complexes or derivatives with sorbed cations (selective elution) or solutions capable of keeping the solute in a complex form during the whole chromatographic process.
263
ION-EXCHANGE CHROMATOGRAPHY
TABLE 10.5 CHOICE OF THE MOBILE PHASE FOR ION-EXCHANGE CHROMATOGRAPHY USING ANION EXCHANGERS ~~
Matrix and mobile phase
Form of ion exchanger OH-
Mixed form (OH- + another anion)
Buffered columns
Composition of the matrix
Stable resins
Resins
Resins, cellulose, polydextran
Usual aqueous mobile phases
Aqueous solutions of bases or their mixtures with increasing concentration or basicity
Aqueous solutions of acids or their mixtures with increasing concentration or acidity
Mixed mobile phases
Solutions of polar organic solvents in water or dilute bases
(1) Cationic buffer with constant pH and low ionic strength (2) Sequence of cationic buffers with constant pH and increasing ionic strength (3) Sequence of buffers with increasing acidity and increasing ionic strength Admixture of small amount of organic solvent influences the relative sorption of s o h tes
Special mobile phases
Addition of organic solvents to mobile phase in instances when the desorbed acid is not soluble in the aqueous phase Solutions of substances capable of forming or decomposing complexes or derivates with sorbed anions (selective elution) or solutions capable of keeping the desired solute in a complex form during the whole chromatcgraphic process.
tion proceeds. In some instances, a lower ionic strength (e.g., I-( = 0.01) is used when substances are sorbed on ion exchangers or in the first stages of the chromatography. Some types of flexible ion exchangers with a low degree of cross-linking (e.g., polydextran ion exchangers) swell considerably in the range I-( = 0.1 -0.05 and lower, so that column processes are not possible with these ion exchangers in this range. The ionic strength of a buffer solution can be increased by the admixture of some nonbuffering soluble salts, e.g., sodium chloride or potassium chloride. This mixing with a salt is often used for selective elution and can be realized either stepwise or continuously with a gradient device (cf:, Chapter 8). Choice of the mobile phase
There are three possibilities for operating with ion-exchange columns as follows. (1) Columns in the H'or OH- form can be used. After the sorption of the sample, the References p.277
264
MOBILE PHASES
column is washed with water and then elution is started, gradually increasing the concentration of the acidic or basic solution. This procedure is possible with chemically stable ion-exchange resins only. It is essentially a simple regeneration of the exchanger accompanied by chroniatographic separation because the displaced ions moving down the column are continuously sorbed and desorbed. (2) Buffered columns can be used. After the equilibrium of the column with a buffer with a sufficiently low ionic strength and a suitable pH, the sample is sorbed and elution is begun with a system of buffers starting with the buffer of the lowest ionic strength. (3) Complexing agents can be used for the selective elution of the sorbed ions. The choice of a suitable mobile phase depends upon the operating conditions and is summarized in Tables 1.0.4 and 10.5.
Increasing concentrations of acids and bases This type of chromatography can be used for the separation of some inorganic ions and the separation of simple organic acids and bases. The sorbed cations are gradually eluted from cation-exchange columns in the H'form with several solutions of increasingly concentrated acid or with a series of different acids with increasing dissociation constants. For example, for orienting experiments, solutions can be used beginning with 0.001 N and finishing with 6 N hydrochloric acid. A sequence of increasing concentrations of acetic, formic, chloroacetic, hydrochloric and sulphuric acids is also possible. H'ions compete with other cations in this type of chromatography. Gradient elution can be used in order to change gradually the composition of the mobile phase. In special instances, dilute bases (e.g., solutions of ammonia) can be used for the selective separation of zwitter-ions (e.g.,amino acids) or weaker bases from inorganic cations bound on the strongly acidic cation-exchange column. The sorbed anions (or zwitter-ions) can be eluted from an ion-exchange column in the base form in a similar manner by using a series of increasingly concentrated solutions of a base or a series of several bases with increasing dissociation constants. For example, 0.01-2 M solutions of pyridine, collidine, amino alcohols, ammonia or aliphatic amines and their mixtures can be used for orienting experiments. In other instances the anionexchange column is used in a mixed salt form (e.g., OH- plus C1-, HCOO- or CH3COOform). The sorbed anions (e.g., mixture of organic acids) are gradually eluted with a sequence of eluting solutions containing an increasing concentration of acids (e.g., from 0.01 N to 6 N hydrochloric acid) or a sequence of solutions of weak and stronger acids of various concentrations.
Mobile phases for chromatography on buffered columns Chromatography on buffered columns is usually used for the separation of zwitterions, especially in biochemistry, but also for other purposes when very fine separations are required. The ion exchangers are used in certain mixed forms. They are equilibrated with buffers of a certain pH value and a known, low, ionic strength. For cation exchangers, the starting pH is low (cJ:,Table 10.3) and the subsequent buffers have higher pH values. The chromatography on anion exchangers begins with a high pH and the following buffers
265
ION-EXCH ANGE CHROMATOGRAPHY
have lower pH values. In both instances, the subsequent buffers have hgher ionic strengths. For the chromatography of proteins, the changes in pH of eluting buffers are of limited use. In many separations, they are omitted because the variation in the dissociation of the amphoteric counter-ions allowing selective desorptidn are compensated by variations in charge of the functional groups of the exchanger, which increase the sorption, and vice versa. Therefore, chromatography at a constant pH and increasing ionic strength only is often used in this instance. The appropriate pH value should be at least 1 pH unit below the isoelectric point of the protein when cation-exchange chromatography is used and at least 1 pH unit above the isoelectric point for anionexchange chromatography (Boman). The final decision depends upon certain special conditions, and in particular the stability of the protein should be considered. After the production of fractions containing many inorganic and non-volatile organic buffers, a large amount of salts contaminates the product. From the point of view of further processing of the fractions of the sample, volatile buffers may be important (Holeyiovsky et al., Keilovi and Keil, Rudloff and Braunitzer, Schroeder et al., Tomilek et al., VangEek et al., and others). Their components are only weakly mutually bound to form salts, w h c h are decomposed and escape when the water is evaporated in a rotating evaporator or by lyophilization. The pure solid substances of the chromatographic peaks are then obtained. The most usual volatile buffers contain aliphatic m i n e s or amino alcohols, pyridine, a-picoline, 2,4,6-collidine or Nethylmorpholine and formic or acetic acid. The disadvantage of using trimethylamine buffers is their offensive odour. Other semi-volatile buffers (ammonium carbonate, ammonium acetate) are decomposed in a h g h vacuum and at elevated temperatures and can be removed by sublimation. Examples of buffers are given in Tables 10.6 and 10.7. TABLE 10.6 VOLATILE BUFFERS FOR THE CHROMATOGRAPHY OF PEPTIDES FROM TRYPTIC DIGEST OF S-SULPHOTRYPSINOGENON A STRONGLY ACIDIC CATION EXCHANGER (HOLEYSOVSKY et al.) The solution of pyridine was adjusted with acid to the given pH and the volume was then made up to the final volume with water. No.*
Pyridine
Acid
PH
0.10 0.10 0.15
Formic
2.85 3.0
0.20 0.40 0.80
Acetic
(M)
S
1 2 3 4 5 ~
3 .O
4.0 5.0 7.0
_ _ ______ *S = buffer for dissolving the sample and application on the column, Zerolite 225, 100-200 mesh,
Length 1 5 0 cm, equilibrated with buffer No. 1 . 1-5 = buffers used for subsequent gradient elution. The fractions were evaluated by paper chromatography or paper electrophoresis.
References p.277
266
MOBILE PHASES
TABLE 10.7 VOLATILE BUFFERS FOR THE CHROMATOGRAPHY OF PROTEINASES FROM ASCITES FLUIDS AND ASCITES CELLS (KEILOVA AND KEIL) The solution of trimethylamine was adjusted with acetic acid to the given pH and then the volume was made up t o the final volume with water.
CM-cellulose column No.
Trimethylamine
DEAE-cellulose column No.
pH
(M> 1
0.005
2 3
0.01 0.01
4 5
0.02
6
Trimethylamine
pH
(M)
,
1
0.005
I
2
0.02 0.02 0.1 0.1 0.1
6 6 5
3
4 5
0.02 0.04
6
7
5
(+0.1 M NaCI)
TABLE 10.8 ANTIMICROBIAL REAGENTS FOR MOBILE PHASES IN ION-EXCHANGE CHROMATOGRAPHY Substance
Caprylic acid (octanoic acid) Chioretone (trichlorobutanol) Hibitane (chlorohexidine) Merthiolate (thimerosal, ethylmercury(I1) thiosalic ylate) Pentachlorophenol Phenylmercury(I1) salts Sodium a i d e
Butanol, carbon tetrachloride, chloroform, toluene
Type of ion exchanger often used
Cation-exchange resins (amino acid analysis) Ion-exchange derivatives of cellulose and polydextran Anion-exchange derivatives of cellulose and polydextran Cation-exchange derivatives of cellulose and polydextran Cation-exchange resins (amino acid analysis) Anion-exchange derivatives of cellulose and polydextran Cation-exchange derivatives of cellulose and polydextran Ionexchange cellulose
*Used in the form of a 0.5% (w/v) solution in 95% ethanol.
Concentration in the solution
(%I
Type of medium where the agent is active
0.01
Weakly acidic
0.05
Weakly acidic
0.002
Weakly acidic, neutral, weakly basic Weakly acidic
0.005
0.0005* 0.001
Weakly alkaline
0.02
Weakly acidic, neutral, weakly basic -
Traces
ION-EXCHANGE CHROMATOGRAPHY
267
When choosing buffers for ion-exchange chromatography, the following recommendations can be made. The buffering ions should not react with the functional groups of the exchanger. With cation exchangers, anionic buffers should be used when possible, and with anion exchangers, cationic buffers are recommended. If !he buffering-active ions interact with the functional groups of the exchanger, there is a danger of the formation of steps in gradient elution, because the continuity of the gradient is lost. However, there are examples in the literature in which these recommendations were not followed and good separations were nevertheless achieved. Anionic buffers are those with which the buffering activity is caused by the anionic component (e.g., acetate, citrate, phosphate and also glycine). In cationic buffers, the active components are ammonia and amines, aminoethanol, imidazole, pyridine and Tris. Barbital displays anionic properties and, below pH 7 . 5 , also cationic properties. Special additives to buffers In some instances when ion exchange is used for analytical purposes, certain substances are added to the mobile phase in order to improve the chromatography. A small admixture (up to 5%) of methanol, ethanol, tert-butanol, benzyl alcohol, methyl Cellosolve or phenetol influences the relative positions of amino acids, and several of these compounds have been used by some workers for accelerated amino acid analysis (DCvCnyi, Ertingshausen et al., Hubbard, Moore and Stein, Nauman, Pobel, Vritny and Zbroiek, Zuev et al., and others). Detergents, e.g., Brij 25, are sometimes used for improving the wetting of the resins by the mobile phase and for preventing the formation of bubbles when warming the mobile phase before colorimetry. Antioxidants are added in order to protect sensitive solutes against the influence of air (e.g., thiodiglycol to prevent oxidation of methionine). Antimicrobial agents (cf:, Table 10.8) keep the mobile phases and ion exchangers free from bacteria and other microbial contamination*. Urea at a concentration up to 8 M in the buffers is used t o dissolve less soluble modified protein or large peptide fragments and to keep them in solution during chromatography. All of these additives should be chosen so that they are not firmly bound to the exchanger. Complex-forming mobile phases and phases that alter the solutes Some organic acids (citric acid, ethylenediaminetetraacetic acid, lactic acid, oxalic acid, a-hydroxybutyric acid, nitrilotriacetic acid, uramildiacetic acid or their salts) and thenoylnitrofluoroacetone display special properties for the selective elution of inorganic cations in the complex form from cation exchangers. If some of them are used for the desorption of a very complicated mixture (e.g., fission products from atomic reactors), groups of similar ions are gradually desorbed and these methods can be used for the first fractionation. Complexing agents are also suitable for the selective desorption of individual inorganic ions. One of the first successes of ion-exchange chromatography with inorganic substances was the separation of rare earths with citrate solutions. *From the point of view of microbial contamination, the most dangerous is the long presence of the substrate in contact with the ion exchanger in phosphate buffers.
References p.277
268
MOBILE PHASES
Some inorganic acids and their salts (e.g., phosphoric acid, cyanides) also display complex-forming activity when used for the elution of cations of heavy metals. In other instances the complex-forming ability of concentrated hydrochloric acid (or other halogen acids), which forms chloride complexes with transition metals, was used for fractionation. They can then be separated on anion exchangers using a decreasing concentration of hydrochloric acid as the eluting agent, which gradually decomposes the complexes. Other examples of the use of complex-forming mobile phases in inorganic chemistry have been described by Helfferich. A similar principle of elution is also sometimes used in ion exchange in organic chemistry. The chromatography of saccharides or other non-ionic hydroxy compounds containing vicinal hydroxy groups (e.g., glycols) can be carried out on anion exchangers using buffers containing boric acid. These compounds undergo the following reactions: I
I
-C-OH I
I
I
-C-OH
-c-0,
HO\
+
/
B-OH-+
HO
I
-c-0 I
-C/
B-OH+
-"."I-O/
'OH
The complex anions formed are much stronger acids than the less dissociated forms of boric acid or of the transient form. Their dissociation constants differ with individual organic compounds, w h c h leads to the possibility of effective ion-exchange chromatography (Khym and Zill, 1951, 1952; Khym et al.; Sargent and Rieman; Zager and Doody). In a similar manner, hydrosulphite buffers can be used for the ion-exchange chromatography of aldehydes and ketones, which in other buffers are not retained on ion exchangers. The following reactions permit the binding:
R
I
C = 0 + HSO;
I
R
I
* HO -C-
H
H
R'
R'
I
SO;
I
I
C = 0 + HSO; =+HO-C-SO;
I
R
I
R
The hydroxysulphonic acids of ketones can be eluted from anion exchangers with hot water, while the hydroxysulphonic acids of aldehydes are eluted with solutions of alkalis or salts. There is also the possibility of the chromatographic separation of individual derivatives (Gabrielson and Samuelson, Ruff, Sherma and Rieman). The so-called ligand-exchange chromatography is mentioned elsewhere (Chapter 6).
ION-EXCHANGE CHROMATOGRAPHY
269
Mixed and non-aqueous phases Some considerations concerning non-aqueous phases for ion exchange have already been discussed in Chapter 6. The resins prefer more polarsolv,ents and therefore after equilibrium of ion exchanger particles with the mixed solution the actual concentration of the organic solvent (e.g., alcohol) will be greater in the aqueous phase than in the resin phase. The swelling and the useful exchange capacity of the resin will be decreased. On the other hand, the sorption of polar non-electrolytes (e.g., sugars, polyalcohols) from the solution will be greater in comparison with their uptake from the aqueous solution. The sorption of less polar solutes (ketones, esters, phenols, hydrocarbons), which are sorbed by Van der Waals forces, is influenced in the opposite way. The presence of organic solvents influences the selectivity coefficients for ions, sometimes very substantially. However, the time required to attain equilibrium is much longer, and this effect is increased with increase in the concentration of the organic solvent. In pure organic solvents, the rate of exchange can be about three orders of magnitude less than in water. The most commonly used organic solvents for studies of mixed or non-aqueous phases are methanol, ethanol, propanol, acetone, ethyl methyl ketone, dioxane, ethylene glycol and glycerol. The admixture of organic solvents with the mobile phase is also used in instances when the desorbed substance, after having lost its electrical charge, is not soluble in aqukous solution. In other instances, the addition of a suitable organic solvent can lead to the separation of ions, which could not be separated in aqueous solutions only. The altered solvation of ions in mixed solutions can be reflected in differences in the chromatographic separation. Because of the presence of two phases with different solvent compositions (gel and outer solution), the factors of partition chromatography may play a role.
Temperature of mobile phases Most chromatographic experiments are carried out at room temperature and many biochemical separations are carried out at 1-4OC. The lability of chromatographed substances does not allow work at normal temperature. Temperatures of up to 100°C are sometimes used with aqueous solutions, because the higher temperature accelerates the achievement of the equilibrium of ion exchange, diminishes the viscosity of the solvent and makes the whole chromatographic process much quicker. The elevating of temperature is of course possible only when stable substances are treated. The influence of temperature on the relative positions of the peaks of separated substances is not too important if true i m exchange is considered. The temperature usually does not change the exchange potential substantially, but if adsorption of the sorbed substances due to Van der Wads forces takes place in parallel with ion exchange, an increase in temperature in general diminishes the sorption. References p.2 77
270
MOBILE PHASES
CALCULATION OF GRADIENTS General aspects Elution chromatography is the most widely used form of liquid column chromatography. In simple elution, the solvent entering the column has a constant composition. If a series of solvents with different compositions is used for the elution of particular components of a complex chromatographed mixture, the procedure is called stepwise elution. Tiselius and his collaborators (Alm, Alm er al., Hagdahl et al., Williams) proposed a chromatographic procedure in which elution is carried out with a solvent the composition of which is changed continuously and they proposed the term gradient elution chromatography for this procedure. The shortened term gradient elution is also used. Other workers have discussed or used a similar principle independently, although they did not consider it to be so (Busch et d.;Busch and Potter, 1952, 1953; Donaldson el a!.; Mitchell et al.; Nervik; Strain, 195 1, 1960; Synge). Gradient elution suppresses the tailing of zones and hence improves their symmetry. In addition, it improves and refines the separation process, especially for biopolymers. The first thorough theoretical discussion of concentration gradient elution was published by Drake, and of pH gradient elution by Piez. The method was rapidly accepted and widely used and has been the subject of several reviews (Dorfner, Henry, Lebreton, Mikes“, and others), and an extensive paper by Snyder (1965) gives a detailed discussion. Gradient elution chromatography is described in this book from the point of view of methods and apparatus in Chapter 8. The aim of this chapter is to present equations for the calculation of single types of gradients.
Classification of gradients Gradient elution chromatography is divided into concentration gradient elution, the aim of which is to change the polarity or ionic strength of the eluent caused by a continuous change in component concentration in the eluent, and pH gradient elution, aiming at a continuous and defined change of pH of the eluent. In view of various devices used today for the creation of gradients various types and shapes of gradients are also known. They are summarized schematically in Fig. 10.6, where the terminology of Snyder (1965) is used. As regards the suitability of the shapes of gradients for chromatography, linear gradients are generally most suitable for first experiments. For repeated chromatography of the same substances, it is most convenient to find experimentally the optimum gradients (so-called “custom gradients”), which may have a complex course (“compound gradients”). For the formation of a gradient, not only its shape but also its steepness is of importance. A gradient that is too mild often leads to broad zones, while a gradient that is too steep makes the zones narrow but also brings closer together zones which would otherwise be well separated with an optimum gradient.
27 1
CALCULATION OF GRADIENTS GRADIENT
TYPES CONTINUOUS
DlSCONTlNUOUS
Slepwise
,
h
rounded swpw,ae
exrendad (CO”t,nUn”*
simple CO”t~”“O“6
stepwise,
Fig. 10.6. Limit and transitional types and forms of gradients. Vertical axis: concentration of the component or pH in the eluent running through the column. Horizontal axis: eluent volume.
Gradient formation Gradient elution (see also Chapter 8, p. 113) can be carried out easily by the continuous mixing of two or more solutions before they enter the column. According to the method of mixing, four different methods of gradient preparation can be distinguished and are represented schematically in Fig. 10.7 and discussed below. (1) “Exponential” methods ofmixing two liquids are illustrated in Fig. 10.7a and b. From the reservoir, A, of an optional volume, the effective “solvent” flows through a mixer, B, of constant volume or through two mixers, B, and B2,of constant volumes. The mixer B contains an ineffective “diluent” with which the column is also filed at the start of chromatography. When two mixers are used, they may contain the same diluent or an active solvent diluted to various degrees. The corresponding device is called a “mixer of constant volume”. The only mixer with a constant volume is the device originally proposed for gradient elution. It is still often used even though it generally does not give an optimum gradient. Its convex course is shown in Fig. 10.60. The use of two mixers is more suitable because an almost linear gradient can be obtained (Drake). References p.277
27 2
MOBILE PHASES
a
b
C
C
d
f
e
i C
h
i
Fig. 10.7. Principles of gradient formation. a, b: device for forming “exponential” gradients; c, d: device for forming “proportional” gradients; e, f, g: device for forming “disproportional” gradients; h, i: device for forming multicomponent gradients. A, reservoirs containing an actively eluting component (“solvent”); A, -A,, reservoirs with liquids of increasing elution strength; B, mixers with liquids of minimum elution strength (“diluents”) used for the initial column (C) equilibration; M, magnetic stirrer; P, laboratory ipump; S, six-way valve;V, and V, ,suitably conjugated valves; I-IX, mixers as connected vessels.
(2) “Proportional” methods of mixing two liquids based on the principle of connected vessels are shown in Fig. 1 0 . 7 ~and d. The device represented in Fig. 1 0 . 7 ~is also called an “open mixer”. Reservoir A and mixer B are open connected vessels, and A contains the solvent and B the weaker solvent or inactive diluent. When the solution is pumped on to the column, the levels of the liquids in both vessels decrease at the same rate. Depending on the ratio of the cross-sections of the vessels, the liquids are mixed in different but constant proportions, giving gradients of various shapes. If the cross-sections of the vessels are equal (A = B), a linear gradient is formed (Fig. 10.6n), if A > B a convex gradient is obtained (Fig. 10.60), while if A < B a continuously increasing concave gradient results
CALCULATION OF GRADIENTS
273
(Fig. 10.6m). In a similar manner, a series of connected vessels (Fig. 10.7d) can be joined in series and stirred with the same motor (for example, the nine-chamber Technicon Varigrad according to Peterson and Sober (1958, 1959) and Peterson and Rowland). (3) ‘‘Disproportional’’ methods of mixing two liquids (Fig. 10.7e, f and g) lead to various forms of gradients. The principle of the method according to Bock and Ling is based on two vessels, A l and A*, to which the required profiles were given (Fig. 10.7e). From these vessels, the liquids flow into a small mixer, for example a funnel filled with glass beads. The shape of the vessels enables a ratio of mixing of the liquids to be used that changes with time and thus creates the special gradient required. Other arrangements of the two vessels according to Bock and Ling (a cone within a cylinder) are represented in Fig. 10.7f. The same effect can be achieved, according to Zahn and Stahl, by regulating the effluent from two vessels, Al and A ? , with suitable conjugated valves, v1 and v2 (Fig. 10.7g). Instead of valves v1 and v 2 , two programmable, reversely proportional pumps can be used, the total output of which is constant, and partial outputs can be regulated with time with an optical device reading the required gradient from a pre-drawn curve. By these methods, gradients of virtually any shape may be obtained. (4) The last method is the formation of gradients by mixing several liquids. When a series of liquids that differ slightly in composition are introduced on to the column, a rounding of the sharp steps in the composition of solutions typical of stepwise elution takes place as a consequence of mixing in the dead spaces of the leading tubes and under the effect of the pump (Fig. 10.6, “rounded stepwise”). A further rounding of the steps takes place on the particles in the upper part of the column. If a small mixer of constant volume is inserted into the tubing leading the eluent (Fig. 10.7h), a still coarser rounding takes place. If, according to Anderson et al. (Fig. 10.7i), the solution is mixed gradually with half of the preceding and then with half of the following solution, an extended gradient (continuous stepwise gradient) is obtained (Fig. 10.6). In ion-exchange chromatography, the buffering function of the ion exchanger causes the total equalization of the rounded stepwise to a continuous gradient (Mik& et al.). By gradual mixing of solutions of similar composition (Fig. 10.6a-d) and by using a suitable arrangement, any of the continuous gradients may be obtained (Fig. 10.6m-p).
Calculation of concentration gradients For the sake of simplicity, in the following calculations the liquid with a lugh elution strength in the reservoir A (Fig. 10.7) is designated as the “solvent” and the liquid with a minimum elution strength in the mixer B as the “diluent”. A mixture of solvent and diluent flowing on to the column is indicated as the eluent. “Exponential ”gradients
For the gradient of the solvent in the liquid flowing out of a single closed mixer, Alm et al. derived the equation V
- = 2.303 log
V
References p.277
X
-
x-x
(1 0.45)
274
MOBILE PHASES
where x is the concentration of the solvent in the mixer B after a volume v has entered the column, X is the concentration of the solvent in the reservoir A and V is the volume of the mixer. Cherkin et al. derived an equation expressing the dependence of the outflowing solvent on its original concentration and the ratio of the volume that has flowed through to the mixer volume. Solving the corresponding differential equation, they obtained the expression
c -eK-1 c0
eK
(10.46)
where C i s the concentration of the solvent flowing out of the mixer, Co is the concentration of the solvent in the reservoir, and K is the ratio of the total volume v of the consumed eluate to the volume V of the diluent in the mixer (K = v / V ) . Only when K does not exceed unity has the gradient an approximately linear shape. If a linear course is required, the volume of the diluent in the mixer should be at least equal to the total volume of the consumed eluate. In this case, however, the final concentration of the solvent from the reservoir cannot be attained in the column during elution. This must be kept in mind in advance and therefore a higher concentration chosen. If the total eluate volume v exceeds the original diluent volume V in the mixer more than two-fold ( K > 2), the gradient effect of further elution is small or negligible. Both eqns. 10.45 and 10.46 are more or less identical. A similar equation was proposed by Mader: A X=Bln ( 10.47) A -Y where X i s the eluate volume that has flowed out of the column, B is the mixer volume, A is the solvent concentration in the reservoir andy is the solvent concentration at the column inlet. Bock and Ling, and Drake derived for this type of gradient equations formulated in a different way, which, however, are also virtually identical. If q,, is the inlet concentration of the solvent introduced into the column, then
qn = C, (c,- c,) . e-('/'m) -
(10.48)
where C, is the concentration of the solvent in the reservoir, C, the starting concentration of the solvent in the mixer, v the volume of the liquid that has passed through the column and V, the volume of liquid in the mixer. The solution of the gradient of one closed mixer and of its practical testing was also studied by Donaldson et al. The calculation of the gradient with the use of two closed mixers (Fig. 10.7b) was carried out by Drake. If the mixers have different volumes (B1 # B z ) , then the equation
applies, where , C and C, are as in eqn. 10.48, V1 and V z are the volumes of the two mixers and C1 and C, are the starting concentrations of the solvent in the two mixers. The gradients with a smaller ratio of the volumes Vz/Vl are less steep. For the special case of eqn. 10.49 for which both mixers have the same volume ( Vl = V z ) ,eqn.
275
CALCULATION OF GRADIENTS
( 10.5 0)
In t h s case, in the interval C, = 0 to C , = C,/2, the course of the gradient approaches linearity for v = 2.5 V2 (maximum).
"Proportional '' gradients Lakshmanan and Lieberman investigated the formation of gradients in which the liquid would flow from the reservoir into the mixer at a constant rate, R l , different from the rate at which it would flow out from the mixer into the column ( R 2 ) .If V o is the original volume of the diluent in the mixer, C, the concentration of the solvent in the reservoir and C the concentration of the solvent in the mixer at time c, then (1 0.5 1) where a = V o / R 1and b = 1 - (R2 / R I ). If b < -1 or R 2 > 2R1, the dependence of the concentration on time (or on eluate volume) is concave, if b = -1 or R 2 = 2 R 1 ,the gradient is linear, and if b > -1 or R 2 < 2 R 1 ,the curve is convex. At R 1 = R 2 ,the curve is exponential because in fact it is a case of the solutions in eqns. 10.45- 10.48. The realization of this method by the procedure mentioned would, of course, be subject to difficulties connected with fluctuations of the level in the mixer. This principle, however, can be easily carried out according to Fig. 1 0 . 7 ~By . choosing the ratio of the cross-sections of the two cylinders, the ratio of the in-flowing and outflowing rates can be controlled in the mixer, because the levels must decrease uniformly. In this manner, all of the gradients mentioned can be obtained. Drake, and Bock and Ling deduced for this type a formally identical equation
c.in = rc- ( C r - C,)
( 1 0.52)
in which the symbols have the same meaning as in eqns. 10.48-10.50, V, is the starting volume of the liquid in the reservoir, V, is the starting volume of the liquid in the mixer and Yot = V, + V,. If the cylinders A and B (Fig. 1 0 . 7 ~ are ) equally wide, i.e., V , = V,, eqn. 10.52 becomes linear: C,=Cr-(Cr-Cm)
( L) 1
--
=Cm+(Cr-C,)-
V
40t
(10.53)
and this gradient is therefore called linear. The composition of the buffer entering the column can easily be found on the straight line connecting the ordinates of a simple rectangular graph constructed on the basis of both starting concentrations Cr and C ,. The volume whch has flowed through v is read on the abscissa of total length Vtot.This graphical method is most often used. If the mixer has a larger volume than the reservoir (Fig. 10.7c), i.e., V, > 5,a continuously increasing concave gradient is formed (Fig. 10.6m):However, if the reservoir is References p.277
276
MOBILE PHASES
larger than the mixer ( V , > V ) a decreasing convex gradient is obtained. Both cases can m : be expressed by eqn. 10.52; Kocent published a nomogram facilitating the calculations. A similar nomogram was published by Warner and Lands. If a large number of equal-sized vessels are connected in series (Varigrad, Fig. 10.7d), of which any internal one contains the solvent of concentration L and the others contain the diluent, then the concentration gradient C of the out-going liquid can be calculated according to the equation (10.54) where N is the total number of vessels in the system, n is the serial number of the vessel containing the solvent of concentration L, v is the volume of liquid entering the column and V is the original volume of liquid in the mixing system. “Disproportional ” gradients The gradient of the system illustrated in Fig. 10.7e is pre-determined by its geometry and, in principle, it can be read from the profile of the vessel. For the system according to Fig. 10.7f, the following equation applies: (10.55)
where C1 and C, are the concentrations of the solvent in compartments 1 and 2 in Fig. 10.7f, v is the volume of liquid that has flowed through and V is the total volume of the system. If the internal vessel of the system (Fig. 10.70 has a cone, the square of the radius of which is equal to the product of the height and the jacket radius (i.e., r2 = k . h ) , then a linear gradient is achieved, characterized by the equation
c=c1+ ( C 2
-C1)-
V
V
(1 0.56)
The calculation of the formation of a gradient obtained by the gradual mixing of several liquids can be simplified by dividing the process into several stages according to some of the above described methods; in this procedure, only two mixing liquids are considered at once and the gradients can therefore be calculated in stages.
pH gradients The calculation of pH gradients was described by Piez, who differentiated between three cases: (1) the gradient is prepared by mixing one buffer with the other; ( 2 ) it is prepared by mixing a weak base or acid with the buffer; (3) it is obtained by mixing a strong base or acid with the buffer. The calculation of pH gradients is more complex and the range of validity is limited. Buffers that consist of a monovalent acid and its salt effectively buffer only over a range of two pH units and the calculated gradients are also formed in this range only. Buffers of polyvalent acids (e.g., citric acid) would buffer over
REFERENCES
277
a range of up to four pH units. We do not consider it necessary to mention the corresponding equations here, and we refer to the original paper by Piez. In ion-exchange chromatography, the most convenient systems are generally those in which a concentration and a pH gradient take place simultaneously and where the concentration gradient is linear and the pH gradient continuously increasing (concave). This can easily be achieved with two equally broad, connected vessels (according to Fig. 10.7c), when a more concentrated buffer from a reservoir is mixed into the more diluted buffer in the mixer. For the separation of proteins, an ionic strength gradient (ie.,concentration gradient) is often used with a constant pH throughout the whole experiment.
Theory of gradient elution The aim of this chapter is to explain the calculation of gradients and not the theory of gradient elution. However, we consider it appropriate to summarize in t h i s last section the theoretical papers concerning this topic. The bases of a general theory were forwarded by Drake, and general equations for the understanding of elution were also derived by Said and by Snyder (1964a, b). A theory of ion-exchange chromatography for discontinuously and continuously changing eluent compositions was published by Freiling (1955, 1957), and a theory of gradient elution in ionexchange chromatography was developed by Schwab ef al. The latter authors, as well as Maslova et al., also compared theoretical calculations with experimentally observed peak positions of separated compounds. The theory of ion-exchange chromatography was further developed by Koguchi et a/, and Ohashi and Koguchi. An extensive discussion of the theory of gradient elution was published by Snyder (1 965) (cf. also Snyder and Saunders). A series of valuable considerations on gradient chromatography of proteins was summarized by Peterson and Sober (1960), Sober et al. and Sober and Peterson; Novotnjr et al. described the linear dependence of the specific electric charge of immunoglobulin fragments and the ionic strength of the buffer at which the fragments are eluted from QAESephadex. Novotnp published an equation permitting the optimization of the elution of proteins and their fragments on ion exchangers when an ionic strength gradient is used.
REFERENCES Alm, R. S., Acta &hem. Scand., 6 (1952) 1186. Alm, R. S., Williams, R. J . P. and Tiselius, A., Acta &hem. Scand., 6 (1952) 826. Anderson, N. G., Bond, H. E. and Canning, R. E., Anal. Biochem., 3 (1962) 472. Bock, R. M. and Nan Sing Ling, Anal. Chem., 26 (1954) 1543. Boman, H. G., in K. Paech and M. V. Tracy (Editors), Modern Methods in Plant Analysis, Springer, Berlin, 1962. Brunauer, S., The Adsorption o f Cases and Vapours, Clarendon Press, Oxford, and Princeton Univ. Press, Princeton, 1945. Brunauer, S., Deming, L. S., Deming, W. E. and Teller, E., J. Amer. Chem. Soc., 6 2 (1940) 1723. Burrel, H., Inrerchem. Rev., 14 (1953) 3 and 31. Busch, H., Hurlbert, R. B. and Potter, V. R., J. Biol. Chem.. 196 (1952) 717. Busch, H . and Potter, V. R., Cancer Res., 12 (1952) 660.
27 8
MOBILE PHASES
Busch, H. and Potter, V. R,, Cancer Res., 13 (1953) 168. Cherkin, A,, Martinez, F. E. and Dunn, M. S., J. Amer. Cbem SOC.,75 (1953) 1244. Crowley, J. D., Teague, Jr., G. S . and Lowe, Jr., J. W.,J. Paint Tecbnol., 34 (1966) 269. Deal, C. H., Derr, E. R. and Papadoupoulos, M. N., fnd. Eng. Cbem., Fundam., 1 (1962) 17. Derr, E. L., Deal, C. H. and Pierotti, G. J., Amer. Soc. Test. Mater., Spec. Tech. Publ., NO. 244 (1957) 111. De Vault, P., J. Amer. Cbem. Soc.,65 (1943) 532. Devenyi, T., Acta Biocbim. Biophys., 3 (1968) 429. Donaldson, K. O., Tulane, V. J. and Marshall, L. M., Anal. Cbem., 24 (1952) 185. Dorfner, K., Cbem. Ztg., 87 (1963) 871. Drake, B., Ark. &mi, 8 (1955) 1. Ertingshausen, G., Adler, H. J . and Reichler, A. S., J. Cbromatogr., 42 (1966) 355. Flory, P. J., J. Pbys. Qzem., 10 (1942) 51. Freiling, E. C., J. Amer. Chem. Soc., 77 (1955) 2067. Freiling, E. C., J. Phys. Cbem., 61 (1957) 543. Gabrielson, G. and Samuelson, O., Sv. Kem. Tidskr., 62 (1950) 214. Gardon, J. L., J. Paint Tecbnol., 38 (1966) 43. Glueckauf, E., Nature [London), 156 (1945) 748. Glueckauf, E., Nature (London), 301 (1947a) 301. Glueckauf, E., J. Cbem. SOC.,(1947b) 1302. Glueckauf, E., J. Chem. SOC.,( 1 9 4 7 ~ )1321. Glueckauf, E., J. Chem. SOC.,(1949) 3280. HagdaN, L., Wdiams, R. J. P. and Tiselius, A., Ark. Kemi, 4 (1952) 193. Helfferich, F. G., fon Exchange, McCraw-Hill, London, 1962. Henry, R. A., in J. J. Kirkland (Editor), Modern Practice of Liquid Chromatography, WileyInterscience, New York, 1971, pp. 80-89. Hildebrand, J. H. and Scott, R. L., Regular Solutions, Prentice-Hall, Englewood Cliffs, N.J., 1962. Hildebrand, J. H. and Scott, R. L., Solubility of Non-ElectLolytes, Dover, New York, 1964. Holeyiovski, V., Alexijev, B., Toma'gek, V., Mike:, 0. and Sorm, F., Collect. Czech. Cbem. Commun,, 27 (1962) 2665. Hubbard, R. W . , Biocbim. Biopbys. Res. Cvmmun., 19 (1965) 679. Huber, J. F. K., Meijers, C. A. M. and Hulsman, J. A. R. J., Anal. Cbem., 44 (1972) 111. Huggins, M. L., Ann. N. Y. Acad. Sci., 43 (1942) 1. Keilovi, H. and Keil, B., Collect. Czech. Cbem. Commun., 27 (1962) 2193. Keller, R. A., Karger, B. L. and Snyder, L. R., in N. Stock and S . G. Perry (Editors), Gas Chromatograpby 1970, Institute of Petroleum, London, 1970, p. 125. Keller, R. A. and Snyder, L. R., J. Cbromatogr. Sci., 9 (1971) 346. Khym, J. X. and Zill, L. P., J. Amer. Cbem. SOC.,73 (1951) 2399. Khym, J. X. and Zill, L. P., J. Amer. Cbem. SOC.,74 (1952) 2050. Khym, J. X., Zill, L. P. and Cohn, W. E., in C. Calmon and T. R. E. Kressrnan (Editors), f o n Exchangers in Organic and Biochemistry, Interscience, New York, 1957. Kirkland, J. J., J. Cbromatogr. Sci., 9 (1971a) 206. Kirkland, J. J., in J. J. Kirkland (Editor), Modern Practice of Liquid Chromatography, WileyInterscience, New York, 1971b, p. 161. KoEent, A., J. Cbromatogr., 6 (1961) 324. Koguchi, K., Waki, H. and Ohashi, S., J. Cbrornatogr., 25 (1966) 398. Kudchadker, A. P., Alani, G. H. and Zwolinski, B. J., Cbem. Rev., 68 (1968) 659. Lakshmanan, T. and Lieberman, S., Arch. Biocbem. Biopbys., 45 (1953) 235. Lebedeva, N. P., Frolov, I. I. and Jashin, J.,J. Cbrornatogr., 58 (1971) 11. Lebreton, P., Bull. Soc. Cbim. Fr., (1960) 2188. Lieberman, E. P., Ofj: Dig., Fed. SOC.Paint Tecbnol., 34 (1962) 30. Locke, D. C. and Martire, D,. E., Anal. Cbem., 39 (1967) 921. Macek, K. and Prochizka, Z., in I. M. Hais and K. Macek (Editors), Paper Chromatography, Academic Press, New York, 1 9 6 3 , ~115. .
REFERENCES
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Mader, C., Anal. Chem., 26 (1954) 566, Maggs, R. J., 1. Chromatogr. Sci., 7 (1969) 145. Martire, D. E., in A. B. Littlewood (Editor), Gas Chromatography 1966, Elsevier, Amsterdam, 1967, p. 21. Martire, D. E. and Locke, D. C., Anal. Chem., 43 (1971) 68. Maslova, G. B., Nazarov, P. P. and Khmutov, K. V., Ionoobmen. Sorbenty Prom., (1963) 103. Mike;, O., Chem. Listy, 54 (1960) 578. Mike;, O., TomGek, V. and Holeys’ovskL, V., Chem. Listy, 53 (1959) 609. Mitchell, H. K., Gordon, M. and Haskins, F. A., J. Biol. Chem., 180 (1949) 1071. Moore, S. and Stein, W. H, J. Biol. Chem., 192 (1951) 663. Morrison, G. H. and Freiser, H., Solvent Extraction in Analytical Chemistry, Wiley-Interscience, New York, 1957. Nauman, L. W.,J. Chromatogr., 36 (1968) 3118. Neher, R., in G. B. Marini-Bettolo (Editor), Thin-Layer Chromarography, Elsevier, Amsterdam, 1964, pp. 75-86. Nervik, W. E., J. Phys. Chem., 59 (1955) 690. Novotny, J., FEBS Lett., 1411971) 7. Novotny, J., Fran*ek, F. and Sorm, F., Eur. J. Biochem., 16 (1970) 278. Ohashi, S. and Koguchi, K., J. Chromatogr., 27 (1967) 214. Peterson, E. A. and Rowland, J., J. Chromatogr., 5 (1961) 330. Peterson, E. A. and Sober, H. A., Fed. Proc., Fed. Amer. SOC.Exp. Biol., 17 (1958) 288 and 1116. Peterson, E. A. and Sober, H: A., Anal. Chem., 31 (1959) 857. Peterson, E. A. and Sober, H. A., in E. W. Putnam (Editor), The Plasma Proteins, Vol. I, Academic Press, New York, 1960, pp. 105-141. Pierotti, G. J., Deal, C. H. and Derr, E. L., Ind. Eng. Chem., 51 (1959) 95. Pierotti, G. J., Deal, C. H., Derr, E. L. and Porter, P. E., J. Amer. Chem. SOC.,78 (1956) 2989. Piez, K. A.,Anal. Chem., 28 (1956) 1451. Pobel, E. S., Anal. Biochem., 18 (1967) 406. Polak, J., Collect. Czech. Chem. Commun., 31 (1966) 1483. Rudloff, V. and Braunitzer, G., Hoppe-SeylerS Z. Physiol. Chem., 323 (1961) 129. Ruff, E., Anal. Chem.. 31 (1959) 1626. Said, A. S., AIChE J., 2 (1956) 477. Sargent, R. and Rieman, 111, W., Anal. Chim. Acta, 16 (1957) 144. Schmit, J. A., Henry, R. A., Williams, R. C. and Dieckman, J. F., J. Chromatogr. Sci., 9 (1971) 645. Schorn, P. J., 2. Anal. Chem., 205 (1964) 298. Schwab, H., Rieman, W. and Vaughan, P. A., Anal. Chem., 29 (1957) 1357. Scott, R. P. W. and Lawrence, G. J., J. Chromatogr. Sci., 8 (1970) 619. Sherma, 3. and Rieman, 111, W., Anal. Chim. Acta, 19 (1958) 134. Shroeder, W. A., Jones, T. R., Cornick, J. and McCalla, K., Anal. Chem., 34 (1962) 1570. Snyder, L. R.,J. Chromatogr., 13 (1964a) 415. Snyder, L. R., Advan. Anal. Chem. Instrum., 3 (1964b) 25 1. Snyder, L. R., Chem. Rev., 7 (1965) 1. Snyder, L. R., Anal. Chem., 39 (1967) 698. Snyder, L. R., Principles ofAdsorption Chromatography, Marcel Dekker, New York, 1968. Snyder, L. R., J. Chromatogr. Sci., 7 (1969) 352. Snyder, L. R. and Saunders, D. L., J. Chromatogr. Sci., 7 (1969) 195. Sober, H. A., Gutter, F. J., Wyckoff, M. M. and Peterson, E. A., J. Amer. Chem. SOC.,78 (1956) 756. Sober, H. A. and Peterson, E. A., in J. T. Edsall (Editor), Amino Acids, Proteins and Cancer Biochemistry, Academic Press, New York, 1960, p. 61. Strain, H. H., Anal. Chem., 23 (1951) 25. Strain, H. H., Anal. Chem., 32 (1960) 3R. Synge, R. L. M., Discuss. Faraday SOC.,7 (1949) 167. TomGek, V., Holey:ovskf, V., Mike:, 0. and Sorv, F., Eiochim. Biophys. Acta, 38 (1960) 570. VanBEek, J., Meloun, B., Kostka, V., Keil, B. and Sorm, F., Biochim. Biophys. Acta, 37 (1960) 169.
MOBILE PHASES VrBtn?, P. and Zbroiek, J., J. Chromatogr., 76 (1973) 482. Warner, H. R. and Lands, W. E. M., J. Lipid Res., 1 (1960) 248. Williams, R. J. P., Analyst (London), 77 (1952) 905. Williams, R. J. P., Hagdahl, C. and Tiselius, A., Ark. Kemi, 7 (1954) 1. Young, D. H. and Crowell, A. D., Physical Adsorption of Gases, Buttenvorths, London, 1962, p. 4. Zager, S. E. and Doody, T. C., fnd. Eng. Chem., 43 (195 1) 1570. Zahn, P. K. and Stahl, I., Hoppe-Seyler’s Z. Physiol. Chem., 302 (1955) 204. Zucv, S. N., Kozarenko, T. D. and Chernov, A. B., Zh. Anal. Khim., 25 (1970) 2039.
PRACTICE OF LIQUID CHROMATOGRAPHY
This Page Intentionally Left Blank
Chapter 11
Operation of a modern liquid chromatograph R. VESPALEC and M. KREJCi
CONTENTS Preparation of the apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sorting of sorbents acc Classification of silica gel particles according to size by sedimentation. . . . . . . . . . . . 289 Regeneration of silica gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Determination of the a Column preparation. . . . . . . . . . . . . . . . . . . . . . . . ........................ 291 Sample preparation and application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General comments . . References . . . . . . . .
PREPARATION OF THE APPARATUS For the preparation of an apparatus for chromatographic measurements, the first aspects to be considered are the demands of the problem being examined. Firstly, suitable dimensions for the column should be chosen and it should be filled by using one of the methods described below. Depending on the character of the packing, it must be decided whether or not a pre-column will also be necessary and the type of material that would b a suitable for filling it. If it is necessary to change the composition of the mobile phase during the analysis, then a device for the production of a gradient should be chosen and connected with the column, and its shape and time programme selected. In this case, a pre-column is usually not connected. However, if a pre-column is indispensable (for example, for complete drying of some of the mobile phase components), it should be inserted before the gradient device. A suitable detector should be connected to the column outlet, followed by a flow meter. Finally, a fraction collector or a waste-container is connected. Further procedures depend on whether the analysis is carried out with a constant or changing mobile phase composition. First, work with a mobile phase of constant composition will be described (Fig. 11.1). From the reservoir, 1, and degasser, 2, the old mobile phase is emptied through the stopcock, 3 (in some instances, the reservoir also functions as a degasser) and the reservoir is filled with a new phase. The performance of the pump, 4, is then chosen, and if the construction of the pressure valve, 5, permits it, the maximum permissible pressure in the apparatus is set. This operation is especially important if the mechanical strength of some components of the chromatograph is limited (for example, when working with glass columns), The pump should be protected by an independent break-through membrane so as to prevent its damage. The pressure pulse attenuator, 6, is set so that it operates with References p.300
283
284
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
Fig. 11.1. Schematic diagram of a liquid chromatograph during operation with a mobile phase of constant composition. 1 = Reservoir;2 = degasser; 3 = emptying and switching valve; 4 = pump; 5 =pressure trap; 6 = pressure damping; 7 = manometer;8 = thermostat;9 = pre-column; 10 = injection port; 11 = column; 12 = detector; 13 = recorder; 14 = flow meter; 15 = fraction collector or waste container.
maximum efficiency at the pump outlet pressure. In chromatographs with pulse-free pumps, the damper is not used. The inlet pressure on the column is read from the control manometer, 7 . The temperature of the thermostat, 8, in which the pre-column, 9, the injection port, 10, and the chromatographic column, 11, are placed, is then set. The whole chromatograph should be carefully washed with the mobile phase so as t o remove the residues of the old mobile phase and air. Therefore, it is useful if all parts are flushed with the mobile phase and if their volumes are as small as possible. Complete deaeration of the column and pre-column is important, especially if detectors with a flowthrough cell are employed. After de-aeration, the detector is switched on. The injection of the sample can be started only when equilibrium is attained in the column, which is often signalled by the disappearance of the baseline drift of the detector. If the detector response is independent of the mobile phase composition, the equilibrium in the column can be ensured by repeated injection of a model mixture. Perfect conditioning of the column is important, especially in adsorption chromatography with a multicomponent mobile phase and when the so-called demixing effects take place (see Chapter 10). Also, when the temperature of the column changes, perfect attainment of the equilibrium state should be achieved. The time necessary for the washing of the chromatograph, degassing and conditioning of the column can be shortened by increasing the flow-rate. After equilibrium in the column has been attained, the pump performance is regulated, the flow-rate of the mobile phase measured and the detector sensitivity and recorder chart speed are set. Unless the total volume of the mobile phase that has flowed through the column is recorded, the stability of the flow should be controlled even during the analysis,
SORTING OF SORBENTS ACCORDING TO PARTICLE SIZE
285
either by measuring the flow-rate or by reading the pressure meter data. During prolonged measurements, periodic refilling of the pre-column is necessary. When concentration gradients are used, the chromatograph must be provided with several reservoirs. Usually, a different reservoir is used for each component. The preparation of the apparatus for measurement is, in principle, the same as that used in work with a simple mobile phase. Increased attention must be paid to column regeneration after each analysis. It is not sufficient if only the mobile phase in the column is replaced, but equilibrium must be attained between the stationary and mobile phases in the whole column. Inadequate column regeneration substantially impairs the reproducibility of the measurements. If mobile phases with large differences in elution strength are employed for gradient preparation, the regeneration may be time consuming, but the time required can be shortened by increasing the rate of flow of the mobile phase and by changing (usually increasing) the temperature. In work with concentration gradients in the mobile phase, it is important that the volume between the outlet of the gradient-forming device and the column inlet should be as small as possible. Large volumes might affect the shape and the time course of the gradient.
SORTING OF SORBENTS ACCORDING TO PARTICLE SIZE In other parts of this book, the importance of particle size (Chapter 3) and the homogeneity of its distribution (Chapter 3) for liquid chromatography has been stressed. The preparation of efficient columns of various types and diameters is connected with the use of sorbents with a particle size of ca. 5-500 pm (Table 1 1.1). Although commercially available sorbents and supports, recommended for certain uses, often have a satisfactory particle-size distribution, in many instancesit is necessary to sort out and remove particles that are either too small or too large. Laboratory methods can be divided into basic groups: classification with sieves and fluid methods. Materials up to ca. 25 pm particle size can theoretically be classified by sieving. When sieving in the dry state is carried out, the uppermost sieve has the largest mesh diameter, while the lowest sieve has smallest mesh size. The material passes through the sieves by its own weight. In order that the fine fractions may fall through the sieves, the sorted filling should be stirred continuously. The sieves are agitated either by hand or on a vibrator. An excessively high vibration frequency causes a strong erosion or fragmentation of the material with ensuing redistribution of particles. Vibrations not exceeding 5 cycles/sec are satisfactory. A receiver is placed under the sieves, in which the material from the finest sieve is collected. When sieving is carried out in the wet state, water flows in the same direction as the classified particles. The receiver must have a sufficiently large diameter that even the finest particles that have passed through all of the sieves may sediment. During the sieving, especially in the dry state, the particle-size distribution of the sorted material is often changed owing to mutual erosion or fragmentation. The powder and very fine particles formed adhere to larger particles from which they are separated with great difficulty. Some materials, especially organic, may have an electrostatic charge on References p.300
286
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
TABLE 11.1 CONVERSION OF U S . AND BRITISH SIZES OF SIEVES INTO METRIC SIZES Taken from the catalogue+ofE. Merck (Darmstadt, G. F. R.) with their kind permission. Sieve opening (mm)
US. Standard, ASTM E 11-61
Number of meshes (Tyler mesh/in.)
0.037 0.044 0.045 0.05 3 0.063 0.074 0.075 0.088 0.090 0.105 0.125 0.149 0.150 0.177 0.180 0.210 0.250 0.297 0.300 0.354 0.355 0.420 0.500 0.595 0.600 0.7 07 0.710 0.841 1.00 1.19 1.20 1.41 1.68 2.00
400 325
400 325
270 230 2 00
27 0 250 200
170 140 120 100 80 70 60 50
170 150 115 100
-
-
45 -
40 35 30 -
25 20 18 16 14 12 10
-
80 65 60 48 42 -
35 32 28 24 20 16 14 12 10 9
British Standard,
BS 410: 1962 (mesh/in.)
350 300 240 200 170 150 120 -
100 85 72 60 52 -
44 36 30 -
25 22 16
-
14 10 8
their surface, which causes aggregation of the particles. The aggregates are retained on the sieves, so that the size of the conglomerates and not the true size of the particles corresponds to their mesh size. Agglomeration occurs particularly in the separation of small particles (approximately 10 pm diameter and less). Many of these disadvantages are avoided in fluid methods, which are based on Stokes' law, from which the rate of movement of particles in a fluid medium can be deduced. At a critical linear rate of the fluid, u,, moving upwards through the separator, the rate of movement of a spherical particle moving under the effect of gravity in the opposite direc-
287
SORTING OF SORBENTS ACCORDING TO PARTICLE SIZE
tion to that of the fluid flow is equal to the rate of fluid flow if (1 1.1) where R is the diameter and L the length of the separator, 77 is the dynamic fluid viscosity,
PI and P2 are the inlet and outlet pressures in the separator, Po is the pressure at which the flow through the compressible fluid (gas) is measured, p the fluid density, dp the particle diameter, s the particle density and g the gravitational acceleration. At a fluid flow-rate of less than u k , equal-sized spherical particles (s and d , constant) move against the liquid flow, while at rates above uk they are conveyed in the direction of the flow. Fluid methods of separation are based on this principle. From eqn. 1 1 . I , it can be deduced that in a moving fluid, spherical particles of diameter dp will float if 9R2
dp’
= 4(s - p)gL
.-P: - P:
(1 1.2)
Po
Particles of larger diameter sediment, while particles of smaller diameter are conveyed from the separator. If the classified particles have irregular shapes, their dimensions can be replaced by an equivalent diameter dp e q u i v . , for which dp equiv.
- 8tpReq2L .R2p
Po
(11.3)
P: --P$
where the Reynolds number Re = ukdpp/q.The values of the correction factor, q, depend on the particle size (see Table 11.2). From eqns. 11.2 and 1 1.3, it is evident that the material can be classified, i.e., fractions of a definite dp can be obtained, by changing the rate of fluid flow (which is achieved by changing P I and Pz)or by changing the radius of the grading device R,if other conditions are constant. TABLE 11.2 DEPENDENCE OF CORRECTION COEFFICIENT, Re2
8000 10,000 20,000 50,000 100,000 200,000
ON Re2
~p,
Particle shape Globular
Round
Angular
Oblong
Flat
1
0.805 0.80 0.79 0.755 0.753 0.74
0.68 0.678 0.672 0.65 0.647 0.635
0.61 0.595 0.59 0.564 0.562 0.560
0.45 0.44 1 0.43 0.42 0.408 0.392
1 1
I 1 1
Tesah’k and NeSasova described a simple laboratory device with which they achieved good results, in which both a gas (air) and a liquid were used as the carrier fluid. A scheme of the device is shown in Fig. 11.2. The basic separation unit consists of a glass tube of I.D. R = 16 mm and length L = 640 mm. In the bottom part of the tube, a porous glass plate is sealed, which ensures a homogeneous distribution of the introduced carrier References p.300
288
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
Fig. 11.2. Scheme of the grading device according to Tesah'k and NeEasovi. 1= Membrane pump; 2, 3 = flow regulation elements; 4 = pulse dampers;5 = grading tube; 6 = porous plate; 7 = fraction collector; 8 = water pressure regulator.
medium over the whole cross-section of the tube. When gas is used as the carrier medium, air is led from an efficient membrane pump (outlet pressure 2 kp/cm2) through a conical regulation valve, a flow meter, a drying column and pressure pulse attenuators (capillaries serving as a pneumatic resistance connected in series and bottles of 250 ml volume serving as pneumatic capacities) into the grading tube. Parallel with this tube, a simple water manostat is connected, which permits the fine setting of accurate flow-rates of air through the grading tube. When water is used as the carrier medium, the use of a manostated gas of precisely known constant pressure is recommended for expelling the water from the reservoir. The grading tube remains unchanged. The classification proper is carried out by introducing the sample of adsorbent (carrier, etc.), up to 3 ml in volume for the given experimental device, on to the porous plate in the grading tube. Then a stream of carrier medium is intfoduced at such a rate as to convey the smallest particles and the particles with lowest density in the direction of fluid flow. They are collected in the fraction receiver. As soon as only those particles are present in the tube w h c h are not carried off by the carrier medium, the flow of the carrier fluid is increased either by increasing the water level in the manostat connected in parallel with the grading tube (if gas is used), or by increasing the gas pressure above the water in the reservoir (if water is used as the grading medium). Then the sorted out fraction that has a larger particle diameter or a higher specific weight is collected in another fraction receiver. The classification process is repeated if necessary until the whole of the volume has been classified. If the technique described is employed and the fluctuation in the gas flow-rate is 4%, then the standard deviation, d,,, of the size of spherical particles is about 2%, assuming that the density of the material is homogeneous. However, the sizes of the particles may differ by up to 50%in instances when the value of s - p varies within the range 0.8-2.1 g/cm3. With particles of irregular shape, the standard deviation of the particle diameters may increase up to 25%on changing the flow-rate (in agreement with Table 1 1.2). Today, various types of grading devices can be purchased. The most widely used are fluid graders based on the principle described by Rumpf and Leschonski, which use separators, the shorter part of which, adjacent to the membrane gas inlet, is cylindrical and the longer part is conical with a diameter that decreases in the direction of gas flow. This
289
SORTING OF SORBENTS ACCORDING TO PARTICLE SIZE
grading vessel vibrates at a suitable frequency and amplitude. At constant flow-rate of the carrier medium, i.e., gas, the material can be sorted into two fractions. The principle of the classification also follows from eqns. 1 1.2 and 1 1.3. In a simplified manner, the grading process may be viewed so that the classified material is mechanically thrown by the vibration into the conical part of the separator (smaller R ) , and, depending on the particle radii and their density, it either falls back on the membrane or is conveyed out from the device into the collector of the fine fraction. The performance of this apparatus is higher than that of the above described laboratory device, but its construction is difficult under laboratory conditions. The particle-size distribution can be determined by measuring a sufficient number of particles microscopically. The resulting dependence is shown in the form of a histogram, showing the number of particles of a given radius present in the measured mixture. The lowest number of measured particles should be 100-500.
Classification of silica gel particles according to size by sedimentation For a single classification of 2-3 kg of crude silica gel, five 10-1 broad-necked flasks and one ca. 20-1 vessel may be used. In flask No. 1 , the added silica gel is stirred with 9-10 1 of water and then allowed to sediment for 1 min. The suspension is decanted (in thirds of the volume) into flask No. 2 , where silica gel is allowed to sediment for 2 min. The suspension is decanted gradually from the full flask No. 2, again in thirds of the volume, up to flask No. 5, allowing sedimentation in flask No. 3 to take place for 4 min, in flask No. 4 for 8 rnin and in flask No. 5 for 16 min. From the last flask, the suspension is decanted (in halves of the volume) into the larger vessel, where the fines are allowed to sediment. This procedure is continued until 40-50 1 of water have passed through the sedimentation vessel. Single silica gel fractions from flasks Nos. 1-5 are then filtered off under suction and dried at 120°C for 12 h. The fine powder from the large sedimentation vessel can be used for thin-layer chromatography, after drying under the same conditions. The procedure is summarized in Table 11.3. It should be noted that, in order to economize with the use of distilled water, the first three runs can be carried out with tap water and only the last two runs with distilled water. For stirring, a strong rod (preferably made of a hard polyvinyl plastic) is used, which is rinsed with water after each stirring. TABLE 11.3 CLASSIFICATION BY SEDIMENTATION
Number of flask
Time of sedimentation (rnin)
End of stirring (min)
Time of decantation (min)
5 4 3 2 1
16 8 4 2 1
0 8.5 13 15.5 17
16 16.5 17 17.5 18
References p.300
290
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
Regeneration of silica gel The silica gel is boiled with a 5-10-fold amount of 1% sodium hydroxide solution for 30 min. After checking that the solution is strongly alkaline to phenolphthalein, the suspension is filtered hot and washed three times with distilled water. Further boiling is carried out with a 3-6-fold volume of 5% acetic acid for 30 min. After filtration, the particles are washed with distilled water until neutral, then with methanol, and twice with distilled water. After filtration under suction, the material is dried. Activation is carried out at 120°C for 12 h.
DETERMINATION OF THE ACTIVITY OF ALUMINA BY THIN-LAYER CHROMATOGRAPHY HeTmanek et al. spread alumina (ca. 10 g) on a glass plate (for example, 20 X 10 cm) and smoothed the surface by rolling it with a glass rod, 0.6 mm of each end being strengthened with insulation tape. The length of rod between these tapes is about 4 cm, which produces on the plate a layer of this width and 0.6 mm thick. Solutions (0.02 ml) of standards (azo dyes: azobenzene (30 mg), p-methoxyazobenzene, Sudan yellow, Sudan red and p-aminoazobenzene (20 mg each) in 50 ml of dry, distilled tetrachloromethane) are applied at a distance of 3 cm from the edge of the plate, which is then developed in a slightly oblique position in a low tank containing tetrachloromethane. The RF values of single azo dyes (measured from the centres of the spots) are correlated with the activities as determined by Brockmann and Schodder (see Table 11.4). For other standardization methods, see Engelhardt and Wiedemann. The activity of commercial alumina is usually less than I. If alumina of this activity is needed, it is heated at 350°C for 6-8 h , or at 120°C in a vacuum (oil pump) for 2-3 h. Lower activities are achieved by adding the corresponding amount of distilled water to this most active alumina (see Table 1 1S ) .
TABLE 11.4 RF VALUES (t0.04) O F INDIVIDUAL AZO DYES ON ALUMINA OF DIFFERENT GRADES
Azo dye
Azobenzene p-Methoxyazobenzene Sudan yellow Sudan red p-Aminoazobenzene
Grade of alumina according to Brockmann and Schodder
I1
111
IV
V
0.59 0.16 0.01
0.74 0.49 0.25 0.10
0.85 0.65 0.57
0.95 0.89 0.7 8 0.56 0.19
0.00 0.00
0.03
0.33 0.08
COLUMN PREPARATION
29 1
TABLE 11.5 ACTIVITY OF ALUMINA, SILICIC ACID AND MAGNESIUM SILICATE DEPENDING ON WATER CONTENT The values given are averages depending on the type and the manufacturing procedure used for individual adsorbents. Activity grade
I I1 I11 IV V
Water added (70)
To alumina 0
3 6 10 15
T o silicic acid 0 5 15 25 38
To magnesium silicate 0
I 15 25 35
COLUMN PREPARATION Today, the factors are known that determine the efficiency of regular filled columns in which the ratio of the column diameter, d,, to the mean particle diameter of the filling, d p , is d,/d, > 10. Although columns can be prepared reproducibly that are comparable with gas chromatographc columns with respect to their HETP, development in this area is still continuing. The technique of filling the columns can be divided into two basic groups: (1) filling in the wet state and (2) filling in the dry state. (1) When the column is filled in the wet state, a suspension of the column filling in a suitable liquid is prepared, which is then introduced into the column in such a way that a bed that is as homogeneous as possible is obtained, in which the particles are settled as densely as possible. The prepared suspension should be stable and no agglomeration or fractionation of solid particles during the preparation of the suspension and filling it into the column should take place. Both of these effects lead to inhomogeneity of the bed, whch impairs the quality of the prepared column. Therefore, in an optimum case, the particles in the slurry should float (the density of the suspending liquid, preferably a binary mixture, must be equal to the mean density of the support particles) and the column should be filled as rapidly as possible. The suspending liquid must also often induce the necessary swelling of the filling particles. In a glass vessel, the column filling is stirred with an amount of a binary liquid such that the resulting suspension contains 10-25% (w/w) of particles. Perfect mixing and degassing of the slurry is achieved by ultrasonic stirring for 2 min. If the particles sediment or if they aggregate at the surface, the density of the suspending solvent is adjusted by adding the heavier or the lighter component until the entire filling remains suspended for at least 10 min. The balanced slurry is then stirred again and transferred rapidly into the reservoir (Fig. 1 1.3), to which a column filled with the suspending solvent is connected. In the reservoir, the suspension is carefully overlayered with an immiscible liquid that functions as a piston. As the filling should be carried out as rapidly as possible, a column connection should be chosen with as large a diameter as possible, and a fritted glass septum closing the column outlet with References p.300
292
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
9
Fig. 11.3. Schematic arrangement for packing the column with equilibrated suspension. 1 = Reservoir containing hexane; 2 = high-pressure pump; 3 = manometer; 4 = high-pressure valve; 5 = reservoir with suspension; 6 = suspension; 7 = water; 8 = hexane; 9 = column; 10 = waste container.
maximum admissible pores (with respect to the grain size of the filling) and the maximum possible working pressure (up to 340 atm) should be used. The reservoir containing the suspension is connected with the pump after the required pressure at the pump outlet has been achieved. The liquid flow-rate'through the column decreases during the filling and therefore the pump speed should be regulated. The column is filled when a calculated volume of the suspending solvent or the first fraction of the liquid serving as a piston has passed through it. After the column has been filled, the introduction of the mobile phase should be stopped until the pressure drops spontaneously. If the pressure is decreased before entering the column, the homogeneity of the bed is seriously disturbed and the column efficiency decreases. This effect applies generally. Classical methods of filling columns with suspensions (sedimentation, filtration with increasing pressure) are not of much use in practice owing to the low efficiency of the
COLUMN PREP AR AT1 ON
29 3
columns so prepared. Later methods involving the use of a balanced density solvent permit the preparation of high-quality , high-efficiency columns even when very fine materials are used, which so far cannot be filled in the dry state. The whole procedure is, however, tedious and time consuming, although the filling can be carried out very rapidly. Fillings are not applicable that would be changed in an undesirable manner by the suspend. ing solvent or the liquid used for the subsequent purification and adjustment of the activity of the filling of the prepared column (changes in the degree of wetting in work with stationary liquids, reactions with some types of chemically bound stationary phases, etc.). In order to illustrate the results that can be achieved by the described method of column packing (in the literature, it is indicated as the “balanced slurry column packing procedure”), several examples are presented here. When a material the particles of which adhere to each other in dry state was packed, for example Zipax with a chemically bonded stationary phase, Kirkland (1971b) acheved with particles of size 37-44 pm an efficiency 30-50% better than when the dry packing procedure was used. With finer particles, the increase in efficiency was even higher. The efficiency of the packed columns was reproducible to +lo%. For the suspension of the packing, he used a mixture of 21 parts of tetrabromomethane and 15 parts of Perclene (DuPont, Wilmington, Del., U.S.A.). The suspending liquids were eluted from the column with methanol, which was then expelled by the mobile phase. When packing the column with silica gel of 5-10 pm particle diameter, Majors suspended 1 g of the packing dried at 200°C for 2 h in 10 ml of a solvent consisting of 60.6% (w/w) of tetrabromomethane and 39.4% (w/w) of tetrachloroethylene; undried silica gel agglomerates in this liquid. For the elimination of the remainder of the suspending liquid and for adjusting the activity of the silica gel, he used a procedure of activation by solvent, proposed by Snyder. At a linear flow-rate of the mobile phase of 1.18 cm/sec, the column had an HEi’P of 0.1 mm. The best results so far were obtained by Kirkland (1972b). Spherical silica gel particles with a regulated porosity of 5-6 pm diameter were packed as an aqueous suspension stabilized by the addition of 0.001 M ammonia solution. For the component with capacity factor k’ = 12, at a linear flow-rate of the mobile phase of 0.44 cmlsec, he measured with a 250 X 3.2 mm column an HETP of 0.038 mm. Tlus efficiency is comparable with the efficiency of capillary columns in gas chromatography. (2) When columns are filled in the dry state, small portions of sorbent are introduced into a tube that is closed at the bottom with a metallic, glass or PTFE filter disc, glasswool or a filter-paper disc. The homogeneity of the sorbent bed can be improved by providing for the movement of the support particles, which permits their subsequent orientation. The amount and the method of supplying the mechanical energy for moving the added particles (shaking down, vibration, compression) should be chosen so that no fractionation of the particles according to their size or even the disturbance of the formed bed occur. Hence, it is advisable to add the packing slowly, most often discontinuously and in small portions, so that the amount of packing that has to be moved each time is as small as possible. Each portion of the material added should be such that its packed length does not exceed 1-5% of the length of the prepared column. The amount of mechanical energy introduced by shaking, vibration or compression should be regulated according to References p.300
294
OPERATlON OF A MODERN LIQUID CHROMATOGRAPH
particle size, shape and mean density. Therefore, a series of variants of thts method have been described for various granulations and types of materials, as illustrated in the examples below. It is important that the packings should have as narrow a particle-size distribution as possible. The larger the distribution, the easier is the separation of the particles according to their size along the column. The smaller the granules used, the more carefully the column should be packed and the longer is the time necessary for the packing. The basic limitation of all procedures in which dry materials are used is that they cannot be utilized for packings of low specific weight, with increased adhesion of particles, for packings that change their volume under the effect of the mobile phase, and for very fine granules (the lower limit varies from 20 to 50 pm). In spite of these disadvantages, a number of workers agree that whenever possible it is preferable to use the dry column packing procedure. A few examples of packing chromatographic columns are presented below, from which differences connected with various materials follow. Halrisz and Naefe showed that for silica gel particles, the surfaces of which were esterified with polyethylene glycol 200 and which were larger than 50 pm, the same method can be used as in gas chromatography. Silica gel was added continuously under simultaneous vibration (60 cycles/sec). Then the packing was shaken down by allowing the tube to fall from a height of 20 cm and the filling was completed. For particles of 10-50 pm diameter, i t was preferable if the packing was added in such amounts as would give a column (zone) about 1 cm long after each addition. After each addition, the column was vibrated and finally filled to completion as in the preceding case, tapping the sides to effect the packing. The efficiency of such columns was reproducible to +-lo%. Kirkland (197 la) stated that relatively light materials (silica gel, Kieselguhr) with nonspherical shapes cannot be packed in the dry state satisfactorily if their diameter is less than 50 pm. He recommended that the support should be added in such a manner as to increase the bed height after each addition by 0.4 cm, with continuous vertical tapping on the floor (2-3 taps/sec) and also tapping the side of the column at the level of the filling. The fdling of the column should be completed after tapping for 5-10 min. The packed column is then run for half an hour at a pressure exceeding the operating pressure during the measurement. If, during this interval, the bed is shortened the column was not packed well and it must be remade. A 1 m X 2 mm column can be packed by this technique in 15-30 min, depending on the particle size and the type of packing. Kirkland (1972b) mentioned that for packing a column with Zipax without a bound stationary phase, the most advantageous procedure is the “modified tap-fill” procedure. The packing is added in aliquots of 100-200 mg and, after each addition, the column is tapped vertically on the floor and also on the side (80- 100 times, with a frequency of 2-3 taps/sec). Then the packing is consolidated by gently tapping the column on the floor for 15-20 sec, without tapping the side. The procedure can be also used for Corasil, Durapak and Porasil, and for particles smaller than 37 pm. Zipax wetted with the stationary phase was packed by Done and Knox. A column of 2.1 mm I.D. was placed into a mechanical apparatus which rotated the column at 180 rpm and simultaneously allowed it to fall from a height of 1 cm 100 times per minute. During the filling, the column was tapped gently on the side at the level of the packing. The packing was introduced into the column continuously with a stream of dry nitrogen. Karger
SAMPLE PREPARATION AND APPLICATION
29 5
et el. recommended filling the column with surface-etched glass beads by tamping with a glass rod. When packing large diameter columns, Sie and Van den Hoed found that rotating the column was advantageous. Randau and Schnell compared packing columns by sedimentation with three methods of dry packing: vibration on a vibrational table, mechanical tamping and tapping on the base. The best columns were obtained by simple tapping, the column prepared by vibration was less satisfactory and the column obtained by sedimentation was the worst (Table 11.6). TABLE 11.6 EFFICIENCY OF COLUMNS PACKED BY VARIOUS METHODS (RANDAU AND SCHNELL) Silica gel of particle diameter 60 A (40-63 wn), coated with 40% (w/w)of 1,2,3-tris-(2-cyanoethoxy)propane; column length 50 cm, flow-rate 0.68 cm/sec. Method of packing
Tapping Vibration Tamping Sedimentation
HETP (cm) (2-cyanoethoxy) 0.80-0.84 0.85 - 1.27 0.96-1.08 1.46-1.94
SAMPLE PREPARATION AND APPLICATION When samples are prepared, two fundamental criteria must be considered: the detector sensitivity and the capacity of the column packing indicating the maximum amount of sample that can be introduced into the column. In liquid-solid systems, the concept of the linear capacity of the packing is common. It is usually considered to be equal to the amount of solute that would result, during its introduction into the column, a maximal 10%decrease in the specific elution volume of a zero amount of a given solute. This specific elution volume of the zero amount of solute is obtained by extrapolation of a graph of the specific elution volume plotted against the amount introduced; it is usually given in grams of solute per gram of support. In adsorption systems, the capacity of the packing is critical, especially in work with non-deactivated adsorbents with large surface areas or with adsorbents with very low specific surface areas. When the support capacity is exceeded, the non-linearity of the distribution isotherm is reflected in the asymmetry of peaks. The occurrence of peak broadening, either at the front or back of the zone (the latter is more common), leads to a poorer resolution of the components and, from the point of view of separation efficiency, it is therefore undesirable. In systems with a linear distribution isotherm, when the support capacity is exceeded, an anomalous spreading of the zones occurs, even when their symmetry is preserved. In partition chromatography, the capacity of the support is the limiting factor at low wetting, for example, if glass beads or porous layered beads are used as supports. Hence, an amount of the separated component should be injected per plate of the chromatographic column such that the capacity of the plate is not exceeded, which differs References p. 300
296
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
for various materials. However, the total amount injected should be such as would permit the detection of the injected component after its passage through the column. For qualitative analysis, it is required that the response should be equal to at least double the noise, while for quantitative purposes the response should exceed the noise by at least five times. This condition, however, would not be fulfilled in many instances, especially for components with a high retention, if the sample were to be introduced only on to the first plate of the column. In addition to the capacity of the stationary phase, the solubility of the sample in the mobile phase also can be a factor that limits the maximum utilizable sample concentration. For a maximum vo!ume of the injected sample, ‘V (which would cause at most a 5% increase in the zone width), Klinkenberg derived the relationship (1 1.4)
where N i s the number of theoretical plates of the column used, vm the volume of the mobile phase in one plate (HETP), vs the volume of the stationary phase in one plate, V, the total volume of the mobile phase in the column, V, the total volume of the stationary phase in the column and K the distribution coefficient of the injected sample. For a component with zero retention, the equation assumes a simpler form:
vs= 1 . 1 v,@=
1 . 1 .-Vm
fl
(11.5)
which represents the strictest condition for the introduced sample volume. The term v, can be made equal to the ratio of the dead volume V,, to the number of plates of the column N only if the volume of the connections is negligible with respect to V,. From eqns. 11.4 and 11.5, it follows that the better the column of given dimensions is prepared, i.e., the more efficient it is, the smaller is the injection volume that can be used. Scott has shown that the sample can be introduced dissolved in the mobile phase, as a solution in the stationary phase (if the stationary phase is a liquid), or as a solution in another, better solvent. The possibility of using an auxiliary solvent with a better dissolving ability than that of the mobile phase for the dissolution of the solute follows from the fact that in the chromatographic column a decrease in the concentration of the solute in the mobile phase takes place as a consequence of its partitioning between the stationary and mobile phases. In the case of a solute characterized by a capacity factor k’, of the total amount injected into the first plate, only a fraction 1/( 1 + k’) remains in the mobile phase. Therefore, for the injection, a concentration of the solute in the auxiliary solvent can be used that exceeds (1 + k’) times its solubility in the mobile phase. It is therefore evident that the utilization of an auxiliary solvent is important in practice and is possible only when solutes with a higher retention are injected. For a component with a low retention (small k’), a decrease in its solubility in the mobile phase and its separation from the mobile phase can easily take place after the substitution of the mobile phase for the auxiliary solvent. The same phenomenon can be observed with excessively large differences in the solubilities in the mobile phase and the auxiliary solvent, even for solutes with a higher k‘, if the solubility of the solute in the auxiliary solvent is higher than (1
+
GENERAL COMMENTS
297
k') times its solubility in the mobile phase. The precipitate is then dissolved slowly in the pure eluting agent and the solution introduced into the column. This phenomenon is equivalent t o the injection of an excessive volume of a dilute sample. The result is a very broadened asymmetric zone. The basic requirements for the injection can be summarized as follows. The accurate measurement of a known volume must be ensured, and this volume must be introduced into the column as rapidly as possible. As diffusion coefficients in liquids are low, it is important that the sample should be introduced accurately into the centre of the column, as an excentric injection decreases the efficiency. High inlet pressures on columns require the use of high-pressure injection ports or special techniques of sample introduction. The choice of organic solvents or of aqueous solutions of acids and bases as mobile phases places appreciable requirements on the construction of the injection ports and the materials used. A detailed description of these devices is given in Chapter 8. However, it can be stated that no system described so far in the literature is universally satisfactory if the following requirements are placed upon it simultaneously: high accuracy and reproducibility of the injection with low consumption of the sample; the possibility of injecting against pressures of several hundreds of atmospheres; ensuring a rapid transport of sample into the centre of the column without diluting the mobile phase or loss of the sample; universality (with respect to the injected volumes) within a range of three orders of magnitude; operational reliability; and simplicity of maintenance.
GENERAL COMMENTS A good result in chromatographic analysis is not dependent only on a suitable choice of the chromatographic system, i.e., by an appropriate combination of stationary and mobile phases, and on the separation conditions, such as temperature, mobile phase flowrate and the use of gradients of various types. The preparation of a sufficiently effective chromatographic column and, last but not least, the choice of an optimal and sufficiently sensitive detector, are also of particular importance. The choice of components is determined by the types of separations or chromatographic methods and techniques that most often come into consideration (ion-exchange chromatography, gel chromatography, chromatography on adsorbents or stationary phases, preparative chromatographic procedures), and thus also by whether the apparatus will be used for a single or a few routine analyses or for a more demanding research work. The choice of detectors depends firstly on the type of substances to be separated and the requirements placed on the sensitivity of their detection. Usually, the composition of the mobile phase must also be taken into consideration, and also the changes in its composition during the analysis when gradients are used. The choice of a particular detector is usually a compromise between the requirements placed on the sensitivity and the universality of the detector, but often the possibility of working under different experimental conditions (flow-rate, temperature, mobile phase composition, etc.) must also be envisaged. It is necessary for all materials with which the mobile phase passing through the apparatus will come into contact to be perfectly inert towards it. %s condition is usually References p.300
29 8
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
fulfilled by stainless steel, PTFE and glass, which are used as basic constructional materials. The resistance of rubbers, which are used as packings at some points in the apparatus, depends on the composition of the mobile phase. So far, no type of rubber that would be perfectly suitable in all instances is known. Efficient degassing of all spaces through which the mobile phase flows is always useful. It can be enhanced by various methods: preliminary evacuation of the chromatograph, its flushing with a gas that is soluble in the mobile phase used, increasing the pressure of the mobile phase in the system, which enhances the dissolution of gases, or degassing the mobile phase before it enters the pump. As the degassing of the mobile phase is also useful during the operation, the degasser is usually an integral part of liquid chromatographs run at higher pressures. In an apparatus that is not provided with a degasser, the formation of bubbles often occurs, which disturbs the operation of detectors with a flow-through measuring cell. A dynamic resistance at the outlet, which increases the pressure in the system and thus suppresses the formation of bubbles, can be connected only if the detector can operate at least at a mildly increased pressure. In some instances, the degassing of the mobile phase may be useful even from the point of view of long-term stability of the column packing. When the mobile phase is changed or when a concentration gradient is employed, efficient washing of the whole system and bringing the column into the original state are essential. Imperfect exchange of the phases or poor equilibration in the column results in irreproducibility of the elution data. If the detector response is dependent on the mobile phase composition (in the case of binary detectors, for example, refractometers), an imperfect phase exchange may also affect the quantitative data. The requirement of perfect sealing of the whole system is obvious. Leakages in components inserted before the column cause a pressure decrease in the system and a disturbance in the correlation between the pump speed and the mobile phase flow-rate through the column. A leakage of liquid after the injection site is also reflected in irreproducibility of the detector response. Leakages also allow the penetration of air into the apparatus, thus facilitating the undesirable formation of bubbles in the detector. In work with organic liquids, there is also a risk of ignition of the escaping vapours. In addition to the input pressure, the flow-rate of the mobile phase through the column should also be followed. The device for the measurement of the flow-rate does not always constitute a part of the chromatograph. Of various methods of measuring flow-rate (syphons, integral measure: ments, weidung of the liquid passed; see Chapter 8), only the determination of the immediate flow-rate is usually suitable for work with narrow analytical columns; the flow-rate is measured on the basis of the time of passage of an air bubble, introduced into the current of liquid behind the detector, through a calibrated volume of a narrow tube; this method was proposed by Gerding and Hagel. Commercial flow meters also operate on t h l s principle, enabling an integral measurement of the passed-through volume of the mobile phase to be made. When working with the degassed mobile phase the air bubble is significantly dissolved in it. The error in the determination of the flowrate caused by this effect, which may be up to 30%, can be eliminated by substituting the air with a liquid immiscible with the mobile phase used. For accurate quantitative work, it is important to know, in addition to the immediate flow-rate, the total volume passed as a function
GENERAL COMMENTS
299
of time. The accuracy of the calculation of the passed-through volume from the flow-rate and time is dependent on the constancy of flow-rate, conditioned by the stability of the pump performance and the pressure drop in the column. The pressure drop in the column of a perfectly sealed apparatus usually changes only if the column is obstructed by pieces of the eroded septum during the introduction of the sample with syringes. The resistance of the septum depends not only on the material used but also on the composition of the mobile phase and the diameter of the syringe needle used for injection. An important condition for accurate work, especially for the measurement of the retention data and during analyses utilizing these data, is to ensure a known, constant temperature of the column to an accuracy of several tenths of 1°C. The effect of temperature.on retention, demonstrated by a number of workers, is comparable, in principle, with the effect in gas chromatography if the system consists of a simple mobile phase. Maggs, and Scott and Lawrence found that in multicomponent mobile phase systems, where the conditions for the attainment of partition equilibria are much more complex, an increase in temperature may cause not only changes in relative retention or the reversal of elution sequences, but even an increase in the retention of a certain component. The need for thermostatic control of column temperatures should also be stressed because, by tradition, it is often still not carried out in liquid column chromatography. However, it is desirable that with the development of modern instrumentation, permitting the analysis of complex mixtures and work at temperatures other than room temperature, it should become equally common as in gas chromatography. In view of the effect of the mobile phase on retention and separation selectivity, and sometimes of the response, increased attention must be paid to the composition and purity of the mobile phase. In analyses carried out without a concentration gradient, it should be ensured that the composition of the mobile phase does not change during the whole series of measurements. Therefore, it is advisable to use as mobile phases either individual chemicals or to prepare large amounts of mixed mobile phases of known composition from components of known or at least defined compositions. In accurate and demanding measurements, it is useful if the purity or the composition of the mobile phase is controlled. The elimination of constituents (impurities) that could function as moderators or substances that appreciably affect the elution strength of the mobile phase is of special importance. In practice, this usually means the elimination of constituents that have a chemical nature different from that of the mobile phase or its components (for example, trace amounts of polar substances from hydrocarbons). If a concentration gradient is used, it is desirable that the changes in concentration with time should be reproduced as accurately as possible during individual analyses. The purity of all of the components must satisfy the requirements mentioned in the preceding case. Only under these conditions can qualitative and quantitative reproducibility of the analysis be achieved that would be comparable with the results obtained with a mobile phase of constant composition. In conclusion, it should be stressed that the successful utilization of narrow and especially short columns of high efficiency is dependent on the minimum volume of the connections between the injection port and the detector. The detector volume is also important in these instances. The dependence of the column efficiency on extracolumnar parameters is discussed in Chapter 8, KrejCi and PospiiilovP also found that the contribution to the References p.300
300
OPERATION OF A MODERN LIQUID CHROMATOGRAPH
zone width, given by zone spreading in the connections, depends not only on the magnitude, but also on the shape, of these volumes. For example, the contribution caused by the spreading of the zone'in a 4O-pl volume, corresponding to 45 cm of a capillary of 0.25 mm diameter, is virtually equal to the contribution created in 176 p1 of free volume of a capillary of 45 cm X 1 mm, packed with glass beads. Scott and Kucera derived equations that permitted the calculation of the dimensions of empty tubes connecting the columns that would prevent an increase in the width of each eluted zone by spreading in these connections above 5%.
REFEREh i;iS Brockmann, H. and Schodder, H., Ber. Deut. Chem Ges., 74 (1941) 73. Done, J. N. and Knox, J. H., J. Chromatogr. Sci., 10 (1972) 606. Engelhardt, H. and Wiedemann, H., Anal. Chem., 45 (1973) 1641. Gerding, J. J. Th. and Hagel, P., J. Chromatogr., 31 (1967) 218. Halhz,I. and Naefe, M., Anal. chem., 4 4 (1972) 76. Hermbek, S., Schwarz, V. and Cekan, Z., Collect. Czech. Chem. Commun., 26 (1961) 3170. Karger, B. L., Conroe, K. and Engelhardt, H., J. Chromatogr. Sci., 8 (1970) 242. Kirkland, J. J., in J. J. Kirkland (Editor), Modern Practice of Liquid Chromatography, WileyInterscience, New York, 1971a, p. 178. Kirkland, J. J.,J. Chromarogr. Sci., 9 (1971b) 206. Kirkland, J. J., J. Chromatogr. Sci., 10 (1 972a) 129. Kirkland, J. J., J. Chromatogr. Sci.,10 (1972b) 593. Klingenberg, A., in R. P. W. Scott (Editor), Gas Chromatography 1960, Butterworths, London, 1961, p. 182. KrejEi, M. and PospiiiJovi, N., J. Chromatogr., 7 3 (1972) 105. Maggs, R. J.,J. Chromatogr. Sci., 7 (1969) 145. Majors, R. E., Anal. Chem, 4 4 (1972) 1722. Randau, D. and Schnell, W.,J. Chromatogr., 57 (1971) 83. Rumpf, H. and Leschonski, K., Chem.-hg.-Tech., 39 (1967) 1231. Scott, R. P. W., J. Chromatogr. Sci.,9 (1971) 449. Scott, R . P. W. and Kucera, P., J. Chromatogr. Sci., 9 (1971) 641. Scott, R. P. W. and Lawrence, J . G., J. Chromatogr. Sci., 7 (1969) 65. Sie, S. T. and Van den Hoed, N., J. Chromatogr. Sci., 7 (1969) 257. Snyder, L. R.,J. Chromatogr., 25 (1966) 274. TesGik, K. and NeEasovi, M.,J. Chromatogr., 75 (1973) 1.
Chapter 12
Practice of gel chromatography J. COUPEK, M. KUBIN and Z. DEYL
CONTENTS Choicc of gel packing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choicc of solvent and operating temperature. . . . . . . . . . . . . . . . . . . . . . . . . Apparatus for gel chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pumping systems and injection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... ........ ....... Columns . . . . . . . . . . . Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Columnpacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .... Special gel chromatographic techniques . Evaluation of gel permeation chromatographic data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of molecular weight and molecular-weight averages . . . . . . . . . . . . . . . . . . . . . . Calculation of molecular-weight distribution from gel permeation chromatographic data. Simultaneous determination of polydispcrsity in molecular weight and the chcmical heterogeneity of copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attempts t o determine the degree of branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of molecular weights of naturally occurring macromolecular compounds by molecular sieve chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
301
306 307 308 310 311 3 12 3 12
3 16 316 317 321
CHOICE OF GEL PACKING The choice of the gel packing, together with an adequate design of the apparatus, plays a decisive role in gel chromatographic separations. Besides fulfdling basic requirements towards the gel, viz., its chemical inertness, minimal adsorption of separated compounds, swelling capacity or at least wettability with an eluent, etc., the gel packing must also have an adequate resolution as required by the given problem. Heitz et al. discussed the factors affecting the resolution, defined by R = AV,/4u, where AV, is the difference between the elution volumes and u is the mean width of the chromatographic zone of compounds being separated that do not differ much in their molecular weights. The u values of such compounds can be regarded as being approximately identical and can be calculated from the relationship suggested by Van Deemter or Giddings (Heitz and Coupek). The dependence of u on particle size, flow-rate of the eluent, diffusion coefficients of the compounds used and column length was investigated. AV, depends on the physical structure of the gel packing or, more exactly, on the pore-size distribution of the gel in the swollen state. This dependence is shown in Fig. 12.1 ;it can be seen that the basic information on the application of the gel for a given problem can be read off conveniently from a calibration graph of the logarithm of the molecular weight of standard test compounds plotted against their elution volumes. The differences between the References p.321
301
PRACTICE OF GEL CHROMATOGRAPHY
302
log
M
Ve
Fig. 12.1. Relationship between log M and the elution volume, Ve (M= molecular weight). 1 = Tailormade gel with a suitable molecular-weight range; 2 = gel with an unfavourable pore distribution; 3 = universal gel with a molecular-weight range that is too wide for the given separation problem.
elution volumes of two compounds with molecular weights Mi and M 2 ,and thus also their resolution, will be the greater the smaller is the slope of the calibration curve defined by the relationship dlog M,/d V,. For a gel with good separation properties, the calibration graph must be linear over the largest possible range of elution volumes (curve 1). It may be expected from the non-linear form of curve 2 that the separation properties of a gel characterized by this curve will not be satisfactory. Curve 3 is the calibration graph of a gel with a wide range of applications, which, in spite of its poorer resolution, may be used successfully for preliminary analyses of samples that have an unknown molecularweight distribution. The emphasis that used t o be laid on the use of spherical packing particles for gel chromatographic elution is nowadays somewhat obsolete, because virtually all gel producers meet this requirement. However, another problem remains. namely, that of the particle size and particle-size distribution of gel packings. The column separation efficiency increases with decreasing particle diameter according to known relationships; on the other hand, analyses become too time consuming, and also the techniques used for the fractionation of the gel particles become more difficult, thus raising the price of the packing material. If the particle-size distribution of the packing used for separation becomes broader, the separation efficiency may be considerably impaired. The apparatus used for separation is another important factor, and its effect on the choice of the gel must not be neglected. The simple technique involving the use of a glass column with elution by hydrostatic pressure, which is still frequently used for preparative separations in aqueous systems, has much lower requirements towards the geometrical and mechanical properties of a gel packing than modern high-speed, high-resolution chromatographic procedures with columns that have separation efficiencies of thousands of theoretical plates per metre of the column length. It is therefore desirable that all of the factors outlined above should be taken into account when choosing the packing in order to achieve a compromise between the separation efficiency of the packing and the time of analysis, and between the use of a universal
CHOICE OF SOLVENT AND OPERATING TEMPERATURE
303
gel and a “tailor-made” gel; it is also useful to consider the solvation of the gel with an eluent and the possible adsorption effects that may arise here and affect considerably the elution volume of the compound, thus causing undesirable deformations of the chromatographic zone (tailing). No lesser importance should be assigned t o the chemical and mechanical stabilities of the gel, which play a decisive role in the lifetime of the packing and the stability of the flow-rates through the column. For exacting separations, ready made columns are commercially available (Waters Ass., Framingham, Mass., U.S.A.; Knauer, Berlin, G.F.R., etc.) with a comparatively broad (virtually universal) range of applications. Gel packings with a rigid structure, such as porous glass or porous silica gel, are less exacting than organic gels with respect to the precise experimental conditions that must be used and are therefore simpler to use.
CHOlCE OF SOLVENT AND OPERATING TEMPERATURE When choosing the solvent, it must be borne in mind that the preparation of a highquality gel chromatographic column is time consuming and requires experimental dexterity. It is therefore desirable that the lifetime and application range of columns should be as high as possible. The solvent should be of a universal character, dissolve the greatest possible number of compounds, be readily available and easy to be freed from impurities. During analysis or storage, it should not undergo any chemical changes, should not be aggressive towards the materials of construction of the chromatographic apparatus and must not react chemically with the gel packing or with the compounds being separated. The requirement that the eluent must be perfectly inert should be observed as strictly as in other chromatographic methods. The eluent must also meet some other requirements as t o its physico-chemical and physical properties. Firstly, it must be a good swelling agent for the gel packing, or at least perfectly wet its surface (macroporous inorganic packings). The viscosity of the solvent should be as low as possible under the conditions of analysis and it must not be too volatile. Special requirements for the eluent ensue from the detection system used. Spectrophotometric detectors require the use of solvents that have a minimal absorbance at the wavelengths used; differential refractometers call for solvents with the lowest possible refractive index or, on the contrary, a very high refractive index - in general, it can be said that the refractive index must differ from that of the sample as much as possible (the detector response should have the same polarity and a high value for all components of the mixture being separated); finally, wire or chain liquid chromatographic detectors with flame ionization units require a certain volatility of the eluent as well as certain chemical properties. The eluent used for gel chromatographic analyses of polymers must be a thermodynamically good solvent. Some polymer systems necessitate the use of elevated temperatures, as the polymer is insoluble in any solvent at room temperature. With increasing temperature of analysis, the requirements for accurate thermostatting increase correspondingly, and the apparatus becomes more complicated. It can be seen from the requirements outlined above that there is no ideal solvent that References p.321
304
PRACTICI OF GEL CHROMATOGRAPHY
meets all of them, and that there will be only a few solvents that meet most of them satisfactorily. For hydrophilic systems, suitable solvents are water, various buffer solutions and polar organic solvents such as methanol, ethanol, acetone, dimethyl sulphoxide and dimethylformamide. For organophilic compounds, the most universal eluent (which, however, forms explosive peroxides in contact with air) is tetrahydrofuran, which has a low viscosity and refractive index and no absorption at the UV wavelengths commonly used for detection (250 and 280 nm). Benzene, toluene, chlorinated aromatic hydrocarbons, chloroform, tetrachloroethane, rn-cresol and trifluorocthanol are also frequently used. Apart from its effects on the solubility of the sample, the temperature also affects other operating,variables. The elution volume, depending on the hydrodynamic volume of the molecule, is'a function o6,teyperature. The dependence of the elution volume of polystyrene fractions on temperature has been measured by Cantow et al. The decrease in the viscosity of the eluent with increasing column temperature results in a lower pressure drop for a given flow-rate. The equilibrium degree of swelling of homogeneous gels varies with temperature; the gels dilate with increasing temperature, which in turn leads to a reduction in the interstitial volume and to a possible change in the distribution of the packing. However, these effects probably play only a minor role compared with the changes in the hydrodynamic volume of the molecule. Little is known about the effect of changes in the diffusion coefficients of compounds with temperature on the separation parameters. Theta-temperatures are recommended for use in theoretical studies (Moore and Arrington).
APPARATUS FOR GEL CHROMATOGRAPHY A reliable apparatus is necessary for successful gel chromatographic separations, in addition to hgh-quality gel packings. Descriptions of equipment that operates without pressure can be found in a number of papers devoted mainly to the preparative aspects of the problem. In this section we discuss in more detail the design of a gel chromatograph that meets the requirements for an apparatus operating with elevated overpressures of the eluent, i.e., high-speed systems. Even if high-resolution columns are used, the result of the analysis may be impaired by the use of an inadequate experimental apparatus (zone spreading in dead spaces of the apparatus, strong pulses of the pump and irregularities in the flow, imperfect thermostatting of the temperature-sensitive detection system, inadequate injection valve, etc.). We shall describe here an apparatus that consists of the necessary parts, and also the principles that should be observed if the apparatus is not to have an adverse effect on the separation process. For general considerations regarding this theme, see Chapter 8. The basic scheme of a gel chromatograph is shown in Fig. 12.2: The gel chromatograph consists of three main parts, viz., a pumping and injection system, columns, and a recording detector system. The eluent is stored in solvent tanks under an inert atmosphere (l), which prevents reaction with oxygen and the access of air moisture. The solvent then passes into a degasser ( 2 ) , where it is freed from dissolved gases. The degasser consists of a boiler (2a),
305
APPARATUS FOR GEL CHROMATOGRAPHY
II I1
II II I1 II
qo
II1
1L
.....
II
01
1 I II
IIt
p7= 0
It II II II
IJ
I1
I1
4q I1
=sm 16
117
Fig. 12.2. Scheme of a gel chromatograph. For a description of the components, see the text.
heated either with a liquid or electrically, a water or air cooler (2b) and a filter (2c). The boiling of the solvent, and thus its degassing, is facilitated by the presence of a boiling centre (a glass-wool or sintered filter) inserted in the lower part of the heater (2d). The fdter may consist of a glass tube provided with fused-in plates of sintered glass, or is made of metal with asbestos padding. From the degasser, the solvent enters the pump (3), which must be very reliable, with minimal pulses, a minimal dead space and a controllable amount of delivered liquid. The dosed amount in rhythmically operating pumps is controlled by the length af stroke of the plunger or piston, or by the revolutions of the driving motor. The pressure of the liquid leaving the pump is read off on a manometer (4); the pump pulses are balanced by a pulse damper, beyond which the flow of the eluent splits in two streams and passes through regulation throttles (5a, 5b); manometers (6a, 6b) are built-in in the individual branches. One branch is led through the injection valve (7) into a system of columns (8), while the other leads to a reference column (9). If the arrangement is to be further simplified, the reference column can be omitted, and the solvent may pass through the reference cell after throttling with the regulation valve (10) directly from the lower part of the heater of the degasser; or the reference cell may be filled with the eluent and then closed (care should be taken that the solvent does not evaporate if the analysis takes a long time). A safety membrane or globe valve (1 1) prevents damage to the apparatus if the pressure becomes too high. In the columns (8) the components of the mixture are separated into chromatographic References p.321
117
306
PRACTICE OF GEL CHROMATOGRAPHY
zones. From the last column, the effluent is led into the detector or the detector system (12), from where the electrical signal reaches the recorder (13). An integrator (14) used for the quantitative evaluation of the chromatogram may serve as an additional device. From the measuring cell of the detector, the solvent passes into an integral volume meter (1 5), which is used for following the volume of mobile phase passing through the columns and whose pulses are recorded on the chromatogram (marking). As a rule, systems are employed that record the overflowing of the measuring siphon or, more simply, the drop counters. If the individual fractions of the sample to be analyzed must be preserved, a fraction collector controlled by an integral volume meter can be added. In other instances the solution is led into a waste solvent tank (17). The first commercial gel chromatograph produced by Waters Ass. (Framingham, Mass., U.S.A.) was described by Maley. Other firms that produce gel chromatographs are Varian Aerograph (Palo Alto, Calif., U.S.A.), DuPont (Wilmington, Del., U.S.A.), Siemens (Karlsruhe, G.F.R.) and recently also Knauer (Berlin, G.F.R.) and others.
Pumping systems and injection The importance of the pumping system becomes more pronounced with increasing requirements for a constant flow-rate and increasing inlet pressures on the column. The flow-rate of the eluent should be adjustable over the range ca. 0.1-5.0 ml/min at a backpressure of up to 30-50 atm. For these purposes, there are a large number of commercially available devices in the form of plunger pumps, membrane pumps or pumps based on linear dosers’which are able to pump the eluent without undesirable pulses. In common pumps with a rhythmic operation, the pulses are damped in most instances by dampers consisting of stainless-steel bellows connected in parallel with the capillary and pressurized to the required value with gas or saturated vapour in equilibrium with the liquid at a suitable temperature. Liquid (mercury) pulse dampers operate in a similar manner (Mulder and Buytenhuys). Good prospects seem to be offered by a pulse-free double-acting rhythmic pump with a programmed plunger movement (Waters Ass., Knauer). Two glass plungers are driven by an electronically controlled step motor in such a manner that the sum of the amount dosed is constant at each moment. Digital adjustment of the electronic control allows the eluent to be dosed within the range from 0.1 ml to 9.9 ml/min with high-reproducibility at an overpressure of up to 400 atm. The operation of the pulse-free double-acting pump is shown in Fig. 1 2 . 3 . The sample may be introduced into a pressure-free column by using the procedure described by Determann. The top of the gel column is covered with sample solution, the sample solution is allowed to soak into the gel bed while the column outlet is left half opened, the gel is then covered with the pure eluent and the sample is eluted. Two types of injection valves are used in devices that operate under an overpressure of the eluent, uiz., multi-port valves and injection with a hypodermic syringe. In the first instance, the sample is inserted into a sample loop and, at the moment of injection, the loop volume is placed in a stream of eluent by turning the valve. In the second instance,
APPARATUS FOR G E L CHROMATOGRAPHY
307
Pulseless pumping
Fig. 12.3. Operation principle of a Knauer pulse-free double-acting high-pressure pump.
the sample is placed in the stream of eluent by perforating a rubber septum with the syringe. The advantages of a multi-port valve are the perfect reproducibility of the injected volume and the feasibility of automation of the injection within exactly defined time intervals in routine continuous analyses. An obvious disadvantage of the septum system is that the exactness of the amount injected is insufficiently guaranteed for quantitative analyses; on the other hand, a much smaller amount of sample is usually needed for the procedure, because the losses caused by filling and washng the loop are avoided.
Columns The column design depends on the manner in which gel chromatographic separation is performed. The simplest procedure can be carried out by using a straight glass tube, 15-25 mm in diameter and 300-500 mm long (Fig. 12.4a), tapered at the lower end. The tapering is such that it enables the minimal dead space of the column below the gel bed to be filled with sand, small glass beads or glass-wool, and also enables the column to be connected with the detector through a plastic tube 1 mm in diameter. A detailed description of such a column has been given by Determann, together with a survey of types and producers. In order to eliminate wall effects, it is recommended that the column should be silylated when working in aqueous systems. More efficient columns (Fig. 12.4b) for low-pressure gel chromatography have mobile plungers that allow contractions or dilations of the gel bed without the risk of damaging the gel bed or the column. High-speed gel chromatography has led to the wide implementation of columns made of stainless steel, 4-25 mm in diameter. By analogy with other chromatographic methods, it would be expected that the column efficiency will continue to increase with increase in the ratio of the column diameter to the particle diameter. The columns most frequently used are 1000-1200 mm long and are connected in series by 'means of small-diameter References p.321
308
PRACTICE OF GEL CHROMATOGRAPHY
supernatant gel bed
gel bed
porous polystyrene plunger glass-beads
glass wool Plug
a
b
Fig. 12,4. Laboratory made and commercial gel chromatographic columns. (a) Commercial column; (b) laboratory made column (proposed by Koch-Light, Colnbrook, Great Britain).
capillaries so as to achieve the required length. This arrangement is particularly suitable for soft gels, which, if subjected to pressure, easily plug the column. For preparative purposes, sectional columns combining the advantages of good resolution and high output have been developed. Carnegie, Horton and Chernoff and Stouffer et al. reported the use of micro-columns 1-3 mm in diameter; Wasteson used polyethylene tubes and glass capillaries for analytical studies. However, with decreasing column diameter, the difficulties connected with a reproducible packing increase, and the columns usually have a lower separation efficiency than the theoretical value. The ultimate in micro-scale arrangements was probably attained by Boguth and Repges, who used individual gel particles. A detailed survey of special designs of gel chromatographic columns has been given by Fischer.
Detectors The fractions leaving the column can be detected by means of a chemical or instrumental analysis of the collected fractions. However, in routine analyses, continuous detection is obviously desirable, whch at the same time may become a source of a signal for an automatic fraction collector. Through-flow ultraviolet spectrophotometers operating with one or more well defined wavelengths or provided with a monochromator have a wide field of application and have become popular. The spectrophotometer is a highly sensitive detector that is particularly suitable for working in aqueous solutions when analyzing natural products. Spectrophotometers are produced as both single-beam and differential analyzers.
309
APPARATUS FOR GEL CHROMATOGRAPHY
A through-flow differential refractometer suitable for work in organic solvents and characterized by a linear concentration response can be used for a variety of purposes. If the work is to be carried out at higher sensitivities, this detector requires accurate thermostatting and a good design of the input-output and geometry of t h e measuring cell, These problems have been solved satisfactorily by Waters Ass. and Knauer. In some instances, an infrared spectrophotometer (Ross and Castro) is recommended; if used for the detection of polyethylene in perchloroethylene, it exhibits a higher sensitivity and a smaller temperature drift than a differential refractometer. Another advantage is the possibility of the quantitative determination of the functional groups in copolymers. Saunders and Pecsok described the design of a simple and very sensitive conductivity detector for inorganic electrolytes; Kondo et af. recommended that colorimetric detection should be used for the analysis of polyether polyols. Jackson measured the difference between the dielectric constants of analyzed compounds as a means of detection; following the concentration of the analyzed compounds by radioactive labelling and polarographcally also proved successful. Very sensitive detecticjn is given by a procedure in which the effluent leaving the column is deposited on a moving wire or conveyor belt, the solvent is evaporated and the non-volatile residue is led into a pyrolysis cell. The products of pyrolysis are detected by a flame ionization detector. Instead of an infinite conveyor or wire, Janak used a rotating stainless-steel grid, thus achieving a quantitative response of the FID. It can be seen from this brief survey of detectors used for CPC (a detailed description is given in Chapter 8) that there is a wide choice. The most suitable method of detection will depend on the properties of the system to be analyzed and on the information required. A combination of a universal detector, e.g., a differential refractometer, with a selective detector seems t o be very useful; it has many advantages and is frequently employed. Both principles of detection have been combined in a single measuring cell by Knauer (Fig, 12.5). The scheme shows the passage of UV and visible light through a single measuring cell.
h m
PHOTOMULTlPLlER
6f
PHOT(3DIOOE
P
4
W
+PP
LAMP
1 LAMP '/
Fig, 12.5. Paths of UV and visible light in a combined UV-RI detector made by Knauer. References p.321
310
PRACTlCE 01:GEL CHROMATOGRAPHY
Column packing According to Van Deemter’s equation, which defines the dependence of the reduced theoretical plate height ( h = H / d p , where H is the height equivalent to a theoretical plate and dp is the diameter of the particle in the swollen state) on the linear reduced’flow-rate (v = vdpfD,where Y is the linear rate of elution and D is the diffusion coefficient of the solute in the mobile phase):
h =a
+ bfv + cv
(12.1)
The constant a depends on the regularity of the column packing, while b reflects the broadening of the zone due to diffusion and c has its origin in non-equilibrium conditions. From this equation it follows that a perfect and reproducible column packing may play an important role in attempts to attain a high separation efficiency. The methods of column packing have been described by Altgelt (1965), Determann, Flodin, Moore, Peaker and Tweedale and by Sie and Van den Hoed. A more detailed investigation of the relationship between the separation efficiency and the experimental conditions of gel chromatography, and also of the problems of reproducible column packing, was carried out by Heitz and Coupek. After comparing various methods of packing columns with gels, they recommend that the column should be packed with dilute gel suspension at a constant flow-rate of the solvent with mechanical vibration of the column. Neither the particle size nor the chemical character of the gel had any effect on the dependence of the separation efficiency on the flow-rate, which, in the case of the optimum packing procedure, is linear over a wide range of flow-rates. The packing equipment shown schematically in Fig. 12.6 consists of a solvent tank (1), degasser ( 2 ) , pump (3), pressure vessel for the gel suspension with a volume approximately I0 times the column volume (4), with a column ( 5 ) attached to it by an extension tube and vibrated mechanically (6). The contents of the pressure container are stirred with a magnetical stirrer (7). The solvent is recirculated, and the flow-rate during packing is approximately
Fig. 12.6. Schematic representation of a column packing apparatus. 1, solvent tank; 2, degasser; 3, pump; 4, stainless-steel pressure vessel; 5, column; 6, vibrator; 7 , magnetic stirrer.
31 I
SPECIAL GEL CHROMATOGRAPHIC TECHNIQUES
I 10
I 15
I 20
-2
Ve.1o
1 25 ,mi
Fig. 12.7. Preparative-scale separation of styrene oligomers. Elution with tetrahydrofuran; column 200 X 5 cm; polystyrene gel containing 2% of divinylbenzene. Detection by refraction.
twice the highest flow-rate actually used for analysis. After packing, the tube used for extension is dismantled and the column is closed with an end-fitting. An ingenious method of packing preparative columns (up to a diameter of 50 mm) has been described by Heitz and Ullner. The container is designed in a similar way, but the column rotates about its own axis instead of being mechanically vibrated. In order to prevent the centrifugation of particles in the column during sedimentation, both the direction and angle of each revolution of the column are random. Such columns exhibited an excellent separation efficiency, and their operation at the optimum elution rate was as good as those of the best analytical columns (Fig. 12.7).
SPECIAL GEL CHROMATOGRAPHIC TECHNIQUES
In order to increase the resolution of gel chromatographic columns, Porath and Bennich used the recycling technique known from gas chromatography. They used a peristaltic pump with a low overpressure and a low flow-rate. Bombaugh et al, and Bombaugh and Levangie applied a small-volume reciprocating pump and obtained optimum conditions with a minimum broadening of the chromatographic zone. By using the recycling technique connected with a concentration of the eluate in a film evaporator without mixing the chromatographic zones, Heitz ef al. separated oligomers into the individual species on a preparative scale. Smith et al. described a technique that eliminates the risk of “overtaking”. The details of the design, mode of operation and some applications of continuous gel chromatography were reported by Fox et al. and by Nicholas and Fox. Barker e t al. also described continuous automatic gel chromatography. In order to increase the flow-rate through the columns with soft homogeneous gels, the upwards-flow technique has been recommended, because it reduces the risk of plugging References p.321
312
PRACTICE OF GEL CHROMATOGRAPHY
the columns. Another possibility is the application of short column sections, which can easily be connected in series and used in large-scale separations on the softest gel types (Type KS 370, Pharmacia, Uppsala, Sweden). Little et al. optimized the operating conditions of a chromatographic apparatus with the aim of increasing the flow-rate, which allowed them to reduce eight-fold the time needed for analysis without any substantial decrease in theseparation efficiency. In order to increase the molecular-weight exclusion limit, Hellsing used a solution of a suitable neutral polymer as an eluent. The h g h loads applied to the column frequently impair the efficiency owing to viscosity effects. Altgelt (1970) found that good separations can be achieved even with overloaded columns on the assumption that solutions that have relative viscosities higher than 2 are avoided and the effects of the difference between the densities of the eluent and solution are reduced by decreasing the diameter of the connecting capillaries. Excellent results can be obtained by an appropriate combination of gel chromatography with another separation or analytical technique such as, for example, a combination of thin-layer gel chromatography with electrophoresis or immunoelectrophoresis, of column gel chromatography with mass, IR or NMR spectrometry, preparative gel chromatographic fractionation followed by analysis of the fractions by GLC, etc. In all combinations, specific features of each of the methods used and of the systems analyzed must be borne in mind. If this requirement is met, gel chromatography will be a valuable addition to other chromatographic methods, thus extending the possibilities offered by other analytical and preparative separations.
EVALUATION OF GEL PERMEATION CHROMATOGRAPHIC DATA Determination of molecular weight'and molecular-weight averages The simplest method for obtaining the average molecular weight from GPC data, uiz., reading directly off a calibration curve the molecular weight corresponding to the retention volume of the maximum on the elution curve, is not recommended for polydisperse samples. It is obviously applicable only when the elution curve has a single maximum and, moreover, this value (MGPc) is an average of an unknown type (it has been shown by Berger and Schultz that the inequality Mn < I V .e
TABLE 12.4 MOLECULAR WEIGHTS, STOKES RADII AND ELUTION CONSTANTS OF PROTEINS ON SEPHADEX (3-100, G-75 AND G-50 GELS
No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
2s 26
Protein
Cy tochrome c Ribonuclease Methaemoglobin Soyabean trypsin inhibitor aChymotrypsin Trypsin Pepsin a-Hydroxysteroid dehydrogenase Peroxidase I Ovalbumin Phosphoglycerate mutase Serum albumin (bovine) Malate dehydrogenase Enolase Creatine phosphate kinase Transferin Glyccraldehyde phosphate dehydrogenase Serum albumin (dimer) Aldolase (yeast) Alcohol dehydrogenase yClobulin (human) Catalase yClobulin Chyrnotrypsinogen A Lima bean trypsin inhibitor Kallikrein inhibitor
r(nm)
1.74a 1.92g -
2.26a 2.289 2.41a -
3.029 2.80a -
3.61a -
-
4.00a
4.30a -
4.55 5.22a -
-
M.10'
13 13.6 17 21.5 22.5 24 35.5 47 40 45 64 67 79 '80 81 88 117 134 147 150 140 225 200 25 8.4 6.5
G200
G1 0 0
G-7 5
v,/vo
G 50
Kd
velvo
Rcyt.m
0.76b 0.76d 0.68d 0.62e 0.S9d
2.61b 2.61d 2.48d
1.00 1.04
-
-
-
2.30e 2.26d
1.20 1.18 0.83 1.40
1.65 1.66b 1.71' 1.40'
-
-
-
-
-
-
-
-
-
2.08'
1.50 1.45
0.43d 0.38e 0.32h 0.31d 0.26d 0.27d 0.26d -
-
-
1.90d 1.80e
1.69 1.66
-
Kd
Kd
x
4 rn P
VeIV,
Kd
VeIVo
5 z P
1.99'
1.35" 1.21b l.lOC -
1.09b
-
-
1.66d 1.54d 1.59d 1.54' 1.61d 1.38' 1.44'
1.81 1.89 2.00, 2.02' 2.19
-
-
-
-
-
-
-
0.12 0.42 0.49
-
-
-
-
-
-
-
-
-
1.20 1.72 1.85
aDetermann and Michel. bDetermann and Michel (data obtained on columns of 300-150 ml and 2 cni-width. The proteins were applied in 1-2 ml of buffer and eluted (10-20 ml/h) with phosphate buffer of pH 7.2 ( I = 0.075) + 0.5 M NaCL). 'Mitaker. dWieland e t a/. eAuricchio and Bruni. fSquire. RLaurent andfillander; hDetermann and Gelotte. 'Calculated from Morris. kAndrews. 'Leach and O'Shea. '"Determann and Michel (data obtained by thin-layer chromatography on Sephades G-200 superfine; bed volume,0.05 X 20 X 40 cm; phosphate, pH 6.6; ionic strength, 0.2; R c y t . = relative mobility to cytochrome c. "Fritz et al.
w e
\D
320
PRACTICE OF GEL CHROMATOGRAPHY
TABLE 12.5 SEPARATION RANGES OF DIFFERENT GELS Trade Name
Separation range for globular materials (mol. wt.)
Sephadex G-10 G15 G25 G 50 G-75 (2-100 G-150 G200
up to 700 up to 1500 1000-5000 1500-3O,OOO 3000-70,000 4000- 150,000 5000-400,000 5000- 800,000
Bio-Gel
200-2600 500-4000 1000-5000 5000-17,000 20,000-50,000 30,000-70,000 40,000- 100,000 50,000-150,000 80,000-300,000 100,000-400,000
P-2 P-4
P-6 P-lo P-30 P-60 P-100 P-150 P-200 P-300 A-0.5 m A-1.5 m A-150 m Sepharose 6B 4B 2B Sagavac
10 9 8 7 6 5 4
3 2 Gelarose 2%, 4%,6%, 8%, 10%
< 10,000-500,000 < 10,000-1.5~106 1.106- > 150'106 up to 2.106
300,000-20. lo6 2'106-25' l o 6 10,000-250,000 25,000-500,000 25,000-7 00,000 50,000-1.5 . l o 6 50,000-2. lo6 50,000-7 * lo6 200,000-15 ' l o 6 500,000-50. lo6 500,000- 150.1 O6 Depends on gel concentration
On the other hand, the proportionality of the elution volume and the logarithm of the molecular weight has been repeatedly verified and is now definitely established (Andrews, 1962, 1964, 1965; Andrews et ~ l; Bess . and Hnilica; Bought et QI. ;Burges er al. ; Burke and Ross; Dellacha er al. ;Dimigen el al. ;Downey and Andrews; Fritz et al. ; Jungwirth and Bodo; Lanchantin er ~ l; Largier . and Polson; Leach and O'Shea; Lisowski; Nakayama and Miyake; Nieschlag and Otto; Ostrowski and Rybarska; Piistoupil; Schane; Selby and Maitland; Whitaker). Granath and Flodin were the first to derive this relationship based on polysaccharide data. Determann and Michel also collected a large series of data related
REFERENCES
321
to the correlation between the elution volume and molecular weight, and the results are presented in Table 12.3. In practice, however, when looking for the molecular weight of an unknown protein, a calibration series is necessary. The most useful in this respect is the list of proteins summarized in Table 12.4. There are, as one would expect from the nature of Sephadex gels, distinct limitations according to the type of gel used. The range within which every gel type is capable of yielding a reliable value of molecular weight can be judged best from Fig. 12.1. In addition to cross-linked dextrans, cross-linked polyacrylamide and agarose are also available today for this type of analysis. The separation ranges and commercial names of these materials are summarized in Table 12.5. However, these data should serve only as a rough guide as exceptions are frequent. Data obtained from different laboratories were summarized by Determann and Michel and fitted the general equations calculated from the actual values remarkably well. Similar linear relationships were published by Leach and O’Shea, but in contrast to the series of equations summarized by Determann and Michel, the former are of only limited validity.
REFERENCES Ackers, G. K., Biochemistry, 3 (1964) 723. Adams, H. E., Farhat, K. and Johnson, B. L., Ind. Eng. Chem., Prod. Res. Develop., 5 (1966) 126. AUiet, D. F., Appl. Polym. S y m p . , 8 (1969) 39. Altgelt, K. H., Makromol. Chem., 88 (1965) 75. Altgelt, K. H., ACS Symposium on Gel Permeation Chromatography, Houston. Texas, February 1970, Waters Ass., Framingham, Mass., 1970. Andrews, P., Nature (London), 196 (1962) 36. Andrews, P., Biochem. J . , 91 (1964) 222. Andrews, P., Biochem. J . , 16 (1965) 595. Andrews, P., Bray, R. C., Edwards, P. and Shooter, K. V., Biochem. J., 93 (1964) 627. Auricchio, F. and Bruni, C. B., Biochem. Z . , 340 (1964) 321. Baijal, M. D. and Blanchard, L. P., J. Appl. Polym. Sci., 12 (1968) 169. Balke, S. T. and Hamielec, A. E.,J. Appl. Polym. Sci., 13 (1969) 1381. Barker, S. A., Hatt, B. W. and Somers, P. J., Carbohyd. Res., 11 (1969) 355. Benoit, H., Grubisic, Z., Rempp, P., Decker, D. and Zilliox, J., J. Chim. Phys. Physicochim. Biol., 63 (1 966) 1507. Berger, H. L. and Schultz, A. R., J. Polym. Sci., Part A , 3 (1965) 3643. Bess, L. G. and Hnilica, L. S . , Anal. Biochem., 12 (1965) 421. Boguth, W. and Repges, R., Z. Wiss. Mikrosk., 68 (1967) 241. Bombaugh, K. J., Dark, W. A. and Levangie, R. F., J. Chromatogr. Sci., 7 (1969) 42. Bombaugh, K. J . and Levangie, R. F., Separ. Sci., 5 ( 1 970) 751. Bought, W., Kirsch, K. and Niemann, H., Biochem. Z . , 341 (1965) 149. Burges, R. A,, Brammer, K. W. and Coombes, L. D., Nature (London), 208 (1965) 894. Burke, D. C. and Ross, J., Nature (London), 208 (1965) 1297.1 Cantow, M. J . R., Porter, R. S . and Johnson, J . F., J . Polym. Sci., Part A - i , 5 (1967) 987. Carnegie, P. R., Nature (London), 206 (1965) 1128. Cervenka, A. and Bates, T. W . , J . Chromatogr.,53 (1970) 85. Chang, K. S. and Huang, R. Y . M., J. Appl. Polym. Sci., 13 (1969) 1459.
322
PRACTICE OF GEL CHROMATOGRAPHY
Chang, K. S. and Huang, R. Y. M., J. Appl. Polym. Sci., 16 (1972) 329. Crammond, D. N., Hammond, J . M. and Urwin, J. P., Eur. Polym. J . , 4 (1968) 451. Crouzet, P., Fine, F. and Mangin, P., J. Appl. Polym. Sci., 13 (1969) 205. Dellacha, J. M., Enero, M. A. and Faiferman, I., Experientia, 22 (1966) 16. Determann, H., Gel Chromatography, Springer Verlag, New York, 1968. Determann, H . and Gelotte, B., in H. M. Rauen (Editor), Biochemisches Taschenbuch, Vol. 2, Springer, Berlin, Gottingen, Heidelberg, 1964, p. 905. Determann, H., Liiben, G. and Wieland, Th., Makromol. Chem., 73 (1964) 168. Determann, H. and Michel, W., J. Chromatogr., 25 (1966) 303. Dimigen, J., Klink, F. and Richter, D., 2. Naturforsch. B, 20 (1965) 924. Downey, W. K. and Andrews, P., Biochem. J., 94 (1965) 642. Drott, E. E. and Mendelson, K. A., J. Polym. Sci., Part A - 2 , 8 (1970a) 1361. Drott, E. E. and Mendelson, R. A., J. Polym. Sci., Part A - 2 , 8 (1970b) 1373. Duerksen, J. H. and Hamielec, A. E., J. PoZym. Sci., Part C, 21 (1968a) 83. Duerksen, J. H. and Hamielec, A. E., J. Appl. Polym. Sci., 12 (1968b) 2225. Fischer, L., An Introduction t o Gel Chromatography, North-Holland, Amsterdam, 1969. Flodin, P., J. Chromatogr.,5 (1961) 103. Fox, Jr., J . B., Calhoun, R. C. and Eglinton, W. J., J. Chromatogr., 43 (1969) 48. Frank, F. C., Ward, I. M. and Williams, T., J. Polym. Sci., Part A-2,6 (1968) 1357. Fritz, H., Trautschold, I. and Werle, E., Hoppe-Seyler's 2. Physiol. Chem., 342 (1965) 253. Granath, K. A. and Flodin, P., Makromol. Chem., 48 (1961) 160. Grubisic, Z., Rempp, P. and Benoit, H., J. Polym. Sci., Part B, 5 (1967) 753. Hamielec, A. E., J. Appl. Polym. Sci., 14 (1970) 1519. Hamielec, A. E. and Ray, W. H., J. Appl. Polym. Sci., 13 (1969) 1319. Heitz, W., Boyer, B. and Ullner, H., Makromol. Chem., 121 (1969) 102. Heitz, W. and Coupek, J., J. Chromatogr., 36 (1968) 290. Heitz, W. and Ullner, H.,Makromol. Chem., 120 (1968) 58. Hellsing, K., J. Chromatogr., 36 (1968) 170. Hess, M. and Kratz, R. F., J. Polym. Sci., Part A - 2 , 4 (1966) 731. Hill, J. A., International Gel Permeation Chromatography Seminar, Boston, 1965, Waters Ass., Framingham, Mass. Hjertin, S., in A. Niederwieser and G . Pataki (Editors), New Techniques in Amino Acid, Peptide and Protein Analysis, Ann Arbor Sci. Publ., Ann Arbor, Mich., 1971, p. 227. Hohn, Th. and Pollmann, W., 2. Naturforsch. B, 18 (1963) 919. Horton, B. F. and Chernoff, A. I., J. Chromatogr., 47 (1970) 493. Ishige, T., Lee, S.4. and Hamielec, A. E., J. Appl. Polym. Sci., 15 (1971) 1607. Jackson, A., J. Chem. Educ., 42 (1965) 447. Jan&, J., private communication. Jungwirth, C. and Bodo, G . , Biochem. Z . , 339 (1964) 382. Kondo, K., Mori, M. and Hattori, M., Bunseki Kagaku (Jap. Anal.), 16 (1967) 414. Lanchantin, G . F., Friedmann, J. A. and Hart, D. W., J. Biol. Chem., 260 (1965) 3276. Largier, J. F. and Polson, A., Biochim. Biophys. Acta, 79 (1964) 626. Lathe, G. H. and Ruthven, C. R. J., Biochem. J . , 62 (1956) 665. Laurent, T. C. and Killander, J., J. Chromatogr., 14 (1964) 312. Leach, A. A. and OShea, P. C., J. Chromatogr., 17 (1965) 245. Lisowski, J., Biochim. Biophys. Acta, 113 (1966) 321. Little, J . N., Waters, J. L., Bombaugh, K. J. and Pauplis, W. J., J. Polym. Sci., Part A-2,7 (1969) 1775. Maley, L. E., J. Polym. Sci., Part C , 8 (1965) 253. Moore, J. C., J. Polym. Sci., Part A , 2 (1964) 835. Moore, J. C. and Arrington, M. C., 3rd International Symposium on Gel Permeation Chromatography, Geneva, May 1966, Waters Ass., Framingham, Mass., 1966. Morris, C. J. 0. R., J. Chromatogr., 16 (1964) 167. Mulder, J. L. and Buytenhuys, F. A., J. Chromatogr., 5 1 (1970) 459. Nakayama, F. and Miyake, H., J. Lab. Clin. Med., 65 (1965) 638.
REFERENCES Nicholas, R. A. and Fox, Jr., J. B., J. Chromatogr., 43 (1969) 61. Nieschlag, E. and Otto, K., Hoppe-Seyler’sZ. Physiol. Chem., 340 (1965) 46. Ostrowski, W. and Rybarska, J., Biochim. Biophys. Acta, 105 (1965) 196. Pannell, J., Polymer, 13 (1972) 277. Pavelich, W. A. and Livigni, R. A., J. Polym. Sci., Part C , 21 (1968) 215. Peaker, F. W. and Tweedale, C. R., Nature (London),216 (1967) 75, Pickett, H. E., Cantow, M. J . R. and Johnson, J . F., J. Appl. Polym. Sci., 10 (1966) 917. Pickett, H. E., Cantow, M. J. R. and Johnson, J. F., J. Polym. Sci., Part C , 21 (1968) 67. Pierce, P. E. and Armonas, J. E., J. Polym. Sci., Part C , 2 1 (1968) 23. Porath, J., PureAppl. Chem., 6 (1963) 233. Porath, J. and Bennich, H., Arch. Biochem. Biophys., Suppl., No. 1 (1962) 152. Porath, J. and Flodin, P., Nature (London), 183 (1959) 1657. Pfistoupil, T. I., J. Chromatogr., 19 (1965) 64. Provder, T. and Rosen, E. M., Separ. Sci., 5 (1970) 437. Rosen, E. M. and Provder, T., Separ. Sci., 5 (1970) 485. Rosen, E. M. and Provder, T., J. Appl. Polym. Sci., 15 (1971) 1687. Ross, J. H. and Castro, M. E., J. Polym. Sci., Part C, 21 (1968) 143. Runyon, J. R., Barnes, D. A,, Rudd, J. F. and Tung, L. H., J. Appl. Polym. Sci., 13 (1969) 2359. Salovey, R. and Hellman, M. Y., J. Polym. Sci., Part A-2,5 (1967) 333. Saunders, D. and Pecsok, R. L., Anal. Chem., 40 (1968) 44. Schane, H. P., Anal. Biochem., 11 (1965) 37 1. Schrager, M., J. Appl. Polym. Sci., 15 (1971) 83. Schultz, A. R., Eur. Polym. J., 6 (1970) 69. Selby, K. and Maitland, C. C., Biochem. J., 94 (1965) 578. Sie, S. T. and Van den Hoed, N., J. Chromatogr.Sci., 7 (1969) 257. Siegel, L. M. and Monty, K. V., Biochim. Biophya. Acta, 112 (1966) 346. Smit, J. A. M., Hoogervorst, C. J. P. and Staverman, A. J., J. Appl. Polym. Sci., 15 (1971) 1479. Smith, J. K., Eaton, R. H., Whitby, L. G. and Moss, D. W., Anal. Biochem., 23 (1968) 84. Smith, W. N., J. Appl. Polym. Sci., 1 1 (1967) 639. Squire, P. G., Arch. Biochem. Biophys., 107 (1964) 47 1. Stouffer, J. E., Oakes, P. C. and Schlatter, J. E., J. Gas Chromatogr., 1 (1966) 89. Strazielle, C. and Benoit, H., Pure Appl. Chem., 26 11971) 451. Sun, K. and Sehon, A. H., Can. J. Chem., 43 (1965) 969. Terry, S. L. and Rodriguez, F., J. Polym. Sci., Part C, 21 (1968) 191. Tung, L. H., J. Appl. Polym. Sci., 10 (1966a) 1271. Tung, L. H., J. Appl. Polym. Sci., 10 (1966b) 375. Tung, L. H., J. Appl. Polym. Sci., 13 (1969a) 775. Tung, L. H., J. Polym. Sci., Part A-2,7 (1969b) 47. Tung, L. H., J. Polym. Sci., Part A-2,9 (1971) 759. Tyle, A. P., Nature (London), 206 (1965) 1256. Van Deemter, J. J., Zuidenveg, F. J. and Klinkenberg, A,, Chem. Eng. Sci., 5 (1956) 271. Van Thoai, N., Kassab, R. and Pradel, L. A., Biochim. Biophys. Acta, 110 (1965) 532. Vladimiroff, T., J. Appl. Polym. Sci., 14 (1970) 1397. Wasteson, A., Biochim. Biophys. Acta, 177 (1969) 152. Whitaker, R., Anal. Chem., 35 (1963) 1950. Wieland, Th., Duesberg, P. and Determann, H., Biochem. Z., 337 (1963) 303. Wild, L. and Guliana, R., J. Polym. Sci., Part A -2,5 (1967) 1087. Yamada, S., Imai, Sh. and Kitahara, S., Chem. High Polym. (Tokyo),24 (1967) 12. Yau, W. W. and Fleming, S. W., J. Appl. Polym. Sci., 12 (1968) 2111.
323
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Chapter 13
Practice of ion-exchange chromatography 0. MIKES
CONTENTS Introduction . . . . . . . . ............................ . . . . . . . . . . 325 325 Choice of suitable ion exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for the fractionation of ion exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 Decantation and cycling of ion exchangers . . . . . . . . Buffering of ion exchangers .................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Deaeration of ion exchangers and filling of chromatographic columns. . . . . . . . . . . . Application of samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 Methods of elution. . . . . . . . . . . . Calculation of flow-rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 Evaluation of fractions. . . . . . . . . . . . . . . . . . . . . . . . .. 3 6 6 . . . . . . . . . . 366 Regeneration and storage of ion exchangers References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
INTRODUCTION The technique of ion-exchange chromatography has rapidly developed from classical procedures to the modern high-speed methods, and has been described in specialized chapters in several books (e.g.: Borman; Dorfner; Hale; Inczedy; Kirkland, 197 1 ; MikeS; Morns and Morris; Samuelson; Scott, 1971; Walton) and also in commercial booklets (e.g. : Thompson, Pharmacia). The procedures considered in this chapter are based on published work correlated with personal experience. The author’s intention was to describe briefly the fundamental techniques for the most important types of ion exchangers used in liquid column chromatography (resins, cellulose and polydextran derivatives).
CHOKE OF SUITABLE ION EXCHANGERS When selecting an ion exchanger for chromatographic purposes, the following factors should be considered: (1) the type of process and (2) the extent of the operation. Ion exchangers can be used in both batch and column processes. The former is essentially static, the exchanger being mixed with the solution in a beaker and, after equilibrium has been reached, it is separated mechanically. The latter process is essentially dynamic; the solution flows through the column, equilibrium being repeatedly renewed, and it can be divided into ion exchange and chromatography. For non-chromatographc processes (deionization of water, separations of cations from anions, exchange of one type of ion for another, etc.), there is a wide choice of ion-exchange resins (Tables 13.1 -13.6), and References p.368
325
326
PRACTICE OF ION-EXCHANGE CHROMATOGRAPHY
TABLE 13.1 CATION EXCHANGERS Lists of producers of the resins are given in Tables 13.5 and 13.6. Trade name
Crosslinking
POUP*
Active
Supplied form
Lattice and particles**
x1
C,H,-SO;
H+
PS
~
Strongly acidic
AG SO or AG SOW (Bio-Rad)
s
x2
H+
x4
Ht
X8
H+
S
x10 x12
H' H+
S
X16 X16
H+ H+
s
S
g
AG MP-SO (Bio-Rad)
Macroporous
C,H,-SO;
Amberlite 200 Amberlite CG-120
C,H,-SO;
Na+
ps PS
g
X8
Amberlite IR-120 Amberlite IR-122 Amberlite XE-69 Bio-Rex 40
X8 X10 X8 Medium pore size
C,H,-SO; C,H,-SO; C,H,-SO; C,H,(OH)CH, -SO,
Na' Na+ Na' H+
PS PS p s PH
s s g g
Bio-Rex RG SOW
X8
C, H, -SO;
H+, NH: or Lit
Dowex SO or Dowex SOW
x1
C,H,-SO;
H+
H'
PS
s g
C, H,-SO;
s
S
PS
s
327
CHOICE OF SUITABLE ION EXCHANGERS
Particle size
Capacity
(u’s‘ mesh dry grading)
Dry resin (mequiv./g)
Resin swollen in water (mequiv./l)
50-100
5 .O
0.4
50-100,100200,200-400 20-50,50100,100-200, 200-400, m
P
Guanidoethyl-c.
-0-C,H4-NH-C
C1-
20-300 50-200
0.5 t 0.1 0.55-0.75
n
0.2-0.3
Cellex T TEAEcellulose
Bio-Rad Serva
TEAEc. TEAEc.
Cellex D (low capacity) Cellex D (standard capacity) Cellex D (high capacity) Cellex PEI
Bio-Rad
DEAEc.
-o-c,H, - ~ H ( c , H , ) ,
CI-
20-300
0.4
f
0.1
Bio-Rad
DEAEc.
-0-C, H, - ~ H ( c , H
s)2
CI-
20-300
0.7
f
0.1
Bio-Rad
DEAEc.
-0-C,H,- ~ H ( c , H , ) ,
ci-
20-300
0.9
f
0.1
Bio-Rad
-(C,H,NH),-C,H4
20-300
0.2
f
0.1
Serva
Polyethyleneiminec. DEAEc.
-0-C, H, - ~ H ( c , H ,),
50-100
0.40-0.55
Serva
DEAEc.
-o-c,H,
- ~ H ( c , H ,),
50-100
0.55-0.75
Serva
DEAEc.
-0-C, H, - ~ H ( c , H ,),
50-200
0.75-0.90
Serva
DEAEc.
-0-C,H,
100-1000
0.30-0.55
Serva
DEAEc.
-o-c,H,-~H(c,H,),
100-200
0.9-1.0
Bio-Rad Whatman Whatman Whatman Whatman
ECTEOLAc. DEAEc. DEAEc. DEAEc. DEAEc.
Mixed amines -o-c,H,-$H(c,H,), -O-C,H4 -$'H(C,HS)I -0-C,H, -YH(C2Hs)2 -O-C2H4 -NH(C,H,),
-fiH(C,H
5
0.1
+
\NH -0-C, H, -$(C H 5)3 - O X , H4 -N(C H s)3
Dimethylaminoethylcellulose SN Dimethylaminoethylcellulose SS Dimethylaminoethylcellulose SH Dimethylaminoethylcellulose GS DimethylaminoethylNeoCel Cellex E DE-1 cellulose DE-11 cellulose DE-22 cellulose DE-23 cellulose
2 0
0.7 0.7 0.7
,kH
\NH
Intermediate basic anion exchangers
1 l.o .o
-NH,
,
)2
c1OHOHOHOH-
20-300 1000 50-250
0.3 f 0.05 1.o 1.0 1.0 1.o
]
0
1 n X
n x
> z
n h
z
Insulin 750 mg/g
w
i%(Continued on p . 350)
TABLE 13.7 (continued) Type
Trade name
Producer*
Nature**
Active groups
Ionic form
Particle size (pm)***
Intermediate basic anion exchangers
DE-32 cellulose DE-52 cellulose ECTEOLAcellulose ET-11cellulose PEI cellulose
Whatman Whatman Serva whatman Serva
No. 7 0 cellulose No. 71 cellulose No. 72 cellulose No. 73 cellulose No. 74 cellulose No. 75 cellulose
SchSch SchSch SchSch SchSch SchSch SchSch
Weakly basic AEcellulose anion exchangers Cellex AE Cellex PAB PAB-cellulose Special ion exchangers
Serva Bio-Rad Bio-Rad Serva
DEAEc.
DEAEc. ECTE0LA-c. ECTEOLAc. Polyethyleneimine-c. DEAEc. DEAEc. DEAEc. ECTEOLAc. ECTEOLAc. ECTEOLAc.
AEc.
AEc. p-Aminobenzoyl-c. p-Amincbenzoyl-c.
BD-cellulose
Serva
BenzoylDEAEc.
BND-cellulose
Serva
BenzoylnaphthoylDEAEc.
-o-c,H,-$H(c,H,), -O-C,H, -NH(C * H 5 ) 2 Mixed amines Mixed amines -(CzH4NH),-C2 H, -NHz -0-C,H, -fi(C2HS), -$(C, H 5 ) 1 -0-C,H, -0-C, H, -N(C, H 5 ) Mixed amines Mixed m i n e s Mixed amines -O-C,H,-NH, -0-C,H,-NH, -O-CH,-C,H,
OHOHOH-
50-200 50-250
100-300 80-240 60-180 100-300 80-240 60-180
Capacity for Notes small ions (mequiv./g)
1 .o 1 .o 0.3-0.4
-NH,g
0
Insulin 850 mg/g
0.9 0.9 0.9 0.3 0.3 0.3 0.4 * 0.1 0.2 * 0.1 0.15-0.20
50-300
u l
0.5
0.3-0.4
20-300 20-300
w
cf, Semenza
Fixation of proteins and nucleic acids Fixation of proteins and nucleic acids
.c
m
b2 3
o .rl
s
Chromatography of ‘f: nucleic acids, cf:, Gillam et al. z Chromatography of 9 z nucleic acids, cf., Gillam et al.
*Producers: Bio-Rad = Bio-Rad Labs., Richmond, Calif., U S A .; Sch-Sch = Schleicher & Schiill, Zurich, Switzerland; Serva = Serva Feinbiochemica, Heidelberg, G.F.R.; Whatman = Whatman Biochemicals, W. & R. Balston, Maidstone, Great Britain. . **c. = cellulose. ***In this column, the length of the particle is given; the average diameter of the rods is 18 pm.
#
z
P 0 3 P
3 n m
%
2
a
$' 2 2 (D
TABLE 13.8 POLYDEXTRAN ION EXCHANGERS Producer: Pharmacia Fine Chemicals, Uppsala, Sweden. Sephadex is the trade name. All derivatives are supplied with particle size 40-120 pm.
Y)
5
Nature
CMSephadex C-25 CMSephadex C-50 DEAESephadex A-25 DEAE-Sephadex A-50 QAESephadex A-25 QAESephadex A-50 SESephadex C-25* SESephadex C-50* SPSephadex C-25 SP-Sephadex C-50
Carboxymethyl Carboxymethyl Diethy laminoethyl Diethylaminoethyl Quaternary aminoe thy1 Quaternary aminoethyl Sulphwthyl Sulphoethyl Sulphopropyl Sulphopropyl
Processed from Sephadex* *
G25 G-50 G-25 G50
I
1
G50
Weakly acidic cation exchangers Weakly basic anion exchangers
Strongiy basic anion exchangers
G 50
G25 G5 0 G25
Type of exchanger
1
1
Strongly acidic cation exchangers Strongly acidic cation exchangers
Functional
Ionic form
Capacity for
Haemoglobin
groups
(counter ions)
small ions
capacity (dg)
-CH,-COO-
N a+
4.5
t
-(CHI 2 &H(C,H,),
c1-
3.5
f
0.5 0.5
0.4
9 0.5 5
0.3
-(CH, )2 t(C,H,), CH,CH,(OH)CH,
c1-
-(CH,), -SO3-
Na'
2.3
f
-(CH,), -SO;
Na'
2.3
* 0.3
*From 1970, SP-Sephadex replaced the earlier SE-Sephadex, which had similar properties.
**Cf., Table 9.5.
(mequiv./g)
3.0
-r
0.4
6
3
% 5
4
2W
r m
;3
z
m
sz 2-
z
n M
'p, 0.3
W ul
TABLE 13.9 ION-EXCHANGE CRYSTALS
h)
These ion-exchang crystals are produced by Bio-Rad Labs., Richmond, Calif., U.S.A. Type
Cation exchangers
Anion and cation exchanger
Trade name
Composition
Bio-Rad ZP-1
Zirconium phosphate
Bio-Rad ZT-l*
Zirconium tungstate
Bio-Rad ZT-2*
Zirconium molybdate
Bio-Rad AMP-1
Ammonium molybdophosphate
Bio-Rad HZO-1
Hydrated zirconium oxide
Particle size (mesh)
Capacity mequiv./g
mequiv./ml
Notes
20-50 50-100 100-200 50-100 100-200 50-100 100-200 Microcrystallbe
1.5
1.5
Strong acid to pH 13
0.6
0.4
Cs' uptake at p ti 4 (the capacity varies with pH from 1 to 4.5) Cs' uptake at pH 4
0.6
0.5
Cs+ uptake at pH 4
PH 1-5
1.2
0.4**
See footnote**
Strong acid to pH 6
20-50 50-100 100-200
Chemical stability
pH 1-6
71
?J
b
1.5
1.4
. *Both crystals are not listed in catalogues issued in 1971 and 1972, but they were listed in older leaflets. **Cs+ uptake at pH 4 of AMP-asbestos ( l : l , w/w).
Anionexchange capacity: Cr,O:uptake at pH 1
pH 1 to 5 N base
n
2
2 %
E ? n X
0
J:
>
z 0
rn
cl 3:
P
0
3 53
METHODS FOR THE FRACTIONATION OF ION EXCHANGERS TABLE 13.10 TYPICAL GRAIN SIZES OF ION EXCHANGERS USED FOR VARIOUS EXPERIMENTS Purpose
Grain size Ctm
Pilot plant experiments Non-chromatographic laboratory preparation experiments Ion sieve process Laboratory chromatographic separation in inorganic chemistry High-sensitivity separations Separations of biochemical materials Ionexchange cellulose rods Examples of special resins for highspeed analysis Lowest limit
850-2000 300-850 > 500 140-300 75-140 40-80 (18-20) X (20-300) 20-30 7-11 2-3
Mesh 10-20
20-50
< 30 50- 100 100-200 200-400
METHODS FOR THE FRACTIONATION OF ION EXCHANGERS Homogeneity of the grain size is best ensured by choosing resins that have been sized carefully by the producer. Refined and carefully sized ion exchangers for chromatographic purposes are obtainable from J. T. Baker (Phillipsburgh, N.J., U.S.A.), Bio-Rad Labs. (Richmond, Calif., U.S.A.), Durrum (Palo Alto, Calif., U.S.A.) and others. If laboratory sizing is necessary, either sieving or a hydraulic method is used. Sieving can be carried out in either the dry or the wet state. For dry sieving, the resin should be air-dried and then separated by means of a set of standard sieves. Dry sieving is simple, but wet sieving is to be preferred because of the better uniformity of the grains obtained. Caution: cation exchangers are never sieved in the H'form owing to extensive corrosion of the sieves and contamination of the resin by heavy metals. The swollen resin is placed on the coarsest sieve in a large funnel and rinsed with a stream of circulating distilled water into a large cylinder. Hamilton's hydraulic method is suitable for the separation of very fine particles (30 K) can simultaneously process data from more than forty chromatographs, mass spectrometers, infrared spectrometers, etc. From the chromatographic viewpoint, it can perform the same service as the smaller computer but, in addition, the large capacity of its quick external memory enables a large library of relative retention data and detector response factors to be stored. Therefore, this system makes it possible to perform effective qualitative analyses and provides for a reduction in the number of calibrations that are necessary for the systems of the lower category.
REFERENCES Bate-Smith, E. C. and Westall, R. G., Biochim. Biophys. A c f a , 4 (1950) 427. Consden, R., Gordon, A. H. and Martin, A. J . P., Biochem. J . , 38 (1944) 224. Haderka, S., J. Chromatogr., 91 (1974) 167. Hais, I. M. and Macek, K. (Editors), Paper Chromatography, Academic Press, London, 1963. Jan&, J.,J. Chromatogr., 15 (1964) 15. Jan&, J., in A. Niederwieser and G. Pataki (Editors), Progress in Thin-Layer Chromatography and Related Methods, Vol. 11, Ann Arbor Sci. Publ., Ann Arbor, Mich., 197 1, p. 63. Kabasakalian, P. and Talmage, I. M., Anal. Chem., 34 (1962) 273. Kovats, E., Helv. Chim. A c f a , 41 (1958) 1915. Macek, K., Hais, I. M., Kopeck?, I . and Gasparit, I. (Editors), Bibliography of Paper and Thin-Layer Chromutography I961 -1965, Elsevier, Amsterdam, London, New York, 1968. Macek, K., Hais, I. M., Kopeck?, I., Gasparit, J., RLbek, V. and ChudEek, J . (Editors), Bibliography of Paper and Thin-Layer Chromatography 1966- 6 9 , Elscvier, Amsterdam, London, New York, 1972. Macek, K., Hais, I . M . , Kopcck?, J . , Schwarz, V.,GaspariE, J . and Churitek, J. (Editors), Bibliography of Paper and Thin-Layer Chrornafography 1970-1973, Elsevier. Amsterdam, London, New York, 1976, in press. Martin, A. J. P. and Synge, R. L. M., Biochem. J., 35 (1941) 1358. N o v i k , J., Advan. Chromatogr., 11 (1974) 1 . Schauer, H . K. and Bulirsch, R., Z . Naturforsch., 13b (1958) 327. Van Dijk, J . H., in E. Kovits (Editor), Column Chromatograph.y,Lausanne, 1969, Sauerlander, Aarau, 1970, p. 234.
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Chapter I 6
Radiochromatographic techniques I. M. HAIS and J . DRSATA
CONTENTS . . . . . . . . . 403
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
Geiger-Muller detectors. . . .
......... a-Radiation detectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,407 Detection modes . . . . . . ... . . . . . . . . . . . . 408 The problcm of low-a ....................... 409 Effluent monitoring and recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 10 Geiger-Muller counting . . . . . . . . . . . . . . . . . . . ...... . . . . . .. 4 1 1 Solid-phase scintillation counting . . . . . . . . . . . . . . . . . . . . . 411 Liquid scintillation counting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 1 Semiconductor counting of 0-parti . . . . . . . . . . . . . . . . . . . . . 412 Radiometry of collected fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 I 2 Semiconductography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 I3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
INTRODUCTION The chromatography of radioactive substances is encountered in many fields, but especially in biochemistry and the biological sciences. In radiochemistry, chromatography is used for the isolation and analysis of the products of nuclear and chemical reactions. Isotope dilution and isotope derivative methods are often combined with chromatography. Column, capillary and flat-bed radiochromatography using a liquid mobile phase generally require a less complicated detector or fraction collector than does the gas-liquid chromatography of labelled compounds (cfi,Schutte and Koenders). The speed of modern liquid chromatography may prove to be of special value in the separation of shortlived isotopes and labile compounds. The chromatographic technique as such, involving sample introduction, columns, sorbents, developers and a pumping system, used for radioactive substances, does not differ in any essential respect from the techniques used for stable nuclides. It should be borne in mind, however, that contamination may present a problem that can be serious owing t o the inherently hgh sensitivity (in terms of the ratio between the signal and the number of molecules of the substance being analyzed) of the technique and the wide range of radioactivity levels that may occur simultaneously in different compounds. In References p.413
403
404
RADIOCHROMATOGRAPHIC TECHNIQUES
this respect, one should ensure the radiochemical cleanliness of the laboratory, glassware (inexpensive items are preferably discarded after use) and the column after completion of the chromatogram. The possibility that radioactive reaction products such as tritiated water from tritiated compounds and I4CO2 from ''C-labelled compounds may be generated should also be considered and atmospheric contamination avoided. When low concentrations of radionuclides of high specific activity are being used with no displacing molecules present and if the sorption isotherm initially rises steeply owing to the presence of a small number of highly active sorption sites, this may lead to the retention of residual radioactivity until a strong displacer is passed through the column. This problem can be av.Jided if a cold carrier is added to the sample before chromatography begins.
DETECTORS Radiation detectors used during or after liquid column chromatography differ according to the type of radiation involved, its energy and sometimes its level.
y-Radiation detectors The usual method for detecting y-radiation involves the use of an aluminium-covered crystal of thallium-activated sodium iodide coupled to a photomultiplier. The counts may be printed digitally at pre-determined time intervals or converted to give an output on a continuously recording rate-meter. The photomultiplier may be connected to either a simple pulse-height analyzer for rapid monitoring or to a multi-channel y-spectrometer if it is intended to use the radiation characteristics of the components of a Aixture for the discrimination and possibly even determination of individual nuclides in the mixture. If Geiger-Muller (GM) counters are used to detect y-radiation, they are provided with a metal radiator to improve their low efficiency for this type of radiation. Semiconductor devices have been used for high-resolution y-spectroscopy in the form of lithium-drifted germanium detectors (Table 16.1).
Geiger-Miiller detectors GM counting has been described for liquid samples, either after fraction collection (Bernhardt) or for continuous effluent monitoring using a cell covered with thin Mylar foil (Jordan) (Fig. 16.1). This method is, of course, suitable only for sufficiently hard 0emitters such as 32 P. However, l4 C- or 35S-labelledcompounds have also been GM counted in paper or aluminium cups after evaporation of the solvent. For tritium, windowless proportional gas-flow counters have been used, but the efficiency is not very
high.
DETECTORS
405
Fig. 16.1. Flow cell for GM counting (Jordan). Dimensions in millimetres. The flow cell proper (in which the spiral groove is machined) and the inlet-outlet tubes are made of PTFE.
Solid-phasescintillation detectors for &particles The scintillator itself may line the walls of a coil-shaped or U-shaped capillary cell. Coils made of NE 102A plastic scintillator (Hertel eta/.) are available as Nuclear Enterprises NE 801 (0.7 mm I.D. tubing, total standard cell volume 0.35 ml). The material is suitable for aqueous solutions and lower alcohols, but is attacked by most other solvents and exhibits about 5% efficiency for l4 C in liquids. The efficiency for less energetic radiation is depressed owing to absorption of particles originating near the axis of the tubular cell. If the phosphor is finely divided, the effluent is forced through the porous mass and shorter path-lengths are thereby achieved. The most important variables include the solubility and the adsorption activity of both the cell material and the phosphor. One of the most frequently used phosphors is anthracene, which has been applied even for tritium (Hunt, Schram and Lombaert). NE 806 and NE 806A are available, 1.2 ml standard spiral cell volume. Anthracene is of course unsuitable for use with organic solvents in which it is soluble or for strongly oxidizing acids (nitric, chromic, etc.). Water or aqueous alcoholic solutions may be used. Many compounds are adsorbed from aqueous solutions, leading to tailing in the “effluent monitoring” mode. Schutte has compared the properties of various materialsjn this respect (Table 16.1). He found that counting efficiencies for organic phosphors in U-shaped tubes were good References p.413
406
RADIOCHROMATOGRAPHIC TECHNIQUES
for 14C but less satisfactory for 3 H . All of the organic materials tabulated are soluble in organic solvents and highly adsorbent. Calcium fluoride (used in NE 808,0.5 ml standard spiral cell volume) is dissolved in solutions of ammonium salts and adsorbs nucleotides. The only universally applicable material is therefore glass powder, as a result of its insolubility and lack o f adsorption. It is marketed as NE 808-modified (0.5 ml standard spiral cell volume). Its principal disadvantage is the high light-induced phosphorescence, which persists for several days to give high background counts. In Schutte's hands the efficiency was better for the U-tube than for the coils, even when smaller cell volumes were involved. For aqueous effluents, Sieswerda and Polak prefer POPOP crystals for their suitable flow properties. A detergent in the mobile phase, such as polyethylene lauryl ether, may reduce adsorption contamination of the scintillator. TABLE 16.1 COUNTING EFFICIENCIES FOR VARIOUS FLOW CELLS (SCHUTTE) Shape of cell
Effective volume
Scintillator
(PI)
U U
coil U
U
U coil U
350 160 500 350 5 00 480 430 190
Anthracene Anthracene Anthracene PPO* Butyl-PBD** Glass*** Glass*** CaF, (Eu-activated)
Counting efficiency (%) ICl C
'H
37 31 20 43 40 17 5 38
1 .o 1 .o 0.6 1.8 1.7 0.2 < 0.1 0.5
*2,5-Diphenyloxazole.
* *2-(4'-rert-Butylphenyl)-5-(4"-biphenylyl)-l,3,4-oxadiazole. ***Cerium-activated lithium glass N E 901 (250- 1000 pm), from Nuclear Entcrprises Ltd., Edinburgh, Great Britain.
Liquid scintillation detectors Liquid scintillation counting seems t o be currently the most generally applicable method, especially after fractionation. Liquid scintillation is also used in the effluent monitoring mode, as will be shown below (Hunt). Commercial scintillation equipment with automatic quench correction (external standard) is available for this purpose. The chromatographic solvent may cause certain problems if it is not removed before counting. Highly quenchng solvents should be avoided. Where aqueous solvents are concerned, either toluene or dioxane scintillation mixtures can be used (Hunt). The addition of polar organic solvents allows one-phase mixtures to be formed with certain proportions of water. Toluene-ethoxyethanol scintillation mixture (Gaitonde and Nixey) consists of 0.1% of PPO and 0.04% of POPOP in a mixture of toluene and ethoxyethanol(7:3, v/v). A 10-ml volume of this mixture will dissolve 0.2 ml of water; 0.5 and 1.O ml of water require the addition of 1.5 and 3 ml of ethoxyethanol, respectively.
DETECTORS
40 7
Dioxane scintillation mixture (Minard and Mushahwar) consists of 4.2328 g PPO and 63.429 g naphthalene per litre of dioxane. According to Gaitonde and Nixey, 10 ml of the mixture should dissolve up t o 4.5 ml of water. Bray's solution contains 4 g of PPO, 0.2 g of POPOP, 60 g of naphthalene, 20 ml of ethylene glycol, 100 nil of methanol and dioxane up to 1 litre: A 10-ml volume dissolves up t o 2.5 ml of water at -5°C. Gaitonde and Nixey have observed a striking loss of efficiency in the scintillation counting of amino acids in the presence of citrate buffers in non-acidic solutions. This efficiency loss, unlike that due to quenching, could not be corrected by using the external standardization facility provided with the counter. One possible cause considered is the precipitation or adsorption of the radioactive solute on the walls of the vial. These observations should be taken as indicating the necessity for periodically checking, by internal standardization, whether quenching correction is adequate for correct results to be obtained. Toluene-based scintillation mixtures are sensitive t o the presence of salts. Emulsions can be counted after stabilization with Triton X-100. Quenching which occurs in strongly acid solutions is suppressed by Triton X-100. Mixtures of the base Hyamine 10-X (Evered, Whyman) are used for solutions that contain protein. Semiconductor detectors for P-particles Partially-depleted surface-barrier silicon semiconductor detectors have been used for the assay of p-emitters, especially if the latter occur in sheet form (Tykva, 197 1). Main advantages are their long lifetime, very low background and high energy-resolution of simultaneously present isotopes. As the noise depends on the overall area of the detectors, the area of the detector and hence the counted sample area are usually small. The set-up consists, in addition t o the detector, of amplifiers, amplitude analyzer and counter. The counts may be recorded in various ways. Standard surface-barrier silicon detectors employed up to now (such as Ortec Model A-018-007-100 or Princeton Model PD-25-18-1000) had to be used in vacuo and preferably at low temperatures. This caused complications in the design of the set-up. Efficient detectors have now been described (Tykva, 1973; Tykva and Votruba, 1974) which detect tritium at atmospheric pressure and room temperature. In practice, the depletion-layer thickness may be about 100 pm for 14C (Tykva et af.) and about 600 pm for 32P (Tykva and Pinek). For improved amplitude resolution, if 32P is measured simultaneously with 14C, deeper layers are preferable. In the example given by Tykva (1971), pulse amplitudes were standardized with conversion electrons emitted from 133Ba, '"Cd and "Co. The window used for counting of 35S in the presence of l4 C corresponded to 156-1 71 keV (Tykva and Prinek); the counting rate in this window was about 0.1% of that in the "overall" 35Swindow (17.2-17 1 keV). a-Radiation detectors Weinlander and Hohlein sited an a-detector directly next t o the end of the effluent tube above the fraction collector (cf:,Fig. 16.2). It consists o f a coated surface barrier References p.413
40 8
RADIOCHROMATOGRAPHIC TECHNIQUES
counter furnished with a 0.1-mm barrier and a 5-mm diameter window. A threshold discriminator is used to suppress the pcount background. The device as reported is not sufficiently accurate for use in quantitative evaluations. Liquid scintillation is well suited for the detection of cw-emitters.
F R O M THE
MEASUREMEN
F R A C T I O N COLLECTOR
ALPHA A N D G A M M A
Fig. 16.2. Schematic diagram of the electronic measuring and recording instruments (according t o Weinlander and Hohlein).
DETECTION MODES The principal detection modes are continuous effluent monitoring and recording, and discontinuous counting of collected fractions. in the latter instance, radiometry can be carried out over longer periods of time than the actual duration of the chromatography. In addition, the effluent can, of course, be also counted discontinuously and individual effluent drops or a streak which have been deposited on a paper band can be counted continuously using a rate-meter. The “sweeping” principle, which is a compromise between the two basic modes, is described below. Flow-through measurements of certain (physical) quantities can be combined with the measurements of other quantities on the fractions collected; with radioactivity, approximate measurements in flow-through cells can be combined with the results of more precise (although more lengthy) measurements on the fractions (Gaitonde and Nixey). If the chromatography is sufficiently rapid for the operator to observe the process, the fractions can be selected manually and their number minimized, thus reducing the workload on the liquid scintillation counter. Continuous flow-through measurements and measurements of fractions can be combined either in parallel, using a stream splitter, or in series. The former method is the method of choice if the technique involves mixing with a reagent that interferes with subsequent measurement, e.g., by excessive dilution, quenching, addition of salts to reduce miscibility with the scintillator mixture, precipitation, etc.
DETECTION MODES
409
The lag-time between measurement of effluent in the flow cell and discharge to the fraction tubes must be determined in order to interpret the respective elution curves correctly. One method is to calculate the inner volume of the tubing, and another is to add a radioactive marker for mutual positioning of the chromatographic curves. The lagtime may change during a series of experiments (or even during a single run) owing to gradual wear and widening of the tubing. It is therefore advisable to check this factor routinely by the marker technique. Correct mutual positioning is, of course, critical when determining specific radioactivities with two flow cells or before and after fractionation. It is also possible to detect the radioactivity while it is still on the column, e.g., by auto radiography.
The problem of low-activity samples The specific activity (i.e., radioactivity in curies divided by the weight in grams) of pure nuclides is a function of their half-life. It is therefore physically impossible, in the case of long-lived isotopes, to obtain low-weight samples of very high radioactivity*. In biological and biochemical experiments, substances undergoing detection (especially if labelled with I4C) are mostly present at low radioactivity levels. This low level is connected with the cost of or difficulties in the preparation of the material, with the radiation hazards of higher radioactivities in biological systems (especially in experiments on human subjects) and the conversion of the original compounds with relatively low yields into a number of products. It is therefore necessary to consider the implications of working with low radioactivities. Firstly, it is necessary to count for sufficiently long periods of time. Both radioactive disintegration and most background counts are random processes and it is therefore impossible to achieve hgh precision if a low overall number of counts is obtained. The standard deviation of the measurement corresponds to the square root of the number of counts. Let us consider a simple example of 10 cpm, disregarding any background. It is clear that if we count for 6 sec, the result is indistinguishable from zero. If one counts for 1 min (10 k 3 counts), the coefficient of variation is about 30%,whch is unsatisfactory. For a 10%coefficient of variation, it is necessary to count for 10 min, and for 3% to count for ca. 100 min. If background counts are considered, low total counts become even less reliable. As only those disintegrations which are counted are relevant, a low efficiency obviously increases the time necessary for a given accuracy. It is therefore clear that where low activities are concerned, the measurement cannot keep pace with the speed with which the zones of substances emerge from a modern highefficiency liquid chromatograph. Several modifications of continuous monitoring systems have been described in the literature and commercial equipment is available. It must be kept in mind, however, that in most instances the chromatographic flow-rate used for these methods was rather low. Effective counting times can be increased if higher cell volumes are used or if the time constant of the rate-meter is increased. Both of these procedures may reduce the chromatographic resolution and cause distortion (tailing) of *E.g., 1 ng of pure cholesterol labelled on one of its carbon atoms with I 4 c 1 ( T L = 5650 years)corre1 sponds to 164 pCi (361 dpm).
References p . 4 1 3
410
RADIOCHROMATOGRAPHIC TECHNIQUES
the peaks. Thus Weinlander and Hohlein ensured that the volume of the cell did not exceed 1-3% of the individual band volume, but this may be too exacting a requirement in the case of very sharp peaks. Schutte indicated that the practical minimum residence time of the measured effluent volume is 0.5 min and the practical detection limit was 2 nCi for I4C and 5 nCi for H. This time may be too long with high-speed chromatographs and these limits too high when smaller samples are being analyzed. Sufficiently high total counts must, of course, be achieved in any of the channels of a pulse-height analyzer. This requirement makes a multi-channel analyzer too insensitive for immediate quantitative effluent monitoring, but spectral data obtained during sufficiently long periods can be used for the characterization of nuclides and the determination of the purity of the fractions. The use of fraction collectors permits an increase in counting time by any factor compatible with the available scintillation capacity. (Bands of paper or other materials that are used to absorb the drops of effluent can be also included under the general heading of fraction collectors.) The counting time could be reduced and thus adjusted to the duration of the chromatographic experiment if subsequent portions of the effluent were counted simultaneously by a number of detectors. The intention is that the effluent would flow through a circular loop along which a number of detectors are situated. The detectors would either move at the same speed as the effluent liquid in the loop or, alternatively, they would be stationary but would be phased in such a way as to obtain integrated values for all counts corresponding to a particular portion of the effluent on its passage through the loop. This “sweeping” principle has not yet been put into practice so far as we know, probably because any advantage in detection speed would be outweighed by the complexity and cost of the equipment required. Each detector unit could be a combination of two or more detectors connected in an appropriate manner. Iqcreasing the number of such units may lead t o considerable complications. Fractionation, on the other hand, makes use of standard collectors and the prolongation of the counting time, compared with effluent monitoring, can be up to several orders of magnitude. One basic technical problem of the “sweeping” principle which would have to be solved is that of ensuring synchronization of the detector phasing with the flow of liquid in the loop.
’
Effluent monitoring and recording This technique has the advantage that data are obtained soon after the effluent has left the column and can be directly compared with the results of other measurements carried out on the same effluent (W absorption, pH etc.; Hertel et d.).If the results of two or more detection methods have t o be evaluated, the time interval between measuring the same effluent portion with different detectors has to be taken into account, as already discussed. If the relative positions of the recorder pens can be vaned at will, they can be exploited for ensuring correct relative positioning of the traces. Large time differences occur particularly if long reaction coils are inserted between the relevant detectors. The disadvantage of effluent monitoring is its low sensitivity when running fast chromatograms, owing to
DETECTION MODES
41 1
the short time available for counting the portions of a chromatogram, as discussed above. When higher radioactivities are involved, with low flow-rates and broad peaks, this principle and commercial equipment based on it may be useful.
Geiger-Mullercounting Jordan described a cell consisting of a spiral groove machined in polytetrafluoroethylene (Teflon) and covered with a Mylar window glued to it (Fig. 16.1). He reported rapid decontamination as checked by 32 P-labelled nucleic acids (90% decontamination was reached after flushing the cell with 0.5 cell volume of effluent). Self-absorption is reduced if samples from which the solvent has been evaporated are counted (the label must, of course, reside in non-volatile compounds). As, in principle, there is no basic difference whether the GM counting proceeds at the same or a slower speed as the emergence of the individual portions from the column, the reader is referred to the section on the radiometry of collected fractions.
Solid-phase scintillation counting Cells for ycounting can be accommodated within a well-type NaI(Tl) crystal. In the combination of instruments used by Weinlander and Hohlein for lanthanides, actinides and fission products, the effluent is forced through a helical flow cell (5 ml volume) made of polythene which is shielded with lead against the remaining tubing system. Two detectors with their respective multipliers are located to face the coil (Fig. 16.2). One of them produces (in addition to the counter) a signal for the immediate record of overall yradioactivity, which is traced in parallel with the volume (drop number) and a-radiation record. The other detector traces a record after passing a supplementary (simple) pulseheight analyzer (2) for provisional identification. The output from both detectors is combined to feed a pulse-height analyzer ( I ) , which consists of two 100-channel sections (for low- and hlgh-energy counts). The respective spectra are recorded simultaneously and punched on to tapes after the storage capacity of the instrument has been reached. These tapes can be evaluated after the separation and assist in the identification of individual fractions. Whereas the first detector operates with constant amplification throughout the chromatography, the amplification can be changed to suit the pulse-height analyzer. For 0-counting, coils made of plastic scintillators as well as porous scintillator materials have been described on p. 405. According to Sieswerda and Polak, the contribution of the porous solid detector to the peak broadening expressed in pI2 is proportional to the flow-rate (ml/h).
Liquid scintillation counting The effluent or aliquot is mixed in a fixed ratio with the scintillation mixture, and a single-phase mixture or an emulsion may result, according to circumstances. In order to ensure good mixing and to prevent distortion and tailing of the peaks due to different laminar flow-rates, the interspacing of bubbles (Le., the Technicon principle, see Chapter 32) may be advantageous, The technicalities of effluent splitting and proportionate References p . 4 1 3
412
RADIOCHROMATOGRAPHIC TECHNIQUES
C'
I
Fig. 16.3. Schematic representation of the homogeneous counting system (Schutte). 1 = Column; 2 = ultraviolet detector, 254 nrn; 3 = two-pen recorder; 4 = liquid scintillation spectrometer; 5 = helical flow cell; 6 = scintillator solution reservoir; 7 = proportioning pump; 8 = mixing spiral; 9 = fraction collector.
mixing are also dealt with in Chapter 32. The diagram of Schutte's equipment is shown in Fig. 16.3. The cell is represented by a spiral (2 mm I.D.) of total volume 1.4 ml; the flow-rate through the cell was 2.3 ml/min in the example given.
Semicoriductor counting of (3-particles As in Geiger-Miiller counting, the effluent would have to be counted in a thin layer to limit self-absorption. For I4C or 35Sthe wall facing the detector would have to be made of very thin material, such as 0.9 mg/cmZ Mylar foil (Tykva, private communication). Tritium cannot thus be counted. Relatively low efficiencies and small sample areas would usually require higher radioactivity. Radiometry of collected fractions The whole liquid fraction or an aliquot of it can be subjected to any radiometric procedure; liquid scintillation counting is the method of choice for most (3-emitters. The problem of aqueous effluents has been mentioned above. If, in order to ensure sufficiently high count numbers, very high sample volumes would have to be measured, concentration by evaporation might be necessary before the sample is mixed with the scintillator; this would complicate the procedure. Fraction collectors are dealt with in Chapter 8. One method of fraction collection is to allow the effluent (either the whole stream or an aliquot of it) to drip on to a suitable advancing medium which is left t o dry*. This can then be separated in portions of suitable length and subjected to liquid scintillation or any other suitable radiometric procedure. Alternatively, the strip need not be separated beforehand but can be counted continuously. Commercial radioactivity scanning apparatus for PC or TLC can be used, equipped with GM or windowless proportional gas-flow counters, which feed a recording rate-meter. The rate of advancement of the strip, the time constant of the rate-meter and the recording range can mostly be varied at will.GM counting is, of course, unsuitable for use with H and of low efficiency for l 4 C and 35S. ~~
*It is assumed that the solvent is volatile and the radioactive components are not.
REFERENCES
413
Semiconductography Among the techniques used for counting of samples deposited on paper or other sheet materials, “semiconductography” (Tykva, 197 1) with its low background may be recommended. The scanning operation may be programmed and the output recorded as a curve (via a rate-meter), numerically or, like in scintigraphy, in the form of (coloured) dots. The set-up published for scanning of paper chromatograms (Tykva, 197 1, Tykva and Pinek), gel electrophoreograms (Tykva and Votruba, 1972) or thin-layer chromatograms (Tykva and Votruba, 1974) may be used with minor modifications. The distance between the sample and the detector window is kept to a minimum (less than 0.1 mm; Tykva and Votruba, 1972). The design is simplified if recently introduced detectors (Tykva, 1973; Tykva and Votruba, 1974) which need not be refrigerated or evacuated are used. Counting efficiencies of 62% for 32 P, 25% for l4 C and 0.5% for H have been assessed by Tykva (1974), the background without any screening usually is less than 0.2 cpm (Tykva and Votruba, 1974). Detectors of very small area would require several scanning operations for each drop, thus increasing total scanning time. Nimarin er al. and Dybczyriski used paper and KyrS and Kadlecova used aluminium as support media. If scintillation counting follows, complications may arise owing to the adsorption or limited solubility of compounds, as is known from PC or TLC (Hais). The low adsorptivity of glass-fibre paper might be of special advantage from this point of view. With substances that contain polar groups, the paper cutting should preferably be first soaked in a small amount of water and a liquid scintillation mixture suitable for aqueous samples should be added afterwards.
REFERENCES Alimarin, 1. P., Miklishanskii, A. Z. and Yakovlev, Yu. V., J. Radioanal. Chem., 4 (1970) 45. Bernhardt, C., Isotopenpraxis, 4 (1968) 143. Bray, G. A., Anal. Bioclreni., 1 (1960) 279. Dybczyhski, R., J. Chromatogr., 7 1 (1972) 507. Evered, D. C., Int. J. Appl. Radial. Isotop., 20 (1969) 608. Gaitonde, M. K. and Nixey, R. W. K., Anal. Biochem., 50 (1972) 416. Hais, 1. M., in K. Macek (Editor), Pharmaceutical Applications of Thin-layer and Paper Qiromatography, Elsevier, Amsterdam, London, New York, 1972, p. 79. Hertel, W., Sacher, V. and Rohrlich, M., Z. Anal. Chem., 252 (1970) 147. Hunt, J. A., Anal. Biochem., 23 (1968) 289. Jordan, B. R., Anal, Biochem., 35 (1970) 244. KyrS, M. and Kadlecovi. L., J. Radioanal. Chem., 1 (1968) 103. Minard, F. N. and Mushahwar, I . K., J . Neurochem., 13 (1966) 1. Schram, E. and Lombaert, R., Anal. Biochem., 3 (1962) 68. Schutte, L., J. Chromatogr., 7 2 (1972) 303. Schutte, L. and Koenders, E. B., J. Chromatogr., 76 (1973) 13. Sieswerda, G. €3. and Polak, H. L., in M. A. Crook, P. Johnson and B. Scales (Editors), Liquid Scintillation Counting, Vol. 2. Heyden & Son, London, 1972, Q. 49.
414
RADIOCHROMATOGRAPHIC TECHNIQUES
Tykva, R., in Advances in Physical and Biological Radiation Detectors, Int. At. Energy Agency, Vienna, 1971, p. 211. Tykva, R., Excerpta Med. Int. Congr. Ser., No. 301 (1973) 455. Tykva, R., Czech. Biochem. SOC.Meet., Prague, February 5,1974. Tykva, R., Kokta, L. and Pa'nek, V., Radiochem. Radioanal. Lett., 10 (1972) 7 1 . Tykva, R. and Pinek, V., Radiochem. Radioanal. Lett., 14 (1973) 109. Tykva, R. and Votruba, I., Anal. Biochenz., 50 (1972) 18. Tykva, R. and Votruba, I., J. Chromatogr., 93 (1974) 399. Weinlander, W. and Hohlein, G., Kerntechnik, 10 (1968) 563. Whyman, A. E., Int. J. Appl. Radiat. Isotop., 21 (1970) 81.
APPLICATIONS
This Page Intentionally Left Blank
Chapter I 7
Hydrocarbons J. CHURACEK
CONTENTS Introduction and general techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on adsorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other methods of chromatography of hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 17
41 7 421 423 429
INTRODUCTION AND GENERAL TECHNIQUES Liquid chromatography is not a very suitable method for the separation of hydrocarbon mixtures. Lower and medium-sized aliphatic hydrocarbons are either gaseous or volatile compounds and therefore the method of choice for their analysis is gas chromatography. Nevertheless, liquid chromatography can be applied successfully to the separation of higher paraffinic hydrocarbons, especially polycyclic aromatic hydrocarbons. This method is mainly used for the pre-separation of such compounds (Hofman et al., Jaworski and Szewczyk, Johnstone and Entwistle). The sorption of aliphatic and cyclic hydrocarbons on polar adsorbents is relatively weak and not very selective. However, a large number of investigators have used these adsorbents for separation purposes. It was shown that the properties of silica gel can be utilized with advantage in combination with temperature programming of the column (Lebedeva e t al.). However, it was found that the use of various gels was more advantageous; on such gels, the separation of hydrocarbons, especially polycyclic hydrocarbons, takes place by the molecular sieving effect. The analysis of polycyclic hydrocarbons is important when considering the problem of their carcinogenic properties. This problem was described in detail by Schaad in an extensive review. Harmful and carcinogenic substances in cigarette smoke have been investigated by several workers (Johnstone and Entwistle, Neurath et al., Stedman et al., Swain e t al.). CHROMATOGRAPHY ON ADSORBENTS
A good mutual separation of single types of hydrocarbons on an alumina column was achieved by Falk and Steiner. Acid-free alumina was washed with diethyl ether, dried and heated at 130°C for 30 h. Alumina treated in this manner contained about 12% of water and another 1.7% of water was added to it. After complete mixing, the material was allowed to stand in a closed container for 12 h. About 50-100 mg of the benzene-soluble References p.429
417
418
HYDROCARBONS
fraction of dust from the air were dissolved in a small amount of chloroform and ca. 1 g of alum’inawas added. The chloroform was then evaporated off and the mixture introduced on to a column (12 X 400 mm), the lower section of which was packed with 9 g of alumina and the upper section with 0.5 g of silica gel. Elution was started with a 100-fold amount of n-pentane containing a small amount of diethyl ether. The concentration of ether in the pentane increased stepwise at levels of 0 , 3 , 6 , 9 and 12%. During this 2-3 h operation, the column was protected from light. Gradually, the following hydrocarbons were eluted: aliphatic hydrocarbons, olefins, benzene derivatives, naphthalene derivatives, dibenzofuran, anthracene, pyrene, benzofluorene, chrysene, benz(a)pyrene, benzoperylene and coronene. Column chromatogr;lphy is fairly often used in the analysis of petroleum products, especially oils and asphalts, which contain paraffinic and aromatic hydrocarbons. Gradient elution has been used with success for this purpose. In this application this technique has the same importance and utilization as temperature programming in gas chromatography. The work of Middleton serves as an example. He separated a mixture of higher aliphatic and aromatic hydrocarbons on a column filled with alumina containing 2% of water (the course of the gradient is represented in Fig. 17.1, while Fig. 17.2 shows the resulting chromatograni of a six-component mixture). A mixture of polynuclear aromatic hydrocarbons was also separated on the same adsorbent, and the course of the gradient and the separation can be seen in Fig. 17.3 (Pop1 et al., 1970). Liebisch and Eckardt separated aromatic hydrocarbons present in oil on a column filled with alumina and picric acid. Asphalt components were well separated by Corbett. Fig. 17.4 represents their separation schematically. The separation of “petrolenes” (components of a heptane extract) and the changes in the elution gradient are shown in Fig. 17.5.
jO
1
2
3
4
5
o
6
7
8
9
10
1
CONSECUTIVE 100-ML INCREMENTS
Fig. 17.1. Composition of eluent gradient (Middleton). Polar solvent added to n-hexane base. DCM = dichloromethane; THF = tetrahydrofuran; MeOH = anhydrous methanol; TOTAL = sum of polar solvents.
419
CHROMATOGRAPHY ON ADSORBENTS I
I
I
I
I
50
40
30
20
10
0
FRACTION COLLECTOR -TUBE INDEX
F
4
D
405nm
START
Fig. 17.2. Separation of a sixcomponent broad-range mixture (Middleton). Column: 20 X 1.8 cm. Sorbent: alumina + 2% of water. Eluent: n-hexane, diethyl ether, chloroform, benzene, methanol. Operatingconditions: gradient elution,see Fig.17.1. Detection: spectrophotometric. A = 1,3-dicyclopentyl-2-dodecylcyclopentane; B = I-phenylpentadecene; C = 2,6-dioctylnaphthalene; D = 1,2,3,-triphenylethane; E = 9,10-dimethyl-l ,2-benzanthracene; F = benz(a)anthracene-7,12-dione.
45
7
60
30
Fig. 17.3. Separation of polycyclic hydrocarbons (Pop1 et al., 1970). Column: 100 X 0.4 cm. Sorbent: alumina + 2% of water. Eluent: n-pentane-diethyl ether. Operating conditions: gradient elution. Detection:'spectrophotometric. 1 = carbazole; 2 = chrysene; 3 = pyrene; 4 = phenanthrene; 5 = fluorene; 6 = acenaphthene; 7 = naphthalene; 8 = indane.
References p.429
420
HYDROCARBONS
f
DISPERSE & PRECIPITATE IN n-HEPTANE FILTER
ASPHALTENES (A)
ELUTION ADSORPTION CHROMATOGRAPHY SATURATES ( 5 ) BENZENE
AWMAT IC S (PA )
;ig. 17.4. Scheme for the separation of asphalt into four generic components (Corbett).
Methanol - benzene
Trlchloroethylene (eluent added)
2.0
-s-
-
VOLUME, mi
NA
-
-PA
-
Fig. 1 7 . 5 . Separation of petrolenes (Corbett). Column: 100 X 3.1 cm. Sorbent: alumina F-20. Mobile phase: n-heptane, benzene, methanol-benzene, trichloroethylene. Detection: eluates evaporated to dryness and weighed. S = saturated; NA = naphthene aromatics; PA = polar aromatics.
42 1
CHROMATOGRAPHY ON GELS
CHROMATOGRAPHY ON GELS Higher hydrocarbon fractions, especially oil fractions, cannot be separated successfully by gas chromatography, and gel chromalography can be used with advantage instead. Klimisch and Reese used gel chromatography for the preparative fractionation of hydrocarbons in cigarette smoke condensate on a 24.5 X 1090 mm column packed with Bio-Beads SX-8 (200-400 mesh). Tetrahydrofuran was used as the mobile phase. A molecular sieving effect was the predominant factar in the separation, and in addition, the separation was influenced by interactions with the gel matrix or reactions with the solvent, depending on the structure of the compounds. A fraction of cigarette smoke condensate was separated into three sub-fractions, one of which contained polycyclic aromatic hydrocarbons composed of up to six rings. For the separation of the fractions of coal tar, gel chromatography on Sephadex LH-20 was found t o be a convenient method (Hsieh e r a / . ) .This sorbent can also be used for the separation of paraffins from cycloparaffins and alkylbenzenes from cyclic benzene derivatives on the basis of selective sorption (Mair et 01.). Higher hydrocarbon fractions also containing polynuclear hydrocarbons were separated on Merckogel (Oelert, Oelert and Weber), and a correlation between the elution volume and the logarithm o f the molecular weight was also found. Elution volumes of the separated hydrocarbons are given in Table 17.1. For the determination of the distribution of hydrocarbons in mixtures according to their molecular weights, Styragel (Coleman eta!.) or Poragel A-1 (Weber and Oelert) can be used. Mate and Lundstrom used gel chromatography for the determination of the molecular mass distribution in fractions of aromatic, naphthenic and paraffinic oils. TABLE 17.1 GEL CHROMATOGRAPHY OF HYDROCARBONS (OELERT) Values are elution volumes (ml) obtained using the following flow-rates of the mobile phases: cyclohexane 0.2 mm/sec; niethylene chloride 0.3 mm/sec; isopropanol 0.1 mm/sec. Column: 60 X 1.0 cm. Gel: Merckogel OR-500 vinyl acetate gel. Temperature: 22°C. Detector: RI. Compound
Mobile phase ~
n-Hexatriacontane n-Hexadecane n-Dodecane n-Heptane Squalene Diphenyl p-Terphenyl Benzene Picene Chrysene Phenanthrene Naphthalene
~
~~
Cyclohexane
Methylene chloride
lsopropanol
17.5 19.3 19.9 20.8
7.7 16.2 17.6 21.6 10.9 21.4 19.0 26.3 20.9 21.8 22.2 23.3
16.8 22.0 22.9 25.0 19.7 24.6 22.0 27.8 36.3 33.4 31.6 29.6
20.9 20.5 22.0 20.8 21.1 21.6 22.8
(Continued on p.422) References p.429
422
HYDROCARBONS
TABLE 17.1 (confinued) Compound
Mobile phase
Rubrer:e Truxene Pyrene Coronene Decacyciene Cyclohexane Decalin Indane Tetrafin Fluoranthene Dihydrophenanthrene Oc tahydrophenanthrene Dihydrotetracene Dioct ylbenzene Tridecylbenzene Diisopropylbenzene Ethylbenzene Dimethylnaphthalene Trimethylnaphthalene Meth ylanthracene Cholesteryl stearate Cholesteryl palmitate Cholesteryl laurate Cholesterol
Cyclohexane
Methylene chloride
19.0 19.0 29.9
15.8 19.1 23.6 32.8 45.5 21.1
20.7 21.4 21.1
lsopropanol
-
47.8 51.8 -
21.3 22.8
-
22.9 22.1 21.3 21.4 21.0 14.5 15.8 19.3 22.7 22.3 21.8 22.0 12.2 12.6 13.2 18.3
-
-
18.5 19.3 20.6 -
22.0 -
25.6 26.0 -
19.3 20.0
TABLE 17.2 GEL CHROMATOGRAPHY O F ALIPHATIC HYDROCARBONS (CAZES AND GASKILL) Column: diameter 3/8 in.; length, four 4-ft. columns in series having exclusion limits of 3 X lo’, 250, 60 and 60 A , respectively. Gel: rigid, cross-linked polystyrene gel (Waters Ass., Framingham, Mass., U.S.A.); Mobile phase: o-dichlorobenzene. Flow-rate: 1 ml/min. Temperature: 130°C. Detection: RI. ~
_
_
_
_
_
_
_
~~
Compound *
Elution volume (ml)
n-Pentane n-Hexane ti-Heptane n-Octane ri-Nonane n-Decane n-Undecane ti-Dodecane r2-Tridecane n-Tetradecane
176.0 172.2 168.0 164.4 161.2 158.4 155.7 153.1 150.7 148.2
1
~
~
I
*Samples were 2-ml aliquots of 0.25-1.2% solutions.
~~
Conlpound*
Elution volume (ml)
n-Octadecane n-Eicosane n-Octacosane n-Pentatriacontane n-Hex triacontane 2,2,4-Trimethylpentane 3-Methylpentane 2-Methylpentene-1 4-Methylpentene-1
141.3 138.3 130.3 125.4 124.5 166.4 173.5 174.5 174.1
423
OTHER METHODS OF CHROMATOGRAPHY OF HYDROCARBONS TABLE 17.3 GEL CHROMATOGRAPHY OF HYDROCARBONS AND SOME ETHERS (HENDRICKSON) Column: 12 ft., except for figures in parentheses, where it was 8 ft. X 3/8 in. Gel: 40 A styrenedlvinylbenzene gel, permeable t o alkanes with mol. wt. < 450. Mobile phase: benzene. Flow-rate: 1 ml/min. Temperature: 24°C. Detection: RI. Compound*
Elution volume (ml)
Peak width** (mi)
Compound*
Elution volume (ml)
Peak width** (mi)
(flC,,H,,),O OiC,)HI d 20 Dioctyl ether*** n-Dodecane** * ri-Decane tt-Heptane** * ti-Hexane n-Pentam*** Diethyl ether*** Dioctyl ether*** n-Dodecane*** n-Nonane n-Hep t a m * * * n-Pentane*** Diethyl ether*** Toluene Ethylbenzene
62.8 67.4 76.0 84.2 88.9 99.2 101.5 105.4 107.5 (53.3) (58.3) (63.0) (67.3) (72.4) (73.6) (81.0) (75.2)
3.60 3.70 3.81 3.96 3.82 4.57 3.89 3.97 4.16 (4.24) (4.25) (4.20) (4.41) (4.48) (4.47)
p-Xylene Cumene Naphthdlene Biphenyl Diphenylmethane Diphenylethane Dicumyl peroxide Anisole Phenetole Cyclohexane** * Cyclohexane* * * Styrene 2-Meth ylpentene-l 4-Methylpentene-1 Heptene-1 Decene-1 Isooctane Methyl methacrylate
(76.21 (7 1.O) (81.5) (75.2) 102.7 96 .O 84.8 114.6 107.6 110.7 (76.2) 114.2 91.8 90.6 (67.3) 82.7 (65.61 98.2
(-) (-)
(-)
(-1
(6.20) (5.92) 5.08 4.94 6.02 4.56 4.43 4.59 (5.44) 4.82 4.85 4.21 (4.41) 4.56 (4.28) 4.95
*Sample sizes were typically 0.1 ml of a 4% solution in benzene. **Peak widths were obtained by drawing tangents t o each side of the curves and are expressed as the number of millilitres of eluent at the base of the triangle. ***Compound run more than once.
Using a cross-linked polystyrene gel and o-dichlorobenzene as the mobile phase, higher aliphatic hydrocarbons can be separated by gel chromatography at an elevated temperature (130°C). Elution volumes of the separated substances are presented in Table 17.2 (Cazes and Gaskill). Medium-sized aliphatic and volatile aromatic hydrocarbons can be separated on styrenedivinylbenzene gels using benzene as the mobile phase (Hendrickson). Table 17.3 lists the elution volumes of the separated hydrocarbons.
OTHER METHODS OF CHROMATOGRAPHY OF HYDROCARBONS The property of olefins t o form complexes with silver ions was exploited by Jan& er al. for their chromatography in a liquid-solid system. Porapak G was used as the sorbent and A g N 0 3 (0.08 g/ml) was added t o the mobile phase, which consisted of n-propanol and water (2: 1). A similar sorbent, Porapak T , was used by Martinb and Janak for the separation of polycyclic hydrocarbons usiiig high-pressure LC. The results of the measurement of relative retention times are given in Table 17.4. A very good separation of phenanthrene derivatives is shown in Fig. 17.6. ,References p.429
HYDROCARBONS TABLE 17.4 RELATIVE RETENTION TIMES, th, OF SOME AROMATIC HYDROCARBONS AND THEIR PARTIALLY HYDROGENATED DERIVATIVES ON PORAPAK T IN A COLUMNAR ARRANGEMENT (LC) (MARTINO AND JANAK) Column: 50 X 0.2 cm. Sorbent: Porapak T (0.853 g). Mobile phase: n-hexane. Flow-rate: 0.3 ml/min. Detection: UV (254 nm). Results are expressed relative to naphthalene = 1.00. Substance
tR
lndene Hydrindene
0.59 0.25
Naphthalene Tetralin
1.oo 0.86
Acenaphth ylene Acenaphthene
1.91 1.10
Anthracene 9,lO-Dihydroanthracene 1,2,3,4-Tetrahydroanthracene Octahydroanthracene Phenanthrene 9 ,I 0-Dihydrophenanthrene Octahydrophenanthrene Fluoranthene 1,2,3,4-Tetrahydrofluoranthene
2.33 1.18 0.82 0.21 2.68 1.44 0.87 3.69 1.47
Tetracene 5,12-Dihydrotetracene
5.33 3.67
Pyrene Dihydropyrene sym.-Hexahydropyrene
3.31 2.24 0.83
I
0
20
40
60
TIME, MIN
100
Fig. 17.6. Separation of a mixture ofphenanthrenes (Janik e t a l . ) .Column: 500 X 2,mm. Sorbent: Porapak T (0.853 g). Mobile phase: n-hexane. Operating conditions: flow-rate 0.15 ml/min. Detection: spectrophotometric. 1 = benzene; 2 = octahydrophenanthrene; 3 = 9,lOdihydrophenanthrene; 4 = phenanthrene.
OTHER METHODS OF CHROMATOGRAPHY O F HYDROCARBONS
425
Polynuclear aromatic hydrocarbons were also separated successfully by high-speed LC using Permaphase ODS and a linear gradient from methanol-water (1 :1) to pure methanol. A very good separation is represented in Fig. 17.7 (Schmit et d.). Reversed-phase chromatography was used for the separation of a series of mediumsized aliphatic and volatile monocyclic aromatic hydrocarbons (Locke). For example, a 3-m column packed with 25% squalene on silanized Chromosorb P was used with acetonitrile as eluent. This chromatographic procedure can be performed at various temperatures, which should not, however, exceed 32°C. The results of the separations are shown in Table 17.5. For the separation of hydrocarbon mixtures (Sergienko ef d.1, the formation
3
11
9
c
1
Z
w
0 2
"
I
2
c
TIME, MIN
Fig. 17.7. Separation of fused-ring aromatics (Schmit et 01.). Column: 1 m X 2.1 mm I.D. precisionbore, stainless steel. Sorbent: Permaphase ODs. Mobile phase: linear gradient from methanol-water (1: 1 ) to methanol. Operating conditions: temperature 50°C; flow-rate 1 ml/min; pressure 100 p.s.i. Detection: UV photometer. 1 = Benzene; 2 = naphthalene; 3 = biphenyl; 4 = phenanthrene; 5 = anthracene; 6 = fluoranthrene; 7 = pyrene; 8 = unknown; 9 = chrysenc; 11 = bcnz(e)pyrene; 12 = benz( 0)pyrene.
References p.429
426
HYDROCARBONS
TABLE 17.5 PARTITION CHROMATOGRAPHY OF HYDROCARBONS (LOCKE) Values are specific retention volumes (ml per gram of squalene). Column: 3 m X f/4 in. Sorbent: 25% (w/w) squalene coated on silanized Chromosorb P (100-200 mesh). Mobile phase: de-aerated acetonitrile. Detection: RI. Cornpound*
Temperature ("C) 20.0
25 .O
35.1
7.40 8.49 8.76 9.50 8.65 10.1 5 13.20 17.75
3.24 4.38 4.60 4.84 5.50 5.79 5.89 6.36 6.70 6.83 7.73 7.95 8.61 8.1 1 9.24 11.80 15.90
2.98 -
2.82 -
3.16 4.12 4.32 4.48 5.15 5.44 5.54 5.90 6.18 6.82 6.32 7.08 7.21 7.82 7.62 8.43 10.65 14.35 2.41 2.67 2.78 2.74 1.91 2.79 1.31 3.73 5.13 4.35 4.09 5.19 5.68 7.29 9.78 2.28 2.92 0.548 0.770 0.989 1.oo 1.08 1.10 1.26
3.01 3.70 3 .84 3.86 4.58 4.88 4.99 5.11 5.29 5.48 5.99 6.00 6.51 6.75 7.09 8.75 11.65 2.40 -
15.05 n-Butane 2-Methylbutane n-Pentane 2,2-Dimethylbutane 2,3-Dimethylbutane 3-hlethylpentane 2-Methylpentane n-Hexane 2,2-Dimethylpentane 2,3-Dimethylpentane 2,4-Dimethylpentane 3-Eth ylpentane
2-Methylhexane n-Hep tane 2,2,4-Trimethylpentane 2,4-Dimethylhexane n-Octane rr-Nonane 2-Methylbutene-1 3-Methylpentene-1 2-Methylpentene-1 2,3-Dirnethylbu tene- 1 Pentene-l Hexene-l 1,s-Hexadiene Hep tenc-1 Octene-1 2,4,4-Trimethylpentene-l Cyclopentane Methylcyclopentane Cyclohexane Methylcyclohexane Ethylcyclohexane Cyclopentene Cyclohexene Benzene Toluene Ethylbenzene o-Xylene m-Xylene p-Xylene n-Propylbenzene
3.33 4.62 4.90 5.24 5.89 6.12 6.23 6.87 7.30 -
-
-
2.10 3.10 1.47 4.23 5.78 4.83 4.55 5.82 6.60 8.16 11.30 2.45 3.26 0.554 0.804 1.04 1.06 1.16 1.17 1.38
-
2.00 2.94 1.38 3.98 5.45 4.58 4.31 5.50 6.1 1 7.71 10.49 2.36 3.08 0.550 0.786 1.01 1.03 1.12 1.14 1.33
-
1.75 2.53 1.17 3.32 4.61 3.94 3.70 4.66 4.93 6.52 8.50 2.14 2.64 0.539 0.738 0.935 0.950 1.02 1.03 1.13
427
OTHER METHODS OF CifROMATOGRAPHY OF HYDROCARBONS TABLE 17.5 (continued) Temperature ("C)
Compound*
20.0
15.05 nButylbenzene n-Pentylbenzene n-Hexylbenzcne n-Oc tylbenzene n-Nony Ibenzene lsoprop yl benzene 1,2,4-TrimethyIbenzene 1,3,5-Trimethylbenzene tert. -Butylbenzene sec. -Butylbenzene o-Diethylbenzene m-Dieth ylbenzene p-Dieth ylbenzene 1,3,5-Triethylbenzene 1 -Methyl.l-isopropylbenzene Tetralin Naphthalene CH ,CI CHCI, CCI " *Samples were 0.01 -1.0
111 of
1.98 2.53 3.89 6.84 9.41
1.90 2.48 3.58 6.37 8.56
-
0.620 1.49
0.643 -
1.52
25 .O
35.1 1.71 2.1 2 2.83 5.18 6.52
1.84 2.38 3.30 5.92 7.80 1.26 1.54 1.57 1.45 1.63 1.71 1.81 1.78 2.88 1.84 1.83 0.593 0.107 0.232 1.45
-
1.39
pure solute.
TABLE 17.6 SURVEY OF DIFFERENT PROCEDURES APPLICABLE TO THE SEPARATION OF HYDROCARBONSANDHALOHYDROCARBONS Chromatographic technique
Compounds
Sorbent
Mobile phase
Refercnce
LC
Hydrocarbons (air pollutants)
Alumina
Cyclohexane
Kotin
LC
Hydrocarbons (air pollutants)
Alumina
Light petroleum Hiros -benzene (10: 1); Light petroleum -methanol (1OO:l)
LC
Polynuclear hydrocarbons
Alumina; silica gel
Methanol-ethanol -water ( 1 : l : l )
Weigert and Mottram
LC
Hydrocarbons
Silica gel
n-Hexane; n-hexane-benzene (4:l)
Hoffman and Wynder
LC
Hydrocarbons
Acetylcellulose
Ethanol-toluenewater (17:4:1)
Spotswood
(Continued on p.428)
References p.429
428
HYDROCARBONS
TABLE 17.6 (continued) Chromatographic technique
Compounds
LC
Sorbent
Mobile phase
Reference
Aromatic hydi ocarbons
MgO-Celite (2: 1 )
n -Aexane -
Lijinsky, Lijinsky et al.
GPC
Hydrocarbons
Sephadex LH-20
Isopropanol; chloroformcyclohexane (4: 1)
Wilk et al.
GPC
Alkanes (application to a rock extract)
Sephadex LH-20
Acetonechloroform (1 :1)
Cooper
GPC
Halogenated hydrocarbons (products of chloroprene production)
Styrene divin ylbenzene cop ol ymer
Tetrahydrofuran
Eoupek and Bouchal
LC
Alkyl chlorides
Silica gel with fluorescent indicator
lsopropanol
Jaworski et al.
LC
n-Halogenated stilbenes (purification and preparation, cistrans isomers)
Alumina
n-Hexane
Krueger and Lipper t
LC
Fluorinated hydrocarbons*
Dowex 50W-X8
Ethanol-water (1:l)
Miller and Key worth
benzene-acetone (3:l:l)
*Determination of F after transforming the bound fluorine into fluoride ion.
of clathrates can be made use of. Oil fractions of higher paraffins and cycloparaffins (boiling range ca. 250-450°C) were separated in the form of clathrates by Stejaru and Popescu. Another method of utilization of clathrates consists in packing the column with a mixture of thiourea and diatomaceous earth and eluting with benzene and methanol. A 120-fold excess of thiourea (relative to the weight of hydrocarbons) was used (Lieberman and Furman). Ion-exchange resins have also been used for the separation of hydrocarbons. Yamada et al. discussed the relationship between the distribution coefficients of benzene derivatives on the hydrogen and sodium forms of styrene-based strong cation-exchange resins (BioRad AG 50 W-Xg) and their chemical structures. A survey of other papers concerning the separation of hydrocarbons and halogenated hydrocarbons both from the point of view of the analyzed substances and that of the sorbents used is given in Table 17.6.
REFERENCES
429
REFERENCES Cazes, J . and Gaskill, D. R., Separ. Sci.,4 (1969) 15. Coleman, H. J., Hirsch, I>. E. and Dooley, J. E., A i d Chem., 41 (1969) 800. Cooper, B. S., J. Chromatogr., 46 (1970) 112. Corhctt, L. W., Anal. Chem., 4 I (1969) 576. Poupek, J. and Bouchal, K.,Macromol. Chem., 135 (1970) 69;C.A. 73 (1970)45865j. Falk, 14. L. and Steiner, P . E., Cancer Res., 1 2 ( I 952) 30. Hendrickson, J., J. Chrornatogr., 32 (1 968) 543. Hdros, M., Rev. Pollut. Arm., 5 (1963) 205. Hoffrnann, D. and Wynder, I:. L., Anul. Chon., 32 (1960) 295. Hofnian, J . , Toniinck, O., VodiEka, L. and Landa, S., Collect, Czech. Chem. Commun., 34 (1969) 1042. Hsieh, B. C. B., Wood, R. E . , Andcrsoii, L. L . and Hill, G . R., Anal. Chem., 41 (196'9) 1066. Janik, J., JagariE, Z. and Drcssler. M.,J. Chromatogr., 53 (1970) 525. Jaworski, M. and Szewczyk, H., Chem. Anal. (Warsaw), I 5 (1970) 53. Jaworski, M., Zielasko, A. and Szcwczyk, H., Chem. Anal. (Warsaw), 14 (1969) 1225; C.A., 72 ( 1970) 106850d. Johnstone, R. A. W. and Entwistle, I. D.,J. Chem. Soc. C.,(1968) 1818. Klirnisch,H. J . and Reesc, D.,J. Chromatogr., 67 (1972) 299. Kotin, P.,Cancer Res., 16 (1956) 16. Kruegcr, K. and Lippert, E., Chem. Ber., 102 (1969) 3233. Lebedcva, N . P . , Frolov, I . I . and Yashin, Ya. 1.,J.Chromarogr., 58 ( 1971) 1 1 . Liberman, A. L. and Furman, D. B., Neftekkzmiya, 8 (1968) 81 1 ; C A . , 72 (1970) 1210.58~. Liebisch, G . and Eckardt, H., Z. Chem., 6 (1966) 377;Anal. Absfr., 15 (1968) 838. Lijinsky, W., Anal. Chem., 32 (1960) 684. Lijinsky, W.,Saffiotti, U. and Shubik, P.,J. Natl. Cancer Inst., 18 (1957) 867. Locke, D. C., J. Chromatogr., 35 (1968) 24. Mair, B. J., Hwang, P. T. R. and Ruberto, R. G . ,Anal. Chem., 39 (1967) 838. Martin;, V. and Janik, J., J. Chromatogr., 65 (1972) 477.
Mate,R.D.andLundstrom,H.S.,J.Polym.Sci.,PartC,No.21(1967)317;C.A.,68(1968)96614f. Middleton, W. R.. Anal. Chenz., 39 (1967) 1839. Miller, M. and Keyworth, D. A., Talanta, 14 (1967) 1287;Anal. Abstr., 16 (1969) 761. Neurath, G., Gewe, J. and Wichern, H., Beitr. Tabakforsch., 4 (1968) 250;Anal. Abstr., 18 (1970) 1863. Oelert, H. H.,J. Chromatogr., 53 (1970) 241. Oelert, H. H. and Weber, J. H., ErdolKohle, Erdgas, Petrochem., 23 (1970) 484; C.A., 7 3 (1970) 791671. Popl, M., Mosteck9, J . and Havel, Z . , J. Chromatogr., 53 (1970) 233. Schaad, R. A,, Chromatogr. Rev.,13 (1970) 61. Schmit, J. A,, Henry, R. A,, Williams, R . C. and Dieckman, J. F,,J. Chromatogr. Sci., 9 (1971) 645. Sergienko, S. R., Chelpanova, M . P . , Aidogdycv, A. and Kuzyreva, A. S., Gazokondens. Neft., Mater. Sredneaziat. N Q U C Soveshch. ~. Neftekhim. Khim. Perezab. Uglevodorov, 2nd. 196 7, 1968, p. 129; C.A., 71 (1969) 126840h. Spotswood, T. M.,J. Chromatogr., 3 (1960) 101. Stedman, R. L., Miller, R. L., Lakritz, L. and Chamberlain, W. J., Chem. Ind. (London), 12 ( 1 968) 394. Stejaru, D. and Popescu, R., Rev. Chim. (Bucharest), 20 (1969) 629; C.A., 73 ( I 970) 1 7 0 3 5 ~ . Swain, A. P . , Cooper, J. I-:., Stedrnan, R. L. and Bock, I;. C., Beifr. Tabakforsch., 5 ( 1 969) 109; C.A., 73 (1970) 6332111. Weber, 1. H. and Oelert, H. H., Separ. Sci., 5 (1970) 669. Weigert, F. and Mottram, J. C., Cancer Rex, 6 (1947) 97. Wilk, M., Rochlitz, J. and Bcnde, H.,J. Chromatogr., 24 (1966) 414. Yamada, M., Nornura, N. and Shiho, D.,J. Chrornatogr., 64 (1972) 253.
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Q a p t e r 18
Alcohols and polyols J. C H U R A ~ E K
CONTENTS Introduction and general techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . High-speed liquid and gel permeation Chromatography of free alcohols . . . . . . . . . . . Chromatography of alcohols on ion exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography of derivatives of alcohols and glycols . . . . . . . . . . . . . . . . . . . . . . . Separation of polyols and polymeric diols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
431
. . 431 435 437 438 439
INTRODUCTION AND GENERAL TECHNIQUES This chapter includes aliphatic, cycloaliphatic and aromatic compounds with one or more hydroxy groups in the molecule. Their chromatographic properties are mainly determined by the number of hydroxy groups present. Sugar alcohols are dealt with in the chapter on saccharides (Chapter 22). Lower monohydric alcohols are best analyzed by gas chromatography, while liquid column chromatography is best used for the separation and determination of higher free alcohols and their derivatives. For the separation of alcohols and glycols by this method, common adsorbents and also ion exchangers and gels are used. New sorbents and stationary phase supports have recently been developed, for example porous silica microspheres, which enable a substantial increase in the separation rate to be achieved (Kirkland). When diols, mainly polymeric compounds of the polyethylene glycol type, are analyzed, the determination of the molecular-weight distribution is the most important task. Gel permeation chromatography is most suitable for this purpose, but silica gel can also be used.
HIGH-SPEED LIQUID AND GEL PERMEATION CHROMATOGRAPHY OF FREE ALCOHOLS High-speed liquid chromatography is now used for the separation of some aromatic and aliphatic alcohols. Highly effective phases on 2 m X 2 mm columns are used as sorbents. For example, using Vydac adsorbent as the packing and elution with 1% amyl alcohol in isooctane at a working pressure of 2800 p.s.i., derivatives of benzyl alcohol and cinnamyl alcohol can be separated, the latter having the longest elution time (Chromatronix) (Fig. 18.1.). When a column packed with Permaphase ETH was used at room temperature and a pressure of 250 p.s.i., these substances were also well separated References p.439
43 1
432
ALCOHOLS AND POLYOLS 1
1
L I
I
TIME,MIN
4
I
I
I
0
5
10
Retentron tlme (mln i
Fig. 18.1. Separation of alcohols (Chromatronix). Column: 2000 X 2 mm. Sorbent: Vydac adsorbent (Chromatronix). Mobile phase: 1%amyl alcohol in isooctane. Operating conditions: flow-rate 3 ml/min; pressure 3000 p.s.i.; temperature ambient. Detection: W at 254 nm. Instrument: Chromatronix 3100 W. Sample: 20 pl. Peaks: 1 = 2-phenyl-2-propanol; 2 = a-methylbenzyl alcohol; 3 = benzyl alcohol; 4 = cinnamyl alcohol.
Fig. 18.2. Separation of a mixture of alcohols (DuPont Instruments). Column: 1 m U-shape; DuPont Model 830 chromatograph. Sorbent: Permaphase ETH. Mobile phase: n-hexane. Operating conditions: column pressure 250 p.s.i.g.; flow-rate 1 m!/rnin. Detection: UV photometer. Peaks: 1 = a-methylbenzyl alcohol; 2 = 01, a’-dimethylbenzyl alcohol; 3 = 2-phenylethanol; 4 = cinnamyl alcohol; 5 = benzyl alcohol.
HIGH-SPEED LIQUID AND GEL PERMEATION CHROMATOGRAPHY OF FREE ALCOHOLS 433
but their elution sequence was different. When n-hexane was used for elution, cinnamyl alcohol had a lower elution time than benzyl alcohol (DuPont Instruments) (Fig. 18.2). In both instances, the time of analysis was less than 8 min. Kirkland separated similar compounds in a liquid-liquid system using porous silica microspheres as the carrier, impregnated with up to 30% of b,$-oxydipropionitrile as the stationary phase. 11-Hexane saturated with the stationary phase was used as the eluent (Fig. 18.3). Under the conditions of liquid-solid chromatography, the same substances were also separated on plain porous silica microspheres as adsorbent. Fig. 18.4 shows how rapid (60 sec) the separation of five aromatic alcohols at 2000 p.s.i. was. A series of homologous alcohols, branched alcohols and some glycols were separated chromatographically on a styrene-divinylbenzene gel using benzene as the mobile phase (Hendrickson), and the results are given in Table 18.1. A styrene gel was also used for the separation of a mixture of alcohols (methanol t o n-heptanol) and diols with o-dichlorobenzene or tetrahydrofuran as eluent (Cazes and Gaskill) (Table 18.2). TABLE 18.1 GEL CHROMATOGRAPHY OF ALCOHOLS (HENDRICKSON) Column: 12 ft. (8 ft.) X 3/8 in. Column packing: 40 A styrene-divinylbenzene gel, permeable t o alkanes of mol. wt. 450. Mobile phase: benzene. Flow-rate: 1 ml/min. Detection: RI. Compound*
Elution volume (ml)
Peak width** (ml)
Methanol Ethanol Isopropanol n-Propanol Benzyl alcohol ferf.-Butanol n-Butanol 3-Heptanol n-Heptanol*** n-Hep tanol* ** Diethylene glycol monomethyl ether 2,2,4-Trimethylpentan01 n-Decanol*** n-Decanol*** lonol Triphenylcarbinol Dipropylene glycol Tripropylene glycol Tetraethylene glycol Ally1 alcohol 2-Butene-1-01
(92.0) 127.3 117.6 (81.3) (82.7) 110.7 112.7 (68.7) (69.8) 101.0 97.3 98.7 93.5 82.2 (60.4) (65.8) (68.1) (61.8) (86.7) 110.0 103.8
(5.47) 4.53 4.52 (5.47) (5.22) 4.60 4.82 4.65 (4.77) 4.40 4.88 4.70 4.70 4.5 1 (5.79) (-)
(7.16) (5.36) (4.77) 5.1 1 5.30
*Sample sizes were typically 0.1 ml of a 4% solution in benzene. **Peak widths were obtained by drawing tangents to each side of the curves and are expressed as the number of millilitres of eluent a t the base of the triangle. ***Compound run more than once.
References p.439
43r
ALCOHOLS AND POLYOLS
2
4
E In
P N
ia 8a m
m
1 I
IMPURITIES
I
I
I
I
I
I
I
2
4
6
8
10
12
14
T I M E , MIN
Fig. 18.3. Separation of hydroxylated aromatics (Kirkland). Column: 250 mm X 3.2 mm. Sorbent: 5-6 p porous silica microspheres (cu. 350 A) impregnated with 30% of p, p'-oxydipropionitrile. Mobile phase: n-hexane (saturated with stationary phase). Operating conditions: flow-rate 1 ml/min; pressure 600 p.s.i.g.; temperature 26°C. Detection: absorbance at 254 nm. Peaks: 1 = a,a-dimethylbenzyl alcohol; 2 = 2,5-dimethylphenol; 3 = a-methylbenzyl alcohol; 4 = 3-phenylpropanol; 5 = p-phenylethanol. I
8.J
1"
0"
TIME, SEC
Fig. 18.4. Separation of hydroxylated aromatics (Hendrickson). Column: 250 mm X 3.2 mm. Sorbent: 8-9 p porous silica microspheres @a. 75 A). Mobile phase: dichloromethane, half saturated with water. Operating conditions: flow-rate 10.5 ml/min; pressure 2000 p.s.i.; temperature 27°C. Detection: absorbance at 254 nm. Peaks: 1 = 2,s-dimethylphenol; 2 and 3 = impurities in standards; 4 = a-phenylbenzyl alcohol; 5 = a,a-dimethylbenzyl alcohol; 6 = benzyl alcohol; 7 = 3-phenylpropanol.
435
CHROMATOGRAPHY OF ALCOHOLS ON ION EXCHANGERS TABLE 1'8.2 CHROMATOGRAPHY OF ALCOHOLS AND GLYCOLS (CAZES AND CASKILL)
Values are elution volumes (ml) obtained under the following conditions. A, column 1, mobile phase o-dichlorobenzene, 130°C; 9,column 2, mobile phase tetrahydrofuran, 25°C; C, column 3, mobile phase o-dichlorobenzene, 130°C. Columns: ( 1 ) four 4-ft. columns in series having exclusion limits'of 3 x l o 3 , 250, 60 and 60 A, respectively; (2) four 4 4 . columns in series having exclusion limits of about 4 0 A; (3) four 4-ft. columns in series having exclusion limits of 3 X l o 3 , 4 5 , 4 5 and 45 A, respectively. Diameter of columns 3/8 in. Gel: a rigid, cross-linked polystyrene gel characterized by Waters Ass., Framingham, Mass., U.S.A. Conditions Compound*
Methanol Ethanol n-Propanol Isopropanol n-Butanol Isobutanol sec. -Butanol t e a - B u tanol Ally1 alcohol n-Pentanol n-Hexanol n-Heptanol 1,2-Ethanediol 1,2-Propanediol 1,4-Butanediol 1,3-Butanediol 2,2-Dimethyl-l,3-propanediol 2,2,4-Trimethyl-1,3-pentanediol
A
B
208.8 196.4 189.7 186.8 183.6 184.4 182.5 181.2 194.4 179.3 174.6 170.6
132.5 131.4 127.3 125.8
c
113.1 -
202.5 186.7 182.9 179.7 175.8 180
*Samples were 2 4 aliquots of 0.25-1.070 solutions.
CHROMATOGRAPHY OF ALCOHOLS ON ION EXCHANGERS Ion-exchange Chromatography is much less important for the separation of free alcohols than other chromatographic techniques. Salting-out chromatography on Dowex 1-X8 (SO:-) columns was used for the separation of less than milligram amounts of diacetyl, acetoin and 2,3-butylene glycol (Speckman and Collins). Sodium or ammonium sulphate solution (0.5 M ) was used for elution. The substances are eluted in order of decreasing polarity, i.e., 2,3-butylene glycol, acetoin and diacetyl. Higher alcohols were separated on a Dowex 50-X8 (H') (200-400 mesh) column by gradual elution with acetic acid of increasing concentration (1 -3 N) (Sherma and Rieman, (Table 18.3). Sherma et al. also studied the possibility of the separation of higher alcohols o n Dowex 1 (CH3COO-) by elution with aqueous-organic salt solutions. In these separations, both the salting out and the solubility play a role and therefore the dependence References p.439
436
ALCOHOLS AND POLYOLS
TABLE 18.3 SOLUBILIZATION CHROMATOGRAPHY OF ALCOHOLS (SHERMA AND RIEMAN) Column: 20 cm x 2 cm. Column packing: Dowex 5O-X8 (H+) (200-400 mesh). Flow-rate: 0.4-0.5 cm/min. Detection: 6-ml frac!ions were mixed with 0.22 M sodium dichromate in concentrated H, SO,, diluted with 25 ml of' water and the resulting Cr(I1l) was measured spectrophotometrically. Acetic acid Alcohol
tert.-Amy1 alcohol n-Amy1 alcohol n-Hexanol n-Heptanol n-Octanol n-Nonanol n-Decanol n-Undecanol n-Dodecanol Benzyl alcohol Cyclohexanol
Water
2.19 4.61 7.97 14.9 27.5 55.1
1M
2M
3IM
4M
1.86 3.85 6.12 10.8 20.7 36.7
3.30 4.88 8.28 13.8 21.6
2.21 2.88 3.81 5.39 7.47 9.60
-
2.53 2.55
1.43 1.46 1.65 I .85 1.98 2.69 3.55 4.77 1.55 1.67
-
5.7 1 4.80
4.30 4.04
-
3.76 3.58
of the logarithm of the distribution coefficient of the separated substance on the electrolyte concentration is not always linear. These workers have shown that the partial separation of higher alcohols (n-hexanol, n-heptanol and n-octanol) with 4M LiCl in methanol as eluent is possible. However, an appreciable broadening of the elution curves was observed. Sl;erma and Lowry studied solubilization chromatography on a macroreticular ion-exchange resin. The separation of a homologous series of aliphatic alcohols by solubilization chromatography on the macroreticular ion-exchange resin Amberlyst 15 was not as successful as that on conventional gel resins. A method of determination of alcohols in aqueous solution by thermodetection liquid chromatography has also been proposed (Suzuki e t a / . ) .A series of sorbents was tested but the best results were achieved on Dowex 50W-X8. The possibility of the separation of organic compounds that are only poorly soluble or are completely insoluble in water can be improved by increasing their sorption affinity to the resin. An anion-exchange resin in the form of amphophilic anions of higher organic acids was used by Small and Bremer for this purpose. On such resins, in addition to the interaction forces between the organic compounds and the resin skeleton, these substances also interact with the amphophilic anion, which increases their sorption affinity to the resin. On such modified resins, some water-insoluble substances of low polarity can be separated more satisfactorily. Amphophilic resins have a greater tendency to swell in organic solvents than in water, and the separation of substances can be considerably affected by temperature. On a Dowex 1-XI column in the form of fatty acid anions [or anions of other acids, for example di-(2-ethylhexylphosphoric acid)] , an almost complete separation of a mixture of propylene glycol and tert.-butanol can be achieved if the elution is carried out with water at 70°C. Satisfactory separations of a mixture of ethanol and n-propanol, and of ethylene glycol and n-propanol, can be achieved by the same
CHROMATOGRAPHY OF DERIVATIVES 01; ALCOHOLS AND GLYCOLS
437
method. Only a weak overlapping of the elution curves took place, while these substances were not separated at all when comparative attempts were made on a chloride form of this resin. A mixture of alcohols was separated on a cation-exchange column of Dowex 50W-X2 (K') with water as the mobile phase (Wu and McCready). Alcohol mixtures (10 11 each of methanol, ethanol, 1-propanol, 2-propanol, 1-butand, 2-butano1, 2-methyl-1-propanol and 2-methyl-2-propanol in 1 ml of water) were applied t o the column and eluted with water at an elution rate of 0.5 ml/min and with fractions of 2.5 ml. The alcohol content in single fractions was determined in 20-11 aliquots with a Beckman Carbon Analyzer.
CHROMATOGRAPHY OF DERIVATIVES OF ALCOHOLS AND GLYCOLS Lower alcohols were separated by liquid chromatography in the form of their p-(N,Ndiniethy1amino)benzene-p'-azobenzoateson a 240 X 2.7 mm column packed with Dowex 50W-X2 (200-400 mesh) cation exchanger (Chura5ek and Jandera). Distribution coefficients for optimum amounts of hydrochloric acid in the eluent were found by elution with aqueous alcoholic hydrochloric acid solution (Table 18.4). A good resolution of these coloured derivatives can also be achieved by adsorption chromatography on a silica gel CH column (5-40 pm) using a cyclohexane-ethyl acetate mixture as the mobile phase. The quantitative micro-determination of alcohols as esters of pyruvic acid 2,4-dinitrophenylhydrazone was carried out on an alumina column (Schwartz). The liquid chromatographic determination of trace amounts of glycols in the form of their 3,5dinitrobenzoates was carried out on a 2 m X 1/8 in. column filled with Corasil 11, with ethyl acetate-n-heptane (1:3, v/v) as the mobile phase, at 1200 p.s.i.(Carey and Persinger). TABLE 18.4 VOLUME DISTRIBUTION COEFFICIENTS, D,,OF SOME ESTERS OF N,N-DIMETHY LPAMINOBENZENEAZOBENZOIC ACID ON THE CATION EXCHANGER DOWEX 50W-X2 IN A 0.925 M SOLUTION OF HYDROCHLORIC ACID IN 80.5% ETHANOL (CHURAtEK AND JANDERA)
D, is defined as the ratio of the amount of compound in a unit volume of the ionexchanger phase to the same volume of external solution. Ester
D,
Methyl Ethyl wPropy1 n-Butyl n-Amy1 ti-Hexyl n-Octyl n-Nonyl it-Decyl Isopropyl Isobutyl
6.3 5.2 4.5 3.9 3.5 3.0 2.3 2.1 1.9 4.3 3.6
References p.439
438
ALCOHOLS A N D POLYOLS
SEPARATION OF POLYOLS AND POLYMERIC DIOLS Silicone-treated Celite 545 was used for the analysis of polyethylene glycol in polyoxyethylene-type non-ionic surfactants. A mixture of n-butanol and water, or n-butanol in 10% aqueous sodium chloride solution, was used for elution (Konishi and Yamaguchi). For the determination 01' the terminal hydroxy group, the possibility of separating the 3,S-dinitrobenzoates of glycols from the excess of 3,5-dinitrobenzoyl chloride and 3,5dinitrobenzoic acid on a silica gel column was made use of by Han. The column was eluted with chloroform and the absorption of the eluate measured at 528 nm. Linear elution adsorption chromatography was also used for the fractionation of polyethylene glycol dei ivatives of the Ph-S-(CH2 -CH2 -O),-CHz -CH2 4 - P h type ( n indicates the degree of polymerization) (Calzolari et d . ) .The effect and the method of activation or deactivation of silica gel with trimethylchlorosilane on the separation was investigated. In column chromatography, the degree of activation of silica gel has a marked effect on the separation of ethylene glycol oligomers. At maximum activation, the wide distribution of the adsorption energy of the active silanol centres that form hydrogen bonds with the ether groups of the oligomers favours tailing. The elimination of the more active centres by partial deactivation with water was used earlier in order to reduce tailing and the retention volume of the peaks. Polyols can also be separated by gel chromatography using a cross-linked polystyrene and 1,2-dichloroethane as eluent. The content of polyether polyols in the effluent was determined colorimetrically, as Co-SCN complexes, at 620 nm. Good linearity was observed between the effluent volume and the logarithm of the molecular weights of the compounds (Kondo ef al.). The chromatographic separation of glycols on hydrophobic gels was described by Cazes and Gaskill (Table 18.2). A column packed with Merckogel PGM-2000 was used for the separation of polyethylene glycols in combination with water or tetrahydrofuran as the mobile phase (Randau ef d.).A 100 X 1.4 cm or 100 X 0.4 cm column was used and the applied pressures were 0, 10 and 15 atm. At 10 atm pressure and a column diameter of 0.4 cm, the time necessary for the separation of four polyethylene glycols of various molecular weights was 20 min, at an effluent flow-rate of 34 ml/h, while at laboratory temperature (20-22"C), the flow-rate, which is given by the resistance of the column packing, was 8 ml/h and the total time of analysis was about 13 h. A method for the chromatographic separation of glycols on a 20 X 2.28 cm column of Dowex 1-X8 (200-300 mesh) in the borate form, using 0.925 M sodium metaborate or 0.02 M sodium tetraborate solution for elution, was also proposed (Sargent and Rieman), and permitted the quantitative separation of diethylene glycol, ethylene glycol, 1,2-propylene glycol, glycerol, nzeso-2,3butanediol and D,L-2,3-butanediol. Propylene glycol is eluted with metaborate together with glycerol, while on elution with tetraborate it is eluted in admixture with meso-2,3butanediol. For the complete separation and determination of all of these glycols, both chromatographic procedures have to be combined. The determination requires 10 min. Chromatography on a cation-exchange column (KU-2) was used for the separation of polyols (xylitols, glycerol and ethylene glycol) and water was used for elution (Dabagov and Balandin). On a Dowex 5O-Xl2 (200-400 mesh) column or a column of KU-2 crosslinked to the extent of 12%, a mixture of polyols was separated with water as the mobile phase at 60°C. The components were eluted in the following order: xylitol, erythritol,
REFERENCES
439
glycerol, ethylene glycol and 1,2-propanediol. The separation required 24-26 h. The fractions were analyzed using an Abbe refractometer or colorimetrically after oxidation with dichromate. The best separation was achieved when the exchanger was in the H' form. On an exchanger in the C;?*+form, a change in the elution sequence was observed. Xylitol is sorbed selectively on this exchanger and is therefore eluted last. The separation of a small amount of xylitol from ethylene glycol on the free H' form of the cation exchanger was also proposed. This method can be used for the analytical control of the large-scale hydrolysis of sugars and polyols.
REFERENCES Calzolari, C., Favretto, L . and Stancher, B.,J. Chromatogr.,47 (1970) 209. Carey, M . A. and Persinger, H . E., J. Chromatogr. Sci.. 10 (1972) 537. Cazes, J . and Gaskill, D. R.,Separ. Sci., 2 ( 1 9 6 7 ) 4 2 6 ; 4 (1969) 15. Chrornatronix, Liquid Chromatography Application, No. 14, Chrornatronix, Berkeley, Calif. ChuriFek, J. and Jandera. P., J. Chromarogr., 53 (1970) 69. Dabagov, N. S. and Balandin, A. A., Izv. Akad. Nauk SSSR, Ser. Khim., (1 966) 1308 and 1 3 15. DuPont Instruments, Product Bulletin No. 830 PBI, DuPant, Wilmington, Del., September 1971. Han, K. W., Analyst (London), 92 (1967) 316. Hendrickson, J . G.,J. Chromatogr., 32 (1968) 543. Kirkland, J. J.,J. Chromatogr. S c i , 10 (1972) 593. Kondo, K., Hori, M . and Hattori, M., Bunseki Kagaku (Jup. Anal.), 16 (1967) 414; C.A., 6 8 (1968) 35636t. Koiiishi, K. and Yarnaguchi, S.,Anal. Chem., 4 0 (1968) 1720. Randau, D., Bayer, H . and Schnell, W . ,J. Chromarogr., 57 (1 97 1 ) 77. Sargent, R. and Riernan, W., Anal. Chim. Acru, 16 (1957) 144. Schwartz, D., Anal. Biochem., 38 (1970) 148. Sherma, J . , Locke, D. and Bassett, D., J. Chromarogr., 7 (1962) 273. Sherma, J . and Lowry, J . D., Anal. Lett.. 1 11968) 707;C.A., 6 9 (1968) 805712. Sherrna, J . and Riernan, W.,Anal. Chim. Acta, 18 (1958) 214. Small, H. and Bremer, D. N., Ind. Eng. Chem., Fundam., 3 (1964) 361. Speckman, R. A . and Collins, E. B . , Anal. Biochem., 22 (1968) 154. Suzulu, Y., Ishii, D. and Takeuchi, T., Bunseki Kagaku (Jap. Anat.), 1 8 (1969) 858. Wu, C.-M. and McCready, R. M., J. Chromatogr., 57 (1971) 424.
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Chapter I 9
Phenols J. CHURAtEK and J. i'OUPEK
CONTENTS Introduction ............. Gel chromato ............. Adsorption chroniato Ion-exchange chroma References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
448
INTRODUCTION The sorption energy of phenols toward aluminium oxide and silica gel is greater than that of alcohols owing to the more acidic character of the phenolic hydroxyl group compared with alcoholic hydroxyl groups. Important factors in chromatographic separations are the number of hydroxyl groups in the phenol molecule, their mutual positions and the presence of other functional groups. These properties can be used not only in the gel chromatography of phenols, but also in liquid and ion-exchange chromatography.
GEL CHROMATOGRAPHY In recent years, an important role in the separation of phenols and their derivatives has been played by gel chromatography on hydrophilic and organophilic gels, and in relatively polar solvents, in addition to the effects of the shape and size of the molecule, the effect of solvation of the phenolic hydroxyl group also strongly influences the separation. When investigating separations of low-molecular-weight compounds on styrenedivinylbenzene gels in tetrahydrofuran, Hendrickson and Moore observed that molecules that contain certain functional groups appear to be much larger than would be expected from their molecular dimensions. This phenomenon was ascribed to the formation of hydrogen bonds between the compound being analyzed and the eluent. Hendrickson (1967) found that chloroform behaved as a hydrogen donor, in contrast with tetrahydrofuran. The elution volumes in benzene, determined by Hendrickson (1968), differed from those measured in tetrahydrofuran. Edwards and Ng, who measured the elution volumes of more than 100 model compounds in tetrahydrofuran, also observed anomalous behaviour with some types of compounds; this behaviour was attributed to the solvation of compounds with solvent and to sorption on the gel. The effect of the formation of the hydrogen bonds between the phenols under investigation and the eluent was shown by Yoshikawa et al. By comparing the theoretical values References p.448
44 1
442
PHENOLS
of the elution volumes of a series of substituted phenols with the measured values, using tetrahydrofuran as the eluent, they concluded that the phenol molecule is not solvated by tetrahydrofuran if the structure of the phenol enables an intramolecular hydrogen bond to be formed. A bulky substituent in the ortho-position limits the solvation with tetrahydrofuran; 2,6-substituted derivatives are not solvated at all. The acidity of the phenolic hydvroxyl group enhances the solvation of the molecule. These conclusions were confirmed by Coupek et al. (1972a), who used a series of 42 monohydric phenols and concentrated their attention on the effects of substitution in order to find a relationship between the contribution of the elution volume and the type and position of the substituent. The elution volumes give information on the steric requirements of the alkyl group on the aromatic ring. By determining the effects of substitution and the character of the connecting bond in thebisphenol series on their chromatographic behaviour, koupek et al. (1973) again demonstrated the important part played by solvation in the gel chromatographic analysis of polynuclear phenolic and extensively substituted compounds. The results obtained were used by Eoupek et al. (1971, 1972b) for gel chromatographic determinations of the content of phenolic antioxidants and light stabilizers in polymers. Additive systems in several commercial polymers were analyzed by Howard in connection with the determination of the effect of the treatment and ageing of polymers on the change in the content of the individual components. Gel chromatography on organophilic packings has also been used extensively in the study of molecular-weight distributions of phenolic resins (Drum, Gardikes and Konrad, Quinn et a/., Varishth et al., Hagen and Schroder).
ADSORPTION CHROMATOGRAPHY The separation of phenols can also be based on the fact that the phenolic hydroxyl group forms bonds with the amide group present in, for example, polyamide gels (Endres and Hormann, Grassmann et al.), The bond strength depends on the number and positions of the hydroxyl groups in the phenol molecule. The capacity of polyamides is usually relatively high, thus making these gels suitable for the separation of isomers. The eluotropic series of solvents used for the elution of phenols from a polyamide column is represented by water, ethanol, methanol, acetone, formamide, dimethylformamide (Horhammer and Wagner). Ligroin (60--70"C) removed mainly ortho,para-substituted phenols, while benzene removed mainly ortho,para-substituted phenols containing a methyl group in the para-position only. Benzene containing 5% of methanol, and methanol itself, removed mainly phenol and parasubstituted phenols. Martin found that methanol removed catechol and its para-substituted derivatives. The sorption properties of phenols on a cross-linked polyamide gel (Bio-Gel P-2 and P-6) were studied by Streuli, who found very good correlations between the molecular structures and the adsorption properties of phenols. The column was calibrated by using a 0.5% aqueous solution of blue dextran to determine the interstitial volume (VJ, and acetone and tetrahydrofuran to determine the void ( V o )plus pore volume. The values of Vo and 5 measured using the above solvents were smaller than those for methanol and larger than those for dimethyl snlphoxide. Acetone and tetrahydrofuran were considered to be molecules with the lowest probability of interaction with gels. A dilute solution of
443
ADSORPTION CHROMATOGRAPHY TABLE 19.1 Kd VALUES FOR BENZENE DERIVATIVES ON CROSS-LINKED GELS (STREULI)
Compound
Benzcne Phenol Benzoic acid 2-Hydroxyphenol 3-H ydroxyphenol
4-Hydroxyphenol 2,3-Dihydroxyphenol 3,s-Dihy droxyphenol 2-Methylphenol 3-Methy Ip hen01 2,4-Dimethylphenol 2,6-Dimethylphenol 2-Carboxyphenol 3-Carboxyphenol 4-Carboxyphenol 2-Chlorophenol 3-Chlorophenol 4-Chlorophenol 2,4-Dichlorophenol 2,4,6-Trichlorophenol 2-Nitrophenol 3-Nitrophenol 4-Nitrophenol 2,4-Dinitrophenol
-~ Bio-Gel P-2 (100-200 mesh) Exp tl.
Calc.
1.69 2.20 1.04 2.81 2.90 2.58 3.4 1 3.50 2.42 2.44 2.48 2.44 1.42 1.40 1.45 3.04 3.40 3.18 4.48 4.98 2.94 3.74 3.75 2.37
2.24 0.95 2.79 2.79 2.79 3.34 3.34 2.39 2.39 2.54 2.54 1.50 1 .50 1 .50 3.26 3.26 3.26 4.28 5.30 3.78 3.78 3.78 5.32
Difference
'
Bio-Gel P-6 Exp tl
Seuhadex G-25 Exptl.
1.43
2.0
-
0.04 -0.09 -0.02 -0.1 1 0.21 -0.07 -0.16 -0.03 -0.05 0.06 0.10 0.08 0.10 0.05 0.22 -0.14 0.08 -0.20 0.32 0.84 0.04 0.03 2.95
I .60
1.9 2.65 2.4 2.4 3.15 2.55
2. I 1.8 1.45 3.4 1.77
2.45 1.94 1.76
sodium chloride was used as eluent. Distribution coefficients (Kd) for all of the phenols studied, calculated from the standard equation Kd = (V, (where 1% is the elution volume), are listed in Table 19.1. The pooled standard deviation for all results was 0.04. Cross-linked dextran gel was used for separation by Gelotte. Woof and Pierce used Sephadex G-25 for the separation of polyhydroxyphenols. This procedure proved to be very effective and can therefore be recommended. With water, elution occurred according to the number of hydroxyl groups contained in the phenol. Adsorption occurred in all instances, and fractionation was therefore achieved not on the basis of molecular size and shape but as a result of differing degrees of bonding between the phenol and the residual carboxyl groups present in the gel matrix. There was little or no difference between ortho- and mefa-substituted compounds (Table 19.2). It will he noted from inspection of the Kd values that the separation of mixtures of these phenols should be possible and water or acid is probably the most suitable medium. Fig. 19.1 shows two traces (a and b) of the elution pattern obtained automatically with an AutoAnalyzer. Fig. 19.la was obtained with water aseluent, and Fig. 19.lb with dilute acetic acid. Fig. 19.1c shows the separation of various phenol derivatives.using acetic acid with NaCl as eluent. Acid was used in this case because of the anomalous behaviour of hydroxy acids in water. References p.448
444
PHENOLS
TABLE 19.2
Kd VALUES FOR PHENOLS IN AQUEOUS SOLUTIONS FROM SEPHADEX G-25 COLUMNS (WOOF AND PIERCE) Compound
Elu tinn medium ~
Phenol Hy droquinone Resorcinol Catechol Phenolglucinol Pyrogallol o-Hydroxybenzoic acid Arbutin o-Nitrophenol Saligenin Guaiacol o-Hydroxybenzaldehyde o-Cresol Orcinol o-Chlorophenol p-Hy droxydiphenyl
~
Water
0.5 M NaCl
0.1 MNH,OH
0.1 M CH COOH
1.8 1.05 2.1 2.05 2.3 2.4 1.9 1.45 1.45 1.85 2.2 2.3 2.6 3.0 3.15 4.8
2.0 2.4 2.05 1.9 3.15 2.4 2.1 1.55 2.45 2.1 1.8 2.3 2.55 2.6 3.4 5.4
1.1 1.7 1.6 1.95 1.1 1.3 1.6 0.85 1.8 1.55 1.45 1.55 1.85 1.5 1.55
1.95 2.4 2.5 2.5 3.25 3.25 3.4 2.4 1.9 2.1 2.3
3.0 -
3.0
2.9
The behaviour even of simple phenols on Sephadex columns is complex and it is difficult to predict Kd values from the structures. The factors that determine elution volume include both the number and positions of hydroxyl groups, substituent groups that affect strength of bonding. and the medium. In addition, steric factors may govern penetration into gel grains. Neither the K , values nor the change in Kd values on passing from neutral to alkaline medium seemed to be related directly to the pKs of the phenols. Only nitro and carboxylic acid groups decreased bonding. In many instances, silica gel (Hanson and Zucker) was also used for preparative separation of a number of phenols. Van Dijk and Mijs separated phenols on silica gel presaturated with solvent vapour, using a mixture of non-polar and polar solvents in a 1: 1 ratio as the eluent. Zakupra et aZ. separated high-molecular-weight alkylphenols on silica gel KSK and aluminium silicate, and achieved a good separation of 2,4- and 2,6dialkylphenols from 2- and 4-monoalkylphenols. However, the procedure is time consuming, a good separation requiring ca. 8 h. The identification of alkyl-substituted phenols present in cashew nut-shell liquid was performed by Murthy ef aZ. using a 150 X 1.5 cm column packed with silica gel and a column packed with silica gel impregnated with silver nitrate. Pure cardanol was also isolated in this manner (De Vries). Mixtures containing 5, 10 and 20% (v/v) of ethyl acetate in benzene were used as eluents. Berrera separated 21 tar-phenols on a column packed with Celite 535 or with a cellulose powder impregnated with formamide. The eluent was cyclohexane and diethyl ether (98:2 or 95:5), saturated with formamide prior to use.
ION-EXCHANGE CHROMATOGRAPHY
44 5
C H A R T DIVISION
Fig. 19.1. Separation of phenols (Woof and Pierce). Column: (a and b) 35 X 2.5 cm; (c) 0.9 X 25 cm; Sephadex (3-25. Mobile phase: (a) water; (b) 0.1 Maceticacid; (c) 0.1 Maceticacid containing 0.05 M sodium chloride. Detection: UV photometric. Traces: (a) 1 = hydroxyquinone; 2 = phenol; 3 = catechol + resorcinol;4 = guaiacol; 5 = pyrogallol + phloroglucinol; 6 = o-cresol; 7 = orcinol. (b) 1 = phenol; 2 = hydroquinone; 3 = resorcinol + catechol; 4 = pyrogallol + phloroglucinol. (c) 1 = saligenin; 2 = nitrophenol + guaiacol; 3 = salicylaldehyde; 4 = catechol; 5 = o-cresol + chlorophenol; 6 = salicylic acid.
ION-EXCHANGE CHROMATOGRAPHY Phenols, which have an acidic character, can be sorbed on anion-exchangers via the mechanism of anion exchange. Their aromatic nuclei exhibit a great affinity for the matrix of styrene-divinylbenzene ion exchangers, and both ionic and molecular sorption are involved. It was found that phenols were also retained by non-ionic sorption on the functional groups of ion exchangers (Anderson and Hansen), which is why both anionand cation-exchange resins can be used for the separation of phenols. A method has been described for the chromatographic separation of mixtures of phenol and m-cresol on a column with the cation-exchanger KU-2 (H?)using water as eluent (Stankevich and Skorochod). Elution with sodium citrate solution of pH 3.42 was used in order to separate 10- 15 mg of a mixture of phenol derivatives on a Dowex 50 References p.448.
446
PHENOLS
column in the following order: phenol, cresols and finally xylenols (Krampitz and Albersmeyer). The quantitative separation of N-acetyl-p-aminophenol from p-aminophenol on a column packed with the cation-exchanger Amberlite IR-120 (H')can be achieved by elution of the N-acetyl-p-aminophenol with water and of the p-aminophenol with a 5% hydrochloric acid. The method was used for the determination of both of these constituents in drugs and urine (Koswy and Lach). Sherma and Rieman used chromatography on a cation-exchange column (Dowex 50) to separate phenols. Pyrocatechol and phenol were eluted with 1.0 N acetic acid, while o-cresol and o-nitrophenol were eluted with 2.0 N acetic acid. Logie described the chromatographic separation and determination of chlorophenols in commercial 2,4-dichlorophenol on a column of the anion-exchanger De Acidite F F (CH3 COO-). Chlorophenols were eluted gradually with solutions of glacial acetic acid in methanol or with buffers consisting of mixtures of acetic acid and triethylamine. The same ion exchanger was used by Thomas and Thomas, who first concentrated on the separation of the ortho-, meta- and para-isomers. The separation of mixtures containing 1 mg of each isomeric nitrophenol is shown in Fig. 19.2 (Thomas and Thomas). A similar separation of isomers of tetrachlorophenol on an anion-exchange column by means of gradient elution with acetic acid solutions was described by Skelly.
Fig. 19.2. Separation of phenols (Thomas and Thomas). Ion exchanger: De Acidite F F (CH,COO-; 3-5% divinylbenzene; > 200 mesh). Mobile phase: 4 % triethylamine in methanol. Flow-rate: 1 ml/min, Detection: Optical density at 275 nm. (A) m-Nitrophenol; (B) o-nitrophenol; (C) p-nitrophenol.
Seki (1954) used elution with a 1:4 mixture of n-propanol and 0.3 N hydrochloric acid for the separation of phenol and substituted phenols on columns of the cation exchangers Amberlite IR-120 and IRC-50 (ITand Na'). In this way, he separated a
447
ION-EXCH ANGE CHROMATOGRAPHY
TABLE 19.3 SURVEY OF DIFFERENT PROCEDURES APPLICABLE TO THE SEPARATION OF PHENOLS Eluent Reference Chromato- Compounds analyzed Sorbent graphic chromatographically technique Soiners Sephadex GPC Phenols LC
Hydroxylated alkylphenols (fractionation)
Silica gel Sh, SM
Chloroform, acetone, methanol and their mixtures
Pozdnyshev and Petrov
LC
Alkylphenols and alkylanisoles, Preparation, purification, concentration
Silica gel KSK (140-180 b m )
Light p e t r o l y m (b.p. 40-60 C)benzene (10:1,4:1 and 1 : l )
Ivanovskaya and Gorfinkel
GPC
Phenolic compounds Fractionation, isolation
Sephadex G-25
Distilled water
Rastorgueva
LC
Dime thylphenols and polynuclear phenols. Preparation and purification
Silica gel (0.2-0.5 mm)
Methylene chloride, te trachloromethane-me thanol ( l : l ) , light petroleum (b.p. 40-60°C)benzene (4: 1)
Mijs er al.
LC
Smoke phenols
Celite impregnated with dimethylformamide Alumina Cellulose
Cyclohexaneethyl acetate saturated with dimethylformamide
Kurko et al.
LC
Alkylphenolethylene oxide adducts in soil
Alumina Silica gel Soda lime
Methanol Chloroform Te trachlorome thane
Weibull and Thorsell
LC
Alumina act. I1 Thymol derivative (rough pre-separation). Preparation
Diethyl ether -light petroleum (b.p. 40-60°C) (1: 10)
Bohlmann et al.
LC
Tropolones (1hyd10xy-6,7,8trim eth ox y naphthalene)
Neutral alumina act. 111, IV
Te trachloromethane, benzenediethyl ether (95:5)
Forbes et al.
LC
3,3'-Dihydroxydiphenoquinone. Purification and preparation after synthesis
Alumina Silica gel
Benzene Benzene, then chloroform
Musso and Pietsch
Aqueous ammonia (pH 9.6) 50% Ethanolic ammonia solution
Nornura et al.
IEC
Phenols, isomeric 0-, Bio-Rad AG SOW-
rn- and p-nitrophenols X8 (Na') halogenated phenols, cresols, isopropylphenols -~
References p.448
Dowex 50-X8 (H')
(Continued on p.448)
448
PHENOLS
TABLE 19.3 (continued) Chromato- Compounds analyzed graphic chromatographically technia ue
Sorbent
Eluent
Reference
BiO-kdd AG 1-X2 (CH,C001
Acetic acidmethanol (5:95), continuous gradient at 280 nrn
Skelly and Crummett
IEC
Halogenated phenols
High-speed I EC
Andres and Latorre Phenols and their Pellicular anion10 mM formic acid derivatives (biological exchange resin (pH 3) containing applications (type LSF) accord- 1 M KCI. Temperature ing to Anal. Chem.,.,80°C, pressure 80039 (1967) 1422) 1000 p.s.i.g.
mixture of phenol, p-cresol and 4-telr. -butylphenol; he also separated a mixture of nitrophenols obtained by the nitration of phenol. A mixture of trinitrophenol, 2,6dinitrophenol, 2,4-dinitrophenol and 0-,rn- and p-nitrophenol was also separated by Seki (1954) by elution chromatography with an acetate buffer of pH 4.5 on a column of the cation-exchanger Amberlite IR-112. For the separation of the isomers of phenol, cresol, dihydroxybenzenes and various nitrophenols, he also used elution with alcoholic solutions of citrate or acetate buffers on columns of finely granulated cation exchangers. Seki (1960a, b) analyzed the individual fractions photometrically after a prior colour reaction. A mixture of acetophenone and &naphthol and a mixture of nitrobenzene and 0-naphthol were successfully separated on a column of the cation exchanger Dowex 50-X4 (H') (200-400 mesh) using an aqueous solution of ethanol for elution (Spitz et al ). The eluate fractions were analyzed photometrically in the UV region. Acetophenone or nitrobenzene appear in the eluate earlier than @-naphthol. Other important papers on the liquid chromatography of phenols, and particularly papers of an applied character published in recent years, are listed in Table 19.3.
REFERENCES Anderson, R. E. and Hansen, R. D., Ind. Eng. Chem., 47 (1955) 71. Andres, M . W. and Latorre, J. P., J. Chrornarogr., 5 5 (1971)409. Berrera, J. B., Rev. Cienc. Apl., 21 (1967) 426; C.A., 68 (1968) 709391. E!ohlmann, F., Niedballa, U. and Schulz, J., Chem. Ber., 102 ( 1 969) 864. Coupek, J., Kahovec, J., K'rivikovi, M. and Pospi81, J . , Angew. Makromol. Chem., 15 (1971) 137. Coupek, J., Pokorni, S. Jir&kovi, L. and Pospih, J., J. Chromatogr., 75 (1973) 87. toupek, J., Pokorni, S . and PospiSil, J., 11th IVPAC Microsymposium on Macromolecules, Prepr. E-2, (1972a);J. Chromatogr., 95 (1974) 103. koupek, J., Pokorni, S . , Protivovi, J., HolEik, J., KarvaS, M. and Pospi8i1, J., J. Chromatogr., 65 (1972b) 279. De Vries, B., J. Amer. Oil Chem Soc., 40 (1963) 184. Drum, M. F., Amer. Chem. SOC.,Div. Org. Coatings Plast. Chem., Prepr., (1 966) 85. Edwards, D. G. and Ng, Q. Y., J. Polym. Sci., Part C, 21 (1968) 105. Endres, H. and Hormann, H., Angew. Chem., 15 (1963) 288. Forbes, E. J., Griffiths, J. and Ripley, R. A., J. Chem Soc., C, (1968) 1149.
REFERENCES
449
Gardikes, J. J. and Konrad. F. M., Amer. Chcm. Soc., Div. Org. Coatingsl’last. Chem., Prepr., (1966) 131. Gelotte, B., J. Chromatogr., 3 (1960) 330. Grassmann, W., Enders. H., Paukner, W. and Mathies, H., Chem. Ber., 90 (1957) 1125. Hagen, E. and Schroder, E., Plaste Kaut., 16 (1969) 335. Hanson, K. R. and Zucker, M.,J. B i d . Chem., 238 (1963) 1105. Hendrickson, J. G., 4th International Gel Permeation Chromatography Seminar. Miami Beach, 1967, Waters Ass., Framingham, Mass., 1967. Hendrickson, J. G., J. Chromatogr., 32 (1968) 543. Hendrickson. J. G. and Moore, J. C., J. Polymer Sci., Part A - I , 4 (1966) 167. Horhammer, L. and Wagner, H.,Phurm. Zt&, 104 (1959) 783. Howard, 111, J. M . , J Chromatogr., 55 (1971) 15. Ivanovskaya, L. Y. and Gorfinkel, M. I., Zh. Org. Khim., 4 (1968) 1227. Koswy, K. T. and Lach, J. L., Drug Stand., 28 (1960) 53. Krampitz, G . and Albersmeyer, W., Experienria, 15 (1959) 375. Kurko, V. I., Kelman, L. F. and Kuznetsova, A. A., Sb. Dokl. Uch. Speta. Myas. Prom. SSSR,(1968) 54;CA., 71 (1969) 79830t. Logic, D., Analyst (London), 82 (1957) 563. Martin, W. N., WoodSci., l(1968) 102;CA., 70 (1969) 1 0 3 5 7 3 ~ . Mijs, W. J., Van Dijk, J. H., Huysmans, W. G. B. and Westra, J . G., Tetrahedron, 25 (1969) 4233. Murthy, B. G. K., Siva Samban, M. A. and Aggarwal, J . S., J. Chromatogr., 32 (1968) 519. Musso, H. and Pietsch, H., Chem. Ber., 100 (1967) 2854. Nomura, N., Hiraki, S., Yamada, M. and Shiho, D. l . , J Chromatosr., 59 (1971) 373. Pozdnyshev, G. N. and Petrov, A. A., Primen. Poverkh. Aktiv. Veschesrv. Neft. Prom., Tr. Vses. Soveshch., 3rd, 1965,(1966) 21: C.A., 69 (1968) 24352s. Quinn, E. J., Ostergouldt, H. W., Heckles, J. S. and Ziegler, D. C., Anal. Chem., 40 ( 1 968) 547. Rastorgueva, L. I., Prikl. Biokhim. Mikrobiol., 5 ( 1 969) 591; C A . , 72 (1970) 39640~. Seki, T., J. Chem. Sor. Jap., Pure Chem. Sect., 75 (1954) 1297. Seki, T., J. Chromarogr., 4 (1960a) 6. Seki, T., J. Chromatogr., 3 (1960b) 376. Sherma, J. and Rieman, W., Anal. Chim. Acta, 18 (1958) 214. Skelly, N. E.,Anal. Chem., 33 (1961) 271. Skelly, N. and C’ruinmett, W. B . , J . Chromatogr., 55 (1971) 309. Somers, T. C.,Nature (London), 195 (1962) 184. Spitz, H. D., Rothbart, H. L. and Rieman, W., Talanra, 12 (1965) 395. Stankevich, 1. V. and Skorochod, 0. R., VestsiAkad. Navuk Belarus. SSR,(1966) 116; C.A., 66 (1967) 98834k. Strculi, C. A., J. ffiromatogr., 47 (1970) 355. Thomas, D. E. and Thomas, J. D. R., Analyst (London),94 (1969) 1099. Van Dijk, J. H. and Mijs, W. J., 2. Anal. Chem., 236 (1968) 419; C.A., 69 (1968) 243592. Varishth, R. C., Schwarz, F. E. and Leong, S. Y., Spectrovision, 20 (1968) 4. Weibull, B. and Thorsell, L., in Asinger, F., Proc. Int. Congr. Chem. and Phvs. Applied Surface-Active Substances, 4th, 1964, Gordon and Breach, London, 1967, p . 523; C.A., 70 (1969) 114178q. Woof, J. B. and Pierce, J. S., J. Chromatogr., 28 (1967) 94. Yoshikawa, T., Kiniura, K. and Fujimura, S., J. Appl. Polym. Sci., 15 (1971) 2513. Zakupra, W. A., Dobrov, V. S., Lebedev, E. V., Blinova, E. K. and Pliev, T. N., Khim. Tekhnol. Topl. Masel., 15 (1970)50;CA., 73 (1970)94418~.
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Chapter 20
Ethers and peroxides J. CHURAEEK
The liquid column chromatography of ethers and peroxides is used predominantly for their purification and isolation; it is used less for analytical purposes. Volatile ethers are analyzed mainly by gas chromatography because they are thermostable. However, other conditions for analysis have to be chosen for thermolabile substances, such as some types of peroxo compounds. The isolation of ethers and peroxides, their separation and their determination in the reaction products or in commercial products is frequently necessary. Six and seven-component mixtures of aliphatic ethers (C, -C,) were separated by salting-out chromatography on Dowex 50-X4 (200-400 mesh) using gradient elution with ammoniumsulphate of decreasing concentration (Sargent and Rieman). The same compounds were separated, using the same principle, with water and acetic acid solutions of various concentrations (1-8 M). The results are summarized in Table 20.1 (Sherma and Rieman). TABLE 20.1 SOLUBILIZATION CHROMATOGRAPHY OF ETHERS (SHERMA AND RIEMAN) Ion exchanger: Dowex 50-X4 (H') (200-400 mesh). Column: 15 X 2 cm. Flow-rate: 0.4-0.5 cm/min. Detection: fractions of 5 ml were mixed with 5 ml of 0.02Msodium dichromate in concentrated H, SO,, the mixture was diluted with 25 ml of water, and the resultant Cr(I1l) measured spectrophotometrically. Compound
Diisopropyl ether Di-ri-propyl ether Ethyl-n-butyl ether Di-n-butyl ether Anisole Diphenyl ether Diisoamyl ether Di-ti-amyl ether
Mobile phase Water
Acetic acid
3.51 4.94 5.25 12.5 15.2
3.1 1 4.16 4.19 9.36 11.6
2.88 3.31 3.60 6.81 9.09 54.6
2.1 3 2.08 2.48 3.90 5.36 19.2
1.30 I .65 1.69 1.83 3.08 6.65 2.28 3.03
Benzoyl peroxide can be adsorbed from non-aqueous media on zirconium oxide (Sokolova and Boichinova). Various non-polar or weakly polar solvents serve as the mobile phase, for example, acetone, benzene, toluene, tetrachloromethane, ethyl acetate, diethyl ether and dichloroethane. The optimum temperature for drying the adsorbent for chromatography is 20-40"C. The adsorption of peroxides is dependent on the amount of water present in the adsorbent and in the solvents which comprise the mobile phase. References p.4.53
45 1
ETHERS A N D PEROXIDES
z
0
L
W J
U.
0" a W
0
s
W 0
K
10
15
20
Fig. 20.1. Separation of ethers (Bomer et al.). Column: 200 X 5 cm. Sorbent: polystyrene gel, crosslinked with 2% divinylbenzene. Eluent: tetrahydrofuran. Flow-rate: 20 ml/h. Oligomeric ethylene glycols of the HO-[CH z-CHz-01 .-H type, where n = 9x and x = 1,2,3, . , . . A discontinuous series of homologous polymers; numbers above peaks designate the degree of polymerization, n. V , = elution volume.
For the separation and the preparation of monodisperse polyethylene oxides, gel permeation chromatography (Bomer et al.) can be used. Pure oligomers were isolated by this method on a preparative scale using a polystyrene gel cross-linked with 2%divinylbenzene. A chromatogram of homologous series of oligomeric ethylene glycols is shown in Fig. 20. I , and demonstrates a good separation of a whole series of oligomers. Newer papers on applications, concerning the separation and preparation of ethers and peroxides by liquid chromatography, are listed in Table 20.2.
453
REFERENCES TABLE'20.2 SURVEY OF PUBLISHED SEPARATIONS OF ETHERS AND PEROXIDES Compounds chromatographed
Sorbent
Mobile phase
Reference
Methyl decenyl ether methyl pentenyl ether (purification)
Neutral alumina
Pen tane
Damico
Diglycidyl ether in epoxy resins (isolation, determination)
Silica gel
Chloroform
Ghosh et nl.
a-and p-lsopropylidene-
Neutral alumina
Benzenechloroform (1 : 1 )
Stevens et al.
Polyethylene glycol monoalkyl phenyl ethers (non-ionic surfactants)
Kie se Iguhr
Tetrachloromethaneisooctane (1:1)
Huber et al.
Peroxo derivatives of benzanthracenc
Alumina
Dichloromethane
Bailey et al.
Compounds resulting from autoxidation of acetylenic hydrocarbons
Silica gel KSK
Light petroleumdiethyl ethermethanol
Chirko et al.
3,3-Di-tert.-bu tyldiperoxyphthalide
Silica gel
Light petroleumdiethylether (3:l)
Milas and Klein
Tetradecalylphenyl peracetate
Alumina
Diethyl ether
Riichardt and Quadbeck-Seeger
cyclo triveratrylenes a-(8)and 048)
REFERENCES Bailey, P. S., Batterbee, J. E. and Lane, A . G.,J. Amer. Chem. Soc., 9 0 (1968) 1027. Bomer, B., Heitz, W . and Kern, W . , J . Chromatogr., 5 3 (1970) 51. Chirko, A. I., Efimova, T. A. and Ivanov, K . I., Khimiya, 4 (1968) 44. Damico. R.,J. Org. Chem.. 33 (1968) 1550. Ghosh, P. K., Bandyopadhyay, C. and Saha, A . N., J. Polym. Sci., Part A - I , 6 (1968) 341 8; C.A., 70 (1969) 480812. Huber, J. F. K., Kolder, F, F. M. and Miller, J. M., Anal. Chem., 4 4 (1972) 105. Milas, N. A. and Klein, R. J., J. Org. Chem., 33 (1968) 848. Riichardt, Ch. and Quadbeck-Seeger, H. J., Chem. Ber., 102 (1969) 3 5 2 5 . Sargent, R. and Rieman, W.,Anal. Chim. Acta, 18 (1958) 197. Sherma, J . and Rieman, W., Anal. Chim. Acta, 20 (1959) 357. Sokolova, 1. A. and Boichinova, E. S., Zh. Prikl. Khim. (Leningrad), 4 3 (1970) 798; C . A . , 73 (1970) 38937f. Stevens, I . D. R., Cookson, R. C. and Halton, B.,J. Chem. Soc., B, (1968) 767.
This Page Intentionally Left Blank
Chapter 21
0 x 0 compounds J. CHURA~EK
CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aliphatic and cyclic aldehydes and ketones . . . . . . . . . . . . . . . . . . Separation of aldehydes and ketones in the form of their derivatives . . Ion-exchange chromatography of free aldehydes and ketones . . . . . . Other chromatographic methods of separation of carbonyl compounds
Quinones
......................................
Applications in lignin chemistry References . . . . . . . . . . . .
........................ ........................
........... ........... ........... ........... ........... ........... ........... ...........
455 456 456 451 458 459 461 463
INTRODUCTION The use of liquid chromatography for the systematic separation of free aldehydes and ketones is not extensive. Most papers deal with applications and mainly concern the isolation and purification of synthetic products. When aldehydic substances are chromatographed on alumina, it should be borne in mind that they may undergo some catalyzed reactions in alkaline media and form various intermediates. These reactions restrict the general utilization of the liquid chromatography of aldehydes, especially if alumina is used as a sorbent. Carbonyl compounds are most often separated in their hydrogen sulphite form by ion-exchange chromatography on basic resins. The reactivity of the carbonyl compound with hydrogen sulphite ions is made use of, which leads to the formation of a-hydroxysulphonic acids. In the liquid chromatography of 0x0 compounds, their derivatization is also made use of: mainly oximes and 2,4-dinitrophenylhydrazones are prepared. The section on quinones includes the liquid chromatography of simple quinones, anthraquinones, ubiquinones and plastoquinones. Liquid chromatography is used mainly for the purification and preparation of synthetic compounds, or for the isolation of active components from natural material. In future, extensive use of the high-speed liquid chromatography of these substances on modern sorbents is to be expected because often they cannot be separated by gas chromatography owing to the low thermal stability of some quinones. The application of liquid chromatography in lignin chemistry is topical at present. Chromatographic papers in this field can be divided into two main groups. The first group deals with the fractionation of polydisperse lignin derivatives and the determination of the molecular-weight distribution on the dextran gel Sephadex. The second group, dealing with the separation of lignin derivatives (mainly.ligninsulphonic acids), References p.463
455
456
O X 0 COMPOUNDS
uses ion exchangers in which molecular sorption plays a role in addition to ion exchange. This chapter also covers papers that concern the identification of lignin derivatives resulting from degradation processes.
ALIPHATIC AND CYCLIC ALDEHYDES AND KETONES Aldehydes and ketones can be separated chromatographically whether they are in the free state or in the form of derivatives. Although carbonyl compounds represent nonionogenic compound:, most investigators use ion-exchange chromatography for their separation. Very often the problem of the group separation of aldehydes and ketones from organic acids and other substances can also be solved. The acids are bound quantitatively on to a column of the strongly basic anion exchanger Amberlite IRA-400 (HCOJ). Aldehydes and ketones pass into the filtrate and the acids are then desorbed with sodium carbonate solution (Gabrielson and Samuelson, 1952a). For the separation of ketones from alcohols, quantitative sorption of ketones on anion-exchange resins in the hydrogen sulphite form can be used, while alcohols pass into the eluate. Ketones can be eluted with hot water or with a solution containing a mixture of carbonate and hydrogen carbonate (Gabrielson and Samuelson, 195213). Samuelson described a complete analysis of a mixture of acetic acid, ethanol, furfural, and acetone, based on the following principle. Acetic acid is bound on a column of anion exchanger in the hydrogen carbonate form, and acetone and furfural are retained on a column containing the anion exchanger Amberlite 1RA-400 (HSO;) (0.12-0.30 mm). They are then.eluted selectively with water and 1 N sodium chloride solution. Acetic acid is eluted with alkali, filtered through a cation exchanger in the H+ form and titrated with alkali; acetone and furfural are determined photometrically by reaction with salicylaldehyde and orcinol, and ethanol is determined by measuring the density of the effluent from the hydrogen sulphite column. A column of anion exchanger in the cyanide form on to which aldehydes and ketones are bound via the formation of addition compounds was not found to be advantageous for their mutual separation, but it can be used for their separation as a group from other types of compounds (Gabrielson).
Separation of aldehydes and ketones in the form of their derivatives Liquid chromatography can be used successfully for the determination of carbonyl compounds in the form of derivatives with hydroxylamine, and the oximes formed can be determined by ion-exchange chromatography. A strongly basic anion exchanger was used with water as eluent (Ebel). Using high-speed liquid chromatography, 2,4-dinitrophenylhydrazones of aliphatic aldehydes and ketones can be separated. The derivatives are orange coloured and absorb strongly in the UV region, producing a strong response during detection. Using a 3 m X 2.1 mm column, packed with Corasil I1 activated by heating in vacuo for 3 h at 110°C, and a mixture of n-heptane and 3% of ethyl acetate, separations were achieved within ca. 35 min (Carey and Persinger) (Fig. 21.1).
ALIPHATIC AND CYCLIC ALDEHYDES AND KET0NT.S
457
I E 0 N ln
8z 4
rn R
$m a
J
Fig. 21 . I . Separation of 2,4-dinitrophenylhydrazones o f aliphatic aldehydes (Carey and Persinger). Column: 3 m X 2.1 mm. stainless steel. Sorbent: Corasil I 1 activated for 3 h at 110°C. Eluent: 9 7 : 3 (v/v) n-heptane-ethylacetate. Operating conditions: flow-rate, 1.7 ml/min; pressure, I 175 p.s.1. Detection: absorption at 254 nm. Peaks: 1 = butyraldehyde; 2 = propionaldehyde; 3 =acetaldehyde; 4 = formaldehyde (all 2.4dinitrophenylhydrazones).
Ion-exchange chromatography of free aldehydes and ketones For the chromatographic separation of aldehydes and ketones, strongly basic anion exchangers in the hydrogen sulphite form were used. On such exchangers, carbonyl compounds are sorbed via the formation of complex a-hydroxysulphonic acids. On the basis of the differences in stability of these complexes, their chromatographic separation can be achieved by using water or carbonate buffers as eluents (Cabrielson and Samuelson, 1950; 1952~).Complexes of aldehydes are more stable than complexes of ketones. The most stable compound is given by formaldehyde, and acetaldehyde, furfural, benzaldehyde, salicylaldehyde, vanillin, glyoxal, acetone and methyl ethyl ketone also form stable, strongly retained complexes. Some ketones can be eluted selectively with hot water and so separated from aldehydes, which remain on the column and which can be eluted with salt solutions (for example, 1 M sodium chloride solution). Thus, for example, acetaldehyde and furfural were separated from acetone and methyl ethyl ketone. Huff separated a mixture of lactic aldehyde, acetone, pyruvic aldehyde and a mixture of formaldehyde and acetaldehyde on a column of Dowex (HSO;) by using gradual elution with hydrogen sulphite solutions of increasing concentration. A mixture of carbonyl compounds was separated chromatographically on a 41 X 1.1 cm Dowex-1 (HSO;) (150-300 mesh) column using the same procedure as above (Christofferson, 1965). After the removal of hydrogen sulphite from the eluate fractions with iodine, the concentration of the carbonyl compounds could be determined photometrically in UV References p.463
458
O X 0 COMPOUNDS
TABLE 21.1. SOLUBILIZATION CHROMATOGRAPHY OF KETONES (SHERMA AND RIEMAN) Column: 20 X 2 cm. Cation exchanger: Dowex 50-X8(H') (200-400 mesh). Flow-rate: 0.4cm/min. Temperature: room temperature. Detection: fractions of 5 rnl were mixed with 5 mI of 0.1 M hydroxylamine hydrochloride and the pH determined after 5 min. The difference between this pH and that of a fraction containing no ketone is proportional t o the amount of ketone present. Samples consisted of 0.2 mrnole of compound dissolved in 1.0 ml of at least 50% of eluent. Ketone
Mobile phase ~~
Aqueous methanol
Methyl n-butyl ketone Methyl n-amyl ketone Methyl n-hexyl ketone Methyl isobutyl ketone Methyl n-heptyl ketone Methyl n-octyl ketone Me thy I n-nony 1 ketone Ace tophenone
Aqueous ethanol
2.0 M
4.0 M
1.OM
2.0 M
4.14 5.95 10.1 3.21 17.1 33.6 63.1
3.04 4.00 6.94 2.76 11.6 21.3 39.6 9.56
4.33 6.13 11.3
3.58 4.96 8.16
12.1
light (Christofferson, 1964). Mixtures containing acetaldehyde, formaldehyde, 5-hydroxymethylfurfura1;furfural and vanillin in amounts of 0.05-0.1 mmole could be separated sharply and the relative error was less than 10%. Ketones were separated on cation-exchange columns of Dowex 50-X8(H') (200-400 mesh) by gradual elution with aqueous methanol or ethanol of increasing concentration (Sherma and Rieman). From Table 21.1, it is evident that a successful separation of various methyl ketones was achieved. The separation of aldehydes and ketones by salting-out chromatography on anion-exchange resins can also be recommended (Breyer and Rieman, 1958; 1960). A good separation of a series of carbonyl compounds is shown in Table 21.2.
Other chromatographic methods of separation of carbonyl compounds Bell et al. separated aromatic ketones from cigarette smoke. By extracting the condensate, acetonitrile fractions were obtained with derivatives of fluoren-9-one, and these fractions were then fractionated on an alumina column by gradient elution. The polarity of the system was gradually increased. The eluent was n-hexane to which benzene, diethyl ether and methanol were added gradually. The final identification of the components was carried out by paper or gas chromatography. In Table 21.3, elution data are given for some carbonyl compounds (and esters), which were separated by gel chromatography on styrene-divinylbenzene gel (Hendrickson).
459
QUINONES
TABLF 21.2 DISTRIBUTION RATIOS OF CARBONYL COMPOUNDS IN SALTINC-OUT CHROMATOGRAPHY (BREYER AND RIEMAN, 1958) Column: 25 X 2 cm. Cation exchanger: Dowex 1-X8 (SO:-) (200-400 mesh). How-rate: 0.2-0.8 cm/min. Tempcraturc: room temperature. Detection: by the differential pH method of Roe and Mitchell in which 5 ml of 0.1 Mhydroxylamine hydrochloride is added to each fraction and the pH is determined after 5 min; the amount of carbonyl compound is related to the pH. Alternatively, fractions are mixed with 5 ml of 0.1 N sodium dichromatc in conc. H,SO,, diluting with 25 ml of water and measuring the absorbance of the resulting Cr(l11). Samples consisted of 0.050-0.1 00 mmole. Compound
Mobile phase Water
Formaldehyde Acetaldehyde Acetone Acetoin Diacetyl 2.5-tIexanedione Diacetone alcohol Propionaldehyde Methy I ethyl ketone Cyclopentanone 2,3-Pen tanedione 2,4-Pentanedione Methyl isopropyl ketone Bu tyraldehyde Methyl rz-propyl ketone Diethyl ketone Cyclohexanonc
Ammonium sulphate solution
0.5 M
I .O M
2.0 M
3.0 M
4.0 M
1.05 0.80 0.70 0.76 1.25 1.06 0.78 1.37 1.28 1.70 1.92 1.90 2.12
0.93 0.98 0.93 0.96 2.06 1.47 1.36 1.86 2.00 2.59 2.59 2.73 3.59
1 .oo
1.22 1.53 I .38 2.20 2.56 2.3 1 2.50 3.02 4.01 4.49 4.34 5.76
0.98 P.52 3.00 2.91 4.60 6.87 7.31 4.58 8.21 8.83 12.8 12.9 16.5
0.97 2.18 6.12 6.1 3 8.50 22.4 22.5 7.70 21.1
0.88 2.92 10.9 10.8 13.9
2.82 2.5 1
4.42 4.56
5.8 I 7.52
2.52 3.09
4.42 5.17
7.16 8.61
-
21.8 -
-
-
19.6 19.6
-
-
-
-
-
-
-
QUINONES Naturally occurring quinones can be isolated from most natural materials by first extracting them with a neutral or alkaline extractant and then fractionating the extracts by column chromatography. Alkali-soluble fractions can be further fractionated on a column packed with deactivated silica gel by elution with benzene (Thomson and Burnett, 1967). Some synthetic quinones of the anthraquinone type were separated by high-speed liquid chromatography on modern sorbents, such as Corasil/CI8 or Permaphase ODs. In the first instance, methanol-water (1 : 1) was used as the polar mobile phase at a pressure of 1200 p.s.i.g. (Fig. 21.2) (Waters Ass.). In the second instance, the separation was carried out at an elevated temperature (60°C) using a methanol-water mixture (45:55) ’ at a pressure of 450 p.s.i.g. (DuPont) (Fig. 21.3). Table 2 1.4 gives a review of some recent applications in which liquid chromatography is used primarily for purification and preparative purposes. References p.463
460
O X 0 COMPOUNDS
TABLE 21.3 GEL CHROMATOGRAPHY OF CARBONY L COMPOUNDS (HENDRICKSON) Column: 12 ft. X 3/8 in. Gel: 40 A styrene-divinylbenzene gel, permeable to alkanes of mol. wt. Mobile phase: benzene. Flow-rate: 1.O ml/min. Temperature: 24°C. Detection: RI.
< 450.
Compound*
Elution volume (V,, ml)
Peak width (ml)**
Ace tone* * * Acetone * * * Ace tone* * * n-Butyraldehyde Methyl ethyl ketone Ethyl acetate Methyl isobutyl ketone Dimethyl terephthalate n-Heptaldehyde Dimethyl adipate Dimethyl sebacdte
(75.6) 115.3 101.5 110.4 110.3 108.6 102.2 100.6 98.1 94.2 73.8
(4.66) 4.34 4.52 4.46 4.10 4.34 4.40 5.30 6.40 4.17 4.62
*Sample sizes were typically 0.1 ml of 4% solute in benzene. **Peak widths were obtained by drawing tangents t o each side of the curves and reporting the number of millilitres of eluent a t the base of the triangle. ***Compound run more than once.
TABLE 21.4 SURVEY OF CHROMATOGRAPHIC PROCEDURES APPLICABLE TO THE SEPARATION OF ALIPHATIC AND AROMATIC O X 0 COMPOUNDS Substances chromatographed
Sorben t
Mobile phase
Reference
Cyclobutanone derivatives
Alumina, act. I
Dichloromethane and methanol
Huisger and F:ei le r
Bis-diazoke t ones
Florisil (60-100 mesh)
Benzene-n-hexane (1:l)
Bien and Ovadia
Dichlorobenzophenone
Florisil
Light petroleum (b.p. 60-80°C) -diethy1 ether (9:l)
Morgan
6,6-Diarylbicyclo[ 3.1.01 hexan-2-one and its derivatives
Silica gel, Celite, silicic acid
n-Hexane-1% diethyl ether
et QI.
Trihydroxycyclo-2,6dien-5-one (purification)
Silicic acid, Dowex AB I-X2
Diethyl ether-benzene (9: 1)
Shaw and Smith
Zimmerman
46 I
APPLICATIONS IN LIGNIN CHEMISTRY TABLE 2 1.4 (corztinued) Substances chromatographed
Sorbent
Mobile phase
Reference
Tropolones (purification)
Silica gel
Chloroform-benzene (1:l)
Forbes and Criffiths
Adaniantanone (preparation)
Silica gel
Light petroleum (b.p. 60-8O0C)-acetone (5:2) and others
Snatzke and Eckhard t
Anthraquinones in natural materials
Silica gel
Benzene
Thomson and Burnett (1 968b)
Florisil
Benzene
Silica gel G
Benzene-light petroleum (b.p. 60-80°C) (1:4)
Thomson and Burnett f 1968~)
Florisil
Light petroleum (b.p. 60-80°C)
Thomson and Burne t t (1968a)
Magnesium oxide, silica gel
Chloroform
Thomson and Brown
Anthraquinone derivatives (purification)
Cristol and Caspar
Alumina
Acid alumina
Benzene
Bredercck et al.
Benzoquinone derivatives (purification)
(a-, p-, y-) Isomers of
rubromycin derivatives of napthoquinone (isolation)
Silica gel
Silica gel C and oxalic acid
Ethanol (extraction from the column with water) Chloroform Chloroform-acetone
(97: 3)
Teuber and Die trich Cuntze and Musso Brockmann and Zeeck
Chloroform-benzene ( I : 1)
APPLICATIONS IN LIGNIN CHEMISTRY In lignin chemistry, liquid chromatography is often used for the fractionation of polydisperse lignin derivatives, mostly on Sephadex gels (Kirk et al. ). For determining molecular-weight distributions, Sephadex G-100 was found t o be the most suitable resin and, when the formamide system was applied, enabled a good separation of single fractions t o be achieved. Aqueous extracts of lignin derivatives were fractionated successfully on a References p.463
O X 0 COMPOUNDS
462
1
J-.
I
I
I
I
0
5
10
I
TIME,MIN
I
20
\
1 I 0
I
5 TIME,MIN
I 10
Fig. 21.2. Separation of quinones (Waters Ass.). Column: 4 ft. X 2.3 mm I.D. Sorbent: Corasil/C,, (reversed phase). Mobile phase: methanol-water (lSO:SO, v/v). Pressure, 1200 p.s.i.g. Peaks: 1 = p-quinone; 2 = 1,4-napthoquinone; 3 = anthraquinone; 4 = 2-methylanthraquinone; 5 = 2ethylanthraquinone; 6 = 2-ferf.-butylanthraquinone. Fig. 21.3. Separation of substituted anthraquinones (DuPont). Column: 1 m X 0.083 in. I.D. Sorbent: Permaphase ODS. Mobile phase: 45:55 (v/v) methanol-water. Operating conditions: column temperature, 60°C; pressure, 450 p.s.i.; flow-rate, 1 cni3/min. Detection: UV photometer. Peaks: 1 = 9,lOanthraquinone; 2 = 2-methyl-9,lO-anthraquinone; 3 = 2-ethyl-9,lO-anthraquinone; 4 = I ,4-dimethyl9,lO-anthraquinone; 5 = 2-fert.-butyl-9,lO-anthrdquinone.
polyamide column using aqueous methanolic solutions for elution (Hostettler and Seikel, Seikel er d.).I t was observed that the gel permeation chromatography of wood components, such as hemicelluloses and lignins, is more effective than column electrophoresis. A very positive effect on the separation of these substances in buffered systems is exerted by the presence of carboxyl groups in the gel, and for this reason the polyacrylamide gel Bio-Gel P and similar gels can be recommended for the fractionation (Simonson).
REFERENCIiS
463
Very often, tasks connected with the fractionation of lignin sulphonates have t o be performed. These substances can be isolated from sulphite liquors (wastes) by means of hexamminocobalt trichloride and conversion into their barium salts on an ion-exchange Using Sephadex G-75 and G-100, fractions of molecular weight column (Alekseev et d.). up t o 100,000 can be obtained. The mechanism of the fractionation of calcium and lithium lignosulphonates by gel chromatography was investigated on a column packed with Sephadex G-25 and C-50 using water, dioxane-water and aqueous solutions of calcium and lithium chlorides as eluents (Stenlund). On Sephadex G-50, lignosulphonates up t o a molecular weight of 40,000 can be well separated, and 011 Sephadex G-75 up to a molecular weight of 80,000 (Forss and Stenlund). When ligninsulphonic acids are sorbed on ion exchangers, molecular sorption occurs in addition t o ion exchange (Seidl). For sorption, the weakly basic anion exchanger Lewatit MP-60 was found t o be the most suitable. The sorption of these acids is partly irreversible. The most efficient desorption agent was a solution of 2 Msodium chloride plus 1.5 M sodium hydroxide. Anion exchangers with a microporous or visibly porous structure were equally efficient. A very common task consists in the separation and isolation of degradation products of lignin derivatives obtained either by degradation with thioacetic acid (Nimz, 1969b) or alkali (Johansson and Miksche), or by new degradation procedures (Nimz, 1969a). In the last instance, Sephadex LH-20 is used as sorbent and dimethylformamide as eluent. In other instances, columns packed with silica gel and eluted with acetone and n-hexane (or cyclohexane) are used.
REFERENCES Alekseev, A. D., Ashina, I. V., Reznikov, V. M . and Sukhaya, T. V., Obshch. Prikl. Khim., (1969) 226;CA., 73 (1970) 132105e. Bell, J . H., Ireland, S. and Spears, A. W . ,Anal. Chem., 41 (1969) 310. Bien, A. and Ovadia, D., J. Org. Chem., 35 (1970) 1028. Bredereck, K., Sornrnerrnann, F. and Diarnantoglon, M., Chern. Ber., 102 (1969) 1053. Breyer, A. and Kiernan, W., Anal. Chim. A c ~ Q18 , (1 958) 207. Breyer, A. and h e m a n , W.,Talanta, 4 (1960) 67. Brockrnann, H. and Zeeck, A., Chem. Ber., 103 (1970) 1709. Carey, M . A. and Persinger, H. E.,J. Chromatogr. Sci., 10 (1972) 537. Christofferson, K., Anal. Chim. Acta, 31 (1964) 233. Christofferson, K . , Anal. Chim. Acta, 33 (1965) 303. Cristol, S. J . and Caspar, M. L.,J. Org. Chem., 33 (1968) 2020. Cuntze, U. and Musso, H., Chem. Ber., 103 (1970) 62. DuPont, Product Bulletin, Liquid Chromatographs, No. 820 PB4, DuPont, Wilmington, Del., 1971. Ebel, S., Arch. Pharm., Berl., 300 ( I 967) 472. Forbes, E. J. and Griffiths, J . , J. Chem. Soc., C , (1968) 575. Forss, K. and Stenlund, B., Pap. Puu, 5 1 (1969) 93 and 97; C.A., 70 (1969) 8 8 9 6 5 ~ . Gabrielson, G.,J. Appl. Chem., 7 (1957) 533. Gabrielson, G. and Samuelson, O., Sv. Kern. Tidskr., 62 (1950) 214. Gabrielson, G. and Samuelson, O . , A C ~Chem. Q Scand., 6 (1952a) 729. Gabrielson, C. and Sarnuelson, O., Acta Chem. Scand.. 6 (1952b) 738.
464
O X 0 COMPOUNDS
Gabrielson, G. and Sarnuelson, O.,Su. Kem. Tidskr., 64 ( 1 9 5 2 ~ )150. Hendrickson, I. G . ,J. Chromatogr., 32 (1968) 543. Hostettler, F. G. and Seikel, H. K., Tetrahedron, 25 (1969) 2325. Huff, E., Anal. Chem., 31 (1959) 1626. Huisger, R. and Feiler, L. A., Clzem. Eer., 102 (1969) 3391. Johansson, B. and Miksche, G. E., Acta Chem. Scund., 23 (1969) 924. Kirk, T. K., Brown, W. and Cowling, E. B.,Eiopolymers, 7 (1969) 135; C.A., 71 (1969) 1 4 3 4 8 ~ . Morgan, N. L., Bull. Environ. Contam. Toxicol., 3 (1968) 254; C.A., 69 (1968) 85459d. Nirnz, H., Chem. Eer., 102 (1969a) 799. Nirnz, H., Chem. Eer., 102 (1969b) 3803. Roe, H. and Mitchell, J., Anal. Chem., 23 (1951) 1758. Saniuelson, O., 2. Elekrrochem., 57 (1953) 207. Seidl, J.,Chem. Prgm., 16 (1966) 273. Seikel, M. K., Hostettler, 1 . D. and Johnson, D. B., Tetrahedron, 24 (1968) 1475. Shaw, S. J . and Smith, P. J . , J . Chem. Soc., C, (1968) 1882. Sherrna, J. and Rieman, W., Anal. Chim. Acta, 19 (1958) 134. Sirnonson, R., Su. Papperstidn., 70 (1967) 711; C A . , 68 (1968) 51090r. Snatzke, G. and Eckhardt, G., Chem Ber., 101 (1968) 2010. Stenlund, B., Pap. Puu, 52 (1970) 197; C.A., 73 (1970) 26793t. Teuber, H. J. and Dietrich, M., Chem. Eer., 100 (1967) 2908. Thornson, R. H. and Brown, P. N., J. Chem. SOC.,C, (1969) 1184. Thornson, R. H. and Burnett, A. R., J. Chem. Soc., C, (1967) 2100. Thornson, R. H. and Burnett, A. R.,J. Chem. SOC..C, (1968a) 850. Thornson, R. H. and Burnett, A. R., J. Chem. Soc., C , (1968b) 854. Thomson, R. H. and Burnett, A. R.,J. Chem. Soc., C, ( 1 9 6 8 ~ )2437. Waters Ass. Inc., Firm Prospects-CorasillCls, Waters Ass., Frarningharn, Mass. Zirnrnerrnan, H. E., Crumrine, D. S., Dopp, D. and Huyffer, P. S., J. Amer. Chem. Soc., 9 1 (1969) 434.
Chapter 22
Carbohydrates K . CAPEK and J . STANkK. Jr . CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid-solid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on charcoal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on silica gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid-liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on ion-exchange resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on silica gel and alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . Gel chromatography ......................................... Ion-exchange chromatography ................................... Chromatography on ion-exchange resins in an H' 01 OH- cyclc . . . . . . . . . . . . . . . Chromatography on ion-exchange resins in a borate cycle . . . . . . . . . . . . . . . . . . . Automated detection methods ................................... Non-destructive methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optical rotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultraviolet spectrophotometry ............................... Refractometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Destructive methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Orcinol-sulphuric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anthrone-sulphuric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenol-sulphuric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potassium hexacyanoferrate (111) assay . . . . . . . . . . . . . . . . . . . . . . . . . . . Cysteine-sulphuric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Periodate oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromic acid and carbazole assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ninhydrin ........................................... Acetic acid-aniline-orthophosphoric acid . . . . . . . . . . . . . . . . . . . . . . . . . Dyed polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flame ionization detector for liquid chromatography . . . . . . . . . . . . . . . . . . . Mono-. oligo- and deoxy saccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on charcoal-Celite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on ion-exchange resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid-liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on ion-exchange resins in the borate form . . . . . . . . . . . . . . . . . Other ion-exchange separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on molecular sieves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino sugars ............................................... Free amino sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ion-exchange chromatography with dilute hydrochloric acid as the mobile phase . . . . .
465
466 467 467 467 467 469 469 469 471 472 472 473 473 474 475 475 475 476 476 476 476 476 477 479 479 480 481 482 482 483 483 483 483 486 487 487 490 492 493 496 496 496
CARBOHYDRATES
466
.
. . . .
. ...
Ion-exchange chromatography in buffered systems . , . . , . . . . . . . . . ... .... . ..... . , ... Mutual separation of amino sugars and amino acids Derivatives of amino sugars and chromatographic methods used in the synthesis of aminosugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Sugar derivatives . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alditols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography on charcoal . .. .. . . .. . ... . . ... . ...... .. Chromatography on cellulose . . . . . . . . .' . . . . . . . . . . . . . . . . . . . . . . . . .. ... ...... ....... Chromatography on ion-exchange resins . . . Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . Glycosides with simple aglycones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complex glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethers and acetals . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . : . . . . . . . . . Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sugaracids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uronic acids , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other sugar acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... .. ..... . ...... ..... , ... . . . .. ... ... Sugar phosphates Ion-exchange chromatography of borate complexes . . . . . . . . . . . . . . . . . . Other ion-exchange separations . . . . . . . . . . . . . . . . . . . . . ........ References . . . . . . . . . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . .
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..
...
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I
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496 499 500 501 501 501 501 503 5 04 504 506 506 507 507 512 513 515 515 517 519
INTRODUCTION It should be said initially that from the viewpoint of carbohydrate chemistry the, review given in this chapter is incomplete because a compromise had to be made between the number of papers published and the space available for this type of compound. As the applicability of column chromatography to carbohydrates has been reviewed several times in the past (Binkley; Lederer; Stankk et al., 1963), we focused our attention mainly on modern methods and on the classical methods in which recent improvements in operating conditions have been responsible for their continuing applicability. Moreover, we have omitted, with a few exceptions, all papers in which separations of different types of saccharides (amino sugars from neutral sugars, sugar acids from their lactones, and similar examples) are described, and all papers in which the chromatography was performed in order to purify the synthesized saccharide from trace amounts of unreacted reagents and from some inorganic compounds. These chromatographic separations are simple and in many instances resemble filtration. The names of compounds mentioned in the separations in this chapter are as given in the original communications; we have changed them only when current nomenclature recommendations dictate that the original name should not be used now. In descriptions of chromatographic separations, saccharides are always listed in the order in which they are eluted from the columns.
GENERAL TECHNIQUES
467
GENERAL TECHNIQUES Liquid-solid chromatography Many adsorbents have been used for the adsorption chromatography of sugars and their derivatives in the last three decades: charcoal, silica gel, alumina, Fuller’s earth clays, calcium acid silicate, hydrated magnesium acid silicate, freshly precipitated calcium carbonate, etc. (Binkley). Nowadays, chromatography on ion-exchange resins and gel permeation chromatography are of major interest and only the first three of the above adsorbents have retained their importance. Chromatography on charcoal As adsorption on charcoal is governed largely by the molecular weight of the sample (see Chapter 9), this sorbent is used mainly in the fractionatiun of monosaccharideoligosaccharide mixtures and in the isolation of sugars and their polar derivatives (alditols, uronic acids, N-acetylated amino sugars, etc.) from natural sources. The advantages of this technique are its simplicity, its ability t o handle large amounts of material and the low cost of the adsorbent. Most of the commercially available charcoals or carbons (Darco G-60, Charcoal BDH, Norit P, Carboraffin, Carbon-Nuchar, etc.) are in the form of fine powders, which require the addition of a filter aid if used in column chromatography. Celite has been found t o be the most suitable for this purpose, but cellulose (Jermyn) or alumina (StefanoviC) is preferred by some workers. The properties of charcoals can sometimes be improved by treating them with borate (Barker et al., 1955), stearic acid ( A h ) or hydrochloric acid (French et al.). Recently, Lammers pointed out that the results obtained with common commercial charcoals are not satisfactorily reproducible when the separation is repeated with the same column. This may be caused by the development of channelling when small particles of charcoal are carried along with the mobile phase, This difficulty can be overcome by flotation of the charcoal in water until it is free from fines. The most suitable mobile phases for carbon column chromatography are water and aqueous alcohols, such as methanol, ethanol, n-butanol and 2-butanol. Taylor and Whelan reported that the pH of the eluting medium has a considerable effect on the manner in which sugars emerge from the column. Non-polar mono- and ohgosaccharides appear as sharper bands when the mobile phase is acidic than when it is neutral.
Chromatography on silica gel As the surface of silica gel is covered with polar oxygen functions, the adsorptive forces between the adsorbent and free sugars or polar sugar derivatives are too high for their successful separation under the conditions of LSC.The wide applicability of silica gel chromatography consists, therefore, in the separation of less polar sugar derivatives, such as sugar esters, ethers and halogen-containing derivatives. Owing to the simplicity and low costs of silica gel chromatography, it is often worthwhile first to convert the References p.519
468
CARBOHYDRATES
polar sugar or sugar derivativesintoless polar (e.g., peracetyl)derivatives, then to separate these lesspolarderivativesonasilicagelcolumn, and finally toregenerate the parentcompounds. The main advantage of this method lies in the possibility of making an appropriate choice of both the mobile phase and the sorbent activity in advance, by means of TLC. As a guide, the following procedure can be followed. (a) The sample is examined on micro-plates coated with silica gel G in order to find the most suitable mobile phase; combinations of one polar and one non-polar solvent are particularly recommended. (b) Next, the sample is tested in the chosen mobile phase on 12 X 20 cm plates coated with the silica gel to be used in the column chromatography. Successful, complete separation of the sample on th? column can be expected only when the spots of individual compounds are at least partly resolved. If the opposite is true, it is recommended that another type of silica gel or solvent should be tried. (c) The chosen silica gel is suspended in light petroleum (b.p. 60-70°C) and loaded into the tube, which is partly filled with the same solvent. The height of the silica gel bed should not be greater than 10 times its diameter. A 30-100-fold excess (related to the weight of the sample) is the most frequently used amount. (d) The sample, highly soluble in a non-polar solvent, is applied on to the column as a solution. If the syrupy sample is insoluble in the desired solvent, the following procedure is recommended. A few millilitres of non-polar solvent are poured into the flask containing the sample; the wet syrupy sample is then absorbed with a piece of cotton-wool rolled on a spatula until all sample is present in the cotton-wool. The silica gel bed is then covered with the cotton-wool containing the sample, and the remaining solvent, if any, is poured on to the column and allowed to drain through. A solid sample can be applied on to the column'in the solid state. (e) If the mobile phase found to be suitable by means of TLC consists of non-polar and polar solvents, the elution should start with the non-polar solvent alone; the concentration of the polar solvent should then increase slowly. (f) For monitoring the sugars in the effluent, any appropriate method of automated analysis can be applied; however, TLC is the most useful in common laboratory practice. In complicated situations, in which the compounds overlap to such an extent that virtually no resolution is observed on TLC, the graph relating the weight of substance in a particular fraction to the fraction number provides invaluable information. Of commercial products, silica gels with particle sizes ranging from 40 to 200 prn have been used most frequently. For the mobile phases, light petroleum, dichloromethane, chloroform and benzene as less polar components, and diethyl ether, ethyl acetate, acetone, methanol and ethanol as more polar components have been used the most. In some exceptional cases, silica gel catalyses the chemical reaction between the solute and the solvent. Thus, methyl 3,4,6-tri-O-benzyl-a-~ -mannopyranoside upon column -mannopychromatography on silica gel affords methyl 2-0-acetyl-3,4,6-tri-O-benzyl-cu-~ ranoside, using ethyl acetate as the mobile phase (Franks and Montgomery). Detritylation of some trityl sugar derivatives on a silica gel column was reported by Trimnell et al. and by Lehrfeld. Kiely and Fletcher observed the decomposition of 3-O-benzyl-l,2-O-isopropylidene~-D-xylo-hcxofurai~os-5-ulose; this decomposition is avoided when silica gel previously washed with acetic acid-benzene (4:96) is used.
GENERAL TECHNIQUES
469
Cliromatography on alumina Chromatography on alumina is, in practice, not very different from the procedures described for silica gel. The preliminary examination of an unknown sample can be performed i n a manner similar to that already described (p.468). In comparison with silica gel, chemical reactions of solutes on alumina columns occur more frequently. Jary et al. observed that during the chromatography of methyl 3-acetamido2,4-di-O-acety1-3,6-dideoxy-a-~ -altropyranoside on an alkaline alumina column using a benzene-ethanol mixture as the mobile phase, partial de-0-acetylation occurred. This phenomenon was later used in the preparttion of several partially acetylated methyl 3acetamido-3,6-dideoxyhexopyranosides(Capek e t a/. , 1966, 1967, 1968a, b, 1970b) and methyl 3,6-dideoxyhexopyranosides(Capek et a/. , 1970a). Ennor et a/. reported that -altropyranoside was completely transformed into methyl 4,6-0-benzylidene-2-O-tosyk~-~ the corresponding epoxide during chromatography on alumina, even if the alumina was acid washed.
Liquid-liquid chromatography There are a vast number of applications of LLC that have been used to solve analytical problems in carbohydrate chemistry over the past 25 years; however, only recently have the exact analytical liquid-liquid method and detailed column efficiency and selectivity studies been carried out. As in GLC, precise retention data and distribution constants form the basis for tabulating carbohydrates and identifying unknown compounds. Chromatography on cellulose Cellulose has been the most frequently used sorbent in the LLC of carbohydrates, and all theories are equally applicable to column and paper chromatography. This analogy is very useful, mainly in the choice of the appropriate solvent system (Hais and Macek). In addition to liquid-liquid partition, the retention of the solute on cellulose is also frequently influenced by adsorption and ion-exchange, and all of these effects contribute to the distribution constant. Whatman No. 1 cellulose (and occasionally also Whatman CF-11, Yoshida etal. (1969b), or CF-I 2 cellulose powder, Otter et a/.) is usually used for the separation of mono- and oligosaccharides and of some of their polar derivatives on a preparative scale. Microgranular cellulose does not give any improvement in separation, tending t o pack too tightly, resulting in a reduced flow-rate. The method of packing the column is important for good separation characteristics (Otter et al.). 4 45 X 1.5 cni column, used for the quantitative chromatography of oligosaccharides (see p.486), is packed to a depth o f 4 3 cm with CF-12 cellulose powder. Prior t o use, the fines are removed from the cellulose by stirring 50 g of the powder with 1 1 of distilled water and allowing the mixture to settle. Then, the supernatant liquid together with the fines is decanted off and this procedure is repeated five times. The column is filled with a slurry of 50 g (less fines) of cellulose in 800 ml of water at 90°C and allowed t o settle References p.519
470
CARBOHYDRATES 2 5
20
-
E
6F P IL
x
to -
Fig. 22.1. Partition chromatography of alditols on Dowex SOW-X8 (Li+, 14-17 pm). Resin bed 131 X 0.26 cm; mobile phase 85% ethanol; flow-rate 3 ml/min.cm2 ; temperature 75°C. 1 = formaldehyde; 2 = ethylene glycol; 3 = glycerol; 4 = erythritol; 5 = ribitol; 6 = arabinitol; 7 = xylitol; 8 = mannitol; 9 = glucitol; 10 = galactitol. (Samuelson and Stromberg, 1968).
100
E E
0'F ~
A
LL
W
n
0 50
I/
150
GENERAL TECHNIQUES
47 1
under gravity with no flow. The packing is consolidated by aspirating the surplus water through the outlet, and this procedure is repeated until the cel1ulose:is packed t o the required depth. When correctly packed, the column should be capable of maintaining a flow-rate of 30-40 ml/h. Water is then displaced by percolating at least 500 ml of waterethanol-a-butanol (24.5:23:52.5) through the column (Otter et al.).
Chromatography on ion-exchangeresins Recently, chromatography on cellulose has been replaced by partition chromatography on ion-exchange resins with different counter-ions, which offers a great variety of experimental conditions. The separation is achieved as a result of the uneven distribution of the solvent components between the resin and the external solution. With aqueous ethanol, for example, the relative amount of water present is greater in the resin phase than in external solution; this explains why polar solutes are preferentially held by the resin (Samuelson). Cation-exchange resins have the advantage over anion-exchange resins that they are readily available commercially as smaller, more uniform resin particles (owing to their use in amino acid analyzers). Dowex 50W-X8, Amberlite IR-120, Aminex A-6 and Technicon TSC, Technicon T4 or Dowex 1 -X8 with a narrow size in the range 5-20 p m are the most frequently used. Lithium, sodium, potassium, barium, calcium, sulphate and chloride are usually used as counter-ions. Of the resin forms studied, the most favourable separations of polyols were obtained with the lithium form (Fig. 22.1) (for the procedure for this separation, see p. 504).Recently, organic base counter-ions, such as piperidinium and methyl-, dimethyl- and trimethylammonium have been used (Hobbs and Lawrence, 1972b), the trimethylammonium ion giving the optimum compromise between elution volumes, peak widths and analysis times. In general, the volume distribution constant, K,, in water-ethanol mixtures increases with increase in the number of hydroxyl groups and decreases with the presence of nonpolar groups in the solute; it may also be influenced by the position of the substituent (Samuelson). The K , values increase markedly with increase in the ethanol concentration, b u t the order of the elution is (within the concentration range of practical interest) independent of this concentration (Samuelson and Stromberg, 1968). The peak elution volume and the width of the elution curves usually decrease with increase in temperature (see Fig. 22.2); in some instances, however, the separation may be jeopardized at very high temperatures because of the decrease in the ratio of the K , values of the separated compounds (Jonsson and Samuelson, 1967b). A factor which limits the applicability of this technique is the extremely low rate of diffusion inside the resin particles. Porous resins and small particles are, therefore, preferable. With such particles, a high-pressure technique must be used in order t o force the solution through the column. A striking example, which shows the influence of the particle size, is given in Fig. 22.3 (Dahlberg and Samuelson). In order to reduce the pressure drop in the column, ion-exchange resins in admixture with Celite 545 were used (Arwidi and Samuelson, 1964). Of course, the solvent-resin interactions and specific interaction forces between polyols and the counter-ion in the resin phase also have a great'influence o n the equilibReferences p.519
CARBOHYDRATES
472 I
I 100
2
-
E
4
E 6 0 '
J I
2
3
F
M u.
0"
100 -.
1
0
\
200 I
1
I I
4
\A, I
600
2 1000
1400
VOLUME, ml
Fig. 22.3. Intluence ofparticle size on the separation of various sugars on Dowex 21K (SO:-) (Dahlberg and Samuelson). Resin bed 75 X 0.8 cm; mobile phase 747bethanol; flow-rate 0.8 ml/min. cm'. Upper chromatogram, 45-75 p m ; lower chromatogram, 15-40 pm. 1 = 2-deoxy-D-glucose; 2 = D-glucose; 3 = sucrose; 4 = raffinose.
rium on the ion-exchange resin; sieve effects also occur, which in some instances result in a reversed order of elution of some sugars when the counter-ion is changed (Samuelson and Strbmberg, 1968).
Chromatography on silica gel and alumina In LLC, silica gel is also sometimes used as a carrier. In this event, ethyl acetate containing dimethyl sulphoxide or dimethylformamide (Otake), methanol-water (7:3) (Heyns et al.) or water-saturated ethyl acetate (Zorbach et al.) are used as mobile phases. When a solvent system containing water, such as water-saturated butanone, is to be used, a 1 : 1 mixture of silica gel and alumina is advantageous owing to a better flow-rate (e.g., Micheel and StanZk). It is for this reason that the bottom layers of 2-propanol-chloroformammonia-water mixtures in various ratios (Cooper et al. ; Capek et al., 1974) are superior. Alumina alone has been used with various water-ethanol mixtures (Bonner et al.). Gel chromatography So far, gel chromatography (for a recent review, see Churms) has been almost exclusively used for the separation of sugars that differ in their molecular size, especially for the
GENERAL TECHNIQUES
473
fractionation of oligo- and polysaccharide mixtures. However, some papers (Brown, 1970b; Marsden) dealing with the examination of the chromatographic properties of monosaccharides and their derivatives, showed that their K , values did not decrease monotopically with increase in molecular weight and that they varied even within the aldopentose or aldohexose series. This phenomenon, which is attributed to the differences in the degree of steric hindrance to the entry of the sugar molecule into the gel phase, indicates further possibilities for the use of GPC in the carbohydrate field. Cross-linked dextran gels (Sephadex) and polyacrylamide gels (Bio-Gel P series) have been the most frequently used so far. Porous glass (Bio-glass) and porous silica beads (Porasil) are now being increasingly applied owing to their resistance to thermal, chemical and bacteriological degradation. Other types of gels, such as agarose gels (Sepharose, Sagarose), cross-linked polystyrene containing hydrophylic groups (Aquapak) and ionexchange resins have been used on a few occasions. Recently, an improvement in the chromatographic properties of Sephadex G-15 has been reported, produced by treating the gel with 1 M hydrochloric acid; this pre-treatment results in increases in the number of theoretical plates, the internal volume and the effective pore size (Goodson and DiS tefano). Concerning the effect of operating conditions on the efficiency of the GPC of saccharides, there are no serious exceptions t o the general procedure (see Chapter 6); gels with particles of a well defined pore size and small diameter, long columns with a small I.D. and very slow flow-rates are recommended. The most suitable mobile phases for the GPC o f sugars are water, aqueous solutions of (odium chloride and various buffers. 1on-exchange chromatography
With ion exchangers, it is usually not easy to decide whether different retention volumes are due merely t o the ion exchange. In practice, there is always some partition effect owing to the partial solubility or adsorption of the compound in the resin matrix, and also molecular sieve eftects cannot be neglected. Separations based predominantly on a change of the counterion or in its chemical identity (complexing) are considered in this chapter. Chrornutogruph?,o t i iotz-exchatige resitis in an If' or OH- cycle In the separation of acidic or basic sugar derivatives on ion-exchange resins, important factors include not only the equilibrium exchange conditions (solute pK), but also the rnolarity of the displacing ion, the pH of the developing buffer and temperature (Brendel er al., 1967a). The differences in peak widths that d o not fit the common pattern that the later a given compound emerges from the column the broader is the peak due t o diffusion, are assumed to be due t o the presence of various species, anomers and conformers and their relative velocities of rearrangement (Brendel er ul., 1967a). Sometimes, the opposite effect, so-called ion-exclusion, may also take place, for instance, with sugar acids, which are excluded from the matrix of Bio-Rad AG 50W-X2 (Lit) containing ionizable groups, thus decreasing the retention volumes (Barker et al., 196%). References p . 5 1 9
474
CARBOHYDRATES
With neutral sugar derivatives, such as glycosides, the separation on strongly basic anion-exchange resins is assumed to be due to an ion-exchange process, involving the loss of a proton from one of the hydroxyl groups, According t o this assumption, the retention volume is proportional to the pK value of the hydroxyl group which is ionized first. This acidity is determined by the number and the proximity of other polar substituents in the molecule, by the relative angles of various dipoles to each other and to the hydroxyl group which ionizes (Neuberger and Wilson). Owing t o the weak acidity of the sugar hydroxyl groups, carbon dioxide must not be present in water used as the mobile phase.
Chromatography on ion-exchange resins in a borate cycle
I
Anion-exchange resins in the borate form are the most common resins amongst ion exchangers with reactive counter-ions. In this method, an anion exchanger in the chloride form is packed in the column and converted several times alternately between the chloride and borate forms until no further settling occurs. All columns prepared in this manner produced a better resolution of sugar mixtures than a column packed once in the chloride form followed by single conversion into the borate form (Kesler). It is also recommended that a large volume of the mobile phase is forced through the column at high speed so as to ensure that the resin bed is closely packed before use (Alfredsson et d). Kesler compared three anion-exchange resins in the borate form and found Technicon 3/28/VI to be far superior to Dowex 1-X8 (200-400 mesh) and Bio-Rad AG 1-X8 (30-40 pm) for the separation of carbohydrates. The use of a resin with a lower degree of crosslinking, Dowex 1-X4, allows equilibrium to be attained at increased rates and greatly improves the chromatographic behaviour of di- and trisaccharides (Walborg and Lantz); for the procedure for this separation, see p. 492. The resolving power is also enhanced by the use of smaller resin particles and a narrower size range (Green), but probably the most critical parameter that affects the resolving power is the ionic strength of the developing buffer (Ohms et d.). For the optimum over-all separation of mono- and oligosaccharides (see p.491,), the following linear buffer gradient at a flow-rate of 1.15 ml/min at 50°C was established. I t is formed in a two-chambered device which contains 325 ml of limiting buffer (38.9 g of NazB407. HzO and 7 g of H3B03 per litre, pH 8.9) in a reservoir and 325 ml of starting buffer (one-fifth dilution of the limiting buffer, 0.042 M> in a mixing chamber. All water used for buffer formulation should be deionized on a mixed-bed resin (Amberlite IRA400 and Dowex 50-X8, both 16-50 mesh, 2 : l ) so as to produce an initial specific conductance of 0.5 . 10-6 R-' cm-' . As the slope of the linear buffer gradient was increased or decreased by varying the ionic strength of the starting buffer from 0.035 to 0.046 M,the resolution of closely emerging doublet peaks was seriously affected. However, the results changed only slightly with a reduction in the pH of the mobile phase to 8.45 (Ohms er al.). It was found that the effect of increasing the column temperature was to shift the eluted bands to longer elution times; simultaneously, the resolution was enhanced (Green; Kesler). However, virtually no further advantage was gained between 55 and 70°C. As the band widths of various sugars do not change under constant elution conditions, the amount of the saccharide eluted bears a linear relationship to the peak height expressed as absorbance (Kesler). Variations in reproducibility, i.e., in the resolving power of the
GENERAL TECHNIQUES
475
system, have been explained as being due t o contamination by metals and silicate. Incomplete conversion of the resin into the borate form also may result in a reduced resolution after re-packing a column (Ohms e f a / . ) . The use of non-alkaline borate buffers, such as boric acid-glycerol (Walborg et a/. , 1965; Walborg and Lantz) or boric acid-23-butanediol (Walborg e l a/., 1969), minimizes the rearrangement processes on the resin, which rnakcs it possible t o achieve absolute saccharide recoveries in the range of 8s-100% (see p. 492).
Automated detection methods
The rapid increase in the number of chromatographic separations of small amounts of multicomponent (2 10) mixtures obtained from natural material regularly necessitated the introduction of automated procedures for monitoring the column effluent. Generally, there are two different types of automated analysis of effluents that contain saccharides. The first type, which could be termed “non-destructive”, is based on the examination of the effluent by physical methods. In the second, “destructive” type, saccharides are detected by means o f a chemical reaction. Both types of method are used mainly for analytical separations. For preparative purposes, either all of the effluent is led to the flow cell, where changes in some physical property are followed (“non-destructive” type, as no changes in the chemical composition of the solute are caused), or an aliquot of the effluent is continuously analyzed for the sugar content by a “destructive” chemical reaction. It should be pointed out that for preparative separations o f sugar mixtures that are carried out only occasionally, manual methods are preferably used in order t o avoid the waste of time resulting from preliminary tests and calibrations.
Nondestructive methods Although less sensitive than colour reactions, these methods have the advantages of being easy t o run with minimal costs of reagents and being highly reproducible. The separated compounds may afterwards be recovered from the eluate, and for preparative purposes, where the highest quantitative accuracy is not demanded, they find wide application.
Optical rotation For qualitative purposes, the optical rotation of the column effluent is monitored. An automatic polarimeter is a convenient and sensitive instrument for following the elution, e.g., of glycosides from resin columns (Evans eta/.),where the anomeric nature of the eluted compound is indicated by the sign of the rotation. The same procedure was also used by Neuberger and Wilson, Yoshida et al. (1969b), and others. With more complicated mixtures, where some overlapping occurs, a disadvantage of this method appears, due t o a broad spectrum of specific rotation values of the compounds in question. The quantitative evaluation of the sugars prcsent is possible only with well separated solutes, for which the specific rotation is known, a constant flow-rate being another necessary condition. References p.519
476
CARBOHYDRATES
Ultraviolet spectrophotornetiy With sugar derivatives that are UV-active, the quality of the separation and the amounts of substances involved can be followed easily by measuring the absorbance at an appropriate wavelength. Unfortunately, there are only a few types of compounds (benzyl and trityl ethers, esters of aromatic acids, aromatic glycosides, arylidene derivatives and some others), which can be considered, and, on the other hand, there are solvents such as benzene and acetone, which are very frequently used in LSC but which cannot be used in this case. For applications of the method, in which W photometers are usually used according to the manuficturers' instructions, see, for instance, Micheel and Pick, Micheel and Stanzk and Neuberger and Wilson. Refractornetry Refractometry is widely used in the sugar industry to measure the concentration of sucrose solutions (Charles and Meads); recently, numerous applications of a differential, automatically recording refractometer in GPC have been described (e.g., Brown, 1970b). A differential refractometer was also used for monitoring the IEC separations of neutral sugars as borate complexes (Liljamaa and HallCn). The sensitivity t o variations in the buffer concentration'together with the presence of false peaks, however, causes some difficulties. Destructive methods When using destructive methods, guidance in the choice in working conditions is usually obtained from the manual methods that are commonly used in sugar analysis (Pigman and Horton, StanEk et al., 1963). Principally, saccharide contained in the effluent is converted into a coloured or UV-active product, which is then determined colorimetrically.
Orcinol-sulphuric acid The orcinol-sulphuric acid method was used in the chromatography of free carbohydrates on anion-exchange resins in the borate form (Kesler). It was also used for the hydrolyzates from wood and wood pulp separated on an anion-exchange resin in the sulphate form with 92% aqueous ethanol as the mobile phase (Arwidi and Samuelson, 1965). The orcinol-sulphuric acid reagent also proved t o be useful in the automated analysis of the effluents from the GPC of polysaccharides (Bathgate). The schematic representation of this system using the Technicon AutoAnalyzer is given in Fig. 22.4. The column effluent is mixed with 1% aqueous orcinol before being mixed with 72% sulphuric acid. The reaction mixture is then heated to 95"C, cooled, and its absorbance is monitored at 420 nm (Bathgate). A double glass coil (24 m X 2 mm I.D.) was used for colour development (John et al.). For other applications of this method, which is probably the. most frequently used, see, for instance, Larsson and Samuelson (1967), Martinsson and Samuelson, and Vrlitny. Anthrone-sulphuric acid The partition chromatography of monosaccharides on cross-linked dextran containing quaternary ammonium ions in the sulphate form (ethanol-water) was followed auto-
477
GENERAL TECHNIQUES PUMPING MANIFOLD F L O W - RATE mllh ELUATE 13 8 (( 0 M HgCI2) O.0055 M HgCI2) SMC
-
AQUEOUS AQUEOUS ORCINOL 11 % %
72% 7 2 1 H2S04
HEATING BATH
9 54
=420nm COLORIMETER
15O
468
15mm
I RECORDER I
AIR
4 8
7r
1
WASTE
46
a
WASTE .( WASTE
I __
Fig. 22.4. Flow scheme for the orcinol-sulphuric acid mcthod (Bathgate). SMC = single mixing coil; DMC = double mixing coil. Tubing that contains sulphuric acid is made from Acidflex, the remainder from polyethylene.
matically using the anthrone-sulphuric acid reagent with an accuracy of 5% (J onsson and Samuelson, 1967b; see also Jonsson and Samuelson, 1966; 1967a). The effluent was mixed with the reagent (2 g of anthrone per 1 1 of sulphuric acid) in the ratio 1 :2. The piston used to feed the reagent into thc analyzing system was covered with PTFE tubing in order t o prevent corrosion. After the eluate and the reagent had been mixed, the colour was developed by passing the solution through PTFE tubing (I.D. 1.2 mm) submerged in a heating bath at 100°C. The time of the reaction was about 1 min. The transmittance was measured at 625 nm in a 2-rnm flow cell and recorded automatically. A modification of this method was developed for cellulose column separations (Otter et al.) because the n-butanol-containing mobile phase (water-n-butanol-ethanol gradient mixtures) interferes with the reaction between a saccharide and anthrone. The most reproducible results were obtained when the mixture was heated for 20 min at 80°C instead of the more usual temperature of 100°C. The reagent used for colour development was a 0.1% solution of anthrone in 85% sulphuric acid, and was freshly prepared at the beginning of each run. The stock bottle containing the anthrone reagent was kept below 4°C before being mixed, at a flow-rate of 14 ml/h, with the effluent which was cooled t o the same temperature. The ratio of the reagent to the column effluent is important and should be maintained at 2 : l . Even small changes in this ratio result in a serious decrease in the absorbance measured at 640 nm (Otter e t a / . ) . For details of this procedure, see the analyzer flow diagram (Fig. 22 S ) . Ph e n d - sulphuric acid The phenol-sulphuric acid method was used in an automated sugar analyzer by Green, Ohms et al. and others. Neutral sugars eluted from the anion-exchange resin with a borate buffer were treated continuously with 5% aqueous phenol and concentrated sulphuric References p.519
CARBOHYDRATES
478
0.1 ‘ A ANTHRONE in 8 5 % H 2 S 0 4
BATH
14.0
SMC
SMC
SMC
ELUATE 1.0 ( WATER-ETH ANOL n - BUTANOL . 4 2 25:33 )
-0--:40 17~ 800
20 min
+--*
-
COLORIMETER
-
IOmm
RECORDER
CAPACITY
7ml (14m) r
+WASTE I
mm
ELUATE 142 (aqueous ethanol)
0 6 N H2S04
2 29
AIR
165
I
HEATING BATH
s~ F
DMC
-
lr
r
+I
WASTE HYDROLYZED ELUATE
0 09% K3Fe(CNI6
2 06 r
COLORIMETER
165
in 2 N N a O H
0 5 % KCN
114
~
-
r
=D,
Fig. 22.6. Flow scheme for the potassium hexacyanoferrate(II1) assay, together with the hydrolysls of oligosaccharides (Samuelson and Swenson). SMC = single mixing coil; DMC = double mixing coil. AU tubing is made from Tygon.
acid, the absorbance of the resulting stream being monitored by a colorimeter with filters at 480,486 and 490 nm. The flow-rates of phenol and sulphuric acid added to the’eluate stream were 0.6 and 3.05 ml/min, respectively (Ohms etal.). Based on this method of detection, a Mark I1 prototype carbohydrate analyzer in which high-resolution ion-exchange chromatography was used for the separation of sugars, with detailed operating conditions, was described by Jolley et al. (1969) and was used in the determina-
479
GENERAL TECHNIQUES
tion of carbohydrates in physiological fluids (Jolley and Freeman; Jolley et al., 1970).
Potassium hexacyanoferra te(1II)assay In 1963, Samuelson and Swenson used potassium hexacyanoferrate(I11) for detection in the Technicon AutoAnalyzer system. As oligosaccharides were involved in the separation on ion-exchange resins with ethanol-water as the mobile phase, a hydrolysis step was also used in the analyzer (see Fig. 22.6). The hydrolysis t o monosaccharides was not complete, but the results were reproducible. Part of the solution obtained after the hydrolysis (the remainder of the solution was discarded) was mixed with a 0.09%solution of potassium hexacyanoferrate(II1) in 2 N sodium hydroxide, 0.5% potassium cyanide solution and air, then heated at 80°C for about 5 min and, after cooling, the light absorption at 440 nm was measured. Differences in flow-rates were produced by using tubing of various internal diameter.
Cvsteine-sulphuric acid In the fractionation of dextran on porous silica beads, an automatic, continuous
.. - -
-
HEATING . .. BATH
FLOW-RATE PUMPING ml/mln MANIFOLD -u SOLUTION I A ID AIR
--
r
WATER
3 min DEBUBBLER SMC
AIR
ELUATE (WATER) AIR SOLUTION 2 SOLUTION 3
AIR SOLUTION 4
WASTE
In
Fig. 22.7. Flow scheme for the cysteine-sulphuric acid and potassium hexacyanoferrate(lI1) assays (Barker et al., 19691-3).SMC ='single mixing coil. Technicon AutoAnalyzer modular equipment was used throughout. Solution I is a 0.0757 (w/v) solution of L-cysteine hydrochloride in 86% (v/v) sulphuric acid. Solution 2 is sodium carbonate (0.53'%) and potassium carbonate (0.065%) in water Solution 3 is potassium hexacyanoferrate(II1) (0.05%)in water. Solution 4 is ammonium iron(lI1) sulphate (0.757,) and sodium lauryl sulphate (0.5%) in 0.05 N sulphuric acid.
References p.519
480
CARBOHYDRATES
cysteine-sulphuric acid assay for the total hexose concentration, in addition t o a reducing end-group assay (using the potassium hexacyanoferrate(II1) reagent mentioned above) was used (Barker et ai., 1969b). The column eluate was mixed with water and added at a flow-rate of 0.1 ml/min t o a 0.07% (w/v) solution of Lcysteine hydrochloride in 86% (v/v) sulphuric acid, which had a flow-rate of 0.53 ml/min. The reaction stream was heated for 3 min at 95"C, cooled, and the absorbance measured at 420 nm. The complete flow scheme of this analyzer is given in Fig. 22.7. The combination of these two a m y s obviates the need for any previous calibration of the column by eluting simples of known molecular weight, as the two assays would give the molecular weight of the species eluted at any point. Care must 3e taken t o keep the waste from the two assays separate, in order t o avoid the productior. of hydrogen cyanide gas.
Perioda te oxida tioti The colour reaction of formaldehyde, resulting from periodate oxidation, with 2,4pentanedione was used in the application of ion-exchange resin chromatography to mixtures of alditols and aldoses (Samuelson and Stromberg, 1966). The flow-scheme for this system is given in Fig. 22.8 (compare also Fig. 22.9). The periodate oxidation of the effluent (flow-rate 0.4 ml/min) is carried out a t pH 7.5 or 1 .O (flow-rate 0.6 ml/min) for about 3.5 min. At pH 1.O, most sugars give rise t o negligible amounts of formaldehyde, 'ING FOLD
FLOW- RATE mllmin PENTANE-2.4-DIONE REAGENT
(2
+ PERIODATE REAGENT
06
__ +
ELUATE 04 __ (AQUEOUS ETHANOL)
-
HEATING
BATH ( 2 5 0 ) DMC
DMC
BATH COLORIMETER
SMC
I RECORDER
-
I
TI
ARSENITE REAGENT
4
WASTE
0 6
__
WASTE
Pig. 22.8. Flow scheme for the assay involving periodate oxidation and formation of formaldehyde (Sarnuelson and Strijniberg, 1966). SMC = single mixing coil; DMC = double mixing coil. Pentane-2,4dione reagent: 2 M ammonium acetate + 0.05 M acetic acid + 0.02 M pentane-2,4-dione. Arsenite reagent: 0.5 M sodium arsenite neutralized with hydrochloric acid to pH 7. Periodate reagent: ( a ) oxidation at ptl 7.5, 0.015 M periodic acid neutralized with ammonia and buffered t o pH 7.5 with a phosphate buffer (100 ml/l); (b) oxidation at pH 1.0, 0.015 M sodium periodate in 0.1 2 M hydrochloric acid.
48 1
GENERAL TECHNIQUES
while the alditols react without difficulty. At pH 7.5, formaldehyde is formed in high yield from both alditols and aldoses. Before the colorimetric determination, the unreacted periodate is reduced t o iodate or iodide (depending on the pH) by the addition, at a flowrate of 0.6 ml/min, of 0.5 M sodium arsenite neutralized with hydrochloric acid to pH 7. This precaution is necessary because periodate destroys the colour obtained with the 2,4pentanedione reagent (flow-rate 1.2 ml/min). The absorbance at 420 nm is measured in a 15-mm flow cell.
For complicate mixtures, a two-channel analyzer, involving the combination of this method with the orcinol-sulphuric acid method (no response with alditols), was used (Saniuelson and Striimberg, 1966). This principle of using several channels, each of which specifically detects only one type of sugar derivative, proved t o be very useful also in other situations, as described below.
Chromic acid arid carbazole assays Samuelson and Thede used a two-channel analyLer i ~ the i chromatography of. sugar acids on Dowex 1-X8 (CH3COO-) (see p. 513). Chromic acid oxidation gives a response with all eluted hydroxy acids, while the carbazole method gives a strong response only with uronic acids and some keto acids, no response with aldonic acids and a weak reaction with aldobionic and lactic acids (Johnson and Samuelson). With complicated mixtures
---(&
SOLUTION 1 04
ELUATE H2S04 SOLUTION 2
0 16
SOLUTION 3
O6
SOLUTION 4
O3
SOLUTION 5
O3 023
SOLUTION6 AIR
SMC
v
WL-
L
023 17
ELUATE
HEATING BATH
023 O3 0 23
SMC
-
I
r
t
-L
-
950
t
1* SMC
!rnin
15rnin
1
SMC
1
40 sec
4 s sec
__ 0.40
I
531 nrn
WASTE
6 rnin 4
1 I
L
4 WASTE Fig. 22.9. Flow scheme for a four-channel manifold, using chromic acid, carbazole and t w o periodate assays for the analysis of ion-exchangc chromatograms of sugar acids, with acetic acid or aqueous sodium acetate as the mobile phase (Carlsson and Samuelson, 1970). SMC = single mixing coil (25"(;alactose
0.27 0.33 0.39 0.58 0.66 0.68 0.69 0.55 0.78 0.7 I
D-Ribose D-XyloSe 2-Deoxy-D-eryrlzro-pentose Methyl*-D-glucoside Me thyl-Ci-I)-gl ucoside Methyl-ol-D-mannoside 1,2-O-Isopropy~dene-a-~-~lucofuranose 1,2: 5,6-Di-O-isopropylidene-a-Dglucofuranose 2,3 :4,5-Di-O-isopropylidene-!3-Dfruct opyranose
0.85 0.67 0.68 0.5 1 0.48 0.67 0.66
D-Maiiiiose
D-Fructose L-R h d m n oSe L-Arabinose D-Ly xosr
0.85 0.89
TABLE 22.1 FRACTIONATION OF VARIOUS CARBOHYDRATES ON BIO-RAD AC SOW-X2 (Lit; 200-400 MESH) (BARKER el al., 1969a) Column, 150 X 0.6 crn; mobile phase, water; flow-rate, 0.23 ml/min; temperature, 25°C. Compound
Retention factor
Compound
Retention factor
Am ylopectin Dextran 80 Li N-Acetylneuraminic acid D-Gluconic acid Clyceric acid Maltohexaose Maltohexaitol Laniinaripentaose Raffinose
0.37 0.37 0.37 0.37 0.37 0.37 0.55 0.55 0.59 0.6 1
Laminaritriose Laminaribiose Maltose DGlucose D-Mannose D-Gakdctose L-Fucose Calactitol
(1.6 I 0.70 0.70 0.78 0.78 0.78 0.78 0.78 0.78 0.78
+
2-Acetamido-2-deoxy -D-glucose
2-Acet amido-2-deoxy-D-mannose
AG 50W-X2 (Li') is eluted with water, neutral saccharides are separated according to molecular size only; polysaccharides emerge from the column earlier than oligo- and monosaccharides. No resolution within a group of sugars of similar molecular weights was observed, even for aldose-alditol pairs. It is of interest that acidic carbohydrates such as N-acetyl-neuraminic acid and D -glucuronic acid were eluted in the same position as compounds of higher molecular weight, apparently due to the ion-exclusion principle. A comparison of several methods for the chromatography of the oligomeric sugars of the p-( 1+4)-linked D-xylose series (Havlicek and Samuelson) revealed that for preparative purposes permeation chromatography on polyacrylamide gel is inferior to the charcoalCelite method and to partition chromatography on ion-exchange resins. In the separation References p.519.
496
CARBOHYDRATES
of lower homologues (up to five residues), however, gel permeation chromatography on ionexchange resins in water was found to give the best results. The charcoalLCelite method was the least satisfactory for analytical purposes; on the other hand, this method was useful for preparative treatment in combination with LLC on ion-exchange resins, because a large amount can be handled and stepwise elution can be applied without disturbances. A disadvantage of partition chromatography is the use of high temperatures for the volatile ethanol-water solvent system; in addition, the reversed relationship between molecular weight of the polysaccharide and its solubility in ethanol limits the use of this solvent system to lower saccharides (Kesler). Disturbances due to the formation of ethyl glycosides occur above 75°C with 2-deoxyaldoses, and above 90°C with some others (Samuelson). Decomposition of ketoses and sucrose was observed when organic-base counter-ions were used (Hobbs and Lawrence, 1972b). The borate method may be more difficult for preparative treatment. Sometimes, transformations of sugars during the borate ion-exchange chromatography were observed (Carubelli, Kesler).
AMINO SUGARS Virtually the only means of separating mixtures of amino sugars and/or complex mixtures of amino sugars and amino acids is ion-exchange chromatography. Elution from the ion-exchange resin column is performed with dilute hydrochloric acid or with various buffers. For this purpose, amino acid analyzers have been often used, the column effluents being assayed by the reaction with ninhydrin (see p. 482). ,
Free amino sugars Ion-exchange chromatography with dilute hydrochloric acid as the mobile phase Hydrochloric acid was used as the mobile phase first by Gardell and later by other workers (Crumpton, Smith). Crumpton examined the effect of the normality of hydrochloric acid on the separation of D-giucosamine and D-gaiactosamine on Zeo-Karb 225 resin. He found that, within the range 0.2-0.9 M, the best resolution of the mixture was obtained with 0.33 M hydrochloric acid. The Rglucosaminevalues of several amino sugars and their derivatives are listed in Table 22.8. The same mobile phase proved to be effective even for the isolation of 3-amino-3,6dideoxy-D-glucose from the hydrolyzate of the phenol-soluble lipopolysaccharide C. freundii 8090 (Raff and Wheat). 3-Amino-3,6-dideoxy-D-glucose was separated from D-glucosamine on a Dowex 50 (H') resin column. The chromatographic properties of the above amino sugar compared with those of other known 3-amino-3,6-dideoxyhexoses (see Table 22.9) supported the proposed configuration.
Ion-exchange chromatography in buffered systems An exhaustive study of the cation-exchange chromatography of amino sugars was made by Brendel et al. (1967a). They examined the behaviour of 29 amino sugars by means of
497
AMINO SUGARS TABLE 22.8 Rglucosamine VALUES OF AMINO SUGARS AND THEIR DERIVATIVES (CRUMPTON) Zeo-Karb 225 (H'; 8% cross-linkage, particle size 0.33 M hydrochloric acid; flow-rate 2 ml/h.
> 200 mesh); column, 4 3 x 0.8 cm; mobile phase,
Compound
Rglucosamine
DGlucosaminuronic acid Isomurarnic acid DGlucosaminc D-Mannosamine Muramic acid DGulosamine D&aiactosdmine D -A II osa rn i ne D-Xy losam ine D-Fructosamine D-Talosamine D-Fucosamine
0.71 0.87 1.00
1.07 1.10 1.20
1.20 1.23 1.43 1 .so
1.60 1.94
TABLE 22.9 Rglucosamine VALUES OF SOME 3-AMINO-3,6-DIDEOXY HEXOSES (RAFI: AND WHEAT) Dowex 50 (H+); column, 50 X 1 cm; mobile phase, 0.33 M hydrochloric acid.
Cornpound
Rglucosamine
3-Amino-3,6-dideoxy-L-glucose 3-Amino-3,6-dideoxy-D-mannose 3-Amino-3,6-dideoxy-D-idose 3-Amino-3,6-dideoxy-D-galact ose 3-Amin~-3,6-dideoxy-L-talose
I .31 1.28 1.44 1 .58 2.37
a Technicon amino acid analyzer, using Chromobeads B resin (1 25 cm X 0.35 cm') and pyridine-acetate buffers at 35°C. It was found that the amino sugars examined emerged from the column in the following order: muramic acids, hexosaminuronic acids, hexosamines, deoxyhexosamines and methyl hexosaminides, diaminohexoses. Satisfactory resolution of amino sugars was achieved only for some of the amino saccharides; a single, complete run for all of the amino sugars and their derivatives examined failed. An almost linear gradient between 0.1 M pyridine-acetate buffer of pH 2.8 (5 .OM acetic acid) and 0.133 M pyridine-acetate buffer of pH 3.85 (0.82 M acetic acid) proved t o be successful for separating some hexosamines and their acid derivatives. Straight pyridineacetate buffers of 0.1-0.2 M, with a pH between 3.85 and 4.15, separated four out of six 2-amino-2-deoxyhexoses examined and were useful even for the separation of 2-amino2,6-dideoxyhexoses or 3-amino-3,6-dideoxyhexoses. 2,6-Diamino-2,6-dideoxyhexoses were partially resolved on a shorter column (20 cm), using 3.1 M pyridine-acetate buffer of pH 4.5. References p.519
CARBOHYDRATES
498
Yaguchi and Perry described the separation of seven 2-amino-2-deoxy-D-hexoses by means of a Technicon NC-1 amino acid analyzer, using a column of Chromobeads B resin (133 X 0.6 cm) jacketted at 60°C. Borate-citrate buffer of pH 7.24 was used for the elution at a flow-rate of 0.5 ml/min and was prepared by adjusting a mixture of 77.9 g of boric acid, 11.8 g of sodium citrate dihydrate, 100 ml of 2 M sodium hydroxide, 40 ml of Brij-35 solution (100 g in 300 ml of water) and 200 p1 of octanoic acid (preservative) t o pH 7.24 at 25°C with 6Mhydrochloric acid, the final volume being 41. Under similar conditions (Chromobeads A), Adams et al. separated all of the possible methyl ethers of 2-amino-2-deoxy-D-glucopyranose (see Fig. 22.1 5). Sodium citrate buffer of pH 4.40 was used for the elution; between the runs, the column was washed with 0.2 M sodium hydroxide for 30 min, and then regenerated with sodium citrate buffer of pH 3.10 (made in a similar manner t o the pH 4.4 buffer except that the pH was adjusted t o 3.1 with 6 M hydrochloric acid). This method could be of particular interest in the analysis of polysaccharides containing D-glucosamine. Bella and Kim studied the separation of amino alditols and amino sugars, and overcame the difficulties that had occurred in previous work on this topic (Donald, Weber and Winzler). The chromatography was performed at 65°C in less than 4 h by means of a Beckman-Spinco Model 120 C amino acid analyzer equipped with a 56 X 0.9 cm column of Beckman UR-30 resin. A citrdte-borate buffer of pH 5.06 (flow-rate 40 ml/h) was used for the elution and was prepared from 0.35 M sodium citrate buffer (pH 5.28) by adding 18.55 g/l of boric acid. The effluent was analyzed automatically (ninhydrin, flow-rate
7
8
9
10
11
12
13
14
15
I
I
I
I
1
I
16
17
18
19
20
21
TIME, h
Fig. 22.1 5 . Separation of methyl ethers of 2-amino-2-deoxy-D-glucopyranose (Adams el d.). Chromobeads A; column, 133 X 0.6 cm; mobile phase, sodium citrate buffer (pH 4.40);flow-rate, 0.5 ml/min; temperature, 60°C.1 = 2-amino-2deoxy-3-0-methyl-D-glucose; 2 = 2-amino-2deoxy-t-0-methyl-Dglucose; 3 = 2-amino-2-deoxyd-0-methyl-D-glucose; 4 = 2-amino-2deoxy-Dgalactose; 5 = 2-arnino-2deoxy4,6-di-O-methyl-D-glucose; 6 = 2-amino-2deoxy-3,4di-O-methyl-D-glucose; 7 = 2-amino-2deoxy-3,4,6-tri-O-methyl-D-glucose; 8 = 2-amino-2-deoxy-3,6-di-O-methyl-D-glucose.
AMINO SUGARS
499
20 ml/h). The following retention times were estimated (relative t o 11-ducosaniine): D-galactosaminitol 0.72, D-glucosaminitol 0.76 and D-galactosamine 1.1 2.
Mutual separation of amino sugars and amino acids Brendel’s method (see p . 4 9 6 ) was extended to the separation of mixtures of amino sugars and of amino acids (Brendel el al., 1967b). The effluent was assayed for amino groups (ninhydrinmethod) as well as for reducing properties (potassium hexacyanoferrate(II1) method). It was concluded that, using pyridine-acetic acid buffers of decreasing acid content and of increasing pyridine molarity and pH, amino sugars and amino acids,
/coo-
R-CK~~,: , emerged
from the column in the following simplified order: R containing - S 0 3 H , -OS03H, --OP03H, groups secondary amino acids muramic acids R con taining -COOH groups R containing alcoholic -OH groups R = alkyl R containing both -NH2 and - W O H groups hexosaminuronic acids R containing aryl groups R containing phenolic -OH groups monoamino sugars R containing -NH2 groups ammonia R containing stronglv basic groups diamino sugars Efficient mobile phases were found only for the resolution of small groups of the substances examined. Thus, 0.1 M pyridine-acetic acid buffer of pH 2.8 (5.OM acetic acid) proved t o be particularly useful for separating the acidic derivatives of amino acids and amino sugars. A gradient of the pyridine-acetic acid buffers of pH 2.8 and 3.85, respectively, allowed the separation of acidic and neutral amino acids from muramic acid and hexosaminuronic acids, as well as ducosamine, galactosamine, quinovosamine and fucosamine. A single buffer consisting of 0.1 33 M pyridine-acetic acid (0.82 M acetic acid), with a pH of 3.85, gave a satisfactory separation of 2-amino-2-deoxy-and 3-amino-3-deoxyhexosesand their 6deoxy derivatives without interference from any amino acids. Fig. 22.16 illustrates the separation of a mixture containing D-ducosamine, D-galactosamine and various amino acids (Monsigny), carried out a t 60°C on a Technicon amino acid analyzer equipped with nine compartments (Autograd) and a 65 X 0.6 cm column of Chromobeads (2-2. Each of the buffers used contained 17.85 g/l of sodium citrate pentahydrate and 5 g/1 of Brij-35 and was adjusted t o the desired pH with 5.6 M hydrochloric acid. The buffer of pH 2.75 contained 10%of methanol and 0.5% of thiodiglycol, buffers of pH 2.875 and 3.80 contained 0.05% of thiodiglycol, and the buffer of pH 6.10 was 1 M in sodium chloride. The gradient used is shown in Table 22.10. References p.519
500
CARBOHYDRATES
9
a
h
n
12
t i g . 22.16. Separation of a mixture of D-glucosamine, D-galactosamine and various amino acids (Monsigny). Chrornobeads C-2; column, 65 X 0.6 cm; mobile phase, see Table 22.10; temperature, 60°C. I = Glycine; 2 = alanine; 3 = Dglucnsaniine; 4 = D-galactosamine; 5 = valine; 6 = cysteine; 7 = methionine; 8 = isoleucine; 9 = leucine; 10 = norleucine; 11 = tyrosine; 12 = phenylalanine; 1 3 = ammonia.
Derivatives ofamino sugars and chromatographic methods used in the synthesis of amino sugars The separation of amino sugars and their isolation from natural or synthetic mixtures can also be achieved in an indirect manner as their derivatives. The chromatographic behaviour of amino sugar derivatives is determined by the number and the nature of the substituents. It is generally true that if the amino group of an amino sugar is blocked, the chromatographic behaviour of the derivative formed no longer resembles that of the parent arninosaccharide. For instance, the chromatographic properties of N-acetylated amino sugars resemble those of free neutral saccharides rather than those of amino sugars. This phenomenon affects the choice of the chromatographic technique t o be used for a particular separation. Whereas for free amino sugars or derivatives that still carry an amino group, cation-exchange chromatography is the method almost exclusively used, carbon column and cellulose column chromatography are of importance in the resolution of N-acetylated sugars (see, for instance, Gibbs et al., Kuhn et al., Perry and Webb). Less polar amino sugar derivatives were mos; frequently separated on silica gel (Albano and Horton, Jeanloz et al.) or on alumina (Capek and Jary). In the synthesis of amino sugars, it is sometimes advantageous to separate the isomers already in form of precursors of amino sugars. For example, the nitromethane condensation of “dialdehyde” formed in the periodate oxidation of methyl a-L-rhamnopyranoside
50I
SUGAR DERIVATIVES
'I'ABI-E 22.10 COMPOSITION OF THE BUFFER GRADIENT ENABLING T H E SEPARATION O r II-GLUCOSAMINE, D-GALACTCSAMINE AND VARIOUS AMINO ACIDS (MONSIGNY) See Fig. 22.16. Compartment
Volume (ml)
NO.
pH 2.75
pH 2.875
pH 3.80
pH 6.10
2.5 M NaCl
Methanol
3
produces a mixture of four methyl 3,6-dideoxy-3-nitroa-~-hexopyrdnosides, 2.5 g of which is than applied on a 40 X 2 cm silica gel column; 100: 1 benzene-ethanol elutes the isomer with the taloconfiguration, 100:1.5 benzene-ethanol elutes the manno- and gluco-isomers (only partially resolved) and 100:3 benzene-ethanol elutes the galactoisomer (Baer and Capek). The alumina column chromatography of azido sugars has been reported, e.g., by Cuthrie and Murphy.
SUGAR DERIVATIVES
Alditols Owing to the number of hydroxyl groups present in the molecule, the methods used for the separation of alditols resemble those for free sugars (see p. 483). In these methods, the most frequently used sorbents are carbon, cellulose and ion-exchange resins. Chromatography on charcoal A carbon column (50% Celite) proved to be satisfactory for the fractionation of the carbohydrate mixture obtained from extracts of Umbilicaria pustulata (L.) Hoffm., when stepwise elution with aqueous ethanol ( 1 -15%) was used (Lindberg and Wickberg). Monitored by optical rotation measurements, fractions containing arabinitol, mannitol, a,&-trdhalose, umbilicin and sucrose were collected.
Chromatography on cellulose Richtmyer (1970a) reported the separation of a mixture of polyhydric alcohols, obtained from avocado seeds, on cellulose columns. For other applications, see Begbie and Richtmyer, Richtmyer (1970b, c), etc. References p.519
v1
TABLE 22.1 1 VOLUME DlSTRlBUTlON CONSTANTS OF VARIOUS ALDITOLS, ALDOSES AND SIMPLE ALIPHATIC CARBONYL COMPOUNDS ON DOWEX 50W-X8 (Li+,Na' AND K') AT 75°C AND VARIOUS ETHANOL CONCENTRATIONS(SAMUELSON AND STROMBERG, 1968) Compound
h)
Volume distribution constants 80% ethanol Li+
Na+
85%ethanol K'
lj+
Na'
90% ethanol
K+
Li+
Na+
95% cthanol
K'
Li'
97% ethanol
Na'
K+ ~
Ethylene glycol Formaldehyde Glycolaldehyde Glyceraldehyde Glycerol Ery throse Ery thri to1 Ribitol Arabinitol Xylitol Mamitol Galactitol Glucitol Xylose Arabinose Glucose Mannose Galactose
0
0.81 0.19 0.37 0.52 1.2 1.0 1.6 2.1 2.5 2.8 3.5 4.1 3.8 1.7 2.0
0.71 0.21 0.45 0.71 1.0 1.4 1.4 2.5 3.2 3.5 4.5 2.2 3.0 3.6 3.6 4.5
0.61 0.18 0.45 0.82 1 .o 1.6 1.6
0.84 0.17 0.37 0.59 1.4 1.1 2.1 3.0 3.7 4.1 5.5 6.7 6.1 1.7 2.2 2.8 2.8 3.4
0.68 0.16 0.41 0.70 1.2 1.7 1.8 2.6 3.4 4.5 5.1 6.7 6.9 2.9 4.2 5.3 5.3 6.7
0.68 0.48 0.92 1.1
1.8 3.1 4.1 4.7 6.0
0.95 0.1 6 0.37 0.67 1.7 1.4 2.9 4.4 5.7 6.4 9.5 11.8 10.4 2.1 2.8 3.8 3.8 4.7
0.72 0.13 0.40 0.75 1.4 2.3 2.7 4.1 5.7 7.9 9.6 13.1 13.4 4.6 6.9 9.7 9.7 12.5
0.66 0.16 0.48 1.1 1.5 2.7 4.4 5.4 7.6 9.1 11.6 12.2
1.1 0.17 0.39 0.80 2.2 1.8 4.3
0.93 0.10 0.48 1.3 2.5 4.2 5.4
~~
1.1 0.25 0.74 2.1 3.0 6.5
Lit
Na'
K'
-
1.2 0.16 0.40 0.87 2.7 2.1 5.5
1.2 0.10 0.67 1.8 3.6 6.3 9 .O
1.3 0.25 0.95
2.9 4.3 10.5
503
SUGAR DERIVATIVES
A mixture of polyhydric alcohols (8.4 g) was mixed with powdered cellulose and transferred to the top of a column (34 X 3.7 cm) containing washed cellulose powder (Whatman); 14-ml fractions were collected as soon as a sample of the eluate left a residue o n evaporation. With water-saturated n-butanol-pure n-butanol (1 :3) as the mobile phase, glycerol (fractions 1 -SO), D-arabinitol (1 26-175), galactitol (301 -625), volemitol and a “perseitol-octitol” fraction were eluted. The last-mentioned fraction (5.9 g) could be re-chromatographed on a larger cellulose column (83 X 5 cm) by elution with watersaturated n-butanol-pure n-butanol (1 : 1 ), giving perseitol, D-etyfhro-D-galacfo-octitol and nzyo-inositol (Richtmyer, 1970a).
Chromatography on ion-exchange resins The separation of alditols by LLC on ionexchange resins has been studied in detail by Samuelson and Stromberg (1966). The successful resolution of alditols on the anionexchange resin Technicon T5B (SO:-) is illustrated in Fig. 22.17. The efficiency of resolution is dependent on the temperature and the exchange capacity of the resin. The great influence of the form of the cation-exchange resin o n the distribution constant was described later by Samuelson and Stromberg (1968). It follows from Table 22.1 1 that the most favourable separations are achieved with the lithium form of the resin.
5
1 200
3M)
400
VOLUME. ml
500
700
Fig. 22.1 7. Separation of various alditols and monosaccharides (Samuelson and Stromberg, 1966). Technicon T , B (SO:-); resin bed, 85.2 X 0.6 cm; mobile phase, 86% ethanol; flow-rate, 2.51 ml/ min . an’; temperature, 75.5”C. 1 = glycerol; 2 = erythritol; 3 = xylitol; 4 = arabinitol; 5 = arabinose; 6 = xylose; 7 = glucitol; 8 = mannose; 9 = galactitol; 10 = mannitol; 11 = galactose; 12 = glucose.
References p.519
SO4
CARBOHYDRATES
Dowex 50W-X8 (Li') with a particle size of 14-17 pm and an exchange capacity of 5.1 mequiv./g of dry resin (H') in a water-jacketted glass column (100-150 cm length, 1.2-2.6 cm I.D.) at a flow-rate of 1-5 ml/min .cm2 is used for separations such as that shown in Fig. 22.1. The chromatogram in Fig. 22.1 was obtained from a run at 75°C in 85% ethanol, which took ca. 5 h. Before this type of column is packed, the resin is slurried with boiled aqueous ethanol of the same concentration as that used as the mobile phase, and is kept in this solution so that all air bubbles disappear. After sedimentation, a concentrated slurry (1 :2, v/v) is poured in the column and the mobile phase is pumped through until a uniform resin bed is formed. After the column has been packed, mobile phase should be circulated through it for at least 16 h before a chromatographic run is started. With the use of a column which is about 25 cm longer than the resin bed, the mobile phase can be pre-heated inside the column even when high flow-rates are used, so that a separate pre-heater can be omitted. The sample is applied on to the column as a solution in ethanol of the same concentration as that used as the mobile phase, in order to prevent swelling changes. If the temperature in the column is above the boiling-point of the mobile phase at atmospheric pressure, it is important to allow the temperature t o decrease before removing the top fitting. A stainless-steel piston pump is used to feed the mobile phase into the column. The pressure is followed on a manometer equipped with a circuit-breaker that stops the pump and heating baths if the desired pressure (80 atm) is exceeded or if the pressure decreases because of leakage. Periodate oxidation is used for detection. A frequently used method for the separation of alditols is based on the resolution of their borate complexes on strongly basic anion-exchange resins in the borate form. Thus, Spencer succeeded in separating a mixture of glycerol, threitol, erythritol, xylitol, arabinitol, ribitol, glucitol, galactitol and mannitol, using De Acidite FF (BOi-),and 0.18 and 0.36 M boric acid (adjusted to pH 9 with triethylamine) at 35°C as the mobile phase. For micromolar amounts, a 60 X 0.8 cm column with a flow-rate of 25 ml/h was used. Rhamnitol and fucitol overlapped with arabinitol and glucitol, respectively. A substantial temperature dependence of the resolving power was observed.
Glycosides In the synthesis of glycosides, the most common task is the separation of the resulting mixture of anomers, and/or the removal of the unreacted starting sugar or its derivative. This problem is also very frequent in the Koenigs-Knorr and Fischer syntheses of glycosides and oligosaccharides.
Clycosides with simple aglycones The chromatography of these unsubstituted glycosides is similar to that of free monosaccharides, i.e., LLC and IEC are now preferably used. For partition chromatography, cellulose (for a review see Mowery) is still of great
SUGAR DERIVATIVES
505
interest. Thus, for example, Yoshida etal. (1969b) resolved a mixture (ca. 10 g) of ethyl p-D-galactofuranoside, ethyl a-D -galactopyranoside and ethyl P-D-galactopyranoside (a-furanoside was not isolated) on a 80 X 4.5 cm column of Whatman CF-I 1 cellulose powder, with ethyl acetate-n-propanol-water (5:3:2) as the mobile phase. For detection, the optical rotation of the eluate was measured. The most promising preparative method seems to involve the utilization of a strongly basic ion-exchange resin in the hydroxide form, such as Dowex 1-X2 (OH-; 200-400 mesh); any free sugar present in the mixture is retained by the resin (Roseman ef a].). Water is used as the mobile phase in such a separation, which is rapid and gives high recoveries; in general, furanosides are adsorbed more strongly than pyranosides. The efficiency of Dowex 2, which has a smaller particle size but a higher degree of crosslinkage (8%) than the above Dowex 1 resin, was not sufficient for the complete separation of methyl D-glucopyranosides (Austin et u1. ). Dowex 1 resin (450 g; 2% cross-linked; 200-400 mesh) was recycled twice between the hydroxide and chloride forms with 2 M sodium hydroxide and 2 M hydrochloric acid. The resin was finally washed in an acrylic column (60 X 3 cm) with deionized, distilled, carbon dioxide-free water. The sample (1-3 g) was dissolved in 3 ml of water and washed on to the column with 1 ml of water. The column was then eluted with deionized, distilled, carbon dioxide-free water from a reservoir fitted with a soda-lime trap. After 400 ml of the mobile phase had been consumed (flow-rate 27-30 d / h ; peristaltic pump), 5-ml fractions were collected and monitored by optical rotation (Neuberger and Wilson). The differences between the volume distribution constants of methyl a- and p-Dglucopyranoside, methyl a- and b-D -mannopyranoside and methyl a- and 13-D-gdactopyranoside were discussed in terms of the anomeric effect and other forms of dipole interactions by Neuberger and Wilson (see also p. 474). However, Evans ef al. assumed from the influence of the C5 substituent of the pyranose ring on the K , value that factors other than the sugar hydroxyl acidity can also be important in separations on resins in the hydroxide form. It should be pointed out that the presence of an amino group in the molecule may cause the free aldose to be eluted from the resin, see, for example, the separation of methyl 3,6-diamino-3,6-dideoxy-aand 4-D-glucopyranoside from 3,6-diamino-3,6dideoxy-D-glucose on Dowex 1 -X2 (OH-) with water as the mobile phase (Kovaf and Jary). With the chloride form of the resin and water as the mobile phase, the sorption probably involves Van der Waals and polar interactions (Evans et al.). For the separation of the a- and 0-anomers of methyl D -glucopyranoside, methyl 6-deoxy-D-glucopyranoside and methyl 6-chloro-6-deoxy-~-glucopyranoside,75% aqueous n-propanol was also used instead of water. The K , values decrease with changes in the substituent on the C5 atom of the pyranose ring: -CH20H > -CH2CI > -CH3. As the order of elution is the same with the chloride and the hydroxide forms of the resin, Evans et al. concluded that the separation is mainly due to partition in both instances. These methods can also be used for analytical purposes by combining them with an automated analytical procedure. However, GLC of the trimethylsilyl derivatives seems to be more efficient (see for instance Evans er al.; Smirnyagin and Bishop; Yoshida er al., 1969a). References p.519
506
CARBOHYDRATES
Complex glycosides With complex glycosides, a bulky aglycone becomes one of the dominating factors influencing the chromatography, so that besides LLC even GPC can be used (Table 22.1 2). By this means, the separation of sucrose, D -glucose and their phenolic glycosides from the bark and the leaves of Populus tremula was achieved by gel filtration on Sephadex G-25 and LH-20, using water as the mobile phase (RepaS and Nikolin; RepaS et al.). However, in synthetic applications (Koenigs-Knorr synthesis, etc.) the separation is usually carried out before the removal of the screening groups, by means of LSC; the solvent system chosen usually follows from TLC, which is used also for the detection. For several examples, see Table 22.12. The same is also true for internal glycosides and all anhydro-sugars. The use of cellulose or ion-exchange resins for free anhydro derivatives, and silica gel for substituted derivatives, is advisable (Table 22.12).
Ethers and acetals For the separation of di-, tri- and tetra-0-methyl derivatives of aldoses, ketoses and their glycosides, respectively, silica gel, alumina, cellulose and Celite were used (see Table 22.13). A large number of applications of these methods in the analysis of natural compounds were listed in a book by Lederer; recently, the GLC-MS method, applied after reduction of the compounds to the corresponding alditols and acetylation or trifluoroacetylation, has become the most widely used technique for analytical purposes. Kefurt et al. described the separation of a methylation mixture (1.6 g) of methyl 4,6dideoxy-a-D-xylo-hexopyranoside on a silica gel column (90 g, 40 X 2.8 cm, 70-200 pm) with benzene-ethanol mixtures as the mobile phase. After a volume of 1800 ml (in 100-ml fractions) of the effluent (0.5% ethanol) had been discarded, 50-ml fractions were collected and the di-O-methyl derivative (0.5% ethanol, fractions 19-22), 3-0-methyl ether (0.5%; fractions 30-35), 2-0-methyl ether (1 .O%; fractions 45-50) and starting diol(2.0%; fractions 61-70) were eluted. For the separation of an analogous mixture with the lyxo configuration, the use of an alumina column resulted in better resolution; with silica gel, the mono-0-methyl ethers overlapped t o a serious extent. For the separation of benzyl ethers, LSC on silica gel can always be used (Table 22.1 3). Good resolution can be obtained even when the solutes are very similar in structure, such as per-0-benzyl a p - , and P,P-trehalose, followed by 2,3,4,6-tetra-O-benzyl-D-glucopyranose (Micheel and Pick). For analytical purposes, ca. 20 mg of the mixture were eluted from a 15 x 1.2 cm column of Merck silica gel (0.08 mm) with water-saturated methYlene chloride-ethyl acetate-methanol (100: 1.3:0.75) at a flow-rate of 20 ml/h. The effluent, containing the above compounds in the order given, was monitored continuously for benzyl ethers with a UV photometer at a wavelength of 255 nm; one run required ca. 2 h. By blocking the hydroxyl groups of carbohydrates with acetal groups, e.g., with isopropylidene, benzylidene or ethylidene groups, one can achieve a significant increase in the hydrophobicity of the molecule; thus, LSC is most frequently used for the separation of such compounds (see Table 22.13). In some instances, the resolution of diastereoiso-
SUGAR DERIVATIVES
507
meric -ylidene derivatives can also be achieved, e.g., from the diastereoisomeric benzylidene derivatives of methyl 4,6-0-benzylidene-2,3-di-O-methyla-~-glucopyranoside, the “unusual” (S) isomer being eluted first (silica gel; 4: 1 benzene-diethyl ether; Bagget et al.). Sometimes, alumina is used with an aqueous solvent: see, for instance, Bonner e f al., who separated 2,3-O-butylidene-D-glucitol from D-glucitol with 93-100% aqueous ethanol as the mobile phase.
Esters
I n the synthesis of sugar esters, the starting compound and the product usually differ substantially in their chromatographic mobilities, so that their separation by LSC is not difficult. However, the separation of mixtures arising from the partial acylation of sugars or their derivatives requires more care. Here, the problem of the quantitative separation of substances, starting with per-0-acylated derivatives, followed by different incompletely substituted derivatives and ending with unreacted starting material, is usually solved by utilizing gradient elution. In the chromatography of a partial acetylation mixture of methyl 3-acetamido-3,6dideoxya-D-mannopyranoside on silica gel (70-200 ym), 100:3 benzene-ethanol first elutes the 2,4-di-O-acetyl derivative. Then the 4-O-acetate, followed by the 2-O-acetate, are obtained with 20: 1 benzene-ethanol, and for the elution of the starting compound, 10:1 benzene-ethanol should be used (kapek et al., 1968b). These solvent systems were successfully used in the separation of analogous mixtures of other configurations (Capek etal., 1967, 1968a, 1970b). Some other applications are given in Table 22.1 3. As the K , values of positional isomers differ only slightly, the choice of an appropriate solvent system and sorbent is very important because these systems affect the K , ratios of the different isomers. Thus, 2,3-di-O-tosyl-, 2-O-tosyl-, 3-0-tosyl- and methyl 4,6-0benzylidene-0-D-glucopyranoside, the first two being virtually unresolved in chloroform and 20: 1 chloroform-methanol on alumina (Guthrie er al.), can be separated without difficulty with 10: 1 benzene-diethyl ether on silica gel (Stantk et al., 1974). The same is true for the corresponding benzoates. The fact that alkaline alumina may sometimes cause deacetylation (see p. 469) was used in the partial deacetylation of various per-0-acetyl derivatives; alumina served simultaneously as the reagent and as the sorbent (Jary era).). Recently, GLC (particularly when combined with MS) of volatile derivatives has become more frequently used in analytical studies of partially acylated derivatives of saccharides (see, e.g., De Belder and Norrman).
Sugar acids The method most widely used for the analysis and separation of uronic, aldobiuronic, aldonic and other sugar acids is based on anion-exchange chromatography. This method, in connection with carbon-Celite or cellulose chromatography, is also used for the isolation of these acids from natural sources. References p.519
wl 0
TABLE 22.12 SEPARATION OF GLYCOSlDES AND THEIR DERlVATlVES
00
Compounds separated
Sorbent
Mobile phase
Reference
Note
Anomeric butyl 2-acetamido-2deoxy-3,4,6-tri-O-methyl-Dgl ucopy ranosides
Silica gel, 0.05-0.2 mm (Merck)
Dichloromethanediethyl ethermethanol (20:lO:l)
Salo and Fletcher
The ol-anomer was eluted
Methyl [methyl 3,4-O-isopropylidene-
Silica gel, 75-250 mesh (Merck)
Benzene-acetone (8:2)
Sip05 and Bauer
Silica gel
Benzene-ethyl acetate ( 1 :1)
Chalk et al.
Dichloromethane diethyl ether (20:3)
Shapiro et al., 1967
2-0-(2,3,4,6-tetra-O-acetyI-p-Dglucopyranosyl)-a-D-gdactopyranosid]
first
uronate Corresponding a-D-( 1+2)-linked disaccharide Benzyl 2-0-(2,3,5-tri-O-benzoyla-Larabinofuranosyl)-3,4-O-isopropylidenep-L-arabinopyranoside 1,3,5- Tri-Obenzoyl-p-L-arabinofuranose
2,3,5-Tri-Obenzoyla-L-arabinofuranosyl bromide Benzyl3,4-O-isopropylideneQ-Larabinopyranoside Benzy12-0-(2,3,5-tri-Obenzoyl-a-Lara binofuranosy1)Q-Larabinopyranoside
2-O-Acetyl-l,6anhydro-3-0-(3,4,6-tri-OSilica gel, benzoyl-2deoxy-2dichloroacetamido-p-D-Davison grade 950, glucopyranosyl)$-D-galactopyranose Corresponding 1-4-linked disaccharide
60-200 mesh
Dry column method was used
%a 2 3
2
l,$-Anhydr0-2,3-O-isopropylidene-p-Dlyxofuranose Methyl 2,3-O-isopropylidene-p-Lribofuranoside Methyl 2,3-O-isopropylidene-D-lyxoside Methyl 2.3-O-isopropylidenea-Lribofuranoside and p- anoiners of 3p-(2deoxy-D4yxohexopyranosyloxy )-I 40-hydroxydpcard-20(22)-enoCde
Silica gel (15 x 2.5 cm)
Toluene -acet one (17:3)
If
Brimacombe et al., 19681,
0 m
Silica gel (50-200 Nm), 220 g premixed with 100 ml of water
B t d . aq. ethyl acetate
Zorbach ct al.
I ,2-O-Ethylenea-D-glucofuranose 1.2-O-Ethylene4-D-glucopyranose 1,2-O-Ethylene-cu-D-glucopyranose 2-042-Hydroxyethyl)-D-glucose
Whatman No. I cellulose (69 x 7 cm)
Satd. aq. n-butanol
Srivastava et al.
Sucrose, D-glucose, salicin, tremuloidin
Sephadex G-25 (106 x 1 cm)
Water
Repa's and Nikolin
1,4-Anhydro-D-altritoI and 1,4-anhydroD-mannitol 3,6-Anhydro-D-altritol 1,s-Anhydro-L-glucitol
Whatman (standard grade) cellulose (50 X 4 cm)
Acetone-water (9:l)
Buchanan and Edgar
1 g of the sample was applied
Dowex 1-X2 (OH-) (50 ml)
Water
Buchananand Edgar
0.4 g of the sample was applied
Cellulose (150g)
Satd. aq. n-butanol
?ern$ e t a [ .
1 g of sample
0-
1,6-A nhy dr 04d e o x y -p-D-ribohexopyranose 4-Deoxy-D+ibo-hexose
wl
TABLE 22.13 SEPARATION OF ETHERS, ACETALS AND ESTERS
c.
0
Compounds separated
Mobile phase
Sorbent
Reference
Methyl 3,6-anhydro-2-O-methyl-fl-D-galactopyranoside Methyl 2,3,6-tri-O-methyl-fl-D-galactopyranoside Methyl 2,3di-O-methyl-fl-Dgalactopyranoside
Ethyl acetate Ethyl acetate Methanol
Silica gel
Brimacombe and Chmg
Mixture of methyl terminal4-O-methylmalto-oligosaccharides
Water -ethanol (1 -15% ethanol)
Deactivated carbon-Celite ( 1 : l )
BeMiller and Wing
Various methyl ethers of D-galactose and L-arabinose
Light petroleumn-butanol(7:3) satd. with water
Cellulose
Anderson and Cree
Benzyl alcohol Benzyl 2,3,4-tri-O-benzyl-fl-D-ribopyranoside 2,3,4-Tri-O-benzyl-D-ribose
Benzene-diethyl ether (9: 1)
Silica gel
Tejima ef al.
Benzyl ethers of 1,6-anhydro-fl-D-glucopyranose
Chloroform-ethyl acetate ( 4 : l )
Silica gel
Seib
1,2 :3,5-Di-Oisopropylidenea-Dapio-L-furanose 1,2 :3 ,S-DiQisopropylidenea-Dapio-D-furanose
Diethyl ether-nhexane (1:l)
Silica gel
Ball et a1
3-Deoxy-l,2:5,6di-O-isopropylidene-D-x~vZo-hexofuranose Benzene-diethyl ether 3-Deoxy-3-fluoro-l,2 :5,6di-O-isopropylidene-a-Dmixtures galactofuranose
Silica gel
Brimacombe et al., 1968a
I ,2 :3,4-Di-O-isopropylidene4-O-(nwthylthio)methyla-D-
Silica gel
Benzene-diethyl ether galactopyranose (9:l) 6-O-Acetyl-l,2:3,4di-O-isopropylidenea-D-galactopyranose
c1 9
Godman and Horton
5 d P
1CCyclohexyl-2,3 :4,5di-O-isopropylidene-Dg~uco-pentitol 1CCyclohexyl-2,3 :4,$-di-0-isopropylidene-Dmnno-pentitol
Diethyl ether-light petroleum (3:17)
Silica gel
Inch et al,
3-0-Benzyl-6 ,7dideoxy-l,2-O-isopropylidene~-Dglucohept-6-ynofuranose
Dichloromethane diethyl ether ( 3 : l )
Silica gel
Harton and Swanson
~
2 c
3-0-Benzy1-6,7dideoxy-1,2-O-isopropylidene-p-L-id~hept-6-y nofuranose
P
2
c
2,4-Di-O-acetyl-l,6-anhydro-/.3-D-glucopyranose 3,4-Di-O-acetyl-l,6-anhydro-p-D-glucopyranose 2,3-Di-O-acetyl-l,6-anhydro-p-D-glucopyranose
Methylene chlorideethyl acetate ( 1 : l )
Silica gel
Shapiro et al., 1970
Oc ta-0-ace ty I-p-ma1t ose 1,2,6,2' ,3 ' ,4'.6'-Hep ta-0-ace t y I$-ma1 tose
Benzene-ethyl acetate (1:l)
Silica gel
Dick et al.
Silicic acid
Tulloch and Hill
Methyl Methyl Methyl Methyl
%
2.3-di-O-acety14.6-0-benzyLidene-p-D-glucopyranaside n-Hexane-chloroform (1 :1) 2-0-acety14,6-O-benzylidene-fl-D-glucopyranoside (1:l) 3-0-acetyl4,6-0-benzylidene-fl-D-glucopyranoside Chloroform 4,6-O-benzylidene-~-D-glucopyranoside Chloroform
E
512
CARBOHYDRATES
Uronic acids The first successful separation of uronic acids on anion exchangers was reported by Khym and Doherty, who separated galacturonic acid and glucuronic acid on Dowex 1 (CH3COO-) with 0.15 M acetic acid as the mobile phase. Free saccharides (arabinose and galactose), which were not adsorbed, were collected in the first fraction. Larsen and Haug described the separation of glucuronic acid and mannuronic acid from each other and from a partially resolved mixture of guluronic and galacturonic acids on a Dowex 1-X8 (CH,COO-) column (45 X 2 cm), with a linear gradient of acetic acid (from 0.5 to 2.0 M) at a flow-rate of 0.3-0.5 ml/min. Johnson and Samuelson successfully separated a mixture of 4-0-methyl-D-glucuronic, D-galacturonic, L-gului onic, D-glUCUrOniC and D-mannuronic acids at 3OoC on a Dowex 1-X8 column (88 X 0.6 cm), using as the mobile phase 0.05 M sodium acetate solution buffered with acetic acid a t pH 5.9 (flow-rate 1.06 ml/min). The effluent was analyzed with a two-channel detector (carbazole and chromic acid methods). In another paper, Carlsson and Samuelson (1970) established the distribution constants of various uronic acids (Table 22.14). A four-channel detector (see p. 481) was used for the analysis of the effluent. The separation of oligogalacturonic acids (products of the enzymatic hydrolysis of pectic acid) was reported by several workers. Derungs and Deuel separated a mixture of mono-, di-, tri- and tetragalacturonic acids on Dowex 3 (HCOO-) with a formic acid gradient (0.1-2.5 M ) . Reid separated galacturonic acid and di- and trigalacturonic acids on De Acidite FF resin (HCOO-) with a linear formic acid gradient (0.2-0.5 hf). Nagel and Wilson resolved a series of oligogalacturonic acids, including di-, tri-, tetra-, penta-, hexa-, hepta- and octagalacturonic acids, on a Dowex 1-X8(HCOO-) column (100 X 3 cm), using 8 1 of 0.2-0.9 M sodium formate (linear gradient). The carbazole method was used to analyze the effluent. Nagel and Wilson also described the separation of unsaturated di-, tri-, tetra- and pentagalacturonic acids, which was performed under similar conditions. However, stepwise elution was preferred in this separation. For instance, for the resolution of this mixture of unsaturated acids, the sodium formate elution scheme was as follows: 0.06 M , 1.5 1; 0.08 M , 1.5 1; 0.1 M , 1.5 1; 0.2 M , 0.9 I; 0.3M,1.5 1 ; 0 . 4 M , 3 1;0.5M,2.81;0.6M,2l;and0.7M,2l.Theunsaturatedacids were detected by measuring the absorbance at 232 nm. The formation of sugar-borate complexes was used by HallCn for the separation of galacturonic and glucuronic acids on Dowex 2-X8 resin. Mannose, fucose, galactose and glucose were also present; these neutral sugars were eluted at room temperature with 0.01 M borax in 0.2 N sodium hydrogen carbonate, and, after the appearance of the last neutral saccharide peak, the uronic acids were eluted with 0.03 M borax in 0.6 M sodium hydrogen carbonate (flow-rate 1.5 ml/min). The analytical methods mentioned above were applied by Carlsson et al. (1969) to the separation of the acids obtained by the isomerization of D-glucuronic acid in neutral aqueous solution; D Jyxo-5-hexulosonic acid, D -alluronic acid, the unresolved mixture of D-altruronic and D-mannuronic acids, D-glucuronic acid and ~-ribo-5-hexulosonicacid were eluted with 1 M acetic acid from Dowex 1-X8 resin. The unresolved mixture of D-altruronic and D-mannuronic acids was re-chromatographed with 0.08 M sodium acetate.
513
SUGAR DERIVATIVES
TABLE 22.14 VOLUME DISTRIBUTION CONSTANTS OF ALDOBIURONIC, HBXURONLC AND HEXULOSONIC ACIDS (CARLSSON AND SAMUELSON, 1970) Dowex 1-X8 (24-27 p m ) ; column, 76 X 0.6 cm; mobile phase, 0.5 Macetic acid, 1 Macetic acid or 0.08 Msodium acetate adjusted with acetic acid to pH 5 . 9 ; flow-rate 4.4 ml/min .cm2; temperature, 30"C. ~~
Acid
Volume distribution constant Acetic acid (0.5 M)
Acetic acid (1 M )
Sodium acetatc (0.08 M ) ~
2-O-(c~-D-Caldctopyran~syluronic acid)L-rhamnose 4-0-(a-DGalactopyranosyluronic acid)D-Xylose 6-O-(p-D-Glucopyranosyluronicacid)-Dgalactose 2-O-(4-O-Methyla-Dplucopyranosyluronic acid)-D-xylose Cellohiuronic acid Alluronic acid Galacturonic acid Glucuronic acid Guluronic acid Mannuronic acid Taluronic acid 4-0-Methyl-D-plucuronicacid arab i n 0 5 -H ex ulosonic acid 17x0-5-Hexulosonic acid rzbo-5-Hexulosonic acid x.ylo4-Hexulosonic acid
8.4
4.16
12.0 15.5
13.2 26.0
6.0 17.1 11.0 22.4 12.3 18.6 14.2 18.0
13.2 15.4 28.2 22.3
9.4 12.8 14.2 8 .o
17.4
Other sugar acids Samuelson and Thede reported the distribution constants of 16 aldonic acids and some other sugar acids, separated at 30°C on Dowex 1-X8 (Table 22.15). Acetic acid (1 M) or sodium acetate (0.08 M) buffered with acetic acid t o pH 5.9 were used for elution. It can be seen that, within the aldonic acid series, acids that contain a greater number of hydroxyl groups appear in the sodium acetate effluent earlier than those with a smaller number of hydroxyl groups. This elution pattern, in which mannonic and Dglycero-L-manna-heptonic acids are exceptions, could be explained by the assumption that the hydrated ionic volumes have a predominating influence on the uptake of the anions, those with a smaller hydrated volume being held more firmly by the resin. The dissociation constants of particular aldonic acids probably determine their elution pattern in acetic acid. This mobile phase gives more favourable separation factors and is therefore more suitable for the fractionation of isomers. A mixture of the non-volatile monoprotic acids isolated from a hydrolyzate of unbleached cotton (Larsson and Samuelson, 1969) was analyzed by the above method; 2-0{4-0-methylReferences p.519
CARBOHYDRATES
514
‘TABLE 22.15 VOLUME DISTRIBUTION CONSTANTS O F VARIOUS SUGAR ACIDS (SAMUELSON AND THEDE) Dowex 1-X8 (26-32 bm);column, 135 X 0.6 cm; mobile phase, 0.5 M acetic acid or 0.08 M sodium acetate adjusted with acetic acid t o pH 5.9; flow-rate, 2.8-7.7 ml/min .cmz;temperature, 30°C. Acid
Volume distribution constant ~~
Acetic acid
Sodium acetate
18.6 20.2 18.8 19.5 14.2 19.5 9.1 7 15.7 11.3 12.5 13.5 17.5 6.25 14.2 10.8 19.3
14.8 11.9 10.6 8.89 10.3 9.24 8.19 7.51 7.21 1.70 9.49 7.37 7.70 6.63 10.9
Aldobionic acids. Cellobionic Lactobionic Maltobionic Melibionic
5.86 5.09 7.35 4.62
3.71 3.15 3.69 2.64
Methylated aldonic acids 2,3,5-Tri-O-methyl-D-galactonic 3,5,6-Tri-O-methyI-D-gluconic 2,3,4,6-Tetr~-O-methyI-D-gluconic
5.38 7.1 2 4.76
2.64 3.90 2.1 8
Saccharinic acids 2-Hydroxypropionic (lactic) D-tfrreo-2,3-Dihydroxybutyric 2,4-Dihydroxybutyric 3.4-Dihydroxybut yric 2-Methyl-2,3dihydroxypropionic D-threo-2,4,5-Trihydroxyvaleric ol-D-GI ucoisosaccharinic P-DGlucoisosaccharinic ol-D-Glucosaccharinic ol-D-Glucornetasaccharinic p-D~~ucomeldsdccharinic
15.1 16.9 14.6 3.39 14.4 11.8 6.09 14.8 5.41 6.84 9.56
13.8 12.0 11.7 9.31 11.1 9.1 7 6.06 6.46 6.72 7.16 7.59
Aldonic acids Glycolic Glyceric D-Erythronic D-Threonic D-Arabinonic D-Lyxonic D-Ribonic D-Xy Ionic DGalactonic DGluconic D-Gulonic D-Mannonic D-Talonic D-gl.ycero-L-manrio-tl ep tonic D-glycero-D-gulo-Heptonic 6-Deoxy -D-mannonic
10.4
515
SUGAR DERIVATIVES
TABLE 22.15 (continued) Acid
Volume distribution constant ~
~
Acetic acid Uroiiic acids D-Galacturonic D-Glucuronic L-Guluronic L-lduronic D-Mannuronic Aldeh.vdo aiid keto acids Glyo~ylic 1,aevulinic 2-Ket o-D-gluconic 5-Keto-D-gluconic
21.4
~
~
~
~~-
Sodium acetate
8.40
44.1
11.7
24.0 29.9 36.5
10.7 12.7 12.9
65.6
20.8 13.2 13.7 13.9
3.64 95 .o 38.8
a - D -glucopyranosyluronic acid)-D -xylose, cellobiuronic acid, 4-0-rnethylglucuronic acid,
gluconic acid, galacturonic acid, anhydrosaccharinic acid, glucuronic acid and laevulinic acid were detected in the hydrolyzate. An example of a synthetic application is the separation of epimeric pairs of 3-deoxyL)-hexulosonic acids resulting (together with pyruvate) from the condensation of oxaloacetic acid with D-glyceraldehyde. The chromatography was performed on Dowex 1 (HCOO-; 200-400 mesh, 75 X 3.8 cm) with a linear gradient of formic acid, 0.23-0.46 M (Portsmouth).
Sugar phosphates As with other sugar derivatives of an ionic character, ion-exchange chromatography is the most valuable method for the analysis of sugar phosphate mixtures and for their isolation from natural sources.
Ion-exchange chromatography of borate complexes Khym and Cohn first used the complexing of sugar phosphates with borate for their chromatographic separation; glucose-1 -phosphate, glucosed-phosphate, fructose-6-phosphate and ribose-5-phosphate were separated by this procedure. Fractionation was performed or 2.5 * 1 O S 3 M)-ammonium chloride on Dowex 1 (Cl-), using ammonia solution ( (2.5 . lo-* M )buffers with varying stepwise concentrations of borate (lo-* -lo-' M potassium tetraborate) as the mobile phase. Later, Lefebvre et al. separated a mixture of glucose-1 -phosphate, galactose-1 -phosphate, fructose-6-phosphate, fructose-1-phosphate, glucose-6-phosphate and fructose-l,6-diphosphate. This was first adjusted t o pH 8 with ammonia solution and then applied t o a 45 X 0.5 cm column of Dowex 1-X4 (BO;-; 200-400 mesh). After washing the column with water, References p.519
516
CARBOHYDRATES
the sugar phosphates were eluted with a linear gradient (0.1-0.4M) of triethyl ammonium tetraborate solution at a flow-rate of 1 .O- 1.5 ml/min). This solution was prepared by mixing a freshly made boric acid solution with triethylamine. For instance, 0.4M triethylammonium tetraborate was prepared by dissolving 99.2g of boric acid and 112 ml of triethylamine in water and making the volume up to 1 1. The same mobile phase (linear gradient from 0.1 t o 0.14M)proved to be useful for the resolution of a mixture of N-acetylglucosamine-1 -phosphate and N-acetylgalactosamine-1-phosphate. Bedetti ef ai. used a concave gradient of ammonium chloride and potassium tetraborate for the separation of labelled compounds, listed in Fig. 22.18,on the anion-exchange resin Bio-Rad AG 1-X4(Cl-; 200-400 mesh). After applying the mixture of sugar phosphates, the resin bed (30 X 1 cm) was eluted with 100 ml of M ammonium hydroxide in order to remove free sugars that may be present. The concave gradient used for the elution of sugar phosphates consisted of 2.5 . M ammonia solution 2.5 .lo-' M ammonium chloride in the reservoir (1.95 1 in a 2.5-1erlenmeyer flask) and of 2.5 Mammonia solution -t 5.10-3M potassium tetraborate 3.10-' M ammonium chloride in the mixing chamber (2 1 in a 4-1Mariotte bottle). The radioactivity in the effluent was monitored (flow-rate 0.5 ml/min). For preparative purposes, borate was removed from the eluted compounds by means of three or four evaporations with methanol, and ammonia by passing the solution down a 10 X 1 cm column of the resin Bio-Rad AG 50W-X4(H'). The substantial differences observed in the chromatographic behaviour of
+
+
E
I Z
6
FRACTIONS
Fig. 22.18. Separation of some sugar phosphates on Bio-Rad AG 1-X4 (Cl-; 200-400 mesh) (Bedetti et d.). Resin bed, 30 X 1 cm; mobile phase, concave gradient of ammonium chloride and potassium tetraborate; flow-rate, 0.5 ml/min; room temperature. 1 = glucose; 2 = lactate; 3 = pyruvate; 4 = glucose-1-phosphate; 5 = dihydroxyacetone phosphate; 6 = glucosed-phosphate; 7 = glyceraldehyde3-phosphate; 8 = fructose&-phosphate; 9 = 3-phosphoglyceric acid; 10 = phosphoenolpyruvate; 11 = fructose-l,6-diphosphate;12 = 2,3-diphosphoglyceric acid.
517
SUGAR DERIVATIVES
members of the same series (e.g., glucose-1-phosphate, glucose-6-phosphate and glucose1,6-diphosphate) are attributed to the resultant effect of pK values of the phosphate groups and the stability constants of the borate complexes. Moreover, the steric hindrance of the phosphate group can diminish the stability of the borate complex and thereby lower the binding of the sugar phosphate to the anion exchanger. This effect, together with the participation of hydroxyl groups arising from the formation of a furanose ring in reactions with boric acid, could be responsible for the higher mobility of aldose phosphates compared with ketose phosphates. The above method does not permit the chromatographic separation of 1,3diphosphoglycerate because of its decomposition under the conditions used.
Other ion-exchange separations Another very useful method for the separation of sugar phosphates was developed by Bartlett (1968a), who used Dowex 1-X8 (HCOO-) and formate buffers as the mobile phase. The linear gradient of 0-5 M ammonium formate proved to be the most successful of the various formate buffers of different concentrations and pH values tested. This buffer was prepared by mixing four parts of 5 M formic acid with one part of 5 M ammonium formate (pH ca. 3 at 1 M ) . Table 22.16 lists some of the compounds examined together with their elution positions. TABLE 22.16 ELUTION PATTERN OF SOME SUGAR PHOSPHATES ( R ARTLETT, 1968a) Approx. 1 0 pmole each of known compounds wcrc chromatographed on Dowex 1-X8 (HCOO-; 100-325 wet mesh); column, 20 X 1 cm; mobile phase, 4 I of a linear gradient of 0-5 M ammonium formate (formic acid-ammonium formate, 4: 1 v/v); flow-rate, 0.5-0.8 ml/min. Compound Octulose-8-phosphate Sedoheptulose-7-phosphate Glucose-1 -phosphate Glucosed-phosphate Fructose-1 -phosphate Fructosed-phosphate Deoxyribose-5 -phospha te Ribose-5-phosphate Ribulose-5-phosphate Xylulose-5 -phospha te 6-Phosphoglucona t e Octulose-1 Jdiphosphate 5-Deoxyoctulose-l,8diphosphate Glucose-I ,6diphosphate Mannose-1.6-diphosphate Sedoheptulose-l,7-diphospha tc €:ructose-l,6diphosphate Bbose-1 ,Sdiphosphate Inositol-hexaphosphate
Elution position* 6 /
I .5 8 8 8.5 8 .I 9 9 .s 9.7 18 26 26 26 27.3 21.5 29 30 100 ~
*The figures give the centre of the elution position as a percentage of the total clution volume
References p.519
518
CARBOHYDRATES
This method has been found advantageous in the elucidation of biochemical problems. For example, it has been found that the metabolism of deoxyinosine and deoxyadenosine by fresh or stored erythrocytes caused the accumulation of large amounts of fructose diphosphate, triose phosphate and deoxyribose-5-phosphate;in addition, two unexpected intermediates, 5-deoxyxylulose-1-phosphateand 2-keto-5-deoxyoctulose-l,8-diphosphate, were determined (Bartlett, 1968b). For similar examples, see Bartlett (1968c),Bartlett and Bucolo, Itasaka, etc. Some workers used dilute hydrochloric acid for the elution of sugar phosphates from anion-exchange resins. For example, Hashimoto and Yoshikawa separated a mixture of D-glucose-1-phosphate and D-glucose-l,6-diphosphateusing Dowex 1 (Cl-). The former compound was eluted with 1.5 1O-* M hydrochloric acid, whereas the diphosphate was eluted with lo-’ M hydrochloric acid. When further purification of the latter compound was required, a linear gradient elution with ammonium formate by the method of Bartlett (1959) was applied. Cosgrove used this method for the separation and identification of some inositol tetra- and pentaphosphates, formed during the hydrolysis of the four naturally occuring inositol hexaphosphates. For instance, when myo-inositol hexaphosphate was hydrolyzed at 110°C and pH 4 for 100 min, the resulting myo-inositol phosphate mixture was first resolved on Dowex AG 1-X2 (Cl-), by gradient elution with hydrochloric acid, in four fractions: tetraphosphate, two peaks of pentaphosphate and hexaphosphate. Re-chromatography of each of the pentaphosphate fractions, using the same ion exchanger and 0.48 M hydrochloric acid, led to the isolation of all four possible pentaphosphates. One of these was shown to be identical with “bird-blood phytate”, i.e., with 1,3,4,5,dmyoinositol pentaphosphate.
TABLE 22.17 DISTRIBUTION CONSTANTS OF myo-INOSITOL POLYPHOSPHATES ON SEPHADEX G-50 AND G-25 (100-300 pm) (STEWARD AND TATE) Mobile phase, aqueous solutions of lithium chloride; room temperature. Com p ound
Molarity of mobile phase (M) 0.01
0.20
Sephadex G-25 myo-Inositol hexaphosphate myo-Inositol tripyrophosphate* nip-Inosi to1 pentaphosphdte* myo-Inositol tetraphosphate** myo-Inosit ol t riphosphate* * myo-Inositol diphosphate** myo-lnositol monophosphate myo-Inositol *Johnson and Tate. **Tomlinson and Ballou.
0.06 0.06 0.06 0.1 2 0.19 0.3 1 0.44 0.75
0.01
0.10
Sephadex G-50 0.1 9 0.19 0.1 9
0.25 0.33
0.45 0.48 0.54 0.57
0.44 0.59
0.64 0.70 0.86
0.75
0.83
0.6 1 0.61 0.64 0.68 0.75 0.79 0.86 0.86
REFERENCES
519
Recently, the successful use of GPC in the examination of inositol phosphates was reported by Steward and Tate. They established the distribution constants of several myo-inositol phosphates, listed in Table 22.17. It follows from Table 22.1 7 that, in general, the distribution constants decrease as the degree of' phosphorylation of the inositol increases. However, the anion-exclusion effect contributes t o the observed K , values of charged compounds to a substantial extent; this follows from the change in K , values when mobile phases of different molarity are used. The anion-exclusion effect can be diminished by increasing the concentration of the eluent to 2.0M.
REFERENCES Adams, G. A., Yaguchi, M. and Perry, M . B.. Curbohyd. Res., 1 2 (1970) 267. Albano, E. L. and Horton, D., Curbohyd. Res., 1 I ( I 969) 485. Alfredsson, B., Gedda, L. and Samuelson, O., Anal. Cllim. A m . , 27 ( 1 9 i 2 ) 63. A h . R. S., Actu Chem. Scund., 6 (1 952) 1186. Anderson, D. M. W. and Cree, G. M., Curbohyd. Res., 6 ( 1 968) 385. Anderson, D. M. W . , Hendrie, A. and Munro, A. C . , J . Chromurogr., 44 ( 1 969) 178. Arwidi, B. and Samuelson, O., A n d . Chim. Acta, 31 ( 1 964) 462. Arwidi, B. and Samuelson, O.,Svensk. Pupperstidti., 6 8 (1965) 330. Aspinall, G . 0. and McKenna, J . P., Curbohyd. Res., 7 (1968) 244. Austin, P. W., Hardy, 1:. E., Buclianan, J . G . and Baddiley, J.,J. Chem. Soc., (1963) 5350. Bacon, J. S. D.,Biochem. J . , 57 (1954) 320. Baer, B. H . and Capek, K., Can. J. Chem., 47 (1 969) 99. Baggett, N., Mosihuzzaman, M. and Webber, J . M., Curbohyd. Res.. I 1 (1969) 263. Ball, D. H., Carey, F. A., Klundt, I. L. and Long, L., Jr., Curbohyd. R e r , 10 (1969) 121. Barker, S. A , , Bourne, E. J. and Theander, O., J. Chem. Soc., (1 955) 4276. Barker, S. A,, Hatt, B. W., Kennedy, J . I;. and Somers, P. J., Curbohyd. R e f , 9 (1969a) 327. Barker, S . A,, Hatt, B. W.and Somers, P. J., Carbohyd. Rex, 1 1 (1969b) 355. Bartlett. G. R.,J. Biol. Chem., 234 (1959) 449. Bartlett, G . R., Biochim. Biophys. Acru, 156 ( 1 968a) 221. Bartlett, G. R.,Biochim. Biophys. Acta, 156 (1968b) 254. Bartlett, G. R., Biochim. Biophys. Actu, 156 ( 1 968c) 231. Bartlett, G. R. and Bucolo, G., Biochim. Biophys. Acru. I 56 ( 1 968) 240. Batligate, G . N.,J. Chromutogr., 47 (1970) 9 2 . Bedetti, G., D'Agnolo, G. and Pocchari, F . , J . Chromufogr.,49 (1970) 53. Begbie, R . and Richtrnyer, N. K.. Curbohjd. R e x , 2 (1966) 272. Bella. A . M,. Jr. and Kim. Y . S.. J. Chrornutogr.. 5 I ( 1970) 3 14. BeMiller, J . N. and Wing, R. E., Curbohyd. Res., 6(1968) 197. Binkley, W . W., Advun. Curbohyd. Chem., 10 (1955) 55. Birch, G. and Richardson, A. C., Carhohyd. Res., 8 (1968) 41 1. Bonner, T. G., Bourne, E. J., Cleare, P. J. V. and Lewis, D., J. Chem. SOC., B, (1968) 822. Brendel, K., Roszel, N. O., Wheat, R. W. and Davidson, E. A , , Anal. Biochem., 18 (1967a) 147. Brendel, K., Steele, R. S . , Wheat, R . W. and Davidson. E. A , , Anal. Biochem., 18 ( I 967 b) 16 I . Brimacornbe, J . S. and Clung, 0. A., Curbohyd. Res., 9 ( I 969) 287. Brimacornbe, J. S., Foster. A. B., Hems, R . and Hall, L. D., Curbohyd. Res., 8 (196th) 249. Brimacornbe, J . S., Hunedy, F. and Tucker, L. C. N . , J . C/zem. Soc., C, (1968b) 13x1. Brown, W., J . Chronwrogr., 52 ( 1 970a) 273. Brown, W., J. Chrotnurogr., 53 (1970b) 572. IJuchanan, 4. C . andyEdgar, A. R., Curbohyd. Res.. 10 i 1969) 295. Capek, K., Capkovi-Steffkovi, J. and Jary, J . , Collecr. Czech. Chem. Commun., 35 ( I 970a) 321.
520
CARBOHYDRATES
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y.
Chapter 23
Polysaccharides K. 6APEK and J. STANkK, Jr.
CONTENTS
.................................................................. 523 .................................................... 524 ................................................... 525 .................................................................... 527
Introduction Ion-exchange chromatography. Gel permeation chromatography References
INTRODUCTION The chemistry and biochemisty of polysaccharides is an area in which chromatographic separation methods are mentioned in almost every communication, and its recent rapid development is indebted mainly to the application of chromatography. However, the main applicability of chromatography in this area is in isolations from natural material, in which polysaccharides are admixed with various compounds, such as proteins, nucleic acids and substances of lower molecular weight. The last compounds are very different in nature from polysaccharides and these separations are therefore not considered here. There is no general method for the purification of all polysaccharides, and the procedures used in each instance depend on the nature of the particular polysaccharide and on possible contaminants. In a book by Pigman and Horton, the whole of Volume I1 is devoted to the chemistry and biochemistry of polysaccharides and their isolation, fractionation and purification, so that we decided in this review to consider only the mutual chromatographic separation of polysaccharides. The general techniques are similar to those discussed in Chapter 22 for carbohydrates; they include mainly ion-exchange and gel permeation chromatography, and automated detection methods. Moreover, there are many examples of the fractionation of various dextrans, mannodextrins, cellodextrins, etc., which have been described in Chapter 22, in which these compounds were separated together with mono- and oligosaccharides in order to establish the relationship between K , and molecular size. After the first attempts to separate polysaccharides by using common sorbents such as cellulose, alumina, silica gel and calcium carbonate (see Lederer), two types of column chromatography are now in general use: ion-exchange chromatography for the separation of acidic and neutral polysaccharides and for the fractionation of their borate complexes, and gel permeation chromatography for separations according to molecular size. Only recently, an application of affinity chromatography of branched-chain polysaccharides was described by Kennedy and Rosevear. The branched-chain polysaccharides were fractionated by elution from a concavalin A column, immobilised on Sepharose 4B, with aqueous phosphate buffer at near neutral pH values as-eluent. Even the mixtures of References p.527
523
524
POLYSACCHARIDES
saccharides which cannot be separated by the usual complex formation with a concavalin A solution, were separated.
ION-EXCHANGE CHROMATOGRAPHY
’
Some difficulties may arise when separating mixtures that contain several acidic polysaccharides, in which the strongly acidic sulphate group masks other differences in the ionic properties of the polymer and greatly decreases the efficiency of IEC based on these differences. For this reason, the fractionation of mucopolysaccharides is discussed in a separate chapter (ChapLer24). In the pioneering work of Neukom et al., it was shown that basic cellulose ion exchangers, such as DEAE-cellulose, are more suitable for the fractionation of polysaccharides than are the anion-exchange resins that were formerly used (Steiner et al.), because of the larger active surface of DEAE-cellulose. Acidic carbohydrates are adsorbed at neutral pH, and neutral carbohydrates only under alkaline conditions. For a similar type of separation, ECTEOLAcellulose was used (Ringertz and Reichard). Fractionation of a pectin from lemon-peel on DEAE-cellulose (PO:-) gave two main fractions of acidic polysaccharides together with a small fraction of a neutral polysaccharide. Preparatively, this neutral polysaccharide was removed on DEAESephadex A-50 (HCOO-) (Aspinall et al.). Fractionation of the polysaccharide exudate from Araucaria bidwillii on a DEAEcellulose (PO:-) column (30 X 4 cm) with 0.025,0.05,0.1,0.25 and 0.5 M sodium dihydrogen phosphate buffer (500 ml each) at pH 6 gave a different elution pattern than when it was chromatographed on a 60 X 4.5 cm column of DEAE-Sephadex A-50 (HCOO-) with water containing 1% (3 l), 5% (4 1) and 10%(3 1) of formic acid as the mobile phase; phenol-sulphuric acid and carbazole assays were used for detection (Aspinall and McKenna). Neutral and acidic polysaccharides formed by Polyporus fomentarius and P. igniarius were separated by treatment with DEAE-cellulose (CH3 COO-), on which the latter were adsorbed from aqueous solution and subsequently desorbed by treatment with 2 M potassium acetate solution. Sephadex G-150 and Sepharose 4B columns (50 X 0.8 cm) were used for further separation attempts (Bjorndal and Lindberg). A heteropolysaccharide composed of D-mannose, D-galactose, D-glucose and D-glucuronic acid, from the cell wall of Aureobasidium pullulans, was purified from contaminating 0-glucan on DEAE-Sephadex A-25 by Brown and Lindberg, using a linear gradient of potassium acetate (0-2 M); P-glucan was eluted first. Neukom et ai. found that by using the borate form of a cellulose ion exchanger (elution with sodium borate), the fractionation on DEAE-cellulose was increased. However, with Dowex 1-X8(sodium chloride-boric acid mobile phase), no separation of xylan and mannan was achieved as the xylan “smeared” through the whole run (Kesler). Pierce and Liao described the contamination of the effluent with D-xylose artefacts in DEAE-cellulose chromatography. Thus, with increased improvements in analytical methods, care must be taken to ensure that these contaminants from chromatographic media are not to be mistaken for minor constituents of the substance being studied.
525
GEL PERMEATION CHROMATOGRAPHY
GEL PERMEATION CHROMATOGRAPHY GPC is probably the most frequently used method (for a review, see Churms) because it is the simplest method for the fractionation of polysaccharides that have a broad molecular-weight distribution; simultaneously, it provides a means of determining the molecular weights of polysaccharides. As mild conditions are used, the technique is particularly useful for labile biological materials. A selection of several hundred papers that deal with this subject is listed in Table 23.1. GPC on Sephadex G-25 (28 X 2.5 cm) with water as the mobile phase was used by Lornitzo and Goldman for the purification of a soluble, lowmolecular-weight (4400) polysaccharide containing 60% D-glUCOSe, 40% 6-0-methyl-D-glucose and one acid group. Filtration through a column of Dowex 50 in water removed small amounts of biuret- and ninhydrin-reacting materials; the polysaccharide was not retained in this step. TABLE 23.1 GEL PERMEATION CHROMATOGRAPHY O F POLYSACCHARIDES Packing
Mobile phase
Sephadex G-25
Water
Sample
Reference
Fractionation of dextrans,
Granath and Flodin Kochetkov et al.
M, 5000 Water Sephadex G-50
Water
Sephadex G-75
0.2 M Ammonium hydrogen carbonate Water
Sephadex (3-100
Sephadex G-200 Bio-Gel A Bio-Gel P-2 Bio-Gel P-I0
0.1 M Ammonium hydrogen carbonate 0.2 M Sodium chloride Water Water Water
Bio-Gel P-300
1 M Sodium chloride
Bio-Gel A-50m
Water
Sepharose 4B
1 M Sodium chloride 0.25 M Ammonium formate Water Water
Bio-Glass 500 Porasil C and D Aquapak
References p.527
Purification of synthetic arabinan Fractionation of dextrans, M,, 1000-7000 Biosynthesis of mannans
Granath and Flodin Koza k and Bretthauer
Fractionation of starch dextrin's Fractionation of arabinogalactans from larch wood
Nordin
Fractionation of polymolecular dextran Fractionation of dextrans Homologous glucose oligomers Fractionation of degradation products of A . elata gum polysaccharide Determination of mol. wt. distribution of arabinogalactans from plant gums Cuprammonium rayon, CMcellulose Fractionation of arabinogalactans of high mol. wt. Average mol. wt. of Citrus limonia gum Fractionation of dextrans Fractionation of dextrans
Laurent and Granath Bathgate John et al. Churms and Stephen
Ettling and Adam
Anderson et a1 (1968) Petterson Anderson et al. ( 1 969) Stoddart and Jones Barker et al. Bombaugh e f al.
526
POLYSACCHARIDES
Hummel and Smith investigated the fractionation of dextrans in the molecular-weight range 5000 to 300,000 on Sephadex, agar, agarose and polyacrylamide gels, and found that the best fractionation of dextrans of high molecular weight was achieved on 6.7% agar gel at 4°C on a 145 cni X 1.2 cm2 column with 0.2 M Tris hydrochloride buffer (pH 8.0) as the mobile phase. A 4% agarose gel, eluted with water, also proved to be effective. The remaining two gels were found to be unsuitable for the fractionation of dextrans with molecular weights above 100,000. In general, the samples of dextran (10-50 mg) were dissolved in 1.0-1.5 ml of mobile phase and applied to the colnmn; the fractions were detected with phenol-sulphuric acid reagent. Granath and Kvist described a method of determining the molecular-weight distribution of dextrans in the range 10,000-1 50,000. A 75 X 1.4 cm column packed with a mixture of two Sephadex gels, G-200 and G-100, in the dry-weight ratio of 1:2 (so that the two gels occupied equal volumes when swollen) was used. The mobile phase was percolated through the bed at 20°C several days before use so as to ensure complete equilibration. After the column had been calibrated with 17 dextran fractions, the dextrans under examination were chromatographed under the same conditions, ie., using 0.3% sodium chloride solution, containing chlorobutanol as a preservative, at a flow-rate of 6-7 ml/h as the mobile phase. With the automated anthrone-sulphuric acid procedure, amounts of polymer as small as 2 mg (dissolved in 2 ml of water) are detected. The prior calibration of the column is not necessary if the continuous, reducing endgroup assay, in which the response is proportional to the number of molecules, in addition to a cysteine-sulphuric acid assay for total hexose concentration, in which the response is proportional to the weight of the sample, is used. Barker er ul. developed this technique for the fractionation of dextrdn on porous silica beads (Porasil D, 75-100 mesh; column, 82.8 X 1.08 cm). Water was used as the mobile phase and was pumped through the column at a flow-rate of 10 ml/h.cm2. In 1966, Anderson and Stoddart described the use of the polyacrylamide gel Bio-Gel P-300 in the determination of molecular weights of fractions of arabinogalactan gum. The column (50 X 6 cm), pre-treated with 1% dichlorodimethylsilane in benzene at 6OoC,was packed with Bio-Gel P-300 that had been allowed to swell in 1 M sodium chloride for 2 days. hi order to stabilise the soft top-surface, 1-cm layers of Bio-Gel P-200 and Bio-Gel P-10 were applied successively to the column. Sodium chloride (1 M) was allowed to flow for 2 days before the column was calibrated with dextran fractions of known number-average molecular weight. Polysaccharide (cu. 10 mg), dissolved in 1 ml of 1.5 M sodium chloride, was then applied to the column and eluted with 1 M sodium chloride. Fractions (2 ml) were screened by the phenol-sulphuric acid method. Elution volumes were estimated to the nearest millilitre from peak maxima. On calibration of the gel column with dextran fractions of known number-average molecular weight,hi,: the correlation between the elution volume and log M, was found to be linear over the M, range from 5000 t o 125,000. Values of M, within this range could therefore be estimated from elution volumes by reference to this calibration graph. This method was successfully applied in structural studies on various Araucariu (Anderson and Munro) and Acacia (Anderson e l ul., 1968) gums. Bathgate pointed out the disadvantage of Bio-Gel P-300 that considerable compression of this gel occurred when a proportioning pump was used. An agarose gel (Bio-Gel A,
REFERENCES
527
0.5 M ,200-400 mesh) did not compress to the same extent and, despite its being a carbohydrate, proved to be eminently suitable for an automated system for the determination of molecular weights. A 30 X 0.9 cm column, pre-treated with dichlorodimethylsilane, was packed with Bio-Gel A (0.5 M , 200-400 mesh) according t o the manufacturer’s instructions. Before use, the column was eluted for several days with a 0.05 M solution of mercury(I1) chloride in order t o replace the sodium azide preservative on the agarose gel, because azide interferes strongly with the orcinol used for detection. The mercury(I1) chloride fulfills the double role of preservative and mobile phase. The void volume of the column was determined by eluting Blue Dextran 2000 (Pharmacia, Uppsala, Sweden) and the column was then calibrated by using standard polysaccharides. Although requiring careful handling in order to prevent microbial contamination, agarose gel gives high resolutions for a wide range of molecular weights ( I 0,000 t o cu. 100,000) on short columns and at a relatively high flow-rate (13.8 ml/h); a complete determination could be carried out in less than 3 h (Bathgate). It is obvious that the determination of polysaccharide molecular-weight distribution depends on the measurement of elution volumes; they must therefore be completely reproducible. An important study was undertaken by Churms et ul. to investigate the effect of sample concentration on elution volume. They found that, within the concentration range 2-20 mg/ml, the elution volumes, V,, of dextran ofM,,, 500,000,70,000 and 10,000 and that of D-ghcOSe on BioGel P-300 were independent of concentration, within the degree of uncertainty. However, a marked dependence of V, on concentration for D-glucose and dextran of@,,, 10,000 was observed on Bio-Gel P-10. With the dextran, the observed 10% increase in V, would correspond to a decrease of ca. 2% ii; an estimated molecular-weight value. In contrast, the elution volume of dextran of M,,,500,000 was independent of concentration on this gel. Hence molecular weight obtained on Bio-Gel P-10 will be meaningful only if the concentrations of both the sample and the calibration solutes are the same.
REFERENCES Anderson, D. M. W.,h a , 1. C. M. and Hirst, E., Curbohyd. Res., 8 (1968) 460. Anderson, D. M. W.,Dea, I. C. M. and Munro, A. C., Curbohyd. Res., 9 (1969) 363. Anderson, D. M. W.and Munro, A. C., Curbohyd. Res., 11 (1969) 43. Anderson, D. M. W. and Stoddart, J. F., Carbohyd. Res., 2 (1966) 104. Aspinall, G . O., Craig, J. W. T. and Whyte, J. L., Curbohyd. Res., 7 (1968) 442. Aspinall, G . 0. and McKenna, J. P., Curbohyd. Rex, 7 (1968) 244. Barker, S. A., Hatt, B. W.and Somers, P. J . , Carbohyd. Res., I I (1969) 355. Bathgate, G. N., J. Chromntogr., 47 (1970) 92. Bjomdal, H. and Lindberg, B., Olrbohyd. Rex, 10 (1969) 79. Bornbaugh, K. J., Dark, W. A. and King, R. N., J. PoZym Sci, Part C, 21 (1968) 131. Brown, R. G . and Lindberg, B., Acra Chem. Scund., 21 (1967) 2383. Churms, S . C., Advun. Curbohyd. Chem Biochem., 25 (1970) 13. Churms, S. C. and Stephen, A. M., unpublished work. Churms, S. C., Stephen, A. M. and Van der Bijl, P.,J. Chromutogr., 47 (1970) 97. Ettling, B. V. and A d a m , M. F., Tuppi, 51 (1968) 116. Granath, K. A. and Flodin, P.,Makromol. Chem., 48 (1961) 160. Granath, K. A. and Kvist, B. E., J. Chromatogr., 28 (1967) 69.
528
POLY SACCHARIDES
Hummel, B. C. W. and Smith, D. C., J. Chromatogr., 8 (1962) 491. John, M.,TrBnel, G. and Dellweg, H., J. Chromatogr., 42 (1969) 476. Kennedy, J. F. and Rosevear, A.,J. Chem Soc., Perkin naris. I., (1973) 2041. Kesler, R. B., Anal. Chem.,39 (1967) 1416. Kochetkov, N. K., Bochkov, A. F. and Yazlovetsky, I. G., Gzrbohyd. Res., 9 (1969) 49. Kozak, L. P. and Bretthauer, R. K., Biochemistry, 9 (1970) 1115. Laurent, T. C. and Granath, K. A., Biochim Biophys. Acta, 136 (1967) 191. Lederer, E., Chromatographie en Chimie Organique et Biologique, Vol. 2, Masson, Paris, 1960. Lornitzo, F. A. and Goldman, D. S., Biochim. Biophys. Acta, 158 (1968) 329. Neukom, H., Deuel, H., Heri, W. J. and Kiindig, W., Helv. Chim. Actn, 43 (1960) 64. Nordin, P., Arch. Biochem. Biophys., 99 (1962) 101. Petterson, B., Svensk Papperstidn., 72 (1969) 14. Pierce, J. G . and Liao, T.-H., A n d . Biochem., 24 (1 968) 448. Pigman, W. and Horton, D., The Carbohydrates: Chemistry and Biochemistry, Vols. IIA and IIB, Academic Press, New York, London, 1970. Ringertz, N. R. and Reichard, P., Acta Chem. Scand., 13 (1959) 1467. Steiner, K., Neukom, H. and Deuel, H., Chimia, 12 (1958) 150. Stoddart, J. F. and Jones, J. K. N., Carbohyd. Res., 8 (1968) 29.
Chapter 24
Polysaccharide-protein complexes M. JUkrCOVA and Z. DEYL
CONTENTS Glycosaminoglycans (mucopolysaccharides) .......................................... Introduction ................................................................ The cetylpyridinium chloride procedure .......................................... Separation on Dowex 1-X2 (CI-) ................................................ Separation on ECTEOLAcellulose. .............................................. Separation on DEAE-cellulose .................................................. Separation on DEAE-Sephadex ................................................. Sephadex gel chromatography and molecular-weight distribution ....................... Additionalprocedures ........................................................ Evaluation of different chromatographic procedures ................................. Glycoproteins and glycopeptides .................................................. Simple carbohydrate unhydrolyzed peptide analysis ................................. Carbohydrate-peptide-hydrolyzed peptide analysis. ................................. References ....................................................................
529 529 530 530 533 533 535 535 537 537 .538 538 540 541
GLYCOSAMINOGLYCANS(MUCOPOLYSACCHARIDES) Introduction Basically, there are two different approaches for the separation of anionic glycosaminoglycans (acid mucopolysaccharides). The most common technique in clinical analysis is based on the selective dissociation of cetylpyridinium complexes of these compounds in solutions of different salt concentrations. This procedure has been developed by Antonopoulos e t al. (1967), Schiller et al. and Scott. Selective elution from an ionexchange column has recently been used by several investigators and a number of ion exchangers have been used for this purpose, such as Dowex 1, ECTEOLA-cellulose, DEAESephadex and calcium phosphate gels. In model systems, the separations usually dealt with are those of hyaluronic acid, chondroitin4-sulphate (chondroitin sulphate A), chondroitin-6-sulphate(chondroitin sulphate C), dermatan sulphate (chondroitin sulphate B), keratan sulphate and heparin. Anionic glycosaminoglycans are liberated from the tissue by non-specific proteolysis of the contaminating proteins with papain (E.C. 3.4.4.10). Digestion is usually carried out at 65°C for a 3-h period (Antonopoulos et al., 1967). It is surprising that little was known about the optimum conditions for the separation of these compounds until the appearance of recent papers by Antonopoulos et al. (1964, 1967), Braselmann and Ramm and Pearce e t al. References p.541
529
5 30
POLYSACCHARIDE-PROTEIN COMPLEXES
The cetylpyridinium chloride procedure The separation of glycosaminoglycans on cellulose columns on a micro-scale has been described by Antonopoulos and Gardell. To the column (60 X 3 mm, provided with a pear-shaped extension at the top with a volume of about 4 ml), 20 yg of each polysaccharide were added and a stepwise gradient consisting of the following mobile phases was used: (1) 1% cetylpyridinium chloride in water; (2) 0.3 M sodium chloride in water containing 0.05% of cetylpyridinium chloride; (3) n-propanol-methanol-acetic acid-water (40:20: 1.5:38.5) containing 0.4% of cetylpyridinium chloride; (4) 0.75 M magnesium chloride in 0.1 M acetic acid; (5) 0.75 M sodium chloride containing 0.05% of cetylpyridinium chloride. Between the application of solvents 2 and 3 , 3 and 4 and 4 and 5, the column was eluted with a 0.05% aqueous cetylpyridinium chloride solution in order t o remove any solvent that remained from the preceding step. The above procedure gives a very good separation of chondroitin4ulphate, chondroitin-6-sulphateand dermatan sulphate. As cetylpyridinium chloride tends to crystallize (depending on the batch) at about 20-22"C, the eluting solvents that contain cetylpyridinium chloride should be maintained above this temperature and the elution should also be carried out above this temperature. This is particularly important for the alternative version of the cetylpyridinium chloride procedure described by Antonopoulos ef al. (1964), in which elution is carried out with an increasing concentration of neutral salts without introducing an organic solvent. The general experimental procedure is similar; micro-columns are eluted stepwise with 1-ml portions of the following mobile phases: (1) 1% cetylpyridinium chloride; (2) 0.5 M sodium chloride in 0.05%cetylpyridinium chloride; (3) 0.7 M magnesium chloride in 0.05% cetylpyridinium chloride; (4) 1.25 M magnesium chloride in 0.05% cetylpyridinium chloride; (5) 6 N hydrochloric acid. During the separation, hyaluronic acid is eluted with 0.5 M sodium chloride (solvent No. 2), chondroitin sulphates with 0.7 M magnesium chloride (solvent No. 3) and heparin with 1.25 M magnesium chloride (solvent No. 4). The recovery is 80-100% regardless of the composition of the mixture analyzed. As this procedure is not capable of separating chrondroitin4-sulphate and chondroitind-sulphate, the cetylpyridinium chloride procedure with organic solvents is usually preferred.
Separation on Dowex 1-X2(Cl-) On the macro-scale. the separation is carried out on a 0.9 X 44 cm column packed with Dowex 1-X2 (Cl-; 200-400 mesh) (Schiller et d).Anionic glycosaminoglycan (5-10 mg) is applied as an aqueous solution, the loaded column is washed with distilled water and a stepwise gradient of increasing salt concentration is introduced (see Fig. 24.1). This
531
GLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES)
30
i
CSA clOmg1
; 1.5 j m
9 2
1.0
HEPARIN (5mg)
HMS ( 5 mg)
TUBE
I
NaCl 05M
2
1
3
T
125M
T
15M
+/
i i 3
T
2 OM
Fig. 24.1. Elution diagrams for hyaluronic acid (HA), heparin monosulphuric acid (HMS), chondroitin sulphuric acid A (CSA) and heparin chromatographed individually on Dowex 1-X2 (CL-) columns (0.9 x 44 cm) (Schiller er al.). Stepwise elution was used with sodium chloride solutions of increasing concentration (0.5 -2.0 M). The concentration of sodium chloride at which each of the substances was eluted is indicated as well as the number of the tube in which each substance appeared in the effluent.
procedure gives a good separation of hyaluronic acid, heparin monosulphuric acid, chondroitin4-sulphate and heparin. Keratan sulphate is eluted with 3 M sodium chloride solution and can also be completely separated. In the more developed form of this technique described by Pearce er al., micro-scale operation is possible, and a micro-scale column is used as shown in Fig. 24.2. The amount of each glycosaminoglycan to be separated is within the range 3-5 pnole, the amount of Dowex 1-X2 (100-200 mesh) used is 400 mg per column and elution is carried out with a linear salt gradient in 8 M urea. The addition of urea minimizes the non-electrostatic binding of proteins to cellulose-based ion exchangers. The presence of 8 M urea in the salt gradient shifts the peaks of glycosaminoglycans to lower elution volumes, indicating that in the absence of urea hydrophobic contacts play a considerable role in the separation process. The peaks are also better resolved in the presence of urea, which suggests that non-electrostatic binding opposes the separation effect. The micro-scale separation of glycosaminoglycans with Dowex 1 -X2 is, according to Pearce.et al., superior to the classical version of the cetylpyridinum chloride procedure (without organic solvents). References p . 541
TABLE 24.1 SEPARATION OF A N ARTIFICIAL MIXTURE OF CLYCOSAMINOGLYCANS (HYALURONIC ACID, CHONDROITIN-MULPHATE, CHONDROITINd-SULPHATE, DERMATAN SULPHATE AND KERATAN SULPHATE) A 10-mg amount of each polysaccharide was applied t o a 2 X 20 cm ECTEOLAcellulose column; formate solutions contained 1.34 M ammonia (Antonopoulos et al., 1967). Fraction
Recovery mg
Uronic acid*
HCXOsamine*
Hexose*
Gluco=mine**
Galactojarnilre**
Predominating gl y cosaminoglycans in fraction
33.6 24.5
-
100
100
Hyalorunic acid Chondroitin4-sulphate, chondroitindsulphate
26.4 26.3
-
-
-
-
25.0
26.6
%
0.02M hydrochloric acid 1.0 M ammonium formate 1.5 M ammonium formate
9.3 2 .o
93.0 6.7***
31.0
2.0 M ammonium formate 2.75 M ammonium formate 2.0 M sodium chloride
17.1 9.2 9.8
57.0***
29.1 23.3 2.8
-
~
30.7*** 98.0
*Expressed as a percentage of airdried material. **Expressed as a percentage of total hexosamines. ***Calculated on the sum of all gahctosaminoglycans added to the column.
~
-
90
100 100
10
Chondroitin-4-sulphate Keratan sulphate
vl
w N
GLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES)
533
rig. 24.2. Micro ion-exchange column. A 7 c m length of glass capillary tubing, 3 mm I.D., was sealed t o a capillary stop-cock, 1 mm I.D., which was beaten into a goose-neck shape (Pearce et ul.). A 21-gauge disposable needle (Becton-Dickinson) was cut at right-angles 10 mm from the end and cemented to the glass with expoxy cement.
Separation o n ECTEOLA-cellulose This procedure was devised by Ringertz and Reichard (1959, 1960) and developed further by Anseth and Laurent; the microgram- version of this procedure was described by Trudle and Mann. By using an ECTEOLA-cellulose column and eluting with an increasing concentration of sodium chloride in dilute hydrochloric acid, it is possible to separate hyaluronic acid, chondroitin sulphates and heparin. Antonopoulos et al. (1967) used a stepwise gradient for the separation of hyaluronic acid, chondroitin4-sulphate, chondroitin-6-sulphate,dermatan sulphate and keratan sulphate. In general, galactosaminoglycans could be eluted from the column with a lower concentration of ammonium formate in ammonia than that needed for keratan sulphate (Table 24.1).
Separation on DEAE-cellulose Although Pearce el al. reported that in DEAE-cellulose chromatography the peaks of individual glycosaminoglycans are eluted close together and that this ion exchanger cannot be recommended for such a separation, a method that gives fairly good results has been developed by Braselmann and Ramm. As in the cetylpyridinium chloride procedure, elution was carried out with an increasing concentration of magnesium chloride solution. The whole procedure requires not more than 50 mg of material and small columns (50 X 4.5 mm) can be used. A typical run is shown in Fig. 24.3. It is obvious that in this References p. 541
534
POLYSACCHARIDE-PROTEIN COMPLEXES
instance the separation of chondroitin-6-sulphate, chondroitin4ulphate and dermatan sulphate is incomplete, but heparan sulphate and hyaluronic acid are clearly separated. This procedure, as with many others in glycosaminoglycan chromatography, uses a stepwise gradient with magnesium chloride concentrations as indicated in Fig. 24.3. The first elution is carried out with 0.2 M magnesium chloride in 0.002 M acetic acid, which helps to elute hyaluronic acid. This step has been used in several procedures, such as those of Antonopoulos et al. (1967) and Thunell. In summary, the advantages and disadvantages of DEAE-cellulose chromatography are as follows. The capacity of the column is rather high, which means that the procedure would be more suitable for preparative than for analytical purposes. It also allows the accumulation of glycosaminoglycans from very dilute solutions, which occasionally occurs when handling natural material. In some instances, the saccharidic material adheres strongly to the ion exchanger, which causes tailing and disturbances in separation; according to Braselmann and Ramm, this problem can be overcome by using mixed beds of DEAE-cellulose and some other ion exchangers. Braselmann and Ramm believe that these disturbances are not the result of hydrophobic binding, as described by Antonopoulos et al. (1967) for Dowex 1-X2, as the application of urea proved ineffective in DEAE-cellulose chromatography. The nature of the DEAEcellulose separation does not, of course, permit the application of organic solvents as used in the cetylpyridinium chloride separation by Antonopoulos and Gardell.
20-
@
W
u1
z
B
10-
u1 W
a 0
1
10
-
0
'
,
'
1
,
,
,
i
'
l
4
~
l
f
l
~
i
*
l
~
,
OHy -
20-
C-4-S D S + C - 6 - S C-4-S r--
He
t
l
r
,
r
l
Fig. 24.3. Separation of glycosaminoglycans on DEAE-cellulose: (A) glycosaminoglycans from rabbit aorta (43.4 mg); (B) the same glycosaminoglycans, treated with hyaluronidase; (C) standards, Hy = hyaluronic acid, C 4 - S = chondroitin4-sulphate, DS = dermatan sulphate, C-6-S = chondroitin-6-sulphate, He = hcparin (Brasclmann and Ramm).
535
CLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES)
Separation on DEAE-Sephadex For this purpose, DEAE-Sephadex A-25 (Cl-) in columns of dimensions 2 X 20 cm was used by Schmidt. After pre-treatment with 0.5 M sodium hydroxide, 0.5 M hydrochloric acid and 0.1 M sodium chloride, a suspension of the ion exchanger in 0.1 M sodium chloride was packed into the column. A stepwise gradient was then introduced, as follows: (1) OSOM sodium chloride; ( 2 ) 1.25 M sodium chloride in 0.01 M hydrochloric acid; ( 3 ) 1.50M sodium chloride in 0.01 M hydrochloric acid; (4) 2.0 M sodium chloride in 0.01 M hydrochloric acid. Before starting the gradient, the column was washed with 5 ml of distilled water; 5 ml fractions were collected during the separation procedure, the result of which is shown in Fig. 24.4. The isomeric chondroitin sulphates cannot be separated by this system and are eluted as a single peak.
2.5
1 :;:
$1
1.25 M
1.5 M
2.0 M
c
; 2.0 E
4 1 5
c
HYALURONIC ACID (5mgl
CHONDROITIN SULPHATES (5mgl
n
n
i 2
HEPARITIN SULPHATE (5rng)
1.0
HEPARIN (5mgl
0.5
0
0
5
10
15
20
TUBE NUMBER
Fig. 24.4. Stepwise elution of a mixture of acid mucopolysaccharides (Schmidt).
Sephadex gel chromatography and molecular-weight distribution There are a number of diseases in which the metabolism of anionic glycosaminoglycans is deranged and specific anionic glycosaminoglycans may accumulate in organs and tissues or may be excreted in the urine. There is some evidence that these glycosaminoglycans may differ from their counterparts in healthy individuals in their degree of polymerization. References p . 541
TABLE 24.2 EVALUATION OF DIFFERENT CHROMATOGRAPHIC PROCEDURES USED IN GLYCOSAMINOGLYCAN SEPARATIONS
ul
w
m
Chroma tographic procedure
Clycosaminoglycans separated
Pairs of glycosaminoglycans not separated
Reference
Cetylpyridinium chloride procedure (cellulose as sorbent: with organic solvents)
Chondroitin4-sulpha te, chondroitin-6-sulphate, dermatan sulphate
Information not available
Antonopoulos and Gardell
As above: without organic solvents
Heparin, chondroitin sulphates, hyaluronic acid
Isomeric chondroitin sulphates
Antonopoulos et al. (1964)
Dowex 1-X2
Hyaluronic acid, chondroitin sulphates, heparin
Isomeric chondroitin sulphates, keratan sulphate acid, derinatan sulphates A and B
Pearce et a1
DEAE-cellulose
Hyaluronic acid, heparin; chondroitin4-sulphate is partially separated from the mixed peak of dermatan sulphate and chondroitin-6-sulphate
Dermatan sulphate and chondroitind-sulphate
Braselmann and Ramm
ECTEOLA-cellulose
Hyaluronic acid, chondroitin sulphates, keratan sulphate
The separation of isomeric chondroitin sulphates is only partial
Antonopoulos et al. (1967)
DEAE-Sephadex A-25
Hyaluronic acid, heparitin sulphate, chondroitin sulphates, heparin
Isomeric chondroitin sulphates
Schmidt
P
GLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES)
537
Most of the methods other than chromatography are much too time consuming to be of practical value for these purposes. We describe here briefly the method of Constantopoulos et al. A sample of chondroitin-4-sulphate is chroniatographed on a column of Sephadex G-200, giving a broad peak corresponding to the variety of polymers present in the sample. From this separated fraction, narrow cuts are made and the molecular weight of each is determined by a suitable method such as the diffusion sedimentation coefficient procedure (surprisingly, the results indicate that these cuts are not too polydisperse) and the retention volumes are plotted against the estimated molecular weights. The whole procedure is carried out because the calibration graph of the logarithm of the molecular weight versus retention time obtained in this mannei- is not identical with the calibration graph obtained with any standard protein calibration series. The advantage of this procedure, however, is that dermatan sulphate, chondroitind-sulphate and heparitin sulphate also follow the same relationship.
Additional procedures
A chromatographic procedure involving the use of a mixed bed of Celite and calcium phosphate and gradient elution with phosphate buffers of pH 6.5 and ionic strength up to 0.2 M enables chondroitin sulphates to be separated from hyaluronic acid or a sample of hyaluronic acid to be separated into fractions with different molecular weights (Bowness, 1960).
Evaluation of different chromatographic procedures It is obvious that none of the separation prxedures can be generally recommended for an unknown mixture of glycosaminoglycans and that one has to specify what particular type of glycosaminoglycan is being sought before starting the analysis. Some idea of the applicability of particular separations can be gained from Table 24.2. Molecular sieving alone is not suitable for the separation of these compounds. A complex procedure involving a combination of Sephadex G-50 and DEAE-cellulose has been described by Hallen. In addition to their use for purely analytical purposes, chromatographic techniques have also been applied in glycosaminoglycan chemistry to demonstrate their interactions with proteins, namely with collagen (Wasteson and Obrink). As far as detection is concerned, the column effluent can be scanned first by a simple colour reaction, such as the carbazole reaction for uronic acids (Dische; Bowness, 1957). A special modification of this procedure suitable for different types of automated analysis has been described by Balazs e t al. The Elson-Morgan reaction for hexosamines is equally applicable (Boas). Further tests on those fractions which contain glycosaminoglycans are desirable. Also, purified glycosaminoglycans can be obtained from these fractions after dialysis followed by precipitation with ethanol in calcium acetate buffer. The applicability of chromatography analysis in the field of glycosaminoglycan analysis coincide with those generally applicable for macromolecular substances of a hydrophilic nature, mainly polysaccharides. References p . 541
538
POLYSACCHARIDE-PROTEIN COMPLEXES
GLYCOPROTEINS AND GLYCOPEPTIDES Chromatographic separations of glycoproteins are virtually indistinguishable from procedures used in protein and peptide chemistry. Also, there are a number of proteins which, in the usual sense, are not considered to be glycoproteins though they carry one or several glycosidic residues. Of course, there is a wide choice of different applications and many different glycoproteins have been separated; a detailed description of these procedures is not very appropriate from the chromatographer’s point of view. On the other hand, there is a chromatographic procedure that is specific for the structural analysis of glycoproteins and glycopeptides; the specificity is based on the suitable arrangement of the detection system rather than on the separation procedure itself. The method has been described by Brummel et al. and will be briefly discussed here. Carbohydrates and their acyl- and alkyloxy-derivatives react with phenol and sulphuric acid to give a yellow-orange chromogen that exhibits an absorption maximum at 485 nm for pentoses and at 489 nm for hexoses. Furthermore, it has been reported by Montgomery that amino acids and proteins do not interfere in this reaction. As this method is both simple and sensitive and does not require special treatment after the reactants necessary for generating the yellow chromogen have been mixed together, it was the method chosen for analyzing large series of samples generated from proteolytic digests of glycopeptides. Brummel et al. developed an automated version of this procedure that is suitable for the continuous analysis of column effluents. The segmented technique with a Technicon AutoAnalyzer has been used for this purpose. This technique was applied previously to carbohydrate analyses using orcinol (Judd et al., Kesler), anthrone (Garza and Weissler, Syamanada et al.), Molish (Johnson et al.) or phenol (Robyt and Bemis) as colorimetric reagents.
Simple carbohydrate unhydrolyzed peptide analysis The procedure is, perhaps, best understood from the flow-sheet shown in Fig. 24.5. The effluent from the chromatographic column (B) is split into two streams, which feed the carbohydrate and peptide lines. The overflow is collected in a fraction collector through line S . The stream entering the carbohydrate line is mixed with a 2% aqueous phenol solution (line C) containing 0.2% of ARW-7 detergent (Technicon, Ardsley, N.Y., U.S.A.). The carbohydrate line is segmented with air (line D), passed through a mixing coil (F) and then to the time-delay coil (G). The length of the delay coil is adjusted such that corresponding peaks are read in the colorimeters from the peptide manifolds. The detergent is used to maintain an even segmentation in the delay coils. This system can also be used for carbohydrate analysis; the delay coil is then by-passed and the detergent is not needed. The phenol-carbohydrate segments are mixed with concentrated sulphuric a;id at a glass joint (H). An additional device that helps to smooth the base-line is the pulse suppressor (a Technicon PC 1 can be used for this purpose, but any other type will suffice) located in the sulphuric acid line immediately after the pumping manifold (E). The complete reaction mixture is passed through another mixing coil (I) and enters a
539
GLYCOPROTEINS AND GLYCOPEPTIbES
heating bath in which the temperature of the mixture is increased to 96°C. The minimum holding time required is 15 min, but the delay is frequently longer a t the lower pumping speeds used for the peptide analyzer. The resulting solution is cooled (K) and recorded (L) at 490 nm in a 15-mm tubular flow-through cell. An additional pull-through ensures a steady flow through the cell.
c
30 - 40-
I
Fig. 24.5. Automatic continuous carbohydrate analyzer (Brurnmel ef al. ). Components (those which do not appear in Fig. 24.5 occur in Fig. 24.6): A, 0.056-in. Acidflex tubing for concentrated sulphuric acid; B, 0.020 or 0,0075-in. clear standard tubing for column effluent; C , 0.020 or 0,025-in. Solvaflex tubing for 2% (w/v) phenol and 0.2% (v/v) ARW-7 or 1.2% (w/v) phenol and 0.1% ARW-7, respectively; D, 0.035-in. clear standard for air segmentation; E, pulse supressor; F, glass mixing coil; G, time delay coil consisting of about 100 ft. of Intramedic PE 240 polyethylene tubing; H , T-junction for mixing concentrated sulphuric acid with the segmented stream; 1, Glass mixing coil; J , glass coil heating bath (96°C); K, water-jacketed cooling coil; L, 490-nm colorimeter with 15-mm tubular flow cell; M, threepoint recorder; N, standard peptide manifold and apparatus for ninhydrin colour production on basehydrolyzed and unhydrolyzed column effluent; P, single-speed proportioning pump and roller assembly; Q, 0.056-in. Acidflex pull-through; R, drain; S, to fraction collector; T, 0.025 or 0,0075-in. clear standard tubing for column effluent; U, 0.045 or 0.056-in. clear standard for 13%(w/v) sodium hydroxide and 0.4 (v/v) ARW-7 or 10.3%sodium hydroxide and 0.3%ARW-7, respectively; V, 0.025-in. clear standard for nitrogen segmentation; W, 0.045-in. Solvaflex tubing for 0.3% (w/v) ninhydrin in methyl Cellosolve, water and acetate buffer (pH 5 . 5 ) ; X, 0.040-in. clear standard for nitrogen segmentation; Y, 0.049 in. clear standard for base-hydrolyzed desegmented stream; Z, 0.040-in. Acidflex tubing for acetic acid t o neutralize the base; DB, de-bubbler; F', glass mixing coil with side-tap for mixing ninhydrin with the nitrogen segmented stream; J', glass coil heating bath (96°C); I", PTFE coil heating bath (96°C); K', water-jacketed cooling coil; L', 570-nm colorimeter with 15-mm tubular flow cell; P', variable-speed proportioning pump and roller assembly; Q', 0.056-in. Acidflex pull-through.
References p . 541
540 540
POLYSACCHARIDE-PROTEIN COMPLEXES COMPLEXES POLYSACCHARIDE-PROTEIN
In the the schematic schematic representation representation of of this this apparatus apparatusshown shown in in Fig. Fig. 24.5, 24.5,the the peptide peptide line line In in the pumping manifold is omitted for the sake of clarity as it does not differ from the in the pumping manifold is omitted for the sake of clarity as it does not differ from the usual arrangement. arrangement. usual
Carbohyhate-peptide-hydrolyzed hate-peptide-hydrolyzed peptide peptide analysis analysis Carbohy This variation variation involves involvesaa hydrolysis hydrolysis step; step; the the result result of of the the analysis analysis isis then then three three lines, lines, This two ninhydrin ninhydrin detections detections corresponding corresponding to to the the hydrolyzed hydrolyzed and and unhydrolyzed unhydrolyzed sample, sample, two respectively, and and the the third third which which results results from from the the carbohydrate carbohydrate analysis. analysis. The The flow-sheet flow-sheet respectively, 24.6.As As the the system system isis rather rather complex complexand and of the the manifold manifold arrangement arrangement isis shown shown in in Fig. Fig. 24.6. of the use use of of tubing tubing of of appropriate appropriate dimensions dimensions isisof of decisive decisiveimportance, importance,the the values values used used by by the Brummel et etal. al. are are summarized summarized in in Table Table 24.3. 24.3. Brummel DDBB
SS
\ \! \! \
II
TT
RR
--
malonic>tartaric acid. Fumaric acid was bound more strongly than maleic acid. The dependence of the logarithms of the volume distribution coefficients of hydroxy acids on the logarithm of the concentration of the eluting agent is linear. At lower concentrations of borate ions, the separation factors of many acids and their separations are improved. By elution with 0.04 Msodium tetraborate, a, 0-and 0,y-dihydroxybutyric acids were separated quantitatively, while the separation of lactic and glycolic acids was successful when 0.015 M sodium tetraborate was used. For the complete separation and elution of more complex acid mixtures, for example lactic, glycolic and the two dihydroxybutyric acids, as well as acids derived from sugars with five and six carbon atoms, a very large volume of eluting solution is necessary, which prevents practical application. In this event gradual elution with solutions of increasing borate concentration should be applied. In the first step, lower acids are eluted with 0.04 M borate; after elution of p,ydihydroxybutyric acid, the borate concentration is increased to 0.07 M in order to increase the rate of elution of higher acids (Alfredsson et al., 1962). The successful separation of lower acids (for example, glycolic and lactic acid) is simpler and more effective in acetate medium.
ION-EXCHANGE CHROMATOGRAPHY OF CARBOXYLIC ACIDS IN VARIOUS SYSTEMS
563
Elution curves in borate medium are usually broader than those in acetate medium. Excessive broadening of the elution curves is often caused by too slow a n equilibration due t o slow diffusion within the resin particles or t o the slow attainment of complexformation equilibrium. At a n elevated temperature, equilibration is accelerated in both instances, which often leads t o narrowing of the elution curves i n d to an improved separation. I n tetraborate solution, equilibria exist between borate anions with different charges. An increase in temperature causes an equilibrium shift in a 0.1 M solution towards the formation of anions with one negative charge, which are weaker eluents than ions with higher charges. Therefore, the elution volumes of aldonic acids and formic acid increase with an increase in temperature, which contrasts with the behaviour in nietaborate s o h tions. An anionexchange chromatographic method WBS developed for the automatic determination of idonic and gluconic acids on a 200 X 9 mm column of Dowex 1-X8 (Cl-; 400 mesh), in which the elution was performed with a 0.4 M borate buffer of pH 7.35 containing 0.05 M sodium chloride at 30°C (Aoki et a/.).The acids were determined by the periodate consumption method. The addition of sodium chloride and the use of an elevated column temperature favoured the separation of the elution bands. This method was also useful for the separation and determination of S-oxogluconic, arabonic and 2-oxogluconic acids in reaction mixtures.
Chromatography of acids on anion-exchange resins in the formate, nitrate and chloride forms The separation of a mixture of carboxylic acids by displacement chromatography on a column of Duolite A-40 was described by Lesquibe and Lesquibe and Rumpf. The acids were displaced with an acid that was stronger than all of those present in the mixture (0.05 N nitric acid), and they appeared in the.eluate in order of increasing acidity constants. The pH and the concentration of the acids were measured in the eluted fractions by titration with 0.01 N sodium hydroxide. A weak auxiliary acid was added t o the mixture, which was eluted first and reacted with trace amounts of alkalis that remained on the column after incomplete regeneration and which otherwise caused low results in the determination of the weakest acid in the mixture. Before separation, an acid slightly stronger than the strongest acid in the mixture was added in order t o prevent the penetration of a small amount of nitric acid into the strongest acid and thus avoid higher results. Using this method, it was possible t o separate lactic, tartaric and oxalic acids, even when present in a sample a t a concentration of 6.1 O4 mequiv./g, with an error of less than 10%. Lawson and h r d i e studied the conditions for the chromatography of organic acids on anion exchangers using formic acid as eluent. They found that the degree of cross-linking of the strongly basic anion exchanger Dowex 1 does not affect the order of elution of carboxylic acids. The molarity of formic acid necessary for the elution of 94 acids from a Dowex 1 -X 10 column was determined by Davies et al. The elution behaviour depends on the pK of the separated acids, and the solubility of the acids in formic acid is also important, because it affects tailing. By choosing a suitable concentration gradient of References p . 5 72
564
LOWER CARBOXYLIC ACIDS
formic acid, a complete or partial separation of some acids was achieved (malic from mesotartaric, succinic from adipic and tartaric from quinolinic acid) and the tailing was suppressed. The eluate was collected in fractions the composition of which was analysed by paper chromatography after the prior elimination of formic acid by vacuum evaporation to dryness over silica gel. The method of Lawson and Purdie was used with a 200 X 10 mm column containing Dowex 1-X10(200-400 mesh) for the determination of the content of non-volatile organic acids in apples by Salkova and Nikiforova. Using gradient elution with formic acid after the removal of sugars, it was possible to determine the contents of malic acid, citric acid and succinic acid, which are the main acidic components, and the content of some other acids, including chlorogenic, shikimic and quinic acids. On a 12 X 1 cm column of Dowex 1-X8 (200-400 mesh), micromole amounts of quinolinic acid were separated from other pyridine derivatives by gradient elution with 0-4M formic acid. The eluted acid was determined photometrically at 254 nm after decarboxylation to nicotinic acid (Pallini). By elution with formic acid, some uronic and aldobiuronic acids were successfully separated on modified Dowex 1-X4 and Dowex 1-X8 resins by Fransson et al. Egashira (1961) investigated the separation of organic acids on a column of the strongly basic anion exchanger Dowex 1-X8(Cl-). He calculated theoretical elution volumes of the acids from their characteristics and found a linear relationship between the elution volumes and [CI-] z , where [Cl-] is the concentration of chloride ions in the eluent and z is the number of acidic groups in the completely dissociated acid. The elution volume was considerably affected by temperature and the peak width was directly proportional to the square-root of the flow-rate of the eluent. The sodium chloride solutions used for elution were buffered, then the buffer was eliminated from the eluate by means of a cationexchange column (Amberlite XE-64 and Dowex SOW) and the acids were determined by titration with 0.01 N sodium hydroxide or by measuring the coloration produced with bromophenol blue. In this manner, acetic, succinic, maleic, fumaric and citric acids were separated, using 0.01 M sodium chloride solution at pH 2,O.l M sodium chloride at pH 4, or 0.15 and 0.2 M sodium chloride at pH 12 for elution. In view of the linearity of the development o f coloration and the instability of the indicator solution, the determination was not quantitative (Egashira, 1966, 1968). A column of a weakly basic anion exchanger in the chloride form was used for the separation of mixtures of mono- and dichloroacetic acids (Anderson). Sulphurous acid or some other acid with an ionization constant between that of mono- and dichloroacetic acid was added to the sample, which was then passed through the column. The acids being separated were then displaced with 1 N hydrochloric acid. Monochloroacetic acid was eluted first, followed by sulphurous acid and then dichloroacetic acid. The acids were partially separated. Multiple cycles can be used to improve the resolution. Mixtures of mono-, di- and trichloroacetates can be quantitatively separated by stepwise elution with 0.05,O.l and 2 N sodium chloride, respectively; mixtures of monochloroacetate, 2,4-dichlorophenoxyacetateand trichloroacetate can also be separated with the same sequence of sodium chloride eluting solutions (Tsitovich and Kuzmenko). An interesting possibility for the chromatographic separation of some aromatic acids was reported by Lee et al. These compounds are usually strongly retained by anion-
HIGH-SPEED ION-EXCHANGE CHROMATOGRAPHY
565
exchange resins and therefore a high concentration and large volume of eluent and a long elution time are required for such acids. A method was suggested involving the use of iron(II1) chloride-organic solvent solution as eluent, which reacts with some aromatic organic acids such as salicylic acid, aromatic hydroxamic acids and phenols to form coloured, stable, non-adsorbable complexes. The formation of such a complex leads to reduced sorption on the anion-exchange resin.
HIGH-SPEED ION-EXCHANGE CHROMATOGRAPHY OF CARBOXYLIC ACIDS WITH ANION EXCHANGERS OF CONTROLLED SURFACE POROSITY The modern high-speed chromatography of carboxylic acids permits rapid separations, but in fact this method is in principle a highly developed form of ion-exchange chromatography involving the use of modern highly effective carriers and phases. High-speed ion-exchange chromatographic separations can be carried out by using ion-exchange column packings of controlled surface porosity introduced by Kirkland. These anion exchangers consist of hard, spherical siliceous particles with a solid, impervious core that is surrounded by a thin superficially porous shell, about 2 pm in thickness. The ion-exchange medium is a methacrylate polymer containing strongly basic tetraalkylammonium groups. These Zipax-supported strong anion exchangers are of low capacity (about 12 pequiv./g) and are intended for use with small samples in analytical-scale applications with equipment with a low dead volume and with high-sensitivity detectors. The most useful mobile phase for such a column is distilled water, with variations in the pH and ionic strength used to vary the resolution and retention times. Sodium sulphate, sodium nitrate and sodium acetate are usually used to change the ionic strength. The retention times are greatly influenced by very small changes in ionic strength. For instance, terephthalic acid is strongly retained in 0.004M sodium nitrate, but by increasing the salt concentration to 0.012 M, the elution is easy to perform. A mobile phase can usually be found such that the sample will be resolved in a few minutes without changing the mobile phase. In this respect, Zipax ionexchange columns of controlled surface porosity do not behave like a classical ion-exchange column, but rather like adsorptive columns with special affinities for charged solutes. An example of the use of high-speed, high-pressure anion-exchange chromatography for the rapid separation of a binary mixture containing maleic and fumaric acids is shown in Fig. 25.10. The isomeric acids were separated in about 90 sec, which is considerably faster than by conventional gel ion-exchange chromatography. The same column (1000 X 2.1 mm, packed with controlled surface porosity Zipax support coated with anion-exchange resin) was used for the separation of three aromatic carboxylic acids, which were eluted in the order benzoic, toluic, terephthalic acid. Using distilled water buffered to pH 9.2 with the ionic strength adjusted to 0.02 M by the addition of ammonium nitrate, it was possible to achieve baseline resolution of the three acids in less than 10 min (Henry and Schmit). Fig. 25.1 1 demonstrates another example of the anionexchange chromatographic separation of carboxylic acids with an anion exchanger of controlled surface porosity References p.572
LOWER CARBOXYLIC ACIDS
566
A
T
A = 0.002
P
120
60
I
0
1
I
5
10
5
Fig. 25.10. Separation of isomeric acids by controlled surface porosity anionexchange chromatography (Kirkland). Column: 1000 X 2 . I mm. Ion exchanger: anion exchanger of controllcd surface porosity. Mobile phase: 0.01 N nitric acid. Operating conditions: carrier flow-rate, 2.73 ml/min; input pressure, 1900 p.s.i.; temperature, 60°C. Detection: spectrophotometric, t = time (sec); A = absorbancc. 1 = Maleic acid; 2 = fumaric acid. Samplc, 3 g1 of 0.5 mg/ml each in 0.01 N nitric acid.
Fig. 25.1 1. Separation of phthalic acid isomers (Henry and Schmit). Column: 1000 X 2.1 mm. Anion exchanger: controlled surface porosity Zipax. Mobile phase: borate buffer. pH 9.2, containing 0.02 M sodium nitrate. r = Retention time (min); A = absorbance. 1 = Phthalic acid; 2 = terephthalic acid; 3 = isophthalic acid.
(Henry and Schmit). Phthalic acid isomers are separated within 15 min by elution with 0.02M sodium nitrate at pH 9.2. These acids all decompose or rearrange when heated and therefore cannot be vaporised to allow gas chromatographic analysis. pH has a great effect on retention and resolution, and this effect may change the elution order of the sample components. For instance, by adjusting the pH to 2.75, the order of elution of phthalic acid isomers becomes terephthalic, isophthalic, phthalic acid. Longbottom determined nitrilotriacetic acid by high-speed ion-exchange chromatography. A column packed with Zipax support coated with a strong anion exchanger was used, the mobile phase being 0.02 M N a 2 P 4 0 7 .The flow-rate was 0.5 mlimin and the column inlet pressure 1000 p.s.i.
5 67
OTHER SEPARATION TECHNIQUES FOR CARBOXY LIC ACIDS
OTHER SEPARATION TECHNIQUES FOR CARBOXYLIC ACIDS Separation of carboxylic acids on silica gel columns Ion exchangers are not the only packings For chromatographic columns used in the separation of carboxylic acids. In some papers, for example, the use of silica gel is described (Moehler and Pires, Nakajima and Tanenbaum, Stamley and Moseley . Markova and Smirnov separated chloroacetic, acetic and succinic acids in this way. An example of a qualitative separation of organic acids from plant material on silica gel is shown in Table 25.4 (Freeman). TABLE 25.4 SEQUENCE O F ELUTION OF ACIDS FROM A SILICA GEL COLUMN (FREEMAN) The acids were eluted with chloroform containing a progressively increasing proportion of n-butanol. The stationary phase was 0.5 N sulphuric acid. Fraction volume: 2.6 ml. Acid
Elution range (fraction numbers)
Peak maximum (fraction number)
Acid
Elution range (fraction numbers)
Peak maximum (fraction number)
n-Bu t y ric n-Valeric Is0bu t y ric Propionic Acetic Mesaconic Pyruvic Adipic Formic Glu taric Citraconic Itaconic Maleic Fumaric Thymol blue indicator Succinic Lactic a-Ket oglutaric tram-Aconitic Malonic
1-10 1-9 1-12 5-19 22-29 24-30 28-38 28-38 31-38 32-37 34-45 39-47 38-50 38-52 47-53 57-63 57-63 54-67 66-72 64-75
2 2 3 8 25 27 32 32 34 34 39 42 45 45 50 60 60 62 69 69
5 -Pyirolidone-2carboxylic Glyoxylic Diglycollic Oxalacetic Oxalic Tricarballylic Glycollic Nitric cis-Aconitic DL-Malic Citric DLGlyceric DL- Isoc itr ic Sulphuric
65-80 53-81 64 -76 61-19 16-95 80-93 84-98 87-147 97-108 103-120 134-153 152-170 176-195 178-192 193-230 175-206 200-233
69 70 70 73 81 88 92 93 103 111 141 160 183 181 198 198 211
>239
-
-~
~
Shikimic D( +)-Tartaric Phosphoric L G l u tamic Quinic L-Aspartic
I
>252
~~~~
Kesner and Muntwyler developed an automatic method for the analysis of organic acids. The technique consisted in chromatography on silica gel columns with chloroform tert. -amyl alcohol mixtures as eluent. The concentration of terr. -amyl alcohol in the eluent was continually increased in a Varigrad gradient apparatus and the mixture was pumped on to the column. The individual separated acids in the eluate reacted with an indicator (o-nitrophenol in absolute methanol), which was continually fed into the References p . 572
568
LOWER CARBOXYLIC ACIDS
effluent stream and the coloration developed was recorded with a flow-photometric detector operating at 350 nm. This method was successfully applied to the separation of a number of physiologically important acids, such as the Krebs cycle intermediates. A routine separation can be performed with a sensitivity about 40 times higher than that in the conventional manual method. The accuracy is greater than f 3%. Furthermore, no preliminary deproteinization and extraction (with the possible loss of volatiles and formation of artifacts) was required prior to introduction of the sample.
Separation of carboxylic acids on Sephadex columns In this case, the separation is based on the sieving effect and molecular size. In most separations weakly cross-linked Sephadex G-10 was used (Brock, Brock and Housley , Schiller and Chung). Sephadex G-25 was also used by Woof and Pierce for the separation of phenolic acids. Monocarboxylic acids were not separated on Sephadex when eluted with water. As reported by Gelotte, the carboxylic acid group has a “negative sorption effect” and in most instances elution occurred much earlier than would be expected from the parent phenol (Table 25 S).Intramolecular hydrogen bonding is not always responsible as there is no difference between 0-and p-hydroxybenzoic acid in water and the 2,4-dihydroxy acid was eluted early while the 2,3-dihydroxy acid was not. In electrolyte solutions, this TABLE 25.5 GEL FILTRATION OF AROMATIC ACIDS AND OTHER AROMATIC COMPOUNDS (GELOTTE) Column: 35 x 3.5 cm. Gel: Sephadex G-25 with a water regain of 2.9 g of water per gram of dry substance and a wet density of 1.099 (50-100 mesh). The Sephadex was swelled in 0.05 M sodium chloride for 30 min and the fine particles were removed by decantation before packing; the column was equilibrated with the solvent before addition of the sample. Mobile phases: (a) distilled water; (b) 0.05 M sodium chloride; (c) phosphate solution, p = 0.05, pH = 7; (d) 0.01 M ammonia solution, pH = 10.6. Flow-rate: 2 ml/min with a hydrostatic pressure of 60 cm. Temperature: ambient. Compound*
Mobile phase a
b
Kd values Benzoic acid Anthranilic acid Sulfanilic acid Picric acid Cinnamic acid Phthalic acid Phenol Aniline Benzyl alcohol Salicyl alcohol
*0.5-142 mg samples tested.
0.5 0.6 0.3 0.4 0.3 1.1 0.7 1.5 1.3 1.4
-
1.1 2.5
C
d
569
OTHER SEPARATION TECHNIQUES FOR CARBOXYLIC ACIDS
“negative sorption” effect disappeared in all acids. The rate of elution seemed to depend on the number and orientation of free hydroxyl groups (i.e., those not involved in internal hydrogen bonding) and the extent to which these groups are xcessible to the carboxyl groups of the gel. Most of the acids were eluted very early with ammonia solution as they also carry a negative charge and will be repelled by similar acid groups in the gel. The formation of a complex with molybdate resulted in earlier elution, as would be expected, except for the 2,6-dihydroxy acid, which, although both groups can complex, was very strongly adsorbed. Methylation, as in vanillic and syringic acids, resulted in behaviour very like that of a monohydroxy acid. They were not differentiated on Sephadex columns. From Table 25.6, it can be seen that an electrolyte is required if the separation of mixtures of phenolic acids is to be achieved. Fig. 25.12 shows the separation of a mixture of acids already reported to be present in barley. Resolution in the first stage is not complete but fractions can be collected as shown and completely resolved by re-running the samples using water as solvent. Downey et al. used a 54 X 2.4 cm column filled with Sephadex LH-20 with a flow-rate of 1 ml/min for the separation of fatty acids in the presence of phospholipids and chloroplast pigments. Using chloroform only as the column eluent, tristearin, tributyrin and stearic, capric, butyric and acetic acids were separated (Fig. 25.13) into well defined peaks (the elution volumes, V,, were 6 5 , 8 5 , 2 2 5 , 3 2 0 , 4 5 0 and 575 ml, respectively). When linolenic acid was included in this mixture, it was not separated from stearic acid. The capric acid appears to have contained a fatty acid dontaminant, as indicated by the inflection in its elution curve (Fig. 25.13), which was also observed on chromatography TABLE 25.6 Kd VALUES OF PHENOLIC ACIDS IN AQUEOUS ELUTING MEDIA (WOOF AND PIERCE) Column: 35 X 2.5 cm Sephadex G 2 5 (medium). Phenolic acid
o-Hydroxybenzoic acid m-Hydroxybenzoic acid p-Hydroxybenzoic acid 2,3-Dihydroxybenzoic acid 2,4-Dihydroxybenzoic acid 2,5-Dihydroxybenzoic acid 2,6-Dihydroxybenzoic acid 3,4-Dihydroxybenzoic acid 3,5-Dihydroxybenzoic acid 3,4,5-Trihydroxybenzoic acid 2,3,4-Trihydroxybenzoic acid Vanillic acid Syriiigic acid Chlorogenic acid
References p.572
Eluting medium Water
NaCl
Ammonia solution
Na,MoO,
1.o 1 .o 1 .o 2.6 0.85 1.o 0.7 1.7 2.2 1.05 2.05
2.1 1.8 1.45 1.6 2.7 3.0 1.45 2.1 1.9 2.5 2.65 1.95 1.95 3.9
1.6 0.85 0.8 0.4 1.1 1.7 0.4 0.7 0.7 1.05 1.1 0.85
1.2
0.85
0.8 1.6
-
2 .o 1.4 1.4 2.95 -
1.9 2.0 -
0.85
-
1.35
-
570
LOWER CARBOXYLIC AClDS
80 1
0
50
100
150
200
250
0
300
ELUTION VOL..ml
Fig. 25.12. Separation of a phenolic acid mixture (Celotte). Column: 35 X 2.5 cm. Sorbent: Sephadex G 2 5 . Mobile phase: 0.1 M sodium chloride. Detection: spectrophotometric. 1 = vanillic + syringic acids; 2 = 3,4-dihydroxybenzoic acid; 3 = gallic acid;4 = ferulic acid; 5 = sinapic acid; 6 = chlorogenic + caffeic acids.
'--I
I
ELUTION VOLUME tml)
Fig. 25.13. Fractionation and separation of triglycerides and fatty acids (Downey e t d.). Column: 54 X 2.4 cm. Sorbent: Sephadex LH-20; Mobile phase: chloroform. Flow-rate: 1 ml/min. Detection: electrometric titration. 0 , triglycerides; 0 , fatty acids. Elution volumes: tristearin 6 5 ml; tributyrin 85 ml; stearic acid 225 ml; capric acid 320 rnl; butyric acid 450 ml; acetic acid 575 rnl.
OTHER SEPARATION TECHNIQUES FOR CARBOXYLIC ACIDS
57 1
TABLE 25.7 GEL CHROMATOGRAPHY OF ALIPHATIC CARBOXY LIC ACIDS ON POLYACRYLAMIDE GEL (STREULI) Column: 97 X 0.5 cm. Gels: (a) Bio-Gel P-2 (100-200 mesh) polyamide gel, exclusion limit 200-2600; (b) Bio-Gel P-6 (100-200 mesh) polyamide gel, exclusionlimit 1000-5000. Mobile phase: 0.01 M sodium chloride. Compound*
Sorbent a
b
Kd value ~
~
Hydrochloric acid Trichloroacetic acid Chloroacetic acid Acetic acid Lactic acid Acrylic acid Crotonic acid Oxalic acid Succinic acid Malic acid Tartaric acid Maleic acid Fumaric acid Citric acid Glycine 4-Aminobenzoic acid
I .29 1.17 0.96 1.08 0.79 0.97 1.28 1.04 1.22 1.oo 1.08 1.26 0.93 1.19 0.93 2.75
-
0.98 -
1 .oo -
1.03 -
1.1 1 0.96 1.04
*Samples of 50 p l . TABLE 25.8 GEL CHROMATOGRAPHY OF ALIPHATIC CARBOXYLIC ACIDS (CAZES AND GASKILL) Columns: four 4 ft. X 3/8 in. columns in series. Gel: rigid, cross-linked polystyrene gel. Mobile phase: odichlorobenzene. Flow-rate: 1 ml/min. Temperature: 130°C. Acid*
V , (ml)
Acetic Propionic rt-Bu tyric n-Valeric n-Hexanoic n-Heptanoic n-Oc tanoic n-Nonanic n-Decdnoic ti-Undecanoic rt-Dodecanoic Myristic Palmitic Stearic
194.4 184.6 178.4 174.7 168.9 165.8 160.2 157.1 154 .O 150.8 148.0 143.1 139.6 132.8
*Samples are 2 ml aliquots of 0.25 to 1.O% solutions.
References P. 5 72
572
LOWER CARBOXYLIC ACIDS
of capric acid alone. The recoveries of the individual fatty acids and triglycerides were approximately 85%. Monocarboxylic and dicarboxylic aliphatic acids were separated by Streuli on a BioGel P-2 column, as indicated in Table 25.7. The homologous series of Cz-Cls aliphatic acids was successfully separated on crosslinked polystyrene gel using o-dichlorobenzene as the mobile phase at 130°C. The results obtained by Cazes and Gaskill are summarized in Table 25.8.
REFERENCES Alenitskaya, S. R. and Starobinets, G. L., Vestn. Akad. Nauk Belorus. SSR, Ser. Khim. Nauk, (1967) 28; C.A., 6 7 (1967) 5 7 5 2 8 ~ . Alfredsson, B., Bergdahl, S. and Samuelson, O.,Anal. Chim. Acta, 28 (1963) 371. Alfredsson, B., Gedda, L. and Samuelson, O., Anal. Chim. Acta, 27 (1962) 63. Anderson, R . E., U.S. pat., 3,409,667 (1968). Aoki, I., Hori, M. and Matsumaru, H., Bunseki Kagaku [Jap. Anal.), 18 (1969) 346. Bengtsson, L. and Samuelson, O., Anal. Chim. Acta, 44 (1969) 217. Brock, A. J . W., J. Chromatogr., 39 (1969) 328. Brock, A. J. W. and Housley, S., J. Chromatogr., 42 (1969) 112. Calmanovici, B., Rev. Chim. (Bucuresti), 17 (1966) 170. Calmanovici, B., Rev. Chirn. (Bucuresti), 17 (1966) 374. Carlsson, B., Isaksson, T. and Samuelson, O., Anal. Chim. Acta, 4 3 (1968) 47. Carlsson, B. and Samuelson, O., A m l . Chim. Acta, 49 (1970) 247. Carroll, K. K., Nature (London), 176 (1955) 398. Cazes,J.andGaskill,D. R.,Separ. Sci.,2(1967)426and4(1969) 15. ChuriEek, J. and Jandera, P., Chem. Listy, 64 (1970) 756. Courtoisier, A. I. and Ribereau-Gayon, J . , Bull. SOC.Chim. Fr., (1963) 350. Davies, C. W., Biochem. J . , 45 (1949) 38. Davies, C. W., Hartley, R. D. and Lawson, G. J., J. Chromatogr., 18 (1965) 47. Davies,C. W. and Owen, B. D. R., J. Chem. SOC.,(1956) 1681. Downey, W. K., Murphy, R. F. and Keogh, M. K., J. Chromatogr., 46 (1970) 120. Egashira, S., Bunseki Kagaku [Jap. Anal.), 10 (1961) 1225. Egashira, S., Bunseki Kagaku (Jap. Anal.), 15 (1966) 1356, Egashira, S., Bunseki Kagaku (Jap. Anal.), 17 (1968) 958. Erler, K.,Z. Anal. Chem., 131 (1950) 106. Fransson, L. A., Roden, L. and Spach, M. L., Anal. Biochem., 23 (1968) 317; C.A., 67 (1968) 1120342. Freeman, G. G., J. Chromatogr., 28 (1967) 338. Funasaka, W.,Bunseki Kagaku (Jap. Anal.), 15 (1966) 835. Gellotte, B., J. Chromatogr., 3 (1960) 330. Goudie, A. J. and Rieman, W.,Anal. Chim. Acta, 26 (1962) 419. Harlow, G. A. and Morman, D. H., A w l . Chem., 36 (1964) 2438. Henry, R. A. and Schmit, J. A., Chromatographia, 3 (1970) 116. Johnard, B. and Samuelson, O., Sv. Kem. Tidskr., 73 (1961) 586. Johnson, S. and Samuelson, O., Anal. Chim. Acta, 36 (1961) 1 . Katz, S. and Burtis, C. A., J. Chromatogr., 40 (1969) 270. Kesner, L. and Muntwyler, E., ACS Winter Meeting, Phoenix, Ariz., January 17-21, 1966. Khym, J. X.and Zill, L. P., J. Amer. Chem. SOC.,73 (1951) 2399. Kirkland, J . J., J. Chromatogr. Sci., 7 (1969) 361.
REFERENCES
573
Kreshkov, A. P. and Kolosova, I. F., Zh. Anal. Khim., 25 (1970) 1234. Larsson, U. B., isakson, T. and Saniuelson, O., Acra Chem. Scand., 20 (1966a) 1965. Larsson, U. B., Norstedt, I. and Samuelson, O., J. Chromatogr., 22 (1966b) 102. Lawson, G. L. and Purdie, J . W.,Mikrochim. Acta, (1961) 415. Lee, K. S., Lee, D. W . and Lee, E . K . Y., Anal. Chem., 42 (1970) 554. Lee, K. S. and Samuelson, O.,Anal. Chim. Acta, 37 (1967) 359. Lesquibe, F., C R . Acad. Sci., Paris. 251 (1960) 2690. Lesquibe, F. and Rumpf, P., C.R. Acad. Sci.,Paris, 260 (1965) 5006. Longbottom, J . E.,Anal. Chem.,44 (1972)418. Markova, A. V. and Smirnov, V. A.,Zh. Anal. Khim., 24 (1969) 1271. Martinsson, E . and Samuelson, 0.. Chromatographb, 3 (1970) 405. Mehta, M. J., Bhatt, R. A,, Hegde, R. S., Patel, D. J . and Bafna, S. L., J. Indian Chem. Soc., 130. Moehler, K. and Pires, R., Z. Lehensm.-Unters.-Forsc/z., 139 (1969) 337; C A . , 71 (1969) 89992m. Nakajima, S. and Tanenbaum, S. W., J. Chromatogr.,43 (1969) 444. Pallini, V., Boll. Soc. Ital. Sper., 41 (1965) 676; C.A., 64 (1966) 86. Patel, D. J . and Bafna, S. L., Ind. Eng. Chem., Prod. Res. Develop., 4 (1965) 1 . Patel, D. J. and Bafna, S. L., Indian J. Chem., 6 (1968) 199. Robinson, P. A. and Mills, G. F., Ind. Eng. Chem., 41 (1949) 2221. Snlkova, E. G. and Nikiforova, T. A., Dokl. Akad. Nauk SSSR, 179 (1968) 218. Samuelson, O., Sv. Kem. Tidskr., 76 (1964) 635. Samuelson, 0. and Simonson, R., Anal. Chim. Acta, 26 (1962a) 110. Samuelson, 0. and Simonson, R., Sv. Papperstidn.,65 (1962b) 363. Samuelson, 0. and Thede, L.,J. Chromatogr., 30 (1967) 556. Scheffer, I;., Kickuth, R. and Lorenz, H., Qual. Plant. Mater. Veg., 12 (1965) 342. Schenker, H. M. and Rieman, W.,Anal. Chem., 25 (1953) 1637. Schiller, J. G . and Chung, A. E., J. Biol. Chem., 245 (1970) 5857. Scoggins, M. W.,Anal. Chem.,44 (1972) 1285. Seki, T., J. Biochem. (Tokyo),45 (1958) 855. Seki, T., J. Chromatogr., 3 (1960) 376. Seki, T., J. Chromatogr., 22 (1966) 498. Seki, T., Inamori, K. and Sano, K.,J. Biochem. (Tokyo),49 (1959) 1653. Shimomura, K. and Walton, H. €:.,Anal.Chem., 37 (1965) 1012. Skelly, N. E., A w l . Chem., 33 (1961) 271. Skelly, N. E. and Crummett, W . B.,Anal. Chem., 35 (1963) 1680. Skorokhod, 0.R. and Sembur, M.E., loniry, Ionnii Obmen. Akad. Nauk SSSR, Sh. Statei, (1966) 152; CA., 67 (1967) 94319n. Skorokhod, 0. R., and Tabulo, M. L.,lonoobmen. Technol., (1965) 186; C.A., 6 3 (1965) 10648a. Stamley, J. B. and Moseley, P. B., J. Amer. Oil.Chem. Soc.,46 (1969) 241; Index Chem., (1969) 117104. Starobinets, G. L. and Gleim, I. F., Zh. Fiz. Khim., 39 (1965) 2188. Starobinets, G. L., Gleim, 1. F., Alenitskaya, S. R. and Chizhevskaya, A. B., Vestn. Akad. Nauk Belorus. SSR, Ser. Khim. Nauk, (1965) 5; C.A., 64 (1966) 54e. Streuli, C. A,, J. Chromatogr.,47 (1970) 355. Thomas, H.,J. Chromatogr., 34 (1968) 106. Tsitovich, 1. K. and Kuzmenko, E. A., Zh. Prikl. Khim., 42 (1969) 2066. Woof, J. B. and Pierce, J. S., J. Chromatogr., 28 (1967) 94. Zerfing, R.C. and Veening, H..Anal. Chem., 38 (1966) 1312.
This Page Intentionally Left Blank
Chapter 26
Higher carboxylic acids J. POKORNY
CONTENTS Introduction and general remarks .................................................. Separation as fatty acid derivatives ................................................. Chromatography on adsorbents in general use ......................................... Chromatography on specific adsorbents ............................................. Gel and ionexchange chromatography .............................................. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
575 575 576 577 578 580
INTRODUCTION AND GENERAL REMARKS Higher carboxylic acids, usually termed fatty acids, are generally analyzed by gas-liquid chromatography nowadays. Liquid column chromatography is used only for non-volatile or thermolabile derivatives, such as oxidation and polymerization products or.highly unsaturated or polar derivatives. The use of column chromatography for the analysis of common fatty acids was recently reviewed (Kaufmann, Stein and Slawson),
SEPARATION AS FATTY ACID DERIVATIVES Fatty acids are often converted into methyl or ethyl esters before their analysis in order to improve the separation. Treatment with diazomethane or with alcohol and boron trifluoride or sulphuric acid as catalysts are the most commonly used esterification procedures. The following procedure of Schlenk and Gellerman is suitable. The apparatus consists of three test-tubes with side-arms. A stream of nitrogen is saturated with diethyl ether in the first tube (1 50 X 16 mm) and carries diazomethane generated in the second tube (85 X 15 mm) into the third tube (85 X 15 mm) where the esterification takes place. The side-arms (7 mm O.D.) are bent downwards and reach close to the bottom of the next tube. They are drawn out at the ends to an O.D. of ca. 1 mm. Rubber stoppers are used for connections. The flow of nitrogen through the diethyl ether in tube 1 is adjusted to ca. 6 ml/min. Tube 2 contains 0.7 ml of 2-(fi-ethoxyethoxy)ethanol(purified by heating it to 110°C for 1 h with 5% potassium hydroxide solution followed by distillation at ca. 90°C and 12 mm Hg pressure), 0.7 ml of peroxide-free diethyl ether and 1 ml of a solution of 6 g of potassium hydroxide in 10 ml of water. Between 5 and 30 mg of fatty acids dissolved in 2-3 ml of diethyl ether which contains 10%of methanol are placed in tube 3. About 2 mmole of N-methyl-N-nitroso-p-toluenesulphonamide per milliequivalent of fatty acids are disReferences p . 580
57 5
576
HIGHER CARBOXYLIC ACIDS
solved in 1 ml of diethyl ether and the mixture is added t o tube 2. Connection is made immediately with tube 1 while the yellow colour of diazomethane is already appearing in tube 2. As soon as a yellow tinge becomes visible against a white background in tube 3, this tube is disconnected and the slight excess of diazomethane is consumed by adding acetic acid diluted with diethyl ether or is removed by a stream of nitrogen. The whole procedure requires 10- 1 2 min. Chlorophenacyl esters of saturated and unsaturated fatty acids can be separated on polyethylene-coated Celite columns (Kibrick and Skupp). Fatty acid esters can be converted into hydroxamic acids and separated on cellulose powder (Davenport). The separation between saturated and unsaturated fatty acids is improved by preliminary conversion of the latter into mercury adducts by boiling them with mercury(I1) acetate (Jantzen and Andreas), e.g., by the following procedure as modified by Kuemmel. Free fatty acids are converted into methyl esters, 0.7 g of the esters are dissolved in 15 ml of methanol and the mixture is refluxed with an appropriate amount of mercury(l1) acetate depending on their iodine value (1.8, 1.7,1.6 and 1.5 g of mercury(I1) acetate are used for methyl esters with iodine values of 120-135,96-119,83-95 and 70-82, respectively) for 7 0 min on a magnetic stirrer-hot plate. After cooling, 160 ml of water are added to the methanolic solution and the mixture is extracted with five 35-ml portions of diethyl ether followed by two 30-ml portions of chloroform. The combined diethyl ether extracts are washed with three 50-ml portions of water, and the combined water washings are then back-extracted with the combined chloroform extracts. The combined diethyl ether and chloroform extracts are dried and filtered and the solvent is removed. The sample should then be refrigerated until it is subjected to chromatographic separation, which should be carried out within 24 h of the preparation of the mercury derivatives. The yield of mercury derivatives ranges from 97 to 100% of theory. Kuemmel obtained relatively pure saturated, monoenoic and polyenoic fractions by the following method. A 390 X 20 mm column of alumina was used for the separation of adducts of a 700-mg sample. The flow-rate was adjusted to 15-18 ml/min. The saturated acids were eluted with 350-400 ml of light petroleum (b.p. 30-45°C) and the monoenoic acid derivatives with 350 ml of a 5% solution of methanol in diethyl ether. If the polyunsaturated acid content is greater than 40% of the total fatty acids, it is more advantageous to use a 1% solution of methanol in diethyl ether and to continue the elution until a 50-ml fraction of eluate contains not more than 2 mg of the substance. The polyunsaturated acid derivatives retained in the column are then decomposed by leaving the column overnight containing 200 ml of hydrochloric acid-methanol (1 : lo), and recovered from the solution. Bromomercurimethoxy adducts are suitable for the isolation of minor polyenoic fatty acids in mixtures (Craske and Edwards).
CHROMATOGRAPHY ON ADSORBENTS IN GENERAL USE Liquid-liquid chromatographic procedures are still important for the preparation of pure polyunsaturated acids, e.g., esters of arachidonic acid (Privett et d.), and for the separation of oxygenated fatty acids. Gunstone and Sykes separated various mono-, di-, tri- and tetrahydroxy, mono- and diacetoxy, and epoxystearic acids by reversed-phase chromatography on siliconized Hyflo Supercel coated with neutralized liquid paraffin or
CHROMATOGRAPHY ON SPECIFIC ADSORBENTS
577
acetylated castor oil. Etyfhro-and threo-isomers of polyhydroxylic acids were not separated. Radin reviewed various procedures for the separation of hydroxylic fatty acids, and discussed-the advantages and disadvantages of reversed-phase chromatography. He recommended the use of polystyrene beads as the support. Naturally occurring epoxy acids were separated by partition chromatography on Celite using acetonitrile as the stationary phase and n-hexane as the mobile phase (Morris e f al.). Piretti e f al. isolated pure hydroperoxides from oxidized methyl oleate on 100-mesh silica gel (Mallinckrodt, St. Louis, Mo., U.S.A.) containing 5% of water by eluting with n-hexane-diethyl ethermethanol (94:5:1) at 18°C (300 X 30 mm column; 1 g of sample; flow-rate 2.6 ml/min). The present author also obtained an excellent separation on Florisil under similar conditions. The silicic acid chromatography was applied with success to the separation of the products of the interaction of linoleic acid hydroperoxide with tocopherols (Gardner e f al.) and to the separation of some acidic cleavage products of oxidized methyl linoleate (Esterbauer el d.).Mixtures of n-hexane with diethyl ether were used as eluents in both instances. Fatty acid oxidative and thermal dimers were satisfactorily fractionated on silica gel impregnated with a 16% solution of methanol in benzene and eluted with a 2% solution of methanol in benzene (Evans e f al.). Figge used elution with cyclohexane-benzene (6:4) and diethyl ether for the separation of oleic acid thermal dimers. Mounts e f al. described a method for the separation of oxidative dimers on silica gel. Cason et al. used a mixture of Darco G-60 charcoal and Celite 52 1 (1 :2) and elution with 95% ethanol, anhydrous ethanol and mixtures of ethanol and benzene for the separation of fatty acids of tubercle bacillus lipids.
CHROMATOGRAPHY ON SPECIFIC ADSORBENTS A specific adsorbent for the separation of unsaturated fatty acids is silica gel impregnated with silver nitrate, as introduced by De Vries. Silver ions form adducts with double bonds so that the unsaturated acids are retained in the column to an extent that depends on their degree of unsaturation, trans-acids being eluted before the corresponding cisisomers. For the preparation of the silver nitrate-impregnated adsorbent, the following procedure reported by Dolev and Olcott is suitable. A 50-g amount of silicic acid (silica gel G, according to Stahl, 100-200 mesh) is suspended in 100 ml of 40% silver nitrite solution, and the suspension is brought to boiling with stirring, allowed to cool, filtered and dried overnight in an oven at 130°C. The cooled powder is ground in a ball-mill and stored in a desiccator. All operations should be protected from light. Florisil can also be impregnated with silver nitrate (Willner). Artman and Alexander combined a chromatographic separation on silica gel using a stepwise gradient elution with n-hexane-benzene, re-absorption of the fractions, and a further separation with the use of columns of silica gel impregnated with silver nitrate in order to separate fatty acids from heated fats. Chromatography on urea columns is a selective method of separation of straight-chain fatty acids from their branched and cyclic isomers (Cason e f al.) because urea forms adducts only with normal fatty acids. As Cason etal. used a 1% solution of methanol in isoReferences p . 580
578
HIGHER CARBOXYLIC ACIDS
octane as eluent, only saturated acids remained in the column, while both monounsaturated and branched fatty acids passed through into the eluate. According to the present author's experience (Pokorny and El-Tarras) all monounsaturated acids remain in the column if methanol saturated with urea is used as the eluent. Urea chromatography is particularly suitable for the preparation of cyclic or dimeric thermal or oxidation products of fatty acids (Sagredos, 1967, 1969). Molecular sieves of the zeolite type (5A, 1 OX, 13X) are suitable for separations according to molecular size (Martinez Moreno and Lbpez Ruiz).
GEL AND ION-EXCHANGE CHROMATOGRAPHY Gel chromatography iias found many applications in the analysis of fatty acids in the last few years. Chang succeeded in separating tall oil acids into normal fatty acids and monomeric, dimeric and trinieric resin acids by using Bio-Beads SX-2 and SX-8. The
mono
@
mono
i
w
m
5
@
a
n.
m
x)
Y
K
140
mono
5
10
1
200 300 400 500 130 150
180
VOLUME (rnl)
Fig. 26.1. Separation of lipidic oligomers by gel chromatography. A, methyl esters of monomeric and dimeric oleic acid: column, 7 5 0 X 25 mm; Sephadex LH-20; eluent, ethanol (after Aitzetmiiller, 1972a). B , mcthyl esters of fatty acid oligomers: column size, 331 ml: sorbent, Sephadex LH-20; weight of adsorbent, 67.1 g ; eluent, cliloroform~mctliaiiol(7:3); elution-rate, 39 ml/h; detection, gravimetric (after Hase and Harva). C, methyl esters of polymerized soyabean oil: column, 6000 X 8 m m ;adsorbent, Sphcron P; eluent, tetrahydrofuran; flow-rate, 35 ml/h; detection, differential refractometer (after Pokorny el ~ 1 . 1D, . heat-polymerized groundnut oil; conditions as in A. E, methyl esters of fatty acid oligomers: column, 2300 X 23 mm; temperature, 48-51°C; adsorbent, Sephadex LH-20; eluent, dimethylformamide; flow-rate, 20 ml/h; detection, differential refractometer (after Inoue et ul.). F, heat-polymerized soyabean oil; conditions as in C.
GEL AND ION-EXCHANGE CHROMATOGRAPHY
579
trimethylsilyl derivatives of Sephadex G-25 and other resins were very satisfactory for the separation of mixtures containing fatty acid esters of fatty alcohols (Tanaka and Konishi). Zinkel and Zank were able t o separate methyl esters of fatty acids on 2000 X 10 mm Styragel columns using diethyl ether as the solvent, but methyl linoleate and methyl linolenate formed critical pairs with methyl myristate and methyl laurate, respectively. Resin acids were separated from one another and from fatty acids. Aitzetmuller ( 1 9 7 2 ~ )used Merckogel SI-50 columns for the analysis of fatty acid oxidation products, viz., methyl 9,lO-epoxystearate in the presence of methyl oleate or methyl 12-ket0-9-octadecanoate,and ricinoleate in the presence of oleate. Methyl linoleate hydroperoxide was isolated by chromatography on Sephadex LH-20 as the hydroperoxidic fraction was retarded on the column during elution with chloroform (Rubach er d.). The most extensive use of the gel chromatography of fatty acids has been in the analysis of polymerized oils. Inoue et af.separated from monomers up to pentamers of unsaturated fatty acids, methyl esters and the corresponding fatty alcohols (Fig. 26.1). They used 2300 X 20 mm or 1000 X 1 5 mm columns packed with Sephadex LH-20, which was left to swell for 24 h in dimethylformamide before the filling; a differential refractometer was used as the detector; 300 or 50 mg of sample were eluted with dimethylformamide a t a controlled temperature, e.g., 50°C. Aitzetmuller (1972a) tested Bio-Beads SX-I in addition t o Sephadex LH-20. His results and also those of some other workers are'shown in Fig. 26.1. For preparative chromatography, Aitzetmuller (1 972b) used Sephadex SR-25/100 columns in conjunction with an LKB fraction collector. A IOO-pI aliquot of each collector fraction was injected monomers
W
cn z
f cn
dimers
W
a
I
glycerol
I
I
90
80
70
60
50
FRACTION NUMBER
Fig. 26.2. Preparative chromatography of fatty acid methyl esters from frying fats (after Aitzetmuller, 1972a). Column: 800 X 25 mm (Sephadex SR-25/100). Sorbent: Sephadex LH-20. Eluent: ethanol. Operating conditions: see text. Detection: Pye LC detector.
References p.580
580
HIGHER CARBOXYLIC ACIDS
with an Eppendorf pipette into a constant flow of ethanol rinsing the wall of a narrow tube which had an opening to accept the tip of the pipette. The ethanol flow transported the sample and rinsed the tubing and coating block between subsequent injections. The frequency of injection was 2-4 min-' at a flow-rate of 30-60 drops/min. An example of the chromatogram is shown in Fig. 26.2. Only limited use has been made of ion-exchange chromatography for the separation of fatty acids. The procedure of Emken et al. for the separation of cis- and trans-monoenoic acids on Amberlyst XN-1005 treated with silver nitrate was recommended by Applewhite.
REFERENCES Aitzetrnuller, K., Fette, Seifen, Anstrichm., 74 (1972a) 598. Aitzetrnuller, K., J. Chromatogr., 72 (1972b) 355. Aitzetrniiller, K., J. Chrornatogr.,73 ( 1 9 7 2 ~ 248. ) Applewhite, T. H., J. Amer. Oil Chem. Soc., 42 (1965) 321. Artman, N. R. and Alexander, J . C., J. Amer. Oil Cliem. SOC.,45 (1968) 643. Cason, J., Sumrell, G., Allen, C. F., Gillies, G . A. and Elbert, G. S., J. Biol. Chem., 205 (1953) 435. Chang, T.-L., Anal. Chem.,40 (1968) 989. Craske, J. D. and Edwards, R. A., J. Chromatogr., 53 (1970) 253. Davenport, J. B., Chem. fnd. (London), (1955) 705. De Vries, B.,J Amer. Oil Chem. SOC.,4 0 (1963) 184. Dolev, A. and Olcott, H. S., J. Amer. Oil Chem. Soc., 42 (1965) 624. Ernken, E. A., Scholfield, C. R. and Dutton, H. J., J. Amer. Oil Chem. SOC.,41 (1964) 388. Esterbauer, H., Just, W. and Sterk, H., Fette, Seifen, Anstrichm., 74 (1972) 13. Evans, C. D., McConnell, D. G., Frankel, E. N. and Cowan, J. C., J. Amer. Oil Chem. Soc., 42 (1965) 764. Figge, K., Chem. Phys. Lipids, 6 ( 1 97 1) 178. Gardner, H. W., Eskins, K., Grams, G. W. and Inglett, C. E., Lipids, 7 (1972) 324. Gunstone, F. D. and Sykes, P. J., J. Chem. Soc. (London), (1960) 5050. Hase, A. and Harva, O., Kem. Teollisuus, 25 (1968) 134. Inoue, H., Konishi, K. and Taniguchi, N., J. Chromatogr., 47 (1970) 348. Jantzen, E. and Andreas, H., Chem. Ber., 94 (1961) 628. Kaufmann, H. P., Fette, Seifen, Anstrichm., 72 (1970) 505. Kibrick, A. C. and Skupp, S. J.,AnaI. Chem., 31 (1959) 2057. Kuernrnel, D. F., Anal. Chem., 34 (1962) 1003. Martinez Morcno, J. M. and L6pez Ruiz, J. L., GrasasAceites, 22 (1971) 351. Morris, L. J., Hayes, H. and Holrnan, R. T., J. Amer. Oil Chem. SOC., 38 (1961) 316. Mounts, T. L., McWeeny, D. J., Evans, C. D. and Dutton, H. J., Chem. Phys. Lipids, 4 (1970) 197. Piretti, M., Capella, P. and Taddid, M., Riv. Ital. Sostanze Grasse, 46 (1969) 324. Pokorni, J. and El-Tarras, M. F., unpublished results. Pokorn);, J., Kundu, M. K., Payizkovi, H., Luan, N.-T., Coupek, J., Pokornc, S. and Jani?ek, G., Fette, Seifen, Anstrichm., 74 (1 972) 625. Privett, 0. S., Weber, R. P. and Nickell, E. C., J. Amer. Oil Chem. SOC., 36 (1959) 443 Radin, N. S., J. Amer. Oil Chem. Soc., 42 (1965) 569. Rubach, K., Schormiiller, J. and Melchert, H.-U., Lebensm. Ernuhr., (1971) 166. Sagredos, A. N., Fette, Seifen, Anstrichm., 69 (1967) 707. Sagredos, A. N., Fette, Seifen, Anstrichm., 7 1 (1969) 863. Schlenk, H. and Gellerman, J. L., Anal. Chem., 32 (1960) 1412. Stein, R. A. and Slawson, V., P r o p Chem. Fats Other Lipids, 8 (1966) 375. Tanaka, H. and Konishi, K., J. Chromatogr., 64 (1972) 61. Willncr, D., Chem Ind. (London), (1965) 1839. Zinkel, D. F. and Zank, L. C., Anal. Chem., 40 (1968) 1145.
Chapter 2 7
Lipids J. POKORNY
CONTENTS Introduction and general remarks .................................................. Separation of lipids into classes .................................................... Separation of glycerol esters and other neutral lipids. ................................... Separation of phospholipids and other polar lipids ..................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
581 581 585
588 590
INTRODUCTION AND GENERAL REMARKS Ldquid column chromatography and thin-layer chromatography are the most important techniques for the purification and fractionation of lipids, the former being mainly used for preparative and the latter for ahalytical purposes. The immense amount of literature that exists on this subject has been reviewed from various standpoints (Carroll, 1963; Creech; Kaufmann; Rouser et al., 1967a; Stein and Slawson). The sample often has to be purified prior to the fractionation by removing water and non-lipidic contaminants, e.g., by chromatography on a Sephadex G-25 column (Williams and Merrilees), especially if it has been obtained by extraction with chloroform and methanol. Lipid-protein complexes may be present, which can be resolved by chromatography on cross-linked polystyrene gels (Fisher). The separation of highly unsaturated lipids is preferably carried out under nitrogen, as otherwise oxidation could take place in the column (Marinetti). Silica gel can protect polyunsaturated fatty acids and their derivatives against autoxidation (Slawson and Mead). Phospholipids may be hydrolyzed to lysophospholipids during chromatography on silica gel (Renkonen, 1962; Camejo). Passage through Amberlite IRA-400 resulted in losses of some components of natural lipids and incomplete recovery of free fatty acids from the ion-exchange resin (Bottcher et al.). Lipids, even triacylglycerols, can be partially methanolyzed and transesterified (Schlenk and Holman).
SEPARATION OF LIPIDS INTO CLASSES The complex mixture of lipids is usually first separated into classes. Among numerous modifications, three methods are the most widely used. The procedure of Hirsch and Ahrens (Table 27. l), designed especially for the fractionation of neutral lipids and the separation of phospholipids, was reviewed by Creech, and in more detail by Sweeley, who compared the procedure with those of other workers who used silica gel columns. References p . 590
581
LIPIDS
582 TABLE 27.1 SEPARATION OF LIPIDS INTO CLASSES (HIRSCH AND AHRENS) Column 25.0 X 1.3 cm; 10 g of silica gel; 200 mg of sample. Amount (ml)
Solvent
Lipids separated
200
1% Diethyl ether in tight petroleum (b.p. 40-60°C) 4% Diethyl ether in light petroleum (b.p. 40-60°C) 20% Diethyl ether in light petroleum (b.p. 40-60°C) Diethyl ether
Cholesterol esters
250 150 I0
I0 100 100 200
Chloroform Chloroform-methanol (4: 1) Chloroform-methanol (3: 2) Chloroform-methanol (1 :4)
100
Methanol
Triacylglycerols and free fatty acids Free cholesterol Diacylglycerols and carotenoids Monoacylglycerols Phosphatidyle thanolamine Phosphatidylcholine Sphingomyelins, lysophosphatidylcholine Remaining phospholipids
Carroll (1961 and 1963) proposed the use of acid-washed Florisil as an adsorbent (Table 27.2). HIS procedure is suitable for neutral lipids and various applications have been discussed by Carroll and Serdarevich. Radin, in his review paper, pointed out the tenacious adsorption of phospholipids and carboxylic acids on this column, contrary to the situation on silica gel columns. Sulpholipids were eluted readily with chloroform-methanol (3: 1) but free fatty acids could be eluted only with an acidic solvent, eg., diethyl ether-acetic acid (96:4). The procedure of Rouser et al. (1967a, b and 1969) is designed for heterolipids (Table 27.3). Rouser er al. (1961) used chromatography on DEAE-cellulose and on silicic TABLE 21.2 SEPARATION OF LIPIDS INTO CLASSES (CARROLL, 1961) Column A: 17.0 X 2.0 cm; 30 g of Florisil; 40 mg of lipids. Column B: 15.0 X 1.2 cm; 12 g of Florisil; 30 mg of lipids. Amount of eluent (ml) Column A
Column B
50 120 150 150 150 150 150
20 50 75 60 60 75 75
Eluent
Lipids eluted
n-Hexane 5% Diethyl ether in n-hexane 15% Diethyl ether in n-hexane 25% Diethyl ether in n-hexane 50% Diethyl ether in n-hexane 2%Methanol in diethyl ether 4% Acetic acid in diethyl ether
Hydrocarbons Cholesterol esters Triacylglycerols Free cholesterol Diacylglycerols Monoacylglycerols Free fatty acids
583
SEPARATION OF LIPIDS INTO CLASSES
acid columns for the fractionation of brain lipids. Various modifications for the separation and determination of phospholipids and glycolipids were discussed by Rouser et al. (1967a). Wells and Dittmer succeeded in separating brain lipids into 24 classes by a combination of several column-chromatographic techniques. Leeder and Clark reported a micro-procedure for the analysis of serum lipids on silicic acid columns. Hardy et al. used silica gel G columns for the chroniatography of neutral lipids from fish. Several papers have been published on the separation of lipids into classes by gel chromatography. Sephadex LH-20 was the adsorbent most frequently used, and its use has been reviewed by De Lange. Nystrom and Sjovall used methylated Sephadex G-25 and G-50. Davenport reported a satisfactory separation of phospholipids from neutral lipids, contrary to previous results obtained by Berry and Kaye. The separation is probably due to the formation of larger micelles of phospholipids in non-polar solvents (Tipton et al.). The fractionation of neutral lipids on Sephadex LH-20 using chloroform (Calderon and TABLE 27.3 SEPARATION O F LIPIDS INTO CLASSES (ROUSER et af., 1969) Column, 20.0 X 2.5 cm; adsorbent, DEAE-cellulose; 100 mg of sample. Volume (number of bed volumes)
Solvent
Classes of lipids separated
8-10
Chloroform Chloroform-methanol (9: 1)
Neutral lipids Cerebrosides, monogly cosyldiglycerides, diglycosyldiglycerides, phosphatidylcholine, lysophosphatidylcholine, sphingomyelins Ceramide aminoethylphosphonate, ceramide dihexosides, ceramide polyhexosides, phosphatidylethanolamine Lysophosphatidylethanolamine, oxidation products of phosphatidylethanolamine, traces of phosphatidylethanolamine. Oxidation products of phosphatidylethanolamine, salts, phosphatidylethanolamine Free fatty acids, glycin-conjugated bile acids, unconjugated bile acids, pigments, non-lipidic substances soluble in organic solvents Phosphatidylserine, proteins No eluate, t o remove acetic acid from the column only Phosphatidic acid, phospha tidylinositol, phosphatidylglycerol, diphosphatidylglycerol, sulpholipid, cerebroside sulphate, unknown compounds, salts Salts, traces of lipids
9
9
Chloroform-methanol (7: 3)
9
Chloroform- methanol (1:l)
10
Methanol
10
Chloroform-acetic acid (3: 1)
10
Acetic acid Methanol
4
10
Chloroform-me thanolammonia-salt
10
Methanol
References p.590
584
LIPIDS
Baumann, 1970a) and ethanol (Calderon and Baumann, 1970b) as eluents has been described. The first procedure was found to be useful for the separation of glycerol esters from diol esters, ether-esters and other neutral lipids (Fig. 27.1). A combination of gel chromatography and silicic acid thin-layer chromatography was necessary for complete resolution. Calderon and Baumann (1 971) analyzed mixtures of glycol lipids, glycerol lipids, hydrocarbons and waxes. The best results were obtained by a combination of gel chromatography on Sephadex LH-20 with partition chromatography (Bende).
180
-
160
-
-
h
Y
I
3 9 140 2
0 F 120 -
3 W
100
80
1
I
I
I
I
Fig. 27.1. Separation of neutral lipids (Calderon and Baumann, 1970a). Column: 700 X 25 mm. Sorbent: Sephadex LH-20. Eluent: chloroform. Sample: 100 mg. Flow-rate: 0.25 ml/min. Detection: gravimetric detection of 1-ml fractions. The following compounds were separated (molecular weights in parentheses): trioctadecylglycerol(849.5); 1,2-dioctadecyl glycerol octadecenoate (863.5); 1-octadecylglycerol dioctadecenoate (877.5); glycerol trioctadecenoate (891 S ) ; dioctadecyl ethanediol(5 67.0); cholesteryl octadecenoate (653.1); octadecyl ethanediol octadecanoate (581.0); ethanediol dioctadecanoate (595 .O); dioctadecyl ether (523.0); octadecyl octadecanoate (537.0); octadecyl ethanediol acetate (356.6); cholesteryl acetate (428.7); octadecyl acetate (312.5); methyl octadecanoate (298.5); octadecanal(268.5).
The use of gradient elution adsorption chromatography for the analysis of lipid classes has been suggested by Stolyhwo and Privett. The chromatographic system consisted of a pressurized apparatus (inlet pressure 20 p.s.i.) and involved the use 0f.a continuous series of gradient changes of n-pentane, diethyl ether, chloroform, and methanol containing 8% ammonia. The 1000 X 2.8 mm column was packed with Corasil I1 (37-55 pm), modified by treatment with 28% ammonia. The following set of standard lipids was separated: methyl oleate, trioleyl glycerol, cholesterol, 1,3-dioleyl and 1,2-dioleyl glycerol, l-monooleyl glycerol, beef brain cerebrosides, phosphatidyl ethanolamine, phosphatidyl choline, and beef brain sphingomyelins. The procedure has been applied for the separation of lipids of rat red blood cells.
SEPARATION OF GLYCEROL ESTERS AND OTHER NEUTRAL LIPIDS
585
SEPARATION OF GLYCEROL ESTERS AND OTHER NEUTRAL LIPIDS Triacylglycerols are the most difficult lipids to subfractionate within the neutral lipid classes because of the very similar physical properties of the various compounds. Silylated Celite was a satisfactory adsorbent for the separation of trisaturated glycerol esters and for the fractionation of cocoa butter if acetone--n-heptane-water systems were used as eluents (Black and Hammond). The procedure could be useful in combination with modern continuous detectors. Evans et al. used their method of column chromatography on silica gel and mixtures of methanol and benzene as eluents, which they had applied previously to the fractionation of saturated glycerol esters, for the separation of triacyl glycerols of oils that contain hydroxy and keto acids, such as isano, oiticica, castor and kamala seed oils. De Vries utilized the formation of adducts between silver nitrate and the double bonds of unsaturated fatty acids, and succeeded in resolving triacylglycerols that differed by one double bond only (Fig.27.2) using a column of silica gel impregnated with silver nitrate. Dolev and Olcott found the procedure to be efficient, even for the separation of polyunsaturated acylglycerols. Another approach involves the transformation of unsaturated triacylglycerols into the corresponding mercury adducts. The chromatography of the adducts is possible on either silicic acid (Hirayama) or deactivated Florisil (Kerkhoven and Deman). The latter procedure was especially suitable for the determination of trisaturated acylglycerols. Bombaugh el al. fractionated saturated triacylglycerols on Styragel (cross-linked polystyrene) using 48,770 X 9.5 mm columns with a flow-rate of 0.4 mllmin and tetrahydrofuran as the eluent. In spite of the extreme efficiency of the column, the peak width was of the same order as the difference between the elution times of triacylglycerols that differed by six carbon atoms. From this example, it can be seen how difficult it is t o separate triacylglycerols of natural fats. Gel chromatography seems to be promising for the analysis of mixed triacylglycerols (hilulder and Ruytenhuys). The dimerization of glycerides during heating could be foliowed by chromato,:raphy on either Sephadex LH-20 (Aitzetmiiller) or Spheron S-232 (Pokomj, et al., 1972) gel (Fig. 26.1 ). Scharmann and Unbehend described the fractionation of heated fats into monomers, dimers and higher oligomers of triacylglycerols using gel chromatography. Liquid chromatography on silica gel using a Pye LC detector was used for the analysis of oxidized triacylglycerols. The oxidation and polymerization of glycerol esters during heating, especially frying, has often been studied after prior conversion into fatty acids (Kajimoto and Katsunori), as their solubility is better and the differences due to oxidation are more pronounced. Perkins et al. compared the separation of fats heated under the conditions of deep fat frying using Sephadex LH-20 and Bio-Beads SX-1 gels. Chloroform, acetone, chloroformmethanol mixtures, and tetrahydrofuran were used as eluents with a Waters 401 differential refractometer as detector. The efficiencies of the gels S-832 and S-232 for the separation of heated oils and of methyl esters prepared from the heated oils were compared by Pokornp et al. ( I 974) who used tetrahydrofuran as eluent, five 1200 X 5 mm columns and a combination of a differential refractometer and a UV analyzer as detectors. Aitzetmiiller (1973b) reported recently on the estimation of total polar products in frying oils by liquid chromatography. He used a modified Pye LC solute transport References p.590
586
LIPIDS
A
I
2 12
I0
8
6
-
E"
5
n
4
4
v
z
0I- 2 a
a
I-
2 w
$
'
A 10
0 25
B 4
20 I
15
-
1I
2
10
5
5
10
15
20
25
30
35
FRACTION NUMBER
Fig. 27.2. Separation of triglyceridcs (De Vries). Column: 40 X 1.1 cm. Sorbent: silica gel impregnated with silver nitrate. Eluent: fractions 1 -40, benzene; 40-60, diethyl ether. Operating conditions: flow-rate 30 ml/h; 10-ml fractions collected and weighed; temperature 15°C. Detection: gravimetrically; the composition of the whole fraction was determined by GLC. A = palm oil: 1, tripalmitoylglycerol; 2, dipalmitoyloleylglycerol; 3, dipalmitoyllinoleylglycerol; 4, palmitoyldioleylglycerol; 5, mixture of palmitoyloleyIlinoleylglycerol and trioleylglycerol; 6 , glycerides containing linoleic, oleic qnd palmitic acids. B = synthetic mixture: 1, tristearoylglycerol; 2, dipalmitoyloleylglycerol; 3, stearoyldioleylglycerol; 4,trioleylglycerol.
detector, 200 X 4 mm columns filled with Merckogel SI-50 or Porasil A (both 36-75 pm). Usually, 10-30 pl of a 20-30% solution of the sample in n-heptane as injected. The Ultrograd gradient mixer was used to switch the solvents. Combinations of n-heptane, diisopropyl ether, ethanol and water were used as solvents. Similarly, samples of heated
SEPARATION OF GLYCEROL ESTERS AND OTHER NEUTRAL LIPIDS
587
oils may be separated, on 300 X 8 mm columns of silicic acid by suitable elution with various concentrations of diisopropyl ether in n-heptane followed by ethanol in diisopropyl ether, in$o several fractions (Aitzetmuller, 1973a) which are subfractionated by gel chromatography. By analyzing model substances, such as dimeric trioleyl glycerol or monoepoxytrioleyl glycerol Aitzetmuller ( 1 9 7 3 ~ showed ) that both non-polar and polar artefacts in frying oils are eluted in one peak by frontal elution liquid chromatography after a procedure similar t o that given before (Aitzetmiiller, 1973b). A combination of a UV detector and a moving wire detector showed that most UV-active substances in heated oils belong t o the polar artefacts (Aitzetmiiller, 1973d). The amounts of polar and non-polar oligomers could be determined by the combination of liquid chromatography and gel chromatography. Mono- and diacyglycerols are generally determined on silicic acid columns by the method of Quinlin and Weiser. The method was recently adopted by IUPAC as an international standard method. Silica gel, of particle size 0.07-0.1 5 mm,should be free from ethersoluble substances and free from iron, and should contain 5% of water. A slurry prepared from 30 g of silica gel and 50 ml of light petroleum is transferred t o a 19 X 300 mm column. A solution of 1 g of sample in 15 ml of chloroform (the temperature of the solution should be kept below 40°C) is added to the column, and an additional 5 ml of chloroform are used for rinsing out the beaker containing the sample solution. The flowrate is adjusted t o 2 ml/min and the triacylglycerol fraction is eluted with 200 ml of benzene. Benzene-ðyl ether (90: 10) is then used t o elute diacylglycerols and free fatty acids. The monoacylglycerol fraction is then eluted by passing 200 ml of diethyl ether through the column. The amount of free fatty acids in the second fraction is determined by titration, by means of the known medium molecular weight of fatty acids, the weight of free fatty acids is calculated, and the weight of the diacylglycerol fraction is thus corrected. The method gives erroneous results if the free fatty acid content is higher than 5%. If the free fatty acid content exceed$2%, all of the fractions should be tested and corrected for the presence of free fatty acids. The interfering effect of short-chain and hydroxylic acids on the separation was studied by Franzke et al. Because of their different structures, the isomeric acylglycerols can be resolved by gel chromatography. Joustra ef al. studied the separation of 1,3- and 1,2-dipalmitoylglycerols on Sephadex LH-20. Pokorny et al. (1973) studied the separation of 1- and 2-monostearoylglycerol on Spheron S gel using 8 X 6000 mm columns, a flow-rate of 35 d / h , tetrahydrofuran as the eluent and a Waters Model R-4 differential refractometer and a UV flow analyzer as detectors. Sucrose esters of fatty acids were satisfactorily resolved into mono-, di-, tri- and higher esters on Sephadex LH-20 columns using dimethylformamide as eluent (Konishi et al.), sorbitans on silica gel columns (Cedras et al.) and polyethylene glycol esters on silica gel pre-wetted with benzene (Papariello et al. ). Wickbold separated polyethylene glycol esters of fatty acids into free glycol, free fatty acids, monoester and diester by chromatography on silylated silica gel columns, eluting with isopropanol-water mixtures after a modified procedure of C.I.D.* *C.I.D. = Cornit6 International des D6rivBs Tensioactifs, Paris, France.
References p . 590
588
LIPIDS
Natural waxes are fractionated into classes either on alumina of various degrees of activation (Wiedenhof) or on silica gel (Netting). A complicated procedure for the systematic analysis of waxes by chromatography on silica gel, an ion-exchange resin and silica gel impregnated with non-polar solvents was described by Scholz. Waxes of Vernix caseosa and skin lipids were fractionated into sterol ester and wax ester fractions by chromatography on magnesium oxide MX-66 columns with use of n-hexane and a 1% solution of acetone in n-hexane as eluents (Nicolaides et d.).
SEPARATION OF PHOSPHOLIPIDS AND OTHER POLAR LIPIDS The phospholipidic and glycolipidic fractions, isolated by the above methods of separation into lipid classes, can be further sub-fractionated by various column chromatographic procedures. For the major components, combinations of column and thin-layer chromatography can be used, while for the minor components the use of preparative column chromatography is necessary, even for the sub-fractionation. The most suitable technique for separating the sample under study will depend on the composition of the substrate. Various methods based on chromatography on DEAE-cellulose (Table 27.3) or TEAEcellulose (Table 27.4) columns were reviewed by Rouser et al. (1969). The present author considers his system to be the most suitable and adaptable to various substrates. A combination of column chromatography on DEAE-cellulose followed by re-chromatography on silica gel was successful for the separation of phospholipids of rapeseed gum (Weenink and Tulloch). Cerebrosides’were separated after preliminary removal of phospholipids by chromatography on Florisil deactivated with 10%of water using mixtures of chloroform-methanolwater as solvents (Mehl and Jatzkewitz). A similar procedure on silica gel was recommended by Klenk and Schorsch for the analysis of brain cerebrosides. Earlier methods for the analysis of gangliosides were based on DEAE-cellulose chromatography (Trams and Lauter, Wolfe and Lowden). Extracts containing gangliosides were purified by chromatography on Sephadex G-100 columns (Raveglia and Ghittoni). Kwiterovich et al. described the separation of liver glycolipids and phospholipids into classes. Gel chromatography on Sepharose gave good results for the purification of lipopolysaccharides in comparison with ultracentrifugation, and the chromatographic procedure was simple and rapid (Romanowska). Prostaglandins are best isolated from natural biological material by extraction and purification of the extract by silica gel column chromatography combined with preparative thin-layer chromatography (Clausen and Srivastava). The procedure of Rouser et al. (1967a, b) is still the most widely used for the fractionation of phospholipids. Slight modifications were suggested by Hladik and Pokorny for the separation of oxidized phospholipids. Shimojo et al. (1962, 1971) separated phospholipids into 13 fractions by chromatography on cellulose equilibrated with chloroform and eluted with chloroform containing increasing amounts of methanol. Renkonen (1 963) separated serum lipids into neutral lipids, cephalins, lecithins, sphingomyelins and lysolecithin by chromatography on silica gel columns and subsequent sub-fractionation
SEPARATION O F PHOSPHOLIPIDS AND OTHER POLAR LIPIDS
589
TABLE 27.4 SEPARATION OF TOTAL LIPIDS ON TEAE-CELLULOSE (SUITABLE FOR ACIDIC LIPIDS) (ROUSER et al., 1969) Column: 25 X 200 mm. Adsorbent: Selectacel, regular grade (Brown, Berlin,'N.H., U.S.A.), or equivalent TEAE-cellulose; 15 g of adsorbent. Sample load: 100-300 mg in 5-10 ml of chloroform. Flow-rate: 3 ml/min. Bed volume: ca. 7 5 ml. Preparation of column: washing with 4 column volumes of 0.01 N potassium hydroxide in methanol, followed by 6-8 column volumes of methanol, 4 column volumes of methanol-chloroform (1 :1) and 4 column volumes of chloroform. Volume (number of bed volumes)
Solvent
Classes of lipids separated
5
Chloroform
8
Chloroform-methanol (9: 1)
8
Chloroform-methanol (2:l) Methanol
Free sterols, sterol esters, glycerides, hydrocarbons, carotenes, xanthophylls, phytins, chlorophylls Crrebrosides, glycosyl diJycerides, phosphatidylcholine, sphingomyelin, more polar xanthophylls Ceramidepoly hexosides
8 6
Chloroform-me thanol (2: 1) t 1% glacial acetic acid
6 3
Glacial acetic acid Methanol
8
Chloroform-methanol (4: 1) made 0.01 -0.1 M in ammonium or potassium acetate, t o which 20 ml of 22% aqueous ammonia is added Methanol
6
Inorganic substances formed by ion exchange with acidic lipids Phosphatidylethanolamine, lysophosphatidylethanolamine, ceramideaminoethylphosphonate, free fatty acids, free bile acids, glycine-conjugated bile acids, phorbides, xanthophylls containing carboxyl groups Phosphatidylserine, residual protein Only wash for removal of excess of acetic acid Final acidic lipid fractions, such as phosphatidic acid, diphosphatidylglycerol, phosphatidylglycerol, cerebroside sulphate, plant sulpholipid, phosphatidylinositol Remaining lipids
into individual components by further column chromatography, e.g., on DEAE-cellulose or neutral alumina. The chromatography on silica gel columns can be combined with another technique, e.g., ultracentrifugation in the investigation of lipoproteins (Nelson and Freeman). A similar technique on silica gel columns was applied to the analysis of milk phospholipids (Smith and Freeman). A suitable ion exchanger for the fractionation of phospholipids was prepared by treating chlorohydroxypropylated Sephadex and cellulose with ammonia or amines, e.g., the dibutylaminohydroxypropyl derivative (in the acetate form) of Sephadex LH-20 was used for the separation of egg phospholipids (AlmC and Nystrom). Sphingomyelins are isolated by pre-fractionation on silica gel, inter-esterification of ester phospholipids with sodium methylate catalyst and a second fractionation on alumina References p.590
590
LIPIDS
(Hausheer er al.). Plasmalogens are determined after treatment of the column chromatographic fractions with 2,4-dinitrophenylhydrazineand phosphoric acid (Rhee et al.). The 2,4-dinitrophenylated and methylated phospholipids can be fractionated on cellulose columns (Collins and Shotlander). Robles and Roels recommended the preliminary deacylation of phosphogly cerides by alkaline hydrolysis followed by silica gel chromatography. Tipton et al. studied the separation of phospholipids by gel chromatography on polystyrene cross-linked with 2% of divinylbenzene. Egg lecithin was separated reasonably well from neutral lipids. Kisselev fractionated lipids from brain tissue on Sephadex LH-20. In chloroform-methano! (2: l), Sephadex behaved as a weakly basic anion exchanger so that phospholipids were separated according to their basic properties. In chloroformmethanol-water (65:35:8), however, it acted as a molecular sieve so that the substances were separated according to their molecular weights. The use of methylated Sephadex was reviewed by Ellingboe et al. ; Sephadex (3-25 was methylated so as to contain 40% of methoxyl groups, and lipids were eluted with chloroform-methanol-water (85:85:30). Under these conditions, phospholipids were eluted in the first fraction together with lipopeptides and glycolipids. DEAE-Sephadex LH-20 was found to be advantageous for the fractionation of acidic lipids of Escherichia coli (Dittmer). Downey e t al. and Shimojo et al. (1971) also successfully isolated phospholipids by gel Chromatography on Sephadex LH-20.
REFERENCES Aitzetmiiller, K., Fette, Scifen, Anstrichm., 74 (1972) 598. Aitzetmiiller, K., Fette, Seifen, Anstrichm., 75 (1973a) 14. Aitzetmiiller, K.,Fette, Seifen, Anstrichm., 75 (1973b) 256. Aitzetmiiller, K., J. Chromatogr., 79 ( 1 9 7 3 ~ 329. ) Aitzetmiiller, K.,J. Chromatogr., 83 (1973d) 461. A h & ,B. and Nystrom, E . , J . Chromatogr., 59 (1971) 45. Bende, H., Fette, Seifen, Anstrichm., 70 (1968) 937. Berry, J. F. and Kaye, B., Lipids, 3 (1968) 386. Black, B. C. and Hammond, E. G.,J. Amer. Oil Chem. Soc., 40 (1963) 575. Bombaugh, K . J., Dark, W . A. and Levangie, R . I;.,J. Chromatogr. Sci., 7 (1969) 42. Bottcher, C. J. F., Woodford, I;. B., Boelsma-van Houte, E. and Van Gent, C. M., Rec. Trav. Chim. Pays-Bas, 7 8 (1959) 794. Calderon, M. and Baumann, W . J . , J . LipidRes., 11 (1970a) 167. Calderon, M. and Baumann, W. J., Boichim. Biophys. Acta, 210 (1970b) 7. Calderon, M. and Baumann, W. J., Biochim. Biophys. Acta, 231 (1971) 52. Camejo, G . , J. Chromatogr., 21 (1966) 6 . Carroll, K. K.,J. Lipid. Res., 2 (1961) 135. Carroll, K. K., J. Amer. Oil Chem. Soc., 40 (1963) 413. Carroll, K. K. and Serdarevich, B., Lipid Clvomatogr. Anal., 1967-1969, 1 (1967) 205. Cedras, J., Carlier, A., Puisieux, F. and Lettir, A., Ann. Pharm. Fr., 25 (1967) 553. Clausen, J. and Srivastava, K. C., Lipids, 7 (1972) 415. Collins, I;. D. and Shotlander, V. L., J. Lipid Res., 1 (1960) 352. Creech, B. G., J. Amer. Oil Chem. Soc., 38 (1961) 540. Davenport, J. B., Lipids, 4 (1969) 308. De Lange, J. H., Chem. Tech. (Amsterdam), 26 (1971) 276.
REFERENCES
59 1
De Vries, B.,J. Amer. Oil Cbern. Soc., 41 (1964) 403. Dittmer, J . C.,J. Clrrornatogr.,43 (1969) 512. Dolev, A. and Olcott, H. S . , J . Amer. Oil Cbem. Soc., 42 (1965) 624, 1046. Downey, W. K., Murphy, R. F. and Keogh, M. K., J. Cbrornatogr., 46 (1970) 120. Ellingboe, J., Nystrom, E. and Sjovall, J., Methods Enzymol., 14 (1969) 317. Evans, C. D., McConnell, D. G., Hoffmann, R. L. and Peters, H., J. Amer. Oil Cirem. Soc., 44 (1967) 281. Fisher, N., J. Ozromatogr., 47 (1970) 501. Franzke, C., Krctzschmann, F., Kustow, B. and Rugenstein, H., Pbarmazie, 22 (1967) 487. Hardy, R., Smith J. and Mackie P. R.,J. Cbromatogr., 57 (1971) 142. Hausheer, L., Pedersen, W. and Bernhard, K., Helv. Cliim. Acta, 46 (1963) 601. Hirayama, 0.. Nippon Nogei Kagaku Kaisbi, 35 (1961) 437. Hirsch, J. and Ahrens, Jr., E. A.,J. Biol. Cbem., 233 (1958) 311. Hladik, J., JirouSova, J . and PokornL, J., Zeszyty Probl. Postepow Nauk Roln., 136 (1973) 87. IUPAC, Fat and Oil Section, Standard Methods, Metbod //.C 7 , Butterworths, London, 1972. Joustra, M., Soderqvist, B. and Fischer, L.,J. Cbromatogr., 28 (1967) 21. Kajimoto, G . and Katsunori, M., E'iyo-to-Sbokuryo,17 (1965) 319. Kaufmann, H. P., Fette, Seifen, Anstricbm., 7 2 (1970) 505. Kerkhoven, E. and Deman, J. M., J. Cbromatogr., 24 (1966) 5 6 . Kisselev, G . V., Biokbinziya, 34 (1969) 483. Klenk, E. and Schorsch, E. ti., Hoppe-Seyler's Z . Pbysiol. Cbem., 348 (1967) 1061. Konishi, K., Inoue, H. and Taniguchi, N.,J. Cbromatogr., 54 (1971) 367. Kwiterovich, Jr., P. O., Sloan, H. R. and Fredrickson, D. S . , J. Lipid Res., 11 (1970) 322. Leeder, L. G . and Clark, D. .4., Micbrocbem. J . , 12 (1967) 396. Marinetti, G . V . , J . Lipid Res., 3 (1962) 1. Mehl, E. and Jatzkewitz, H., Naturwissenschaften, 50 (1963) 227. Mulder, J. L. and Buytenhuys, F. A., J. Chromatogr., 51 (1970) 459. Nelson, G . J. and Freeman, N. K., J. Biol. Cbem., 235 (1960) 578. Netting, A. G . , J . Cbromatogr., 53 (1970) 507. Nicolaides, N., Fu, H. C., Ansari, M. N. A. and Rice, G. R., Lipids, 7 (1972) 506. Nystriim, 1:. and Sjovall, J., A n d Biocbem., 1 2 (1965) 235. Papariello, G. J., Chulkaratana, S., Higuchi, T., Martin, J . E. and Kuceski, V. P., J. Amer. Oil Cbem. Soc., 37 (1960) 396. Perkins, E. C, Taubold, R. and Hsieh, A,, J. Amer. Oil Cbem. Soc., 50 (1973) 223. Pokornf, S., Coupek, J., LuHn, N.-T. and PokornL, J., J;Chromatogr., 84 (1973) 319. Pokornf, J., Kundu, M. K., Pa'rizkovi, H., Luln, N.-T., Coupek, J., Pokornf, S. and Janizek, G., Fette, Seifetr, Anstricbm.. 74 (1 972) 625. PokornL, J., Kundu, M. K., PokornL. S., Bleha, J . and e o u p e k , J., J. Cbromatogr., (1974) i n press. Quinlin, P. and Weiser, Jr., H . J., J. Amer. Oil Cbem. Soc., 35 (1958) 325. Radin, N. S . , Metbods Enzymol., 14 (1969) 268. Raveglia, I. F. and Chittoni, N. E.,J. Cbromatogr., 58 (1971) 288. Renkonen, O., J. Lipid Res., 3 (1962) 181. Renkonen, O., Acta Cbem. Scund., 17 (1963) 1925. Rhee, K. S., Del Rosario, R. R. and Dugan, L. R. Jr., Lipids, 2 (1967) 334. Robles, E. C. and Roels, G. F. M., Cbem. Pbys. Lipids, 6 (1971) 31. Romanowska, E., Anal. Biocbem., 33 (1970) 383. Rouser, G., Bauman, A. J., Kritchevsky, G . , Heller, D. and O'Brien, J. S., J. Amer. Oil. Cbem. Soc., 38 (1961) 544. Rouser, G., Kritchevsky, G . , Simon, G . and Nelson, G . J., Lipids, 2 (1967a) 37. Rouser, G . , Kritchevsky, G. and Yamamoto, A., Lipid C/zromatogr. Anal., 1 (1967b) 99. Rouser, G., Kritchevsky, G . , Yamamoto. A,, Simon, G . , Galli, C. and Bauman, A . J.,Metbods Enzymol., 14 (1969) 272. Scharmann, H. and Unbehend, M., Hauptversammlutig, Ges. Deut. Cbem.; Angew. Cbem., 83 (1971) 929.
592 Schlenk, H. and Holman, R. T.,J. Amer. Oil Chem. Soc., 30 (1953) 103. Scholz, G. H., Fette, Seifen, Anstrichm., 69 (1967) 565 and 651. Shimojo, T., Kanon, H. and Ohno, K., J. Biochern. (Tokyo), 69 (1971) 255. Shimojo, T., Yokoyama, A. and Ohno, K.,J. Biochem. (Tokyo), 51 (1962) 293. Slawson, V. and Mead, J. F., J. Lipid Res., 13 (1972) 143. Smith, L. M. and Freeman, N. K.,J. Dairy Sci., 42 (1959) 1450. Stein, R. A. and Slawson, V., Progr. Chem. Fats Other Lipids, 8 (1966) 375. Stolyhwo, A. and Privett, 0. S., J. Chromatogr. Sci., 11 (1973) 20. Sweely, C. C., Methods Enzymol., 14 (1969) 254. Tipton, C. L., Paulis, J. W. and Pierson, M. D.J. Chromatogr., 14 (1964) 486. Trams, E. G. and Lauter, C. J., Biochim. Biophys. Acta, 60 (1962) 350. Weenink, R. D. and Tulloch, A. P., J. Amer. Oil Chem, SOC.,43 (1966) 327. Wells, M. A. and Dittmer, J. C., Biochemistry, 5 (1966) 3405. Wickbold, R.,Fette. Seifen, Anstrichm., 74 (1972) 578. Wiedenhof, N., J. Amer. Oil Chem. Soc., 36 (1959) 297. Williams, J. P. and Merrilees, P. A., Lipids, 5 (1 970) 367. Wolfe, L. S. and Lowden, J . A., Can. J. Biochem., 42 (1964) 1041.
LIPIDS
Chapter 28
Steroids
i. PROCHAZKA CONTENTS Introduction .................................................................. 593 General techniques ........................ ......... .. . . . . . . . . . . . . . .594 Introductory and theoretical considerations ........................................ 594 Sample preparation and applic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 Liquid-solid chromatography ......................................... 595 Examples of the preparatio itrate-impregnated or silvered adsorbents ......... 595 Liquid-liquid chromatography ................................................. 597 Hydrophobization of Kieselguhr (Celite) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598 Purification of Hostalen .................................................... 599 Hydrophobization of Sephadex ............................................... 599 Gel chromatography ............................................. Ion-exchange chromatography ..................................... Detection .................................................................. 603 Porter-Silber reagent .................... .-. ............................... 603 Zimmermannreagent ...................................................... 603 Applications .................................................................. 604 Sterols .................................................................... 604 Androgens ................................................................. 604 Estrogens ................................................. . . . . . . . . . . . . . .605 Gestagens (progestins) ....................................... . . . . . . . . . . . . . .613 Corticosteroids .............................................................. 614 Bile acids and other steroid acids ................................................ 617 Steroidal glycosides ........................................................... 618 Steroidal insect hormones ..................................................... 619 References .................................................................... 620
INTRODUCTION The number of papers that describe or mention the use of column chromatography in the separation of steroid derivatives is enormous . It would be interesting to know the percentage of papers on steroids that do not mention chromatography . Taking into consideration that the number and variety of known steroids are also tremendous. it is impossible to review here all. or even most. of the applications of column chromatography in the steroid field . All that can be done is to restrict the survey to more recent papers. to indicate various types of chromatographic methods. t:, compare them (if they are comparable). and to try to recommend some types of chromatographic procedures for certain particular types of steroids or separations . Some steroid mixtures can be. and actually have been. separated satisfactorily by several chromatographic methods that differ in principle . As an illustration. a quotation References p .620
59 3
594
STEROIDS
by Cavilla et al. (1971) may suffice, indicating that similar results can be achieved by two chromatographic methods. They used silicic acid columns (cc, Figs. 28.2 and 28.3) and a gradient of diethyl ether in light petroleum for elution. However, they state: “A recent paper by Fernandez and Noceda describes separations of progestins and estrogens by means of column chromatography on Sephadex LH-20 with methanol-water (85: 15) as eluent; our results compare favourably with those reported by these authors, especially regarding better separations between different progestins”. Experience shows that a synthetically or pharmaceutically oriented chemist will probably prefer the silicic acid method, while some biologically or clinically oriented biochemists will choose the Sephadex LH-20 method. However, the laboratory worker should always keep in mind the purpose the separation serves. For example, a chemist isolating a small amount of a new or unknown steroid from a natural material will probably have a double task: first the eiimination of ballast in a preliminary run (on a large amount of sorbent, but a shorter column), and then the required separation and purification on a small diameter, but very long, column. Finally, a clinical biochemist, often working with minute amounts of labelled steroids present in biological liquids, will have the problem of achieving accurate and economical results with expensive material and sophisticated equipment, especially when his work is routine. All these problems make the recommendation of a general procedure for sample preparation, application, chromatography and detection in the steroid field very difficult, and in the space devoted to steroids in this book impossible.
GENERAL TECHNIQUES lntroduct ory and the oretical considerations For the choice of the most suitable method for a particular separation problem in the steroid field, several criteria must be kept in mind: (a) scale, i.e., the amount of the mixture to be separated; (b) proportions of the required or analyzed steroid or steroids in the mixture, i.e., the number and amounts of components; (c) the physico-chemical character of the steroids to be separated, i.e., polarity, solubility, etc.; and (d) the structures of the steroids to be separated. The differences in polarity among steroids are enormous, ranging from sterols esterified by fatty acids, the lipophilic character of which is similar to those of fats and paraffins, to steroid glycosides or bile acid conjugates, which are appreciably soluble in water. Nonetheless, most steroids are of medium polarity, with a tendency towards lipophilicity owing to their large hydrocarbon skeleton. This is the reason why adsorption chromatography with solvents of low polarity largely prevails in this field, while such methods as gel permeation or affinity chromatography are used much less often. The last criterion, structure, may also become decisive when choosing the most suitable method to use. For example, the use of ion-exchange resins seems t o be suitable for the separation of ionisable steroids, such as bile acids or some ionisable steroid conjugates. It is also known that homologous series are poorly separated on adsorbents, but are well separated in liquid-liquid systems. In the latter case, the steroids that are poorly separated in a system of partly miscible phases may be converted into more
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lipophilic derivatives (for example, by acylation) and separated in a pair of very immiscible solvents, or on very active adsorbents that seem adequate for substances of very weak polarity. The furmation of steroid derivatives is also currently used for making the steroids tractable, for example volatile and thus suitable for gas chromatography.
Sample preparation and application Because of the variety of steroids that exist, no general procedure for sample preparation can be given, but a few hints may be useful. As most steroids are neutral, partition of an extract from natural material between an organic solvent (most commonly toluene, benzene, chloroform, dichloromethane, diethyl ether and ethyl acetate) and an aqueous alkali solution is recommended for the elimination of organic acids or other acidic material. Washing an organic extract with dilute hydrochloric acid is useful in cases when alkaloids or other basic impurities are present. However, highly polar neutral steroids can behave like estrogens, which are weakly acidic owing t o the presence of a phenolic group, or bile acids when partitioned between a non-polar solvent, as for example toluene or chloroform and stronger aqueous alkali (Eberlein). In this case, filtration through an ion-exchange resin column may also lead to a high enrichment of the sample (Hobkirk and Nilsen, 1969a, b). Except for sterol esters and some very unpolar steroids, crude organic extracts from plant or, especially, animal material containing steroids can be pre-purified before application by partition between light petroleum (or n-hexane, n-heptane or other hydrocarbons) and 90-95% methanol. Common steroids remain in the polar phase, while paraffins, fats and the above exceptions pass into the paraffinic solvent phase. The effect of enrichment by such partitioning can be considerably increased by applying the counter-current distribution technique. Application of the chromatographed mixture on t o a classical preparative column can be carried out in several ways (see Chapter 8). Introduction of the sample into the head of an analytical, high-efficiency , micro-column is usually achieved by injection of a solution of the sample in the mobile phase. The heads of such columns are of a special construction (see Chapter 8).
Liquid-solid chromatography The best and most commonly used sorbents for the liquid-solid chromatography of steroids are alumina, silica gel and Florid, with the use of silica gel largely prevailing today. Silver nitrate-impregnated adsorbents have been used successfully for difficult separations of steroids that differ only in the position or the number of double bonds. This method, although used extensively nowadays, is relatively new and therefore deserves the following more detailed description.
Examples of the preparation of silver nitrate-impregnated or silvered adsorbents Silicic acid A 25-g amount of silver nitrate was dissolved in 500 ml of distilled water; 100 g of silicic acid (Unisil; Clarkson Chemical Co., Williamsport, Pa., U.S.A.) were added t o this References p . 620
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STEROIDS
solution, the water was removed from the slurry with a rotary evaporator and the adsorbent was dried overnight in an oven at 110°C. If the adsorbent is not protected from light, it steadily becomes darker, but this effect does not affect its activity to any appreciable extent. (Vroman and Cohen.)
Silica gel G and Celite Silver nitrate (16.8 g), silica gel G (41.6 g) and Celite (41.6 g) were mixed as a suspension in water. (Canonica et al., Galli and Grossi-Paoletti.) Alumina and Hyflo Supercel Neutral alumina AG-7 (30 g) was mixed with Hyflo Supercel (Johns-Manville, Denver, Colo., U.S.A.) (1 5 g), a solution of 9 g of silver nitrate in 75 ml of water was added and the mixture swirled. The suspension was frozen and lyophilized and stored in vacua overnight. Colurnns were prepared from a suspension of the powder in a solvent (usually 98: 2 chloroform-methanol). (Paliokas et al.) Silvered Florisil Florisil (1 0 g, 60-100 mesh) was suspended in a solution of 1.5 g of glucose in 150 ml of water. A solution of 160 mg of silver nitrate in 20 ml of water containing 1 ml of concentrated ammonia solution was then added with stirring at room temperature, and, after heating the mixture at 50°C in a water-bath for 10 min with constant stirring, the silica gel was filtered and washed thoroughly with water. The product was dried at 80°C for 4 0 min. (Ercoli etal.) In the classical .preparative elution chromatography (the so-called Reichstein chromatography) on alumina, a 1 :30 ratio of sample to adsorbent is most commonly used, the elution being started with a non-polar solvent (usually light petroleum), the polarity of the solvent being increased by the gradual addition of a more polar solvent or the solvent being changed to a more polar solvent when solid material can no longer be eluted with the first solvent. With silica gel or Florisil, the ratio of sample to adsorbent is usually between 1 :SO and 1 :loo. For difficult separations or analytical purposes, this ratio ranges from 1 :100 to 1 : 1000. Because of some advantages (rapidity and ease of preparation, and the similarity between the results and those obtained by TLC), dry column chromatography on silica gel has become the method of choice in more difficult preparative separations. As regards the choice of solvents and the volume of the fractions, the method of Joska (Chmel ef al.) is recommended. Employing non-adhering thin layers of silica gel, a solvent system is first found in which the least polar component of the separated mixture has an RF value of ca. 0.5. The same solvent is then used for dry column chromatography, but the volume of the fractions collected (in millilitres) should be about one twentieth of the weight (in grams) of the adsorbent in the column. As a rule, if a good separation is achieved on common thin-layer plates with a particular solvent, then a less polar solvent should be used for column chromatography. For optimization of the composition of the solvent system in TLC, Turina proposed a method in which the best separation can be achieved while carrying out the least number of experiments. This method is based on
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general principles and should also be applicable t o column chromatography. Examples for analytical liquid-solid high-resolution (high-, medium-, and low-speed) chromatography are given in the Applications section of this chapter. At this point the multicolumn high-resolution liquid-solid chromatography systems developed by Vestergaard and his co-workers ( c t , Vestergaard, 1973) should be mentioned. His system is of medium to low speed, but the high resolution and the multiplicity and automation of accessories allow the use of his method for large numbers of routine serial analyses of urinary steroids per day, i.e., up t o 100 analyses or more per day. The main features of his method are: (a) automated dry-filling of 6 ft. X 1.5 mm PTFE capillary columns; 20 columns are filled at a time in 5-20 min. (Vestergaard, 1967); (b) gradient elution on neutral alumina (100-200 mesh) deactivated with 6%water (for 17-ketosteroids), or on silica gel (100-200 mesh) containing 20% water (for corticosteroids), and the use of a battery of 12 or more chromatographic pumps (Vestergaard et al., Vestergaard and Sayegh) or a nitrogen pressure system (Vestergaard and Jacobsen) for chromatography on 25 columns; (c) a battery of needle valves connected with a battery of simple injection ports and PTFE columns (see under a) immersed in a water-bath; (d) a multicollector; (e) a computer; (13 running times from about 6 h up t o 24 h and more. Centrifugal liquid-solid chromatography of steroids on microcolumns of microparticulate silica was described by Ribi et ul. The method seems very rapid (separations in about 7 min) and rather efficient.
Liquid-liquid chromatography Liquid-liquid chromatography is much less frequently used in synthetic organic chemistry. The commonest support for the polar stationary phase is diatomaceous earth (Celite or Hyflo Supercel), but cellulose powder and other carriers have also been used. Various polar organic solvents, often containing water, can be used as the stationary phase. Aqueous methanol or acetic acid (for bile acids) and formamide are among the most commonly used, while various hydrocarbons or their mixtures with other non-polar solvents (benzene, toluene, etc.) serve as mobile phases. For column preparation (Engel e f ul.), Celite or Hyflo Supercel is washed with 6 N hydrochloric acid in order t o remove metals, then washed by elutriation until free from chloride and fines. Finally, it is washed with absolute methanol and light petroleum and dried. The components of the partition system are agitated in a funnel and allowed to equilibrate overnight. Ten parts by weight of the washed Kieselguhr are then treated with 5-6 parts by volume of the lower phase (for example, 90% methanol) and the mixture is stirred until the solvent is uniformly distributed. The upper phase is then added (for example, trimethylpentane) so as t o make a thin slurry. After inserting a small plug of glass-wool into the bottom of the chromatographic column, a small amount of the slurry is added and packed in with a stainless-steel perforated packing disc which fits the tube snugly. The plunger is moved up and down slowly in order t o remove air bubbles, and finally pressed down firmly. Successive portions of slurry are added and packed in. Care should be taken t o exert an even pressure during the packing and t o maintain a head of mobiie phase above the column a t all times. After the column has been packed, the pre-equilibrated upper phase is allowed to flow through References p . 620
598
STEROIDS
the column overnight by gravity in order to condition it. The sample for chromatography is dissolved in a small amount of the stationary phase and a small amount of dry Kieselguhr is added until a powdery material is obtained, and this material is also packed on the top of the column with a small amount of the mobile phase above the packing. According to some workers (Butte and Noble, Dixon, Mickan et al., Siiteri), the columns can be prepared by the socalled “dry-pack’’ procedure. Celite is mixed with the stationary phase (2: 1 to 1 : I ratio) and then added to the column in portions and packed in with a glass rod or plunger. The column can be topped with a small amount (ca. 8%)of dry Celite and a glass-wool plug. In order to minimize the amount of air trapped in the column, the mobile phase is aspirated through the bottom of the column by applying a vacuum to the top. With small automated analytical columns, Meijers found that these conventional methods of loading the stationary phase can conveniently be replaced by the so-called “in situ” loading, which consists in the injection of the equilibrated stationary phase directly into the column containing the dry support. As regards the choice of the phases, the data presented by Hulsman may be useful. He measured and calculated “selectivity factors” (the ratio of the partition coefficients of two components) for many combinations of almost 100 steroids in several common solvent systems (light petroleum-toluene-methanol-water, light petroleum-benzenemethanol-water, benzene-methanol-water, light petroleum-methanol-water, formamidebenzene-chloroform, formamide-benzene, propylene glycol-toluene, formamidecyclohexane-benzene, propylene glycol-ligroin and propylene glycol-cyclohexanebenzene). Retention volumes of some steroids (androstane and pregnane derivatives and bile acids) were tabulated for Sephadex derivatives and several solvent systems in a review by Ellingboe et al. (1969). The ratio of the diameter to height for Kieselguhr columns wits 1 :20 to 1:30. For reversed-phase chromatography, i.e., the fixation of the less polar stationary phase, Celite has also been used (Burstein et al., Burstein and Zamoscianyk), but hydrophobized supports or carriers seem to be more advantageous, such as siliconized Kieselguhr (Bergstrom and Sjovall), Hostalen (polyethylene powder) (Hoshita et al.) or methylated Sephadex (Nystrom and Sjovall). These techniques have been described thoroughly in a review by Eneroth and Sjovall. Other hydrophobized Sephadexes were also found suitable, such as hydroxypropyl Sephadex, Sephadex LH-20 and hydroxyalkoxypropyl Sephadex (Ellingboe et al., 1968, 1969). The latter is hydrophobized by the reaction of the former with an aliphatic olefin oxide. In the following sections, a description is given of the methods of preparation of the above and similar supports.
Hydrophobization ofKieselguhr (Celite) Hyflo Supercel is washed with 6 N hydrochloric acid until a colourless supernatant is obtained, then with distilled water until neutral and finally with acetone. The dried material is placed in dishes in a desiccator together with a beaker containing 50-100 ml of dimethyldichlorosilane or a low-boiling mixture of chlorosilanes (British ThomsonHouston Export Co., Rugby, Great Britain), and is left for 2-3 weeks. The support is then
GENERAL TECHNIQUES
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washed with ethanol or methanol until the filtrate is free from hydrochloric acid, dried at 100°C and stored in a closed vessel. The hydrophobic support thus obtained has been found to,,be superior to supports silanized with solutions of dimethyldichlorosilane. (Bergstrom and Sjovall, Eneroth and Sjovall.)
Purification of Hostalen Hostalen is purified by continuous Soxhlet extraction with ethanol until the extract is colourless. The support is dried below 75°C. (Eneroth and Sjovall.)
Hydrophobizatiori of Sephadex Methylated Sephadex Sephadex G-25 (1 0 g, fine, bead form) and 65 ml of 34% (w/v) sodium hydroxide solution are stirred under a nitrogen atmosphere for 4 h at room temperature. Dimethyl sulphate (90 ml) is added slowly while cooling the reaction vessel in ice, taking care that the reaction temperature does not exceed 25°C. After stirring the mixture for 4 h, the excess of dimethyl sulphate is hydrolyzed by adding a large amount of water. The reaction mixture is left overnight and then neutralized with ammonia solution. The methylated Sephadex is collected on a sintered glass funnel and washed with water and ethanol. The -OCH3 content is 34.6%; a higher degree of methylation (-OCH3 = 43.6%) is achieved, however, when Sephadex methylated with dimethyl sulphate in aqueous sodium hydroxide solution (-OCH3 = 34.6%) is used as the starting material. This two-stage reaction has been used on a 150-g scale and is preferred to the direct Hakomori reaction, which requires large amounts of the methylsulphinyl carbanion. (Nystrom, Nystrom and Sjovall.) Hydroxypropyl Sephadex Sephadex G-50 (20.0 g, fine, bead form) is soaked for 2 h in 4% (w/v) sodium hydroxide solution, after which the excess of the aqueous phase is removed by filtration with suction. The wet Sephadex (190 g) is then suspended in 700 ml of propylene oxide (commercial grade) and refluxed with stirring for 2 h. The product is filtered free from solvent and soluble reaction products, then washed consecutively with acetone, water (until the filtrate is no longer alkaline), acetone and finally light petroleum (b.p. 4 0 4 0 ° C ) . The product is first dried under suction on the filter, then at 80°C until a constant weight of 41.3 g is obtained. (Ellingboe et al, 1968.) Hydroxyalkoxypropy I Sephadex Sephadex LH-20 (100 g, superfine, bead form) is soaked in 150 ml of dry methylene chloride. Boron trifluoride ethyl etherate (2 ml, 48% BF3) is added and the constituents are mixed thoroughly. Stirring is continued at room temperature while an aliphatic olefin oxide (Needox 11 14, 150 ml) is added slowly. The rate of addition is adjusted so that the resulting exothermic reaction does not cause uncontrolled boiling. After the addition of the olefin oxide, ca. 100 ml of dry methylene oxide are added in order to facilitate stirring and the mixture is stirred for a further 20 min at room temperature. The References p . 620
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STEROIDS
product is filtered free from solvent and washed consecutively with chloroform, ethanol and acetone. The solvents are removed by suction on the filter and the product is brought to a constant weight at room temperature in vacuo. The gain in weight is about 125%. The derivative is no longer wetted by water and swells in solvents such as chloroform, benzene and heptane. Acetone and the lower alcohols cause little swelling. (Ellingboe e t nl., 1969.) Carboxymethoxypropyl Sephadex Hydroxypropyl Sephadex G-25 or LH-20 (50 g) is suspended in 750 ml of isopropanol. Sodium hydroxide (60 g) is then added, the suspension is stirred for 15 min and then 60 g of sodium chloroacetate are added in small portions. After continuous stirring for 12 h, the Sephadex derivative is collected on a filter and washed consecutively with water, ethanol, chloroform, ethanol, water, 0.1 N hydrochloric acid, 0.001 N hydrochloric acid, ethanol and diethyl ether. The degree of substitution is estimated by suspending the Sephadex derivative (about 1 .O g) in 10 ml of 0.2 M potassium chloride solution and titrating with 0.2 N potassium hydroxide solution while measuring the pH electrometrically. (Joustra e t a / . )
DiethylaminoethoxypropylSephadex Mydroxypropyl Sephadex G-25 or LH-20 (50 g) is carefully mixed with aqueous sodium hydroxide solution (33.2 g in 142 ml of water) in an ice-cooled flask. After 30 min, 2-chlorotriethylamine hydrochloride (29.2 g in 37.5 ml of water) is added slowly while stirring, and the temperature is then raised to 80-85°C for 45 min. The product is cooled, then filtered free from the aqueous phase and washed with water, 10%ammonia solution, ethanol, 0.1 N hydrochloric acid, 0.001 N hydrochloric acid, ethanol and diethyl ether, and dried at 60°C. The degree of substitution, as determined by the titration procedure described above for carboxymethoxypropyl Sephade,:, is ca. 0.8 mequiv./g. (Peterson and Sober.) Among the advantages of all these lipophilic derivatives are their chemical stability, ease of preparation and handling, and flexibility in application. Variations in solvent systems and in the degree of substitution of the gel can be used to effect different kinds of separations. Columns can be kept in continuous use over long periods. For most purposes, molecular sieving effects are negligible in reversed-phase chromatography on these derivatives, but may be significant in “straight-phase” chromatography. For the above hydrophobized supports, n-heptane alone or in admixture with chloroform is commonly used. Sometimes chloroform-isooctanol or the less polar phase of n-heptaneacetone-water, n-heptane-chlorofoim-isopropanol-water and other mixtures is used (Ellingboe et al., 1968). Aqueous methanol or the more polar phase of the above mixtures are used as mobile phases. For the chromatography of bile acids, the addition of acetic acid in order to suppress dissociation is advisable. According to Eneroth and Sjovall, after equilibration of the solvent mixture in a separating funnel, a volume of the stationary phase (4 m1/4.5 g of hydrophobic Hyflo Supercel; 3 m1/4.5 g of Hostalen; 6 m1/4.5 g of methylated Sephadex) is added to the support and the mixture is thoroughly homogenized with a spatula for a short time (about half a minute so as to avoid evaporation of solvent). The mobile phase is added,
GENERAL TECHNIQUES
60 1
the slurry is homogenized and poured into a chromatographic tube with a diameter that gives columns with a ratio o f height t o diameter of 10:1 t o 20: I . Air bubbles are removed with a perforated plunger and the column is allowed t o settle by gravitation under free solvent flow. Final packing a t the top is achieved by light pressure with the plunger. The sample is applied in a small volume of the mobile phase; a few drops of the stationary phase may be added. According t o Ellingboe ef al. (1968), the columns are packed with a slurry of the lipophilic support under gentle pressure. Most important for easy packing and good column performance is that the lipophilic material should be thoroughly equilibrated with the solvent, preferably by agitation in an ultrasonic bath. The solvent flow-rate with these materials is mentioned in the section Gel chromatography. For automated high-resolution liquid chromatography with reversed phases, Siggia and Dishman studied the use of Amberlite LA-I [ti-dodecanal( trialkylmethyl)amine] as the stationary phase fixed to several supports [Anakrom AB diatomaceous earth, a terpolymer consisting mainly of trifluoroethylene (Plaskon CTFE-2300) and Zipax spherical silica beads] and water and water-methanol mixtures as the mobile phase. The hydrophobic CTFE support exhibited lipophilic adsorption even after coating with the stationary phase, which, however, could be utilized to the advantage of separations. For similar purposes (analytical reversed-phase chromatography), Waters Ass. recommend a novel reversed-phase affinity packing, Poragel PN, w h c h is extremely rigid. In a paper o n the analysis of various types of steroids and their dinitrophenylhydrazones by high-speed liquid chromatography, Henry et.al. described useful separations in liquidliquid partition systems in which Zipax was used as the support and the following “straight” and reversed phases were used for the partition of various steroids: (a) “straight” phases: 1% of P,ij-oxydipropionitrile on Zipax as stationary phase and the following solvent mixtures as the mobile phases: n-heptane, tetrahydrofuran-n-heptane mixtures ( 2 : 8 , 1 :9 and 5:95); 1% ethylene glycol on Zipax as the stationary phase with a moving chloroform-n-heptane mixture (3:97); ( b ) reversed phases: stationary 1% hydrocarbon polymer on Zipax with methanol-water ( 1 5 : S S ) as the mobile phase; 1% cyanoethylsilicone on Zipax as stationary phase and methanol-water (2.5:97.5) as eluent; Permaphase ODS (DuPont, Wilniington, Del., U.S.A.; permanently bonded chromatographic support), i.e., octadecylsilane bonded on Zipax, as the stationary phase and the linear gradient water to 50% aqueous methanol (at S%/min) as the mobile phase. The authors also consider other stationary phases t o be useful, such as tris(2-~yanoethoxy)propane,trimethylene glycol, triethylene glycol, cyanoethylsilicones and Carbowaxes.
Gel chromatography Various types of Sephadex gel are also commonly used for steroid separations. On lipophilic Sephadex gels, separations are governed by two main mechanisms, liquid-gel partition and molecular sieving. However, Gelotte has shown that aromatic and heterocyclic compounds are adsorbed more strongly to the gel matrix than other types of substances. This effect is important in the gel chromatography of some conjugates of aromatic steroids (estrogens). References p . 620
602
STEROIDS
Gel chromatography is complementary t o established techniques for steroid chromatography. The low chemical reactivity of gels and, with lipophilic gels, also the possibility of using non-polar solvents, make these gels highly convenient for the partition or sieving of readily hydrolysable derivatives and also for quantitative analysis. Furthermore, with lipophilic Sephadex gels as a medium for partition chromatography (cf:,preceding section), no solvent mixtures have to be pre-equilibrated and no stationary phase has to be applied to the support. Among hydrophilic gels, DEAE-Sephadex A-25 in combination with a sodium chloride gradient elution and Sephadex G-25 with distilled water as eluent were mainly used for the separation of estrogen conjugates (see below) or protein-bound testosterone (Horton et al., Kato and Horton). Lipophilic Sephadex LH-20 with various mixtures of organic solvents used for swelling and elution is suitable for the separations of various types of steroids: benzene-methanol for estrogens and some other steroids (Mikhail et al.); chloroform-methanol (1 : 1) for various steroids from bile (Laatikainen and Vihko); 99% n-butanol for corticoids and 17-ketosteroids(Seki and Sugase); and dichloromethane for sterols (Van Lier and Smith). With hydrophilic gels, various ratios of height to diameter of the columns are used, from 6: 1 to about 60: 1. With lipophdic gels, a ratio of height to diameter of the column of 30: 1 is common. The ratio of the volume of the solution of the steroid mixture to the volume of gel in the column varies from about 1 : 5 to 1 :40; a ratio of 1:20 seems reasonable for most separations. According to Ellingboe ef al. (1969), the optimum solvent flow-rate for Sephadex derivatives is about 0.1 -0.5 ml/min per square centimetre of column cross-sectional area. Pre-washing of the columns is important, as is the uniformity of particles achieved by the sedimentation method. When thoroughly washed after each run, the columns can be used repeatedly, even for analytical purposes.
Ion-exchange chromatography This type of chromatography is much less used than other types in the steroid field, because most steroids are non-ionizable. However, ion-exchange resins are commonly used for the enrichment of the sample, i.e., its pre-purification. A very good and exhaustive paper (Seki, 1969) has been published on the separation of various types of steroids by means of ion-exchange resins. Henry et al. recently described the analysis of Dexamethasone disodium phosphate on a Zipax support coated with 1% of a weak anion exchanger, viz. an amine-substituted polyamide, and ethanol-water (1 :9>with 0.1 M orthophosphoric acid-0.1 M sodium orthophosphate as the mobile phase, and the analysis of 17P-estradiol-l7/3-glucosiduronic acid on Zipax coated with a strong anion-exchange polymer, viz. a qudternary aminesubstituted lauryl methacrylate polymer, and a linear gradient of pH 9.2 buffer to pH 9.2 buffer + 0.8 M sodium perchlorate (at 5%/min>as the mobile phase.
GENERAL TECHNIQUES
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Detection Steroids are detected in the eluate by submitting single fractions or aliquots to UV absorptiotnetry or colorimetry after addition of an appropriate reagent. 111 automated analytical chromatography, spectrophotometry and refractometry are the most commonly used methods. In classical preparative chromatography, single fractions are evaporated t o dryness and the weight of the residue is determined. Today, the fractions are usually analyzed by TLC, applying a few microlitres of each fraction close t o each other on the start of a broad thin-layer plate. then developing and detecting. For this purpose spraying with concentrated sulphuric acid, or a 30% solution in methanol or diethyl ether, and heating is a universal detection method. For analytical purposes, especially in advanced laboratories, the effluent may also be monitored continuously by means of a hydrogen flame ionization detector, part of the effluent being continuously drawn off. The operating conditions for the Barber-Colman (Rockford, Ill., U.S.A.) Model 5400 liquid chromatographic detector were reported by Cavina et at. (1969). However, for many investigations, labelled steroids are used and are detected and measured in the eluate on the basis of their radioactivity. Estrogens can be determined by measuring their UV absorption at 280 nm (in alcoholic solution), or by fluorimetry (Bates and Cohen, Eechaute e t a / . ) . The use of Kober reagents (phenols in sulphuric acid) (Osawa and Slaunwhite) and heating is a suitable method for the colorimetry of estrogen conjugates. The authors gave the following procedure: 1 g of hydroquinone was dissolved in-14 ml of distilled water t o which 3 5 ml of 98% sulphuric acid were added. The reagent should be prepared freshly every day. All A4 -3-ketosteroids or other steroids that contain an a,&unsaturated carbonyl group in the molecule can be detected and measured on the basis of their UV absorption at about 254 nm. A great number of important steroids and their derivatives, such as testosterone, androstenedione, progesterone and corticoids, belong t o this group. 17-Hydroxysteroids are detected with the Porter-Silber reagent (phenylhydrazine sulphate and sulphuric acid) (Seki, 1967, 1969), while 17-ketosteroids are detected with the Zimmermann reagent (m-dinitrobenzene in aqueous potassium hydroxide solution) and determined with the same reagent by the Epstein method (Epstein; Seki, 1969). Porter- Silber reageti t Phenylhydrazine sulphate (50 mg) is dissolved in a mixture of 21 ml of ethanol (99.5%) and 39 ml of sulphuric acid (1 5 8 ml of sulphuric acid t o 3 7 ml of deionized water). The reagent is added (for example, 1 ml) t o evaporated fractions dissolved in 60%ethanol (for example, 0.5 ml). The mixture is allowed t o stand at room temperature for 15 h and the optical density finally measured at 410 ntn. (Silber and Porter.) Zimmemanti reagetit The residue of the evaporated fraction is dissolved in 0.05 mi of methanol; 0.2 ml of a saturated solution of tn-dinitrobenzene in 5% Hyamine 1622 solution is added, followed by 0.1 ml of 8 N aqueous potassium hydroxide solution with mixing. After the mixture References p . 620
604
STEROIDS
has stood for 30 min, 2.0 nil of 5% aqueous Hyamine 1622 solution are added and the optical density is measured at 510 nm against deionized water. (Epstein, Seki, 1969.) For high-resolution analytical column chromatography, some methods of detection and quantitation in the eluate are already in use (W absorptiometry and refractive index measurement, see above; radioactivity measurement; flame ionization after evaporation of the solvent), but others have still to be devised.
APPLICATIONS Sterols Sterols and their esters are the most lipophilic steroids and are generally easily separated by various types of chromatographic procedures. Solid-liquid adsorption chromatography is the most commonly used technique. In instances when the differences in structures of the components of the mixture are too small (for example, pairs of sterols that differ only in the number and the position of double bonds), separation can be achieved by the so-called argentation chromatography (silver nitrate-impregnated adsorbents) (Canonica et uf., Paliokas and Schroepfer, Vroman and Cohen, Lee er al., Ziller et al.). Table 28.1 gives a short survey of some recent separations by various methods.
Androgens In the synthetic preparative field, Florisil as adsorbent and benzene as eluent were used recently by Janot er al. for the purification of weakly polar androstane derivatives. The same adsorbent and light petroleum (b.p. 30-40°C) as eluent were used by Swann and Turnbull for some 17-mercaptoandrostanes.Grimwalde and Lester separated formylated testosterone derivatives on dry columns of alumina deactivated by the addition of 7% of water, using the mixtures dichloromethane-cyclohexane (1 :1) and dichloromethanecyclohexane-ethyl acetate (20:20: 1 and 5 : 5 :1). The classical method of Dingemanse e t al. for the separation of urinary 17-ketosteroids on alumina is still used and recommended (Forriol). The same is true of the classical Reichstein elution chromatography on alumina, which was used by Nagasawa et al. for the separation of the transformation products of sterols by microorganisms using successively light petroleum, benzene and diethyl ether for elution. Drosdowsky er al. used alumina and n-hexane-chloroform mixtures for the separation of labelled testosterone and epitestosterone. Silica gel was used by McCurdy and Garrett for the separation of 19-nortestololactone from other components. An adsorbent to sample ratio of 100:1 and ethyl acetate as eluent were used. Ambrus and Wix isolated microbial transformation products of 4-androstene-3,17-dioneon silica gel (sample to sorbent ratio cu. 1:70)by gradient elution with dichloromethane with additions of ethanol. For analytical purposes, where labelled steroids are often used, other types of chromatography seem to predominate. Thus Eckstein et al. identified 5a-androstane-3a,
APPLICATIONS
605
17P-diol as a metabolite of pregnenolone using 90% methanol fixed on Celite as the stationary phase and trimethylpentane as the mobile phase. Huang separated the metabolites of [7-3H] 4-androstene-3,17-dione by reversed-phase chromatography on the same support as above, using toluene-70% methanol, and for straight-phase chromatography a solvent system consisting of n-heptane-toluene-80% methanol (4:6: 10) ( c j : , Schneider et al.). Hydrophilic conjugates of androstane derivatives were detected and analyzed in natural material predominantly after their cleavage. However, Dray et al. separated free steroids from their sulphates on Celite with plasma or distilled water as the stationary phase and isooctane-ethyl acetate-n-butanol-methanol-1 M ammonia solution (2:4:1:2:3) as the mobile phase. They separated sulphates from glucuronides on alumina by gradient elution according to CrCpy ef al. For the separation of steroids released from their sulphates by solvolysis, they also used Celite with formamide as the stationary phase and n-hexanebenzene (7:3 and 3:7) for elution. I n their paper on the analysis of steroid hormones by high-resolution liquid chromatography, Siggia and Dishman tested several types of supports for the fixing of Amberlite LA-I [n-dodecanal(trialkylmethyl)amine] as the stationary phase. They also presented useful data for the separation of androgens (see Fig. 28.1) using Plaskon CTFE-2300, a terpolymer consisting mainly of trifluoroethylene, as the support. Applying the technique of Henry et al. (see p. 601) Fitzpatrick et ul. developed the hgh-speed liquid chromatography of derivatized urinary 17-ketosteroids (2,4-dinitrophenylhydrazones). For straight-phase chromatography, Zipax coated with 0.75 or 1.5% of P,P’-oxydipropionitrile was used as the stationary phase and isooctane as the mobile phase; for reversed-phase chromatography, Corasil with permanently bonded CIB-silane was used as the stationary phase and aqueous ethanol (1 :1) as the mobile liquid phase. Good separations were achieved of all four epimeric forms of androsterone from each other and from other androstane derivatives and impurities (from urine and blood plasma). The time required for the separations was about half an hour and the precision was equivalent to a relative standard deviation of about 10%at the microgram and lower levels. Useful information on the determination of androgens, mainly testosterone, in human plasma and urine etc. can also be found in papers by Henry et al., Saez et al., Peng and Munson and Vermeulen and Verdonck. Serial analyses of urinary 1 7-ketosteroids were carried out successfully by Vestergaard and Jacobsen using the multicolumn chromatographic system described in the section on liquid-solid chromatography of steroids.
Estrogens Except for their aromatic A ring and the phenolic character, estrogens do not differ much from androgens or gestagens from the chromatographic point of view, and therefore only a few analytical methods will be described here. Cavina et al. (1 97 1) developed an analytical method for mixtures of various steroids, including estrogens, in pharmaceutical preparations. As the adsorbent they used Bio-Rad References p . 620
m m
TABLE 28.1 SEPARATIONS OF SOME STEROLS
0
Technique
Compounds separated*
Sorbent or ion exchanger
Eluent
Note
Reference
Prep. solidliquid
5OrC-l0l,2~ diol 5 4 - ID ,2pdiol
Alumina + 10% of 10%AcOH Alumina + 10% of 10% AcOH
Benzene-die thy1 ether (9:1) Benzene-diethyl ether (1:l)
200-fold excess of sorbent 200-fold excess of sorbent
Davey et al.
Prep. sotidliquid
30-OH-C-Sene. 3P-OH-C-5,7diene and other pairs
Silicic acid + AgNO,
n-Hexane- benzene (stepwise increase in benzene concentration)
200-fold excess of sorbent
Vroman and Cohen
PEP. sotidliquid
30-AcOC-5ene, 7-ene, and 8( 14)-ene
Alumina Supercel
n-Hexmebenzene (9: 1)
100 X 1 cm column per 6-7 mg of mixture; 2 . 9 4 fractions
Lee et al.
Prep. solidliquid
Coprostanol, C-3P-014-’~C, cholesterol
200 g Silicic acid + 136 g AgNO, (with 20% of water)
Benzene in hexane (1 2% and
200- to 500-fold excess of sorbent
Ziller e t al.
Cholesteryl sulphate, neutral sterol derivatives
Methylated Sephadex G-25, Sephadex LH-20
Chloroform
Solute to gel ratio 1:130 to 1:260; above 1:lOOO; 44 x 0.84 cm column
Eneroth and Nystrom
p-Sitosterol, campesterol
Hydrophobic hydroxyalkyl Sephadex LH-20
Methanolhexane ( 9 5 : 5 )
Gel
+ Hyflo + AgNO,
Davey et QI.
15%)
CQ.
v l
Gel
25 mg on columns of 182- and 432-mI volume
Hyde and Elliot**
3;d
s
FJ
ch9
Gel
3p-OH-C-5en-23-one
Sephadex LH-20
Dichlorome thane
250 mg of mixture on a 2.5 X 6 0 cm column, fraction volume 13.5 ml
Lier and Smith
Liquidliquid
170.200-Dihydroxycho lesterol, 170,20p-dihydroxy-20isocholesterol, cholesterol, 3p,l7wdihydro xypregn-5en-20-one
Celite 545 as support
Heptane or Skelly-Solve Cmethanol-water (10:8:2); heptane- benzenemethanol- water (33: 17:40: 10); toluene-heptanemethanol-water (8:4:4: I ) ; toluene-propylene glycol; hexane-formamide; methanol-n-propanol water-tolueneisooc tane (4: 1 :1.3:2:2)
The last system was used as a reversed-phase system, i.e., 0.3 ml of the upper phase for wetting 1 g of Celite
Burstein et ~ l*** .
Ion exchange
Cholesteryl sulphate
Amberlyst 15 ( H + ) (batch No. 625) converted into NH: form and washed with chloroformmethanol CM-Sep hadex LH-20
Chloroformmethanol (4: 1)
ca. 1000-fold excess of
Eneroth and Nystrom
Chloroformmethanol (4: 1)
Large excess of the ion exchanger, 1.46 x 46.5 cm column
s
a
'p
2 D
~
the resin
_ _ *C = cholestan or cholest-. **Sephadex LH-20 was treated with Needox 11 15 (a mixture of C , , -C,4 olefin oxides) according t o Ellingboe e t n l . (1968, 1969). The preparation had a weight increase corresponding to a hydroxyalkyl content of 49% (w/w), columns 2.5 X 45 and 2.5 X 100 cm. ***Burstein and Zamoscianyk described further mixtures for liquid-liquid chromatography o n Celite.
m
s
608
STEROIDS
1
2
Fig. 28.1. Separation of some androgens (Siggia and Dishman). Column: 0.2 X 48.5 cm I.D. Packing: 23% Amberlite LA-1 on Plaskon CTFE-2300 (see p. 601). Eluent: water. Initial flow-rate: 0.17 ml/min, increased at A to 0.49 ml/min. Detection: battery-operated Beckman DU spectrophotometer equipped with a deuterium source lamp (Beckman Part No. 96280). A Varian G-2000 strip-chart recorder (Varian. Palo Alto, Calif., U.S.A.) equipped with 1-1000 mV variable span was wired across the null meter. Peaks: 1 = 4-androstene-3,11.17-trione; 2 = 4-androstene-1 lp-ol-3,17-dione: 3= 1.4-androstadiene(19-nor17p-ol-3-one; 4 = 19-nor-4-androstene3-17-dione; 5 = 19-nor-4-androstenel7p-ol-3-one 7 = 4-androstene-17p-01-3-one (testosterone). testosterone); 6 = 4-androstene-3,17-dione;
silicic acid, 325 mesh, for lipid chromatography, according to Hirsch and Ahrens. The water content was about 9% (9.38 ml of water per 100 g of silicic acid dried at 125°C for 8 h and cooled in a desiccator). Glass columns were packed with the adsorbent, and when 10 g of silicic acid were used, a filling of 56 cm height was obtained. Silicic acid suspended in light petroleum (b.p. 65-75°C) was gradually poured into the column, which was half-filled with solvent, the solvent being allowed to flow slowly out of the column. Samples of steroids (see Table 28.2) were introduced as a solution in oil or in n-heptane (1-2 mglrnl). The sample, dissolved in as small a volume as possible (1-2 ml), was transferred on to the column by pipette and washed with light petroleum (3 X 1 d), allowing a slow efflux by gravity. The solvent was pumped into the column by a metering pump with an adjustable flow-rate. The gradient of diethyl ether in light petroleum was obtained by pumping diethyl ether from the reservoir into the mixing chamber containing light petroleum. In order to monitor the various fractions eluted from the column as concentration peaks, part of the eluate from the column was transferred on to a chain conveyor, which, after the solvent had been evaporated to dryness, introduced the solid residue into a hydrogen
609
APPLICATIONS
flame ionization detector with a moving chain. The splitting ratio was adjusted t o deliver 6-776 of the total effluent volume to the detector when the flow-rate was 1 ml/min. The fraction collector was regulated t o fractions of 10 min. The spectropfiotometric determination of testosterone propionate or of other steroids that absorb in a similar manner in the UV region, was carried out b y collecting all the fractions corresponding to the peak in a tared 50-ml vessel and adjusting the volume t o the mark with light petroleum. Aliquots were removed, evaporated to dryness under nitrogen, the residue was dissolved in methanol and the UV spectrum in the range 225250 nm measured on a Beckman DU-2 spectrophotometer (1-cm cells). Figs. 28.2 and 28.3 illustrate the separations of some steroids from each other and from carrier oil or impurities.
I
I
I
I
I
I
10
0
I
20
I
I
40
30 F R A C T I O N NUMBER
Fig. 28.2. Chromatogram of an oil solution containing 50 mg/ml of testosterone cyclopentylpropionate (TCPP) and 2.5 mg/ml of estradiol cyclopentylpropionate (ECPP) (Cavina et al., 1971). Sample diluted 1 t o 25 (v/v) with n-heptane; volume analyzed, 1 ml ( 2 mg of TCPP and 0.1 mg of ECPP). Column: 0.6 X 56 cm I.D. Sorbent: 1 0 g of silicic acid. Eluent: diethyl ether in light petroleum (b.p. 65-75°C) (gradient elution). Operating conditions: see text. Detection: hydrogen flame ionization detector with moving chain (Barber Colman Model 5400 liquid chromatographic detector). Peaks: 1 = sterol esters; 2 = triglycerides; 3 = sterols; 4 = 1,3-diglycerides + ECPP; 5 = 1,2-diglycerides; 6 = TCPP; 7 = monoglycerides.
W v)
4
2
B v)
A
d, A 2
W
a L
,
0
I
10
20
I
30
I
I
40
I
50
F R A C T I O N NUMBER
Fig. 28.3. Chromatogram of a mixture of estradiol dipropionate, progesterone, testosterone cyclopentylpropionate and testosterone propionate (Cavina et al., 1971). Sample: 1 ml of an n-heptanc solution containing 1 mg, 1 mg, 2 mg and 1 mg, respectively, of the listed steroids. Operating conditions: As in F'ig. 28.2 except for the detector sensitivity, which in this case was 3 x lo-'". Peaks: 1 = triglycerides (small amount as impurities); 2 = estradiol dipropionate; 3 = estradiol monopropionate (small amount as side product of the dipropionate); 4 = testosterone cyclopentylpropionate; 5 = testosterone propionate; 6 = progesterone.
References p . 620
610
STEROIDS
TABLE 28.2 ELUTION VOLUMES 01’ SOME STEROIDS AND RELATED COMPOUNDS (CAVINA el al., 1971) Compound
Elution volume (ml)
19-Nortestosterone 170-decanoate Testosterone 170-cyclopcntylpropionate 19-Nortestosterone 170-propionate 19-Nortestosterone 170-phenylpropionate Testosterone 17p-propionp .e Testosterone Methyltestosterone 19-Nortestosterone Sterol esters from oil Tocopherol acetate Tocopherol Triglycerides from oif Sterols 1,3-Diglycerides 1,ZDiglycerides Monoglycerides Estradiol dipropionate Estradiol 17pcyclopen tylpropionate Mestranol Estradiol monopropionate Estradiol 3-benzoate Ethynylestradiol Allylestrenol Lyncstrenol Ethylestrcnol Ethynodiol diacetate Vinyl estrenolone Norethinodrel Chlormadinone acetate Nore thindronc Progesterone
310 355 360 360 395 >440 >440 >440
110 110 125 130 250 2 80 315 >440 215 270 275 320 360 385 145 165 165 260 360 395 >440 >440 >440
Hulsman ( c t , Fuber er al.) investigated the determination of estriol in pregnancy urine using liquid chromatography for controlling single steps of the conventional procedure. He concluded that hydrolysis of conjugates and extraction does not cause serious losses of estriol. After preparing the sample as described below, quantitative information can be obtained within 15 min by chromatography. However, the simultaneous quantitative determination of estrone and estradiol has not been possible owing to inadequate prepurification of the complex sample. For pre-purification, Hulsman recommended heating a 50-ml sample of urine to boiling, addition of 7.5 ml of concentrated hydrochloric acid and refluxing for half an hour. After cooling, the mixture is extracted with three 50-ml portions of diethyl ether. The total ethereal phase is treated with 20 ml of concentrated sodium carbonate solution (pH 10.5) and the aqueous layer is discarded. A 4-ml volume of an aqueous solution of 8%(w/w) sodium hydroxide is thoroughly shaken with the
61 1
APPLICATIONS
ethereal fraction. The alkaline layer is not discarded, but its pH is reduced to 10 and its ionic concentration increased by adding 20 ml of 8% sodium hydrogen carbonate solution, and the aqueous layer is then discarded. The ethereal layer is washed with 4 ml of 8% sodium hydrogen carbonate solution and with 3 ml of water. The aqueous layer is discarded and the ethereal fraction is evaporated t o dryness and submitted t o chromatography under the conditions given in Fig. 28.4. Before use, the eluent must be deaerated and saturated with the stationary liquid. To ensure equilibrium between the eluent and the stationary liquid, a pre-column is necessary. The detector unit, which was directly connected t o the outlet of the column, was a Zeiss PhlQ I1 spectrophotometer with a 7 . 5 ~ flow 1 cell. The signal from the detector was fed t o a 2-mV linear recorder (Servogor, Type RE-5 11). Celite partition columns and a number of various solvent mixtures were used by Abdel-Aziz and N'illiams, Smith and Kellie, and Williams and Layne in metabolic studies. In their study on the aromatic ring hydroxylation o f estradiol in man, Fishman et al. carried out chromatographic separations o f extracts by gradient elution partition chromatography on an acid-washed Hyflo Supercel column with 90% aqueous methanol as the stationary phase and 2,2,4-trimethylpentane with a gradient of 1,2-dichloroethane as the mobile phase. The system was proposed originally by Engel er al.
0
5
10
15
T I M E . MIN
25
Fig. 28.4.Chromatogram of (A) pre-treated pregnancy urine and (B) a test mixture of estrogens (Hulsman). Column: 0.27 X 50 cm. Support: diatomaceous earth, particle size 28-32 pm. Stationary phase: water-rich phase of a mixture of water-ethanol-2,2,4-trimethylpentane (composition in molar fractions: 0.229:0.680:0.091). Eluent: water-poor phase of the same solvent mixture (composition in Operating temperature: 22°C.Detection: Zeiss PMQ 11 spectrcmolar fractions: 0.019:0.177:0.805). photometer with a 7.5-p1flow cell. The signal of the detector is fed t o a 2-mV linear recorder (Servogor, Type RE-511).Peaks: Estrone, estradol and estriol (from left t o right).
References p . 620
STEROIDS
612
4
m
U
9 I'
30 40 50 60ml 16,17E,
100
120
140
160 ml
Fig. 28.5. Separation of estradiol and estriol epimers on Sephadex LH-20 (Horst et al. ). Column: (A) and (C) 1 x 60 cm; (13) 0.9 X 25 cm. Packing: Sephadex LH-20. Eluent: 0.02 N NaOH. Operating conditions: Temperature, (A) and (9)25"C, (C) 55°C; sample application in 0.15 M acetate buffer (pH 4.2). Detection: optical density at 254 nm, Uvicord 11. For the signal amplification, a square cell with a 10-mm light path was used (Hellma GmbH, Miillheim-Baden, G.F.R.) Peaks: E, = estrone; E,Q = estradiol-17~;E20= estradiol-170; E, = estriol; 16E, = 16-epiestriol; 16,17E, = 16,17-epiestriol; I7E, = 17epiestriol.
I
1
2
u r/)
z
s2
U
4
5
II 6 7 A
I 10 20
0
R
I
~
30
40
TIME, MIN
60
Fig. 28.6. Separation of estrogens (Siggia and Dishman). Column: 0.2 X 48.5 cm I.D. Packing: 28% Amberlite LA-1 on Plaskon CTFE-2300 (cf., p. 601). Eluent: water adjusted to pH 11.5 with sodium hydroxide. Flow-rates: initial, 0.12 ml/min; (A) increased to 0.145 ml/min; (B) increased to 0.19 ml/ min; (C) increased to 0.49 ml/min. Detection: see Fig. 28.1. Peaks: 1 = 1,3,5(10)-estratriene-3,17p-diol17~-glucosiduronicacid (estradiol-170-glucosiduronic acid); 2 = 1,3,5(10)estratriene-3,16a,l7p-triol (estriol); 3 = 1,3,5(lO)estratrien-3-01-16,17-dione(16-ketoestrone); 4 = 1,3,5(1O)estratriene-3,17pdiol-16ae (16-ketoestradiol); 5 = 1,3,5(1O)estratriene-3,16p,17p-triol (16epiestriol); 6 = 1,3,5(10), 6,8-estrapentaen-3-01-17-one(equilenin); 7 = 1,3,5(1O)-estratriene-3,17p-diol (estradiol); 8 = 1,3,5(10)estratrien-3-0 1-17-one (estrone).
APPLICATIONS
613
A liquid-liquid system for checking the purity of estradiol was proposed by Henry et al. (see p. 601). Estrogens from female urine were chromatographed by Hsu et al. on a nylon powder (60-80 mesh) column (1 X 25 cm) using benzene and benzene-ethanol mixtures as the mobile phase. Detection and colorimetry were carried out after reaction with hydroquinone and sulphuric acid. In their radioimmunoassay of plasma estrone and estradiol, Mikhail e f al. used repeatedly, for a period of 3-6 months, Sephadex LH-20 (1 X 30 cm, 3 g) and benzenemethanol (85:5). Jellinck and Fletcher and Jellinck et al. applied the gel filtration of water-soluble estrogen conjugates on a Sephadex (2-25 column (1.8 X 38 cm) with distilled water as eluent (3-ml fractions). Horst described an assay for the separation of urinary estrogens on a 1.2 X 60 cm Sephadex G-10 column which could be loaded with samples up to 200 ml. Estrogens and a few other urinary ingredients were reversibly adsorbed by the gel matrix if the test sample was saturated with sodium sulphate and the pH adjusted to 4.6 0.2. Almost all non-estrogenic material was eluted before the three estrogens, which left the column in highly purified fractions. A continuous non-linear gradient elution was used. The gradient was produced by mixing a solution of 16% sodium sulphate with 0.1 N sodium hydroxide solution. An automatic estrogen determination could be achieved by fluorimetry in a flow cell. Useful separations of estrogens were also described by Henry et al. (Zipax coated with a strong anion-exchange polymer, pH 9.2 buffer to pH 9.2 buffer + 0.8 M sodium perchlorate gradient for elution), Hobkirk et al., Hobkirk and Nilsen (1969a, 1970; prepurification according to Bradlow) (DEAE-Sephadex A-25,O-0.8 M and 0-0.4 M sodium chloride gradient for elution), Horst et al. (Fig. 28.5), Osawa and Slaunwhite (Amberlite XAD-2,30%ethanol as eluent) and Van Baelen e l al. (Sephadex G-25 and LH-20, large amounts of water as eluent). Siggia and Dishman used a column packed with Plaskon CTFE-2300 (Allied Chemical Co., Morristown, N.J., U.S.A.) on which Amberlite LA-1 (see p. 601) was futed. The particles of the support were 325 mesh. A dilute alkaline solution was used as the mobile phase. Detection was performed by density recording. The separation achieved is illustrated in Fig. 28.6.
Gestagens (progestins) In the two preceding sections, a large number of applications of various types of chromatography have been mentioned, and in this section we shall limit ourselves predominantly to examples of analytical high-resolution liquid chromatography. As regards preparative chromatography (especially the adsorption and liquid-liquid chromatography of progestins), this does not differ essentially from the chromatography of androgens and estrogens (see Fig. 28.3). In fact, in terms of chromatographic character (i.e.,polarity and adsorptivity) progesterone is a homologue and isomer of androstenedione, pregnenolone is very similar to dehydroepiandosterone and a little less to estrone, pregnanediol can be compared to estradiol and androstenediol, and pregnanetriol does not behave very References p. 620
614
STEROIDS
differently from estriol and some androstenetriols. In general, the most frequently used adsorbents have been alumina and silica gel, followed by Florisil and Chromosorb (Cardner et al. ). For liquid-liquid chromatography, Celite has often been used, with aqueous methanol as the stationary phase and mixtures of benzene with other hydrocarbons as the mobile one. Silver-impregnated Florisil was used for the separation of 17a-ethynyl steroids (Kulkarni and Coldzieher). Sephadex LH-20 was also successfully applied with the solvent system chloroform-methanol (1 :1) containing sodium chloride (Laatikainen and Vihko). An example of high-resolution liquid chromatography of gestagens was proposed by Siggia and Dishman. The operating conditions are much the same as those described for androgens and estrogens. Table 28.3 gives retention data and operating parameters for some progestins. Some retention data for gestagens are also given in Table 28.2. Another example of analytical high-speed partition chromatographic analysis may be found in the paper by Henry et al. (see p. 601). TABLE 28.3 RETENTION DATA FOR SOME PROGESTINS (SIGGIA AND DISHMAN) Column, 485 mm X 2 mm I.D.; eluent, 33% (v/v) methanol in water; flow-rate 0.56 ml/min; packing, 28% Amberlite LA-1 on Plaskon CTFE-2300; (p. 601); detection, optical density recording. Steroid
tR* (min)
k**
Testosterone 170-Hydroxyprogesterone Progesterone 4-Premene-20&ol-3-one
4.43 6.15 30.6 30.6
1.74 2.80 17.5 17.9
*rR = retention time. * * k = capacity ratio, defined as the ratio of the weight of a compound in the stationary to that in the mobile phase. Corticosteroids Corticosteroids, even when not conjugated with hydrophilic components such as glucuronic acid, are generally more hydrophilic than other hormonal steroids, owing to their greater oxygen content per molecule. In other respects, they are similar t o more hydrophilic progestins and androgens and therefore we shall treat them very briefly here. For preparative adsorption chromatography, silica gel is probably most often used, followed by alumina and Florisil. Celite with aqueous methanol as the stationary phase and ligroin-benzene (Kelly et al.) or a gradient of 1 ,Zdichloroethane in 2,2,4-trimethylpentane (Dufau and Villee) is used for biosynthetic studies. Reversed-phase chromatography on Sephadex LH-20 is often used for metabolic and general biochemical studies, often with labelled steroids (Seki, 1967; Seki and Sugase; Shapiro and Peron). Corticosteroids are usually detected on the basis of their UV absorption or by the colour reaction with blue tetrazolium.
APPLICATIONS
615
The detection of some corticosteroids is sometimes a problem because of their low concentration in various body tissues or liquids; high-resolution chromatography therefore seems to be the method of choice in this field. Siggia and Dishman separated corticosteroids as shown in Fig. e28.7. Using a DuPont 820 liquid chromatograph, a W photometer as detector, Zipax as the stationary phase support, 0, 0'-oxydipropionitrile as the stationary phase and n-heptanetetrahydrofuran (8:2) as the mobile phase, Henry et a/. achieved good analytical separations of corticosteroids and similar steroids. They also described other phase systems, normal ar-d rzversed, uiz., ethylene glycol on Zipax and n-heptane-chloroform, and a hydrocarbon polymer (HCP) on Zipax and methanol-water (see p. 601). Touchstone and Wortmann used a Perkin-Elmer Model 1240 analytical liquid chromatograph, equipped with a UV detector with a 0.3 X 50 cm column packed with a treated silica gel, either SIL-X from Perkin-Elmer (for liquid adsorption chromatography) or SIL-X (RP) (for liquid-liquid chromatography). In Fig. 28.8, the separation of three corticosteroids and androstenedione by high-pressure liquid adsorption chromatography on SIL-X is shown, while Table 28.4 shows the retention times of some corticosteroids and progesterone rneasured during reversed-phase hgh-pressure liquid chromatography on SIL-X (RP).
1
Fig. 28.7. Separation of some adrenal corticosteroids (Siggia and Dishman). Column: 0.2 X 485 cm I.D. Packing: 23% Amberlite LA-I on Plaskon CTFE-2300 (see p. 601). Eluent: water. Flow-rate: initial, 0.10 ml/min; (A) increased to 0.13 ml/min; (B) increased to 0.21 ml/min; (C) increased to 0.26 ml/min; (D)increased to 0.44 ml/niin. Detection: see Fig. 28.1. Peaks: 1 = 4-pregnene-6p,l7a-2I-triol-3,11,20trione (6P-hydroxycortisone); 2 = 4-pregnene-1 lp,2I-diol-3,20-dione-l8-a1 (aldosterone); 4 = 4pregnene-l7a,21-diol-3,1l-dione(cortisone); 7 = 4-pregnene-21-ol-3,11,20-trione (1I-dehydrocorticosterone); 8 = 4-pregnene-1 lp,21-diol-3,20-dione (corticosterone); 11 = 4-pregnene-l7a,21-diol-3,20dione ( I 1-deoxycortisol); 12 = 4-pregnene-l7a,21-diol-3,11,20-trionc-21-acetate (cortisone-21-acetate); (deoxycorticosterone). 13 = 4-pregnene-2013.21-diol-3-one; 14 = 4-pregnene-21-01-3,20-dione
References p . 620
STEROIDS
61 6
ADN
W u)
z
: E K
E
n
F
J TIME
I
1
0
1
K I
I
2
3
L I
I
I
4
5
6
TIME, MIN
Fig. 28.8. Separation of cortisol IF), cortisone (E), substance S (S) and androstenedione (ADN) by high-pressure liquid adsorption chromatography (Touchstone and Wortrnann). Column: 0.3 X 50 cm I.D. Packing: SIL-X. Eluent: chloroform-dioxan (100:4).Operating conditions: flow-rate, 1 ml per 55 sec; pressure, 350-450 p.s.i.; amount injected, 4 pg each. Detection: UV attenuation, 0.05 O.D.; speed of recorder, 30 cm/h. Fig. 28.9. Chromatogram of a standard mixture of corticosterone and cortisol (Meijers). Column: 0.27 X 10 cm.Packing: diatomaceous earth (dry), particle size 5-10 prn. Stationary phase: water-rich phase of water-ethanol-2,2,4-trimethylpentane (18.5:75.0:6.5). Eluent: Water-poor phase of the same flow-rate, 20 ml/h. solvent mixture (0.1:5.5:94.4). Operating conditions: phase ratio, 28. Detection: Zeiss PMQ I1 UV spectrophotonieter. Peaks: 1 = solvent front; 2 = corticosterone; 3 = cortisol.
Meijers described a system for the separation of corticosterone and cortisol using the phase system water-ethanol-2,2,4-trimethylpentane. The support was diatomaceous earth, which was packed into the column (2.7 mm I.D.) in a dry state. Shaking the column and packing under gentle pressure from a PTFE plunger was necessary. The air was expelled from the column by the mobile phase and the stationary phase was added to the column by injection. Conventional loading of the stationary phase was also satisfactory. The detector was a Zeiss PMQ I1 UV spectrophotometer. An example of the separation of the two steroids is given in Fig. 28.9.
617
APPLICATIONS
TABLE 28.4 SEPARATION OF' ADRENAL CORTICOSTEROIDS AND PROGESTERONE BY REVERSED-PHASE LIQUID-LIQUID CHROMATOGRAPHY ON SIL-X (RP) (TOUCHSTONE AND WORTMANN) Chart-speed: 0.26 cm/min. Steroid Cortisone Cortisol Aldos terone Corticosterone 11-Deoxycortisol, Reichstein's subst. S Progesterone
Retention (crn)
Band width (cm)
1.8 2.0 1.7 3.1
0.9 1 .o 1.o
3.5 15.0
0.9 5.8
1.6
For the determination of methylprednisolone residue in milk, Kreminski e f af. used high-pressure reversed-phase liquid chromatography with a DuPont 820 liquid chromatograph (3-ft. column) equipped with a precision photometer. A hydrocarbon polymer (HCP, DuPont No. 820950008) fixed on Zipax served as the stationary phase and watermethanol (3: 1) as eluent. The sensitivity of their method was in the parts per billion range and the accuracy was 94 k 4%. Beyer and Morozowich developed a semi-automated procedure for the quantitation and characterization of steroid phosphates, such as methylprednisolone 2 I-phosphate, using ion-exchange column chromatography on DEAE-cellulose. Vestergaard and Sayegh described an excellent separation of 17 corticosteroids on silica gel using their medium-speed automated multicolumn chromatography mentioned on p. 595
Bile acids and other steroid acids The ability of bile acids to dissociate, i.e., ionize, leads to new possibilities for their chromatography, but also gives rise to new problems. Except for acid steroid conjugates, the bile acids (and their conjugates) are the only steroids that have also been chromatographed on ion exchangers. On the other hand, their dissociation makes them unsuitable for chromatography unless this dissociation is either suppressed or made complete, as otherwise they will streak. This effect is usually avoided by using acidic systems for their chromatography (cc, Matschiner e f al., who used 70% aqueous acetic acid as the stationary phase fixed on Celite and n-hexane-benzene as the eluent), in which the dissociation of bile acids is supressed. Another solution to this problem consists in the esterification of the bile acids, usually with diazomethane, which renders them neutral and able to be chromatographed in any common system suitable for other neutral steroids. In 1969, an excellent article by Eneroth and Sjovall summarized all methods of analysis in the biochemistry of bile acids. Two tables in that paper described separations References p . 620
618
STEROIDS
on silicic acid and aluminium oxide and also the structure-mobility relationships in the bile acids group. It seems that the use of hydrophobized diatomaceous earth (siliconized) or polyethylene powder (Hostalen) as the support for the stationary phase of a reversedphase system are the most common (see p. 599) for the separation of free acids. Methylated Sephadex has also been used. n-Heptane, mixtures of n-heptane and chloroform and of chloroform and iso-octanol, served as the stationary phase and water-methanol mixtures as the mobile phase. In analytical applications, labelled bile acids were used so that the chromatograms could be recorded on the basis of their radioactivity. Other papers include the separation of two new bile acids from rat bile, using liquidliquid partition on Celite, carried out by Matschiner e t al. (see above), synthetic work by Mitra and Elliot using low-activity alumina, a metabolic study by Norii e t al. making use of hydrophobic Celite, a structural study of steroid acids from Cephalosporiurn acremonium by Chou e t al. and a study on enzyrnatic saponification of steroid acids by Nguyen Gia Chan and Prochazka. In the last two cases, silica gel was used.
Steroidal glycosides Glycosides of various steroids (cardenolides, buffadienolides and spirostanes) are must commonly purified and separated by chromatography on silica gel with chloroformmethanol-water mixtures of various compositions, with chloroform prevailing. However, following the work of Haack e t al., many workers used cellulose powder impregnated with formamide as the stationary phase and various mixtures o’fn-heptane with xylene and ethyl methyl ketone as the mobile phase (cf:,Nover; Nover e t al., 1969a, b, who investigated the relationship between the chemical structure and the chromatographic behaviour of cardiac glycosides). For filling the columns, Haack et al. applied the “dry pack” procedure described on p. 599. Before filling the column, cellulose was first suspended in formamide-diethyl ether (1: l), filtered off under suction, spread on a sheet of paper and allowed to stand until the diethyl ether had completely evaporated. However, Angeliker et al. found that it is better for some purposes if the cellulose impregnated with formamide and suspended in the mobile phase is not packed into the column, but allowed to settle by gravity. They also used silica gel impregnated with formamide (6 parts of formamide per 40 parts of silica gel) and formamide-saturated chloroform plus 1% of n-butanol for the separation of lanatosides. Recently, Singh and Rastogi used formamide fixed on Celite 535 and benzene saturated with formamide as the eluent for the separation of glycosides from Asclepius curassavica L. Mackie and Turner found DEAE-cellulose (Whatman DE-32) to be suitable as the stationary phase and phosphate buffer as the eluent for the separation of biologically active glycoside from starfish (Marthusterias glacialis). These examples should suffice for glycosides. The separation of aglycones is no problem because they behave like other steroids on alumina or silica gel, Florisil, etc. The introduction of high-resolution micro-column chromatography into this field will lead to great progress. So far, only Henry e t al. have described an analytical high-speed micro-separation of digitoxin from digoxin in a reversed-phase system, in which 1% cyanoethylsilicone on Zipax
619
APPLlCATlONS
was used as the stationary phase and aqueous 2.5% methanol as the mobile phase (column pressure 600 p.s.i.g., flow-rate 0.5 ml/min, temperature 40°C, W photometric detection).
Steroidal insect hormones Steroidal insect hormones represent a relatively new group of physiologically active steroids, isolated for the first time from insects, but later discovered also in crustaceans and various plants. They could be defined as polyhydroxylated steroids, mainly characterized as 50-cholestanols with a conjugated 6-keto group and a 20,3P-diol grouping. Little remains to be said about their classical chromatography, i.e., on the common adsorbents which were extensively used for their separation and purification. In addition to adsorbents such as alumina, silicic acid and Florisil, active carbon and aqueous methanol and pure methanol were also used for their pre-purification by Imai et al. They also used automatic liquid chromatography, utilizing an Amberlite XAD-A column and elution with a linear gradient of 20-70% of aqueous ethanol. The eluate was monitored by the absorption of these substances at 254 nm. Gel chromatography (Sephadex C-25, polyacrylamide gel Bio-Gel P-10) was used for the isolation of crustecdysone from crayfish by Horn et al., and hydrophobic Celite impregnated with n-butanol-cyclohexane (9: 1 or 7: 1 ; 15 ml per 20 g) and water saturated with the stationary phase as the mobile phase for synthetic work on crustecdysones by Galbraith and Horn and Galbraith ef al. Highresolution liquid column chromatography was introduced into this field by Waters Associates (Framingham, Mass., U.S.A.), who proposed a novel reversed-phase affinity
ARI
rl-
0,7
z
8
R2
H 11
. ... 0
1. ECDYSONE
JV, 2 5 4 M
Y
2. CYASTERONE
P-OH p-OH OH q > O OH
Fig. 28.10. Separation of three insect moulting steroids (Water? Ass.). Column: 0.785 X 91.5 crn. Packing: Poragel PN. Eluent: methanol-water (7:3). Operating conditions: instrument, ALC-100, flow-rate, 3.1 ml/min; sample load, 50 p g total. Detection: UV absorption at 254 nm.
References p.620
620
STEROIDS
packing, Poragel PN, which is claimed to be extremely rigid and thus suitable for a scale-up even to large diameter columns. The details of this method are represented in Fig. 28.10 (see also Henry et al.). Less common types of steroids, such as aza-steroids (cf.,Mechoulam), various thiosteroids (cf:, Djerassi et al. ), etc., present no special difficulties in their preparative chromatography in various chromatographic systems. I t seems that so far no need has arisen to devise analytical high-resolution liquid column Chromatographic methods for them. I t is probable that with the increasing use of this method in the future, it will be used in all fields of steroid research.
REFERENCES Abdel-Aziz, M. T. and Williams, K. I. H., Steroids, 13 (1969) 809. Ambrus, G. and Wix, Gy., Acta Chim. Acad. Sci. Hung., 55 (1968) 99. Angeliker, E., Barfuss, F. and Renz, J., Helv. Chim. Acta, 4 1 (1958) 479. Bates, R. W. and Cohen, H., Fed. Proc., Fed. Amer. SOC.Exp. BioL, 6 (1947) 236. Bergstrom, S. and Sjovall, J., Acta Chem. Scand., 5 (1951) 1267. Beyer, W. and Morozowich, W., Ann. N . Y. Acad. Sci., 153 (1968) 393. Bradlow, H. L., Steroids, 11 ‘1968) 265. Burstein, S., Kimball, H. L., Chaudhuri, N. K. and Gut, M., J. Biol. Chem., 243 (1968) 4417. Burstein, S. and Zamoscianyk, H.,Sreroids, 15 (1970) 13. Butte, J. C. and Noble, E. P., Acta Endocrinol., 6 1 (1969) 678. Canonica, L., Fiecchi, A., Kienle, M. G., Scala, A., Galli, G., Paoletti, E. G. and Paoletti, R., J . Amer. Chem. Soc., 90 (1968) 6532. Cavina, G., Moretti, G., Mollica, A. and Antonini, R., Int. Symp. V I , Chromatographie, Electrophorkse, Bruxelles, 14,16 September 1970, Presses Acadkmiques Europkennes, Brussels ( 1971). Cavina, G., Moretti, G., Mollica, A. and Siniscalchi, P., J . Chromatogr.,4 4 (1969) 493. Chmel, K., Pihera, P. and Schwarz, V., Chem. Listy, 67 (1973) 649. Chou,T. S., Eisenbraun, E. J . and Rapala, R.T., Tetrahedron, 25 (1969) 3341. CrBpy, O., Jayle, M . F. and M e s h , F., Acta Endocrinol., 24 (1957) 233. Davey, C. W., McGinnis, E. L., McKeown, J. M., Meakins, G. D., Pemberton, M. W. and Young, R. N., J. Chem. SOC.,C , (1968) 2674. Dingemanse, E., Huis in’t Veld, L. G. and Hartogh-Katz, S. L.,J. Clin. Endocrinol. Metab., 12 (1952) 66. Dixon, R., Steroids, 14 ( 1 969) 7 18. Djerassi, C., Lightner, D. A., Schooley, D. A., Takeda, K.,Komeno, T. and Huriyama, K., Tetrahedron, 24 (1968) 6913. Dray, F., Mowszowicz, 1. and Ledru, M.-J., Sferoids, 10 (1967) 501. Drosdowsky, M. A., Nguyen, T. T., Populu, J. and Jayle, M. F., Bull. SOC.Chim. B i d . , 50 (1968) 1723. Dufau, M. L. and Villee, D. B., Eiochirn. Biophys. Acta, 176 (1969) 637. Eberlein, W. R., Steroids, 14 (1969) 553. Eckstein, B., Mechoulam, R. and Burstein, S. H.,Nature (London), 228 (1970) 866. Eechaute, W., Demeester, G. and Leusen, l.,Steroids, 13 (1969) 101. Ellingboe, J., Nystrom, E. and Sjovall, J., Biochim. Biophys. Acta, 152 (1968) 803. Ellingboe, J., Nystrom, E. and Sjovall, J . , Methods Enzymol., 14 (1969) 317. Eneroth, P. and Nystrom, E.,Steroids, 11 (1968) 187. Eneroth, P. and Sjovall, J., MethodsEnzjwol., 15 (1969) 237. Engel, L. L., Cameron, C. B., Stoffyn, A., Alexander, J. A., Klein, 0. and Trofimov, N . D., Anal. Biochem., 2 (1961) 114. Epstein, E., Clin. Chiin. Acta, 7 (1962) 735. Ercoli, A., Vitali, R. and Gardi, R., Steroids, 3 (1964) 479.
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Fernandez, A. A. and Noceda, V. T., J. Pharm. Sci., 58 (1969) 740. Fishman, J., Guzik, H. and Hellman, L.,Biochemistry, 9 (1970) 1593. Fitzpatrick, F . A., Siggia, S. and Dingman, I., Sr., Anal. Chem., 44 (1972) 221 1. Forriol, E. F., 5th h i t . Symp. on Chromatography aiid,~lectrophoresis,1968, Ann ArborHumphrey Sci. Publ., Ann Arbor, Mich., 1969, p . 379; C.A., 7 2 (1970) 3 9 3 3 8 ~ . Galbraith, M. N. and Horn, D. H. S., Ausr. J. Chem., 22 (1969) 1045. Galbraith, M. N., Horn, D. H. S., Middleton, E. J. and Hackney, R. J., Ausr. J. Chem.. 22 (1969) 1059. Galli, G . and Grossi-Paoletti, E., Lipids, 2 (1967) 72. Gardner, J . N., Carlon, F. E. and Gnoj, O., J. Org. Chem., 33 (1968) 1566. Gelotte, B. J.,J. Chromatogr., 3 (1965) 330. Grimwalde, M. J. and Lester, M. G., Tetrahedron, 25 (1969) 4535. Haack, E., Kaiser, F. and Spingler, H., Chem. Ber., 8 9 (1956) 1353. Hakomori, S., J. Biochem. (Tokyo), 55 (1964) 205. Henry, R. A., Schmit, J. A. and Dieckman, J. F., J. Chromatogr. Sci., 9 (1971) 513. Hirsch, J. and Ahrens, E. H., J. Biol. Chem., 233 (1958) 31 1. Hobkirk, R., Musey, P. and Nilsen, M., Steroids, 14 (1969) 191. Hobkirk, R. and Nilsen, M.,Steroids, 14 (1969a) 533. Hobkirk, R. and Nilsen, M., Steroids, 15 (1969b) 649. Hobkirk, R. and Nilsen, M., Anal. Biochem., 37 (1970) 341. Horn, D. H. S., Fabbri, S., Hampshire, F. and Lowe, M. E., Biochem. J . . 109 (1968) 399. Horst, H.-J.,J. Chromatogr., 5 8 (1971) 227. Horst, H.-J., Grunert, E. and Stoye, M.,J. Chromatogr., 69 (1972) 395. florton, R., Kato, T. and Sheridan, R., Steroids, 10 (1967) 245. Hoshita, T., Hirofuji, Sh., Sasala, T. and Kazuno, T., J. Biochem. (Tokyo), 61 (1967) 136. Hsu C.-T., Tung Yih-Chili, Lee Hung-Tu, Hank-Feng, Lo Chi-Ngo, Wu Tsu-Ye and Lee Fang-Zu, Proc. Asia Oceania Congr. of Endocrinol., 3rd, Manila, 1967; C.A., 70 (1969) 74717f. Huang,W. Y . , S t e r o i d s , 9 (1967)485. Huber, J . F. K., Hulsman, J . A. R. J . and Mei.jers, C. A. M.,J. Chrotnatogr., 62 (1971) 79. Hulsman, J . A. R. J., Thesis, University of Amsterdam, Amsterdam, 1969. Hyde, P. M. and Elliot, W. H.,J. Chromatogr.,67 (1972) 170. Imai, S., Hori, M., Fujioka, S., Murata, E., Goto, M. and Nakanishi, K., Tetrahedron Lert., (1968) 3883. Janot, M. M., Milliet, P., Lusinchi, X. and Goutarel, R., Bull. Soc. Chim. Fr., 1967,4310. Jellinck, P. H. and Fletcher, R., Can. J . Biochem.,48 (19701 1192. Jellinck, P. H., Lewis, J . and Boston, F.,Steroids, 10 (1967) 329. Joustra, M., Siiderqvist, B. and Fischer, L.,J. Chromatogr.. 28 (1967) 21. Kato, R. and Horton, R., Steroids, 1 2 (1968) 63 1. Kelly, W . G., Ranucci, S. R. and Shaver, J . C., Steroids, 1 1 (1968) 429. Kreminski, L . F., Cox, B. L., Perrel, P. N. and Schiltz, R. A., J. Agr. Food Chern., 20 (1972) 970. Kulkarni, B. D. and Goldzieher, J. W . , Steroids, 13 (1969) 467. Laatikainen, T. and Vihko, R.,Ettr. J. Biochem., 10 (1969) 165. Lee, W.-H., Lutsky, B. N . and Schroepfcr, G . J . , J . Biol. C%em., 244 (1969) 5440. Mackie, A. M. andTurner, A. B.,Biochern. J . , 117 (1970) 543. McCurdy, J. T. and Garrett, R. D.,J. Org. Chem., 33 (1968) 660. Matschiner, J. T., Mahowald, T. A., Elliot, W. I I . , Doisy, E. A,, Jr., Hsia, S. L. and Doisy, E. A., J. Bid. Chem., 225 (1957) 771. Mechoulam, R., IsraelJ. Chem., 6 (1968) 909. Meijers, C. A. M., Thesis, University of Amsterdam, Amsterdam, 1971. Mickan, H., Dixon, R. and Hochberg, B.,Steroids, 13 (1969) 477. h k h a i l , G . , W u , f . H. and Ferin, M., Steroids, 15 (1970) 333. Mitra, M. N. and Elliot, W. H.,J. Org. Chem., 33 (1968) 2814. Nagasawa, M., Bae, M., Tamura, G. and Arima, K., Agr. Biol. Chem., 33 (1969) 1644. Nguyen Gia Chan and Prochhka, Z., Collect. Czech, Chem. Commun., 38 (1973) 2288.
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Norii, T., Yamaga, N. and Yamasaki, K.,Steroids, 15 (1970) 303. Nover, L.,Arch. Pharm. (Weinheim), 302 (1969) 321. Nover, L., Baumgarten, G. and Luckner, M., J. Chromatogr., 39 (1969a) 450. Nover, L.. Juttner, G., Noack, S., Baumgarten, G . and Luckner, M., J. Chromatogr., 39 (1969b) 419. Nystrom, E., Ark. Kemi, 29 (1969) 99. Nystrom, E. and Sjovall, J., Anal. Biochem., 12 (1965) 235. Osawa, J. and Slaunwhite, W. R., Jr.,Steroids, 15 (1970) 73. Paliokas, A . M., Lee, W. and Schroepfer, G. J.,J. Lipid Res., 9 (1968) 143. Paliokas, A. M. and Schroepfer, G . J., J. Biol. Chem., 243 (1968) 453. Peng, T . C . and Munson, P. L., Steroids, 1 1 (1 968) 105. Peterson, E. A . and Sober, H. A . , Biochem. Prep., 8 (1961) 39. Ribi, E., Filz, C. J., Goode, G., Strain, S. M., Yamamoto, K . , Harris, S. C. and Simmons, H., J. Chromutogr. Sci., 8 (1970) 577. Saez, J. M., Saez, S. and Migeon, C. J., Steroids, 9 (1967) 1. Schneider, J. .I. Crabbd, , P. and Bhacca, N. S., J. Org. Chem, 33 (1968) 3 118. Seki, T., J. Chromatogr., 29 (1967) 246. Seki, T.,Methods Erizymol., 15 (1969) 219. Seki, T. and Sugase, T., J. Chromatogr., 42 (1969) 503. Shapiro, B. H. and PBron, F. G., J. Chromatogr., 65 (1972) 568. Siggia, S. and Dishman, R. A.,Anal. Chem. , 4 2 (1970) 1229. Siiteri, P. K.,Sreroids, 2 (1963) 687. Silber, R. H. and Porter, C. C., Methods Biochem. Anal., 4 (1967) 139. Singh, B. and Rastogi, R. P., Indian J. Chem., 7 (1969) 1105. Smith, E. R. and Kellie, A. E., Biochem. J., 104 (1967) 83. Swann, D. A . and Turnbull, J. H., Tetrahedron, 24 (1968) 1441. Touchstone, J. C. and Wortmann, W., J. Chromatogr., 76 (1973) 244. Turina, S., private communication. Van Baelen, H., Heyne, W.and De Moor, P., J. Chromatogr., 30 (1967) 226. Van Lier, 1. E. and Smith, L. L., J. Pharm. Sci., 59 (1970) 7 19. Vermeulen, A. andverdonck, L., Steroids, 11 (1968) 609. Vestergaard, P., J . Chrornatogr., 31 (1967) 21 3. Vestergaard, P., in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, New York, London, 1973, p.1. Vestergaard, P., Hemmingsen, L. and Hansen, P. W., J. Chrornatogr., 40 (1969) 16. Vestergaard, P. and Jacobsen, E., J. Chromatogr,, 50 (1970) 239. Vestergaard, P. and Sayegh, J. F., Advan. Autom. Anal., Technicon Int. Symp., 1969, Mediad, New York, 1970, p. 327. Vroman, H. E. and Cohen, C. F., J. Lipid Res., 8 (1967) 150. Waters Ass., Application Highlights, Leaflet 3, Steroids (Insect Moulting), Waters Ass., Framingham, Mass., U.S.A. Williams, K . L H. and Layne, D. S., Steroids, 9 (1967) 275. Ziller,S. A., Doisy, E. A. Jr. and Elliot, W. H., J. Biol. Chem., 243 (1968) 5280.
Chapter 29
Terpenes 0. MOTL
CONTENTS
................................................................. .................................................................. ..................................................... ....................................................................... ........................................................... ...................................................................... ...................................................................... ....................................................................
Introduction ..623 Hydrocarbons 624 Ethers, epoxides and furans.. .629 Esters .630 Aldehydes andketones 631 632 Lactones Alcohols 633 Acids ........................................................................ 633 634 References
INTRODUCTION Terpenic compounds occur mainly as plant components and they are often obtained by steam distillation in the form of essential oils, in addition to conventional extraction procedures, or by collection of the exudates of some shrubs and trees (some structurally less common terpenes, or those which contain less common elements, for example bromine and chlorine, have also been found in animal and insect tissues and also as metabolites of moulds). These products are usually complex mixtures of isomeric terpenic substances, in addition to other types of natural substances, comprising monoditerpenic (&), sesterterpenic (C25), triterpenic terpenic (C,o), sesquiterpenic (C (C30), and tetraterpenic (C40) substances*. The complexity of the mixtures, especially of essential oils, which sometimes contain 100-200 components, usually requires a preliminary separation (counter-current distribution, distillation, group separation - see Lawrence, 197 1) before the proper chromatographic separation of single isomeric terpenic substances is carried out. For the analytical separation of lower terpenes (mainly monoterpenic hydrocarbons and oxygen-containing substances - see Guenther et ul.), GLC is mainly used although in many instances TLC is a very useful complementary method (see, for example, Baines and Jones or Lawrence, 1968a). For preparative purposes, liquid column chromatography is the predominant separation method, and for unstable substances it is the only method other than TLC for their separation. For column chromatography, classical adsorbents are used such as silica gel, alumina and silicic acid, more recently also silica gel and alumina impregnated with silver nitrate, and less often Florisil and charcoal. In the study of terpenic metabolites and very polar terpenic substances, various types of ion exchangers, *This chapter does not include terpenic alkaloids and tetraterpenes.
References p . 634
623
6 24
TERPENES
Sephadexes and modified celluloses have also been used. In spite of the fact that up to the present time terpenic substances have been separated predominantly by the methods mentioned above, high-speed liquid column chromatography will evidently soon serve for the separation of higher terpenoids.
HYDROCARBONS For the separation of most terpenic hydrocarbons, silica gel or neutral alumina impregnated with silver nitrate, and also activated alkaline alumina (activity 1-11), can be used; commonly used eluents are n-pentane, light petroleum fractions or benzene, in some instances with the gradual addition of small amounts of diethyl ether (gradient elution). Monoterpenic hydrocarbons usually represent the low-boiling fraction of essential oils or some balsams and resins. The distillation fractions, even when obtained with efficient distillation columns, often contain oxygen-containing substances as impurities. These impurities can be removed by chromatography on alkaline alumina (activity 11-111 according to Brockmann and Schodder; activity determination by a modified TLC method according to Heiminek ef al.) or deactivated silica gel (1 1% of water), or by displacement chromatography on silica gel with a modified surface (0.7% of Emulphore - Kugler and Kovats). Sometimes the high activity of alumina (1-11) may cause the isomerisation of unstable hydrocarbons (for example, sabinene). If the hydrocarbons are present in the extract, they can be isolated by partitioning between light petroleum (b.p. 40-60°C) and aqueous methanol and subsequent further purification of the light petroleum fraction by the above procedures. In the first chromatographic fractions, paraffins are also present in addition to terpenic hydrocarbons if the mixtures obtained by extraction are submitted to separation. The separation of monoterpenic hydrocarbons from paraffins or higher boiling terpenic hydrocarbons (sesquiterpenic and diterpenic) is usually carried out by simple distillation in a Hickmann flask or in a closed system in a high vacuum at room temperature; condensation takes place in a condenser cooled with dry-ice or liquid nitrogen (Bambagiotti er al. ). The paraffins can also be separated from terpenic hydrocarbons by chromatography on silver nitrate-impregnated silica gel (a 20-50-fold excess of adsorbent is usually used). The silver nitrate-impregnated silica gel is prepared as follows. Silver nitrate (45 g) is dissolved in 350 ml of distilled water and the solution is added to 300 g of silica gel. The suspension is evaporated on a rotary evaporator in the vacuum of a water pump. The free-flowing adsorbent is dried to constant weight either in a flask in vucuu (bath at 130°C) or in a drying oven at 130°C. Andersen and Syrdal evaporated the suspension on a rotary evaporator at 70°C for 12 h, then activated it by heating at 80°C (0.13 mm Hg) for 6 h, eventually in a drying oven at 120°C for 8 h. It is recommended that the columns should be protected from light during chromatography in order to prevent the complete darkening of the adsorbent. The solvents used for chromatography on such adsorbents should be freed from traces of sulphur compounds, preferably by filtration through a small amount of the same adsorbent. When less polar substances are to be separated, the sorbent can be regenerated by washing it with anhydrous diethyl ether which has previously been freed from peroxides, for example by filtration through alkaline alumina of activity 1 (ca. 250 ml of diethyl ether can be used per 25 g of adsorbent). The remaining ether is then displaced with light
H Y DKOCARBON S
625
petroleum (b.p. 40-60°C) and the column is ready for further use. In view of the physical and chemical properties of monoterpenic hydrocarbons, the most commonly used method for their separation is GLC (analytical and preparative), but from a study of the behaviour of 14 monoterpenic hydrocarbons (Lawrence, 1968b) on thin layers of silica gel, containing various concentrations of silver nitrate (6.25-25761, it follows that they can be separated on this type of adsorbent. In agreement with this, Andersen and Syrdal, who separated monoterpenic hydrocarbons from the essential oil of Chamaecyparis nootkatensis leaves, eluted a-pinene, A,carene, 0-pinene, two unidentified hydrocarbons, limonene and myrcene consecutively from a column of silica gel containing 15% of silver nitrate. The first two compounds were eluted with cyclohexane, the others with a 0-50% gradient of benzene in cyclohexane. Silver nitrate-impregnated neutral alumina is prepared as follows. Silver nitrate (1 2 5 g) is dissolved in 380 ml of distilled water and the solution is added, with stirring, t o 500 g of neutral alumina. The suspension is evaporated t o dryness on a rotary evaporator at 110- 130°C bath temperature and under reduced pressure (water pump), thus permitting the smooth distillation of water. When the mixture has been dried, it is activated at 130°C (bath temperature) for 15 min under a full vacuum (water pump). The suitability of this argentised neutral alumina follows from the isolation of the hydrocarbon 1-vinyl-5,5dimethyl[2.1.1] bicyclohexane (Hogg and Lawrence) from the essential oil of Mentha cardiaca (Scotch spearmint). The lowest boiling fractions of the essential oil were separated into 10 fractions by fractional distillation on a column of 35 theoretical plates. The ninth fraction (3 g) was chromatographed on a 40-fold excess of neutral alumina (activity I ) by gradient elution (light petroleum-diethyl ether-methanol) into 60 fractions. The combined fractions 1-10 (1.5 g), containing a-pinene, a-thujene and other hydrocarbons, were further chromafographed on a 40-fold excess of neutral alumina containing 15% of silver nitrate, applying gradient elution (light petroleum-diethyl ether-methanol) into 30 fractions of 1 ml. The composition of the fractions was: 9-12,a-pinene; 13 and 14, a-pinene and a-thujene; 17-24, solvents only; 25-28, l-vinyl-5,5-diniethyl[2.1.1] bicyclohexane, camphene, 6-pinene and limonene; and 30, myrcene. It was possible t o isolate the required hydrocarbon from fraction 27 by preparative GLC, although it cannot be isolated easily by this method if a-pinene is present. Owing t o its supposed lower isomerisation tendency, silica gel impregnated with silver borate was employed by VokaE et al. for the separation of monoterpenic hydrocarbons from albene (C IzH18), a hydrocarbon which is probably biogenetically similar t o terpenes. Silver borate-impregnated silica gel is prepared as follows. In a 1-1 flask with a groundglass joint, 1 0 0 g of silica gel are mixed with 200 ml of a saturated solution of silver borate, which is freed from the substances t o be separated before use. The flask with the suspension is heated in a water-bath at 30°C and in a vacuum (water pump) until the swirling of the adsorbent has ceased. Drying is continued in the vacuum of an oil pump (ca. 0.05 m m Hg) at a bath temperature of 40°C. Silver borate is prepared by precipitation of an aqueous silver nitrate solution with a 20% borax solution. The precipitate formed is thoroughly washed with distilled water on a fritted-glass filter. In contrast to monoterpenic hydrocarbons, sesquiterpenic hydrocarbons represent a very rich class of substances of which some are very similar to each other in their physical References p . 634
626
TERPENES
and chemical properties (positional and spacial isomcrs of one basic skeleton). These properties impair their separation, and another difficulty consists in their tendency to isomerise. Their separation is most commonly carried out by fractional distillation of the neutral fraction of the essential oil using an efficient column. The distillation process is controlled by GLC and TLC. Corresponding fractions are combined and single components are separated by combined chromatography on alkaline alumina, silica gel or neutral alumina impregnated with silver nitrate ( c c , Fig. 29.1), usually under GLC control. When three columns are connected in series, with decreasing diameter and increasing length, up to 15 g of a mixture of sesquiterpenic hydrocarbons can be separated on 250 g of alumina containing 25% of silver nitrate (Lawrence er al.) if gradient elution is used. In view of the fact that separations on strongly activated alumina are dependent on the number of the double bonds (cf.,Table 29.1) while on argentised adsorbents the character of the double bonds is more important than their number, a combination of both procedures is a very useful method of separation. Andersen and Syrdal partially separated a complex distillation fraction by this method, eluting first tricyclic hydrocarbons (a-copaene, a-ylangene and longifolene) with light petroleum from a column of alkaline alumina, and then separating all of the remaining hydrocarbons by elution with a more polar system by chromatography on silica gel containing 15%of silver nitrate. The Essential oil from
Zinaiber zerumbet Fractionated & the fractions pooled according to TLC & GLC
Group No.
ill
b.p.Smm
Oh yield
55-75115- 20
1
IV
VI
V
112-113/5
79-95/10 81-112/5
109-113/3-5
11O-ll813
5
10
e
s
12UII
'+
30
I
I
I
I S
118-120/1-3
q
u
i
e
t
r
p
e
n
e
s
Refractlonation
c
Non-adducting
1
1
Humulene Cut: A
B
C
Zerumb0r-e
D
92-I04 b.p.YO.5mm Oxides (Impure)
2.AgN03-Si02
*( I * -
Prep. GLC
I
AgN03-SQ
I
1
I. A1203/11; 2.AgNOg-SiOg I
Dihydro-\y -photozerum
Fig. 29.1. Separation of essential oil from Zingiber zerumbet Smith (Damodaran and Dev).
Alcohols
627
HYDROCARBONS
TABLE 29.1 CHROMATOGRAPHY OF SESQUITERPENIC HYDROCARBONS (8.4 g) FROM ATRACTYLIS OIL (CHOW et al.) Sorbent: alkaline alumina (activity I), 1100 g. Eluent: light petroleum (b.p. 40-60°C). Fraction
1 2 3 4 5 6 7 8 9
16 11 12
Volume (ml)
Weight
100 110 210 100 200 200 200 200 200 200 200 400
0.20 0.85 0.65 0.40 0.55 0.60 0.95 0.45 0.50 0.30 0.20 0.45
[a]g
Main constituent
--57.6 -59.5 +34.7 +63.2 +17.1 -40.6 -45.9 +23.8 +39.9 +40.2 +28.9 -2.52
Tricyclic Sesquiterpene Mixture of two sesquiterpenes
(g)
No. of double bonds
1 -
a-lsovetivene
2
0-Selinene
2
ar-Curcumene
Aromatic
sequence of the eluted components was the remaining longifolene, “calamenenes”, a-alaskene, P-alaskene, 6-cadinene, a-curcumene, 0-curcumene, y-curcumene, y-cadinene, 0-bisabolene and 0-farnesene. Some hydrocarbons are unstable on the adsorbents mentioned and therefore a very mild procedure was used during their isolation. Weinheimer er al., on extraction of gorgonians with hexane and subsequent distillation (up to 35”C), obtained a mixture of sesquiterpenes composed of p-elemene, 0-selinene and germacrene A. The mixture (4 g) was separated on 540 g of adsorbent composed of 70% of Florisil, 30% of powdered saccharose and 3% of maize starch. The mixture was homogenised in a blender in benzene. The column temperature was 5”C, elution was carried out with 2% solution of benzene in hexane, the flow-rate was 5 ml/min, the pressure 2.5 p s i . under nitrogen and the fractions were each of 25 ml. The authors also pointed out that Pelemene is isomerised to Bselinene when Florisil impregnated with silver nitrate is used. Less stable azulenes can also be separated on argentised alumina or on neutral deactivated alumina of activity 11-111 alone. During their preliminary isolation, the formation of the azulenium salts may be utilised, by extracting them with 85% phosphoric acid or dilute hydrochloric acid. The azulenes can be liberated from their colourless salts by dilution with water and extraction with light petroleum (b.p. 40-60°C) or diethyl ether. The separation of the dehydrogenation products of reduced prochamazulenogen was carried out by Herout and Sorm by chromatography on a 100-fold excess of alumina (activity 11-111). Elution with light petroleum gave guaiazulene, while with benzene it gave artemazulene. When using alumina impregnated with 5 % of silver nitrate (610 g), Bertelli and Crabtree separated a mixture of dihydrochamazulenes (5 g) from absinth; elution with pentane gave 3,6-dihydrochamazulene, while a 5% solution of benzene in pentane eluted chamazulene and pure benzene gave 5,6-dihydrochamazulene. Azulenes can be regenerated easily from their addition compounds (which are used for their References p . 634
628
TERPENES
characterisation) with trinitrobenzene by chromatography on alkaline alumina (activity 111-IV) with cyclohexane. In most instances, diterpenic hydrocarbons can be separated successfully on argentised silica gel by gradient elution, depending on the nature of the original mixture. In one of the first papers describing this type of separation, Norin and Westfelt separated the neutral fraction of the ethereal extract ofPinus silvestris wood on an alumina column into three fractions: elution with light petroleum (b.p. 40-60°C) gave hydrocarbons, while benzene gave aldehydes and ethanol gave alcohols. The mixture of diterpenic hydrocarbons thus obtained was separated on a 30-fold excess of silica gel containing 22% of silver nitrate. Elution was carried out with light petroleum (b.p. 40-60°C) and a 0-1% linear gradient of diethyl ether in the same solvent. In the first fractions pimaradiene was eluted and subsequent fractions contained isopimaradiene. If a smaller percentage of silver nitrate was used (ca..9%), light petroleum (b.p. 40-60°C) alone sufficed for the elution, as for example in the isolation of diterpenic hydrocarbons from the leaves of Cryptorneria japonica, carried out by Appleton et al. The light petroleum extract was chromatographed with light petroleum (b.p. 40-60°C)-diethyl ether (10: 1) on neutral alumina of activity
0
Fig. 29.2. Separation of diterpene hydrocarbons formed from 2-[ I4C]mevalonate by enzyme preparation of castor bean seedlings. Column: 1.7 X 27 cm. Sorbent: 30 g of 5% Bio-Sil HA silver nitrate-impregnated silicic acid (-325 mesh) (Calbiochem, Los Angeles, Calif., U.S.A.). Operating conditions: non-linear gradient of increasing concentrations of benzene in n-hexane; starting from fraction 56 (arrow), benzene was replaced with ethyl acetate; fraction volume 5-10 ml; flow-rate lml/ min. Detection: A 0.1 ml aliquot of each fraction was assayed for radioactivity and the amount of radioactivity per millilitre was plotted against the fraction number. A = trachylobane; B = kaurene; C = sandaracopimaradiene; D = beyerene; E = casbene.
ETHERS, EPOXIDES AND FURANS
629
1. The hydrocarbon fraction obtained was re-chromatographed on silica gel impregnated with silver nitrate. Elution with light petroleum (b.p. 40-60°C) gave, consecutively, kaurene, phyllocladene and sclarene. A still lower concentration of silver nitrate (5%) was used by Robinson and West during the isolation of labelled diterpenes obtained on biosynthesis from 2- [I4 C] mevalonate. Their separation is shown in Fig. 29.2. A mixture of 10 triterpenic hydrocarbons from the leaves of the fern Adiantum monochlamys was separated by Ageta e t al. by a combination of repeated chromatography on alkaline alumina (Wako, Osaka, Japan), elution with n-hexane, and silica gel containing 20% of silver nitrate, elution with n-hexane or n-hexane-diethyl ether (9: 1). The course was followed by GLC. The authors were able to identify fern-8-ene, fern-9(1 1)-ene, ferna-7,9( 1 1)-diene, fern-7-ene, adian-Sene, neohop-13( 18)-ene, neohop-l2-ene, filic-3-ene, neohopa- 1 1,13(18)-diene and hop22(29)-ene. If neutral or acid-washed alumina was used, isomerisation of the double bonds was observed.
ETHERS, EPOXIDES AND FURANS The separation of these substances is usually carried out on deactivated neutral alumina of activity 11-111 or deactivated silica gel (seldom impregnated with silver nitrate) or Florisil. In view of the low polarity of the substances and the use of deactivated adsorbents, light petroleum (b.p. 40-60°C) plus a small amount of benzene, diethyl ether or ethyl acetate is most commonly used for elution. Highly activated alumina cannot be used for the separation because isomerisation might take place (see the detailed study by Joshi ef al.). Coates and Melvin purified the reaction product of the dehydration of cis-2,2-dimethyl3-hydroxy-6-methylenecyclohexanemethanol on silica gel, using light petroleum (b.p. 30-60°C) for elution. The separation of very similar sesquiterpenic oxides (differing in the axial and the equatorial positions of the methyl group) obtained on dehydration of 4-hydroxyguaioxide and subsequent hydrogenation was carried out by Ishii et al. ( I 970) on a 200-fold excess of alumina of activity 11. The first fractions, obtained on elution with light petroleum, contained guaioxide, and the last fraction was liguloxide. A more complex example of the isolation of diterpenic isoincensoloxide and incensoloxide was successfully solved by Forcellese et al. in the following manner. The neutral fraction of the resin Boswellia carteri was chromatographed on a 30-fold excess of alumina (activity 11-111); benzene first eluted incensol and then incensoloxide and isoincensoloxide. The oily fraction containing both oxides was further chromatographed on a 50-fold excess of silica gel; elution with benzene containing 5% of diethyl ether gave fractions from which incensoloxide crystallised out. The mother liquors also contained, according to TLC on silver nitrate-impregnated silica gel, oxides that could not be separated well on silica gel alone (TLC on silica gel gave a single spot). The mixture was converted into benzoates (1.25 g) and separated on silica gel (75 g) with benzene containing 3% of diethyl ether, affording isoincensol benzoate, and with benzene containing 15% of diethyl ether, affording incensoloxide benzoate. acetate with The reaction mixture obtained on oxidation of 1 l~-hydroxylmostan-3~-yl lead tetraacetate and iodine was chromatographed on deactivated silica gel (15% of water), References p . 634
630
TERPENES
affording in the first fractions the corresponding iodoether acetate (Roller and Djerassi). On neutral alumina (activity 11), the unsaponifiable part of the light petroleum extract of Polypodium vulgare rhizomes was separated using light petroleum as eluent. TWOtriterpenic hydrocarbons were eluted first, followed by 17,21-epoxyhopane (Berti er al.). The effect of the epoxy group on the chromatographic behaviour of sesquiterpenic furan derivatives is apparent from a comparison of the conditions of the isolation of 8,8a-epoxyfuranoligularanefrom the essential oil of Senecio silvaticus (described by Schild), and furoligularane obtained by degradation. The native substance was isolated by chromatography of the essential oil on a 100-fold excess of neutral alumina (activity 111) with light petroleum (b.p. 40-60°C) containing 2% of ethyl acetate, while furanoligularane was eluted under virtually the same conditions with light petroleum (b.p. 4O-6O0C) alone. Cimino et al. (1972b) separated another mixture of simple sesquiterpenic furans (pleraplysillin and dehydrodendrolasin) on silica gel impregnated with silver nitrate (150 mg of the crude extract on a mixture of 15 g of silica gel and 2.5 g of silver nitrate) in the system light petroleum (b.p. 40-7O0C)-benzene (beginning with a 9:1 mixture and then increasing the content of benzene). In connection with the same theme, viz., the study of the components of sea sponges, Cimino et al. (1972a) separated two Czl furanoterpenes (degraded sesterterpenes) by multiple chromatography on silica gel; using benzene-light petroleum (b.p. 40-70°C) (7:3) they eluted first tetrahydrofurospongin-2 and then dihydrofurospongin-2.
The most commonly used adsorbents for the separation of simple esters are silica gel and neutral alumina (activity 11-HI), as well as silica gel impregnated with silver nitrate. Mixtures of light petroleum (b.p. 40-60°C) and benzene (or a small amount of diethyl ether and benzene) were employed as eluents. With substances that contain several double bonds, which are chromatographed on silver nitrate-impregnated silica gel, more polar elution systems should be used, such as benzene containing a small amount of ethanol. The amount of adsorbent is dependent, as in other instances, on the composition and the number of components of the mixture being chromatographed; it is usually from 30 to 50 times the weight of the mixture for alumina, while for silica gel alone or impregnated with silver nitrate it is usually from 15 to 30 times the weight of the mixture. The oxidation product of dimethyl shellolate was purified on neutral alumina by elution with benzene by Yates and Field. Other esters of sesquiterpenic acids obtained on hydrolysis of Palas seedlac and subsequent esterification were also separated by Singh et al. on alumina (activity 11), using a mixture of benzene and light petroleum (b.p. 40-60°C) (3: 1) for the elution of the ester of laccishellolic acid, and benzene for the ester of epilaccishellolic acid. The separation of four stereoisomeric synthetic geranyl esters of farnesylacetic acid, which was poor by fractional distillation or preparative GLC, was carried out by Pala et al. on a 25-fold excess of very fine silica gel impregnated with silver nitrate. The first fraction eluted with benzene-ethanol(98:2) indicated a separation of isomer I (trans,cis-(4,5) and cis-(8,9)) from isomer I1 (cis-(4,5) and cis-(8,9))in only a 5.5:4.5 ratio. However, by triple re-chromatography, pure isomer I1 could be obtained.
ALDEHYDES AND KETONES
63 1
The last fraction eluted with benzene-ethanol (95:5) was a mixture of I and I1 in a 9.2:0.8 ratio, from which a single additional chromatography gave pure isomer I. In a similar manner, the isomers trans-(4,5), trans-(8,9) and cis-(4,5), trans-(8,9) were also separated. The separation of the methyl esters of diterpenic acids obtained on oxidation of a mixture of six diterpenic aldehydes and esterification with diazomethane, which displayed two main peaks and several by-products on GLC analysis, was carried out by Bruns on a 15-fold excess of silica gel containing 28% of silver nitrate; with light petroleum (b.p. 40-80°C) containing 2%of diethyl ether an ester of dehydroabietic acid was obtained, followed by an ester of isopimaric acid, which was purified by crystallisation and re-chromatography on alumina. Triterpenic acetates (0-amyrenyl acetate and hop-1 7,(2 l)-en3/3-yl acetate) were separated by Arthur e t al. on a 75-fold excess of alumina by elution with light petroleum (b.p. 60430°C)(in the above sequence). Wahlberg e l al. separated the following four triterpenic esters on silica gel and then on silver nitrate-impregnated silica gel with light petroleum-diisopropyl ether: a-amyrin palmitate, lupenyl palmitate, 0-amyrin palmitate and cycloartenyl palmitate, present in the neutral fraction of the acetone extract of the wood of Carphephorus odoratissimus.
ALDEHYDES AND KETONES In view of the chemical properties of these compounds it is possible to use either neutral or acidic alumina, as well as silicic acid, silica gel and silver nitrate-impregnated silica gel for their chromatographic separation. Depending on the number of functional groups and the activity of the adsorbents used, the following elution systems can be employed: light petroleum (b.p. 40-60°C), mixtures of light petroleum and diethyl ether, benzene, mixtures of benzene and diethyl ether, and chloroform. The separation of two sesquiterpenic aldehydes, nuciferal and torreyal, was achieved by Sakai e t al. on a 15-fold excess of silicic acid by elution with a 3% solution of ether in hexane; nuciferal was eluted first. A mixture of three ketones of the elemane type from the essential oil of the Acorus cafamus rhizomes was obtained by Yamamura et af. by chromatography of the whole essential oil on a 15-fold excess of silica gel with light petroleum-diethyl ether (4: 1). This mixture was then separated into single components by repeated chromatography on an 80-fold excess of silica gel, using benzene for elution. The substances were eluted in the following sequence: shyobunone, epishyobunone and isoshyobunone. Canonica et al. used a 100-fold excess of silica gel G (Merck)-Celite (1 :1) for the chromatographic separation of unstable substances in the acetone extract from the bark of Cinnamosmafragrans; elution with benzene and benzene-diethyl ether (9: 1) gave fractions containing cinnamodial (a CISdialdehyde containing a carbonyl and a hydroxyl group). Baker et af. isolated from the reaction mixture of 15a,16-epoxyphyllocladane with boron trifluoride-diethyl ether complex 16-epiphyllocladan-l5-one using alumina deactivated with 5% (v/v) of aqueous 10% acetic acid; elution was carried out with light petroleum. In a study of the unsaponifiable fraction of the ethereal extract of pinus strobus bark, Zinkel and Evans combined chromatography on silicic acid and silicic acid References p . 634
632
TERPENES
containing 40% of silver nitrate with gradient elution with light petroleum-diethyl ether in order to isolate, after manoyl oxide, the diterpenic aldehyde strobal; the aldehydes abietal, dehydroabietal, neoabietal, communal and isopimaral followed. Triterpenic ketones from the light petroleum extract of Quercus glauca leaves were separated by Tachi et al. first on silica gel (elution with an n-hexane-ethyl acetate mixture) and then on silver nitrate-impregnated silica gel (elution with 99: 1 n-hexaneethyl acetate). The first fraction contained cyclobalanone, which was followed by 24methylenecycloartanone. In addition to silica gel, alumina washed with acids is also often used. Djerassi and McCrindle even used alumina of activity I (ratio adsorbent: extract = 40: 1) for the separation of the neutral fraction from the methanolic extract of Tillandsia usneoides. Elution with light petroleum (boiling range 40-6O0C)-diethyl ether (7:3) gave first cycloartenone and then frideline.
LACTONES Adsorbents used for the separation of lactones are of neutral character: silica gel, alumina, or silicic acid and Florisil. As most lactones contain additional oxygen-containing functional groups, the elution systems are usually of a more polar character than those for the groups of terpenic substances discussed above. Often mixtures of benzene and chloroform, light petroleum (b.p. 40-60°C) and ethyl acetate, and benzene and diethyl ether are used in which the more polar components account for 10-50%. Sesquiterpenic lactones, the number of which has increased rapidly in recent years (at present more than 400 of these compounds are known - c.f , Devon and Scott) are separated predominantly on silica gel, silicic acid and neutral alumina. Herz e f al. separated a mixture of six sesquiterpenic dilactones, present in the chloroform extract from the working up of Mikania scandeizs, on silicic acid, using benzene-chloroform (3 :2) for elution. Herz and Srinivasan chromatographed a chloroform extract from Gaillardia amblyodon on a 10-fold excess of alumina (Alcoa F-20); on elution with benzenechloroform (3: l), gaillardipinnatin was obtained, while elution with chloroform gave amblyonin. Diterpenic lactones from the filtrate of Gibberella fujikuroi were isolated by Cross et al. on a 100-fold excess of a mixture of Celite and silica gel (2:l). Elution with light petroleum (b.p. 60-80°C) containing 15- 17% of ethyl acetate gave 7-hydroxykaurenolide, the same mixture, but containing 30-32.5% of ethyl acetate, eluted 7,18-dihydroxykaurenolide, while ethyl acetate-methanol (9: 1) gave 7,16,18-trihydroxykaurenolide.In a similar manner, substances of the same type were obtained by Serebryakov et al. from the metabolites of Fusarium moniliforme when a 6-fold excess of silica gel was used and some fractions were re-chromatographed on neutral alumina (40-fold excess). The separation of triterpenic lactones was also studied by Chanley et al. during the analysis of the components of the toxic principle of holothurin present in sea cucumber (Actinopygga agassizi). This principle is a mixture of about six glycosides; aglycones obtained by acid hydrolysis were chromatographed on a 15-fold excess of deactivated alumina (4% of 10% acetic acid solution). Elution with benzene afforded, in the second fraction, a mixture of 22,25-oxidoholothurinogeninand its 17-deoxy derivative. From
ALCOHOLS
633
several chromatographic runs, a mixture of both substances was obtained, which was acetylated and separated chromatographically on alumina deactivated with 3% acetic acid solution. Elution with a 1 :4 mixture of benzene and Skelly B (a hydrocarbon mixture containing about 50% of n-hexane; Skelly Oil Co., Kansas City, Mo., U.S.A.) gave 17deoxy-22,25-oxidoholothurinogeninacetate in the first fraction, while a mixture of benzene and Skelly B (1 :3-1: 1) gave 22,25-oxidoholothurinogeninacetate. Djerassi et al. also used neutral alumina (a 60-fold excess) for the separation of acetylated aglycones prepared by hydrolysis and acetylation of the neutral fraction from the ethanolic extract of the cactus Lemaireocereus stellatus. On elution with benzene-diethyl ether (1 :1) they obtained thurberogenin acetate, and with diethyl ether-chloroform (3:2)they obtained stellatogenin acetate.
ALCOHOLS For the separation of terpenic alcohols, neutral alumina and silica gel are mainly used, and silicic acid very rarely. In view of the degree of adsorbent activity and the number of hydroxyl or other functional groups in the chromatographed substances, more polar systems serve as eluents, such as benzene-ethyl acetate, benzene-diethyl ether and chloroform-e thanol. The separation of 60- and 70-hydroxyguaioxides from the microbial transformation of guaioxide was performed by lshii e t al. ( I 971) on a 30-fold excess of alumina (activity V); elution was carried out with light petroleum-diethyl ether (98:2 and 95:5), affording the 60-hydroxy derivative. Medium fractions, eluted with 9: 1 light petroleum-diethyl ether, contained the 7a-hydroxy derivative. lguchi et al. separated calamendiol from isocalamendiol by silica gel chromatography using in the first instance light petroleumdiethyl ether (5: 1) for elution, while the second substance was eluted with the same mixture in a 3: 1 ratio. The diterpenic alcohols manool and isopimaradienol from the neutral fraction of the acetone extract of Daciydium bidwillii wood were separated by Grant et al. on a 30-fold excess of alumina, activity 11, by elution with light petroleum (b.p. 60-8O0C)-diethyl ether mixtures. A 4:6 mixture eluted manool, while a 3:7 mixture eluted a mixture of manool and isopimaradienol, which was further separated by TLC. Fujita and Taoka chromatographed an ethereal extract of the leaves of lsodon lasiocarpus on a 40-fold excess of silicic acid; elution with dichloromethane-acetone (8:2) gave lasiokaurin (kaurenetriol) and lasiodonin (kaurenetetraol). The separation of the triterpenic alcohols zeorin and leucotylin from the neutral fraction of the ethereal extract of the lichen Parmelia leucotyliza was carried out by Yosioka et al. by multiple chromatography on alumina and gradual elution with chloroform, chloroform-methanol and pure methanol.
The separation of organic acids is usually carried out on the acids either in a free state or in the form of esters, mainly methyl esters (after esterification with diazomethane). References p . 634
634
TEKPENES
The separation of free acids is carried out by adsorption chromatography on alumina, silica gel on a Celite-charcoal mixture, using a variety of systems of different polarity, ranging from light petroleum (b.p. 40-60°C)-benzene, or benzene-diethyl ether, to chloroform-methanol. When partition chromatography was applied, alumina impregnated with dimethyl sulphoxide and diisopropyl ether-acetone as eluent, or silica gel with anchored formic acid and a gradient of ethyl acetate in n-hexane were used. The separation of esters was generally carried out under the conditions mentioned in the section on esters. Two sesquiterpenic hydroxy acids were separated by Runeberg on a 100-fold excess of dimethyl sulphoxide-impregnated alumina. Elution with diisopropyl ether gave hydroxy acid I, while with acetone, hydroxy acid I1 was eluted. The separation of 33 gibberellins (diterpenic acids, 100 mg) was investigated by Durley et al., using for partition chromatography silica gel (Woelm, Eschwege, G.F.R.) (20 g) on to which 0.5 M formic acid saturated with a mixture of ethyl acetate and n-hexane (1 :9) was fixed as the stationary phase. A gradient of ethyl acetate in n-hexane was used as the mobile phase. This mixture was saturated with 0.5 M formic acid in a Varigrad system with four mixing chambers (1, ethyl acetate: n-hexane 65:35; 2,20:80; 3, 1OO:O; and 4, 1OO:O). The separation achieved was relatively good: groups of 3-6 substances, in some instances even 1-2 substances. Nagai et al. separated two triterpenic acids by chromatography on silica gel using benzene-diethyl ether (9: 1) for elution. The early fractions contained urs-12-en-30-01-27oic acid, and were followed by fractions containing olean-12-en-30-01-27-oic acid.
REFERENCES Ageta, H., Shiojima, K. and Arai, Y., Chem. Commun., (1968) 1105; private communication, 1973. Andersen, N. H. and Syrdal, D. D., Phytochemistry, 9 (1970) 1325. Appleton, R. A., McCrindle, R. and Overton, K. H., Phytochemistry, 9 (1970) 581. Arthur, H. R., Hui, W. H., Lam, C. N. and Szeto, S. K., Aust. J. Chem., 17 (1964) 697. Baines, D. A. and Jones, R. A.,J. Chromatogr., 47 (1970) 130. Baker, K. M., Briggs, L. H., Buchanan, J. G. St. C., Cambie, R. C., Davis, 8. R., Hayward, R. C., Long, G. A. S. and Rutledge, P. S.,J. Chem. SOC.,Perkin Trans. I , (1972) 190. Bambagiotti, M. A., Vincieri, F. F. and Cod, G., Phytochemistry, 1 1 (1972) 1455. Bertelti, D. J. and Crabtree, J. H., Tetrahedron, 24 (1968) 2079. Berti, G., Bottari, F., Marsili, A. and Morelli, I., Tetruhedron Lett., (1966) 979. Brockmann, H. and Schodder, H., Chem. Ber., 74 (1941) 73. Bruns, K., Tetrahedron, 25 (1969) 177 1. Canonica, L., Corbella, A,, Gariboldi, P., Jommi, G., K?epinsk$, J., Ferrari, G. and Casagrande, C., Tetrahedron, 25 (1969) 3895. Chanley, J. D., Mezzetti, T."and Sobotka, H., Tetruhedron, 22 (1966) 1857. Chow, W. Z., Motl, 0. and Sorm, F., Collect. Czech. Chem. Commun.,27 (1962) 1914. Cimino, G., De Stefano, S., Minale, L. and Fattorusso, E., Tetrahedron, 28 (1972a) 267. Cimino, G., De Stefano, S., Minale, L. and Trivellone, E., Tetrahedron, 28 (1972b) 4761. Coates, R. M. and Melvin, L. S., J. Org. Chem., 35 (1970) 865. Cross, B. E., Galt, R. H. B., Hanson, J. R., Curtis, P. J., Grove, J. F. and Morisson, A,, J. Chem SOC., (1963) 2937. Damodaran, N. P. and Dev, S., Tetrahedron, 24 (1968) 4113. Devon, T. K. and Scott, A. I., Handbook of Naturally Occurring Compounds, Vol. II, Terpenes. Academic Press, New York, 1972.
REFERENCES
63 5
Djerassi, C., Lin, L. H., Farkas, E., Lippman, A. E., Lernin, A. J., Geller, L. E., McDonald, R. N. and Taylor, B. J . , J. Amer. Chem. Soc., 77 (1955) 1200. Djerassi, C. and McCrindle, R., J. Chem. Soc., (1962) 4034. Durley, R. C., Crozier, A., Pharis, R. P. and McLaughin, G. E., Phytochemistry, 11 (1972) 3029. Forcellese, M. L., Nicoletti, R. and Petrossi, U., Tetrahedron, 28 (1972) 325. Fujita, E. and Taoka, M., Chem. Phurm. Bull. (Tokyo), 20 (1972) 1752. Grant, P. K., Huntrakul, C. and Sheppard, D. R. J . , Ausr. J. Chem., 20 (1967) 969. Guenther, E., Gilbertson, G. and Koenig, R. T., Anal. Chem., 43 (1971) 45R. H e h i n e k , S., S.hwarz, V. and Eekan, Z., Collect. Czech. Chem. Commun., 26 (1961) 3170. Herout, V. and Sorrn, F., Chem. Listy, 4 8 (1954) 706. Herz, W. and Srinivasan, A., Phytochemistry, 11 (1972) 2093. Herz, W., Subramanian, P. S., Santhanan, P. S. and Hall, A. L.,J. Org. Chem., 35 (1970) 1453. Hogg, J . W. and Lawrence, B. M., Flavourlnd., 3 (1972) 321. Iguchi, M., Nishiyama, A., Koyama, H., Yarnamura, S. and Hirata, Y., Tetrahedron Lett., (1969) 3729. Ishii, H.,Tozyo, T. and Nakamura, M., Chem. Pharm. Bull. (TokyoJ,19 (1971) 842. Ishii, H., Tozyo, T., Nakamura, M. and Minato, H., Tetrahedron, 26 (1970) 291 1. Joshi, V. S., Darnodaran, N. P. and Sukh, D., Tetrahedron, 24 (1968) 5817; 27 (1971) 459 and 475. Kugler, E. and Kovits, E., Helv. Chim. Acta, 46 (1963) 1480. Lawrence, B. M., Perfum. Essent. Oil Rec., 59 (1968a) 421. Lawrence, B. M.,J. Chromatogr., 38 (1968b) 535. Lawrence, B. M., Can. Inst. Food Technol. J., 4 (1971) A44. Lawrence, B. M., Hogg, J . W. and Terhune, S. J., Perfum. Essent. Oil Rec., 60 (1969) 88. Nagai, M., Izawa, K. and Inouve, T., Chem. Pharm. Bull. (TokyoJ,17 (1969) 1438. Norin, T. and Westfelt, L., Acta Chem. Scand., 17 (1963) 1826, 1828. Pala, G., Mantegani, A., Bruzzese, T. and Sekules, G., Helv. Chim. Acta, 53 (1970) 1827. Robinson, D. R. and West, Ch. A., Biochemistry, 9 (1970) 70. Roller, P. and Djerassi, C.,J. Chem. SOC.,C, (1970) 1089. Runeberg, J.,Actu Chem. Scand., 15 (1961) 721. Sakai, T., Nishirnura, K. and Hirose, Y., Bull. SOC.Chim. Jup., 38 (1965) 381. Schild, W., Tetrahedron, 27 (1971) 5735. Serebryakov, E. P., Simolin, A. V., Kucherov, V. F. and Rosynov, B. V., Tetrahedron, 26 (1970) 5215. Singh, A. N., Upadhye, A. B., Wadia, M. S., Mhaskar, V. V. and Sukh, D., Tetrahedron, 25 (1969) 3855. Tachi, Y.,Taga, S.,Kamano, Y. and Komatsu, M., Chem. Pharm. Bull. (TokyoJ, 19 (1971) 2193. VokiE, K., Samek, Z., Herout, V. and b r m , F., Tetrahedron Left., (1972) 1665. Wahlberg, I., Karlsson, K. and Enzell, C. R., Acfa Chem. Scand., 26 (1972) 1383. Weinheimer, A. J . , Youngblood, W. W., Washecheck, P. H., Karns, T. K. B. and Ciereszko, L. S., Tetrahedron Lett., (1970) 497. Yamarnura, S., Iguchi, M., Nishiyarna, A. and Niwa, M., Tetrahedron, 27 (1971) 5419. Yates, P. and Field, G. F., Tetrahedron, 26 (1970) 3135. Yosioka, I., Nakanishi, T. and Kitagawa, I., Chem. Pharm. Bull. (TokyoJ,17 (1969) 279. Zinkel, D. F. and Evans, B. B., Phytochemistry, 11 (1972) 3387.
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Chapter 30
Amines Z. DEYL
CONTENTS
...................................................................
Introduction Aliphatic mono-, di- and polyamines.. Aromaticamines................................................................ Aromatic amines and aliphatic polyamines in mixtures ................................. Tryptophanmetabolites Quaternary ammonium compounds and amino alcohols. ................................ Biogenicamines References
..............................................
.......................................................... ................................................................ ....................................................................
637 .637 643 .645 645 .649 650 655
INTRODUCTION In the separation of amines, several types of compounds have been studied very intensively, particularly catecholamines and tryptophan metabolites, which have been dealt with in many publications using a variety of separation procedures. For other aniines, such as aliphatic amines, polyamines and aromatic amines, the situation is less complex, although the distribution into the above types is sometimes difficult to achieve as different types of amines are frequently chromatographed side by side. Also, some types of amines, such as tryptamine and serotonin, are chromatographed together with amino acids. These types of separations are not listed here or in the chapter on amino acid chromatography. However, it is possible to obtain some idea of these separations from the ion-exchange procedures described in this chapter. Ion-exchange chromatography in nearly all its variations has been widely exploited for liquid column chromatographic separations of amines. High-speed techniques and gel permeation chromatography are not popular at present and it is likely that classical ion-exchange techniques will dominate this area as they give good and rapid separations. Another important factor is the obvious applicability of automated amino acid analyzers for this purpose.
ALIPHATIC MONO-, DI- AND POLYAMINES The first type of ion-exchange chromatography of aliphatic monoamines that proved suitable for automation was that involving the ion-exchange resin Arnberlite CG-120 (Perry and Schroeder). A summary of the elution volumes for a series of aliphatic monoamines on this ion exchanger is presented in Table 30.1. References p . 655
637
638
AMINES
TABLE 30.1 ELUTION VOLUMES OF AMINES CHROMATOGRAPHED ON AMBERLITE C G 1 2 0 (PERRY AND SCHROEDER) Authentic compounds were chromatographed in mixtures on Amberlite CG120 columns 30 cm in length and 0.9- 1.0 cm in diameter, at a flow-rate of 30 ml/h and a temperature of 50°C. Chromatograms were developed with 0.2 N pyridine acetate buffer (pH 3.50) for the first 600 ml, and thereafter with 0.8 N pyridine acetate buffer (pH 5.50). The breakthrough of the second developer occurred at 636 ml. Compound
Range of elution (ml)
Elution Compound peak (ml)
Glucosamine Galactosamine N Ace tyle thylenediamine 0-Methoxye thylamine N-Methylethanolamine 3-Amino- 1-propanol Serinol Dimethylamine 0-Hydroxyprop ylamine N-Methylethylamine Diethylamine Ethanolamine 2-Aminobutanol Pyrrolidine Piperidine Me thylamine Ethylamine
115-132 115-132 145-160 150-165 156-171 175-191 175-191 187-200 195-209 198-215 212-224 210-230 220-236 250-270 250-272 262-280 272-294
121 121 152 157 163 183 183 193 201 206 218 219 228 260 261 273 282
?Me thylmercaptopropylamine sulphoxide Ammonia Propylamine Isobu tylamine Hydroxylamine 1-Methylhistidine C yclopropylamine 3-Me thylhistidine n-Butylamine Isoamylamine ?-Me thylmercaptopropylamine n-Amylamine Histidine Ornithine Lysine Carnosine Arninine
Range of elution (ml)
Elution peak (ml)
290-300 296-345 312-332 316-345 334-371 340-375 350-382 353-388 390-420 430-465 447-490 504-542 591-629 646-656 653-662 657-666 820-855
295 308 322 328 347 36 1 364 370 405 44 5 467 520 600 650 656 66 2 832
11
w
y4: 8.5 0.3 p
0
cn
0.2 0.1
2 TIMEth)
1
2
EFFLUENTtml) 30 60 SOLVENT pH 5.28 Na cltrate
3
4
5
6
90
120
150
180
Na' 0.30M-!-pH
8.02 Na borate Na'O.6OM+pH 11.08 Na salicylate Na' 0 . 2 3 M
Fig. 30.1. Chromatogram of primary monoamines separated at 50°C on an Aminex A-5 column (10 X 0.6 cm) (Miyagi and Ando). Peaks: 1, methylamine; 2, ethylamine; 3, allylamine; 4, n-propylamine; 5, isobutylamine; 6, n-butylamine; 7, dopamine; 8, isoamylamine; 9, histamine; 10, namylamine; 11, tyramine; 12, phenylethylamine; 13, serotonin; 14, n-hexylamine.
639
ALIPHATIC MONO-, DI- AND POLYAMINES
The separation of primary amines, diamines and polyamines on ion-exchange columns of the Aminex type, using small variations of the equipment normally used for automated amino acid analysis, appears to be the most advanced liquid column chromatographic procedure used for these compounds nowadays. Aminex A-4 and A-5 and Bio-Rex 70 were used for this purpose by several workers (Hatano er ul., Miyagi and Ando, Perry and Schroeder, Rosenthal and Tabor, Yoshioka er al.). Evaluation is carried out by the conventional ninhydrin procedure. In practice, the individual systems differ in the Varigrad buffer composition, and in the timing of the sudden buffer changes, which, of course, depends greatly upon the mixture being separated and the necessity to spread a particular region or the chromatogram. The problems met here are similar to those which occur in amino acid analysis, as both the nature of the separation process and the equipment used have much in common. Typical runs on primary monamines and diamines are presented in Fig. 30.1. With Aminex A-4 (Hatano er ul.), elution is carried out with a complex buffer system (Table 30.2). The column size is the same as that with Aminex A-5 (Fig. 30.1); before use, it is packed under 1 atm overpressure and operated thereafter at 50°C. Before application of the sample, the column is conditioned with 0.1 16 M sodium citrate buffer (starting buffer). Buffers I and I1 (Table 30.2) are used for 70 and 150 min, respectively. After the appropriate amount of buffer I1 has passed through the column, the system is connected automatically to a three-chamber Technicon Varigrad system containing 120 ml each of the remaining buffers listed under 111 in Table 30.2. The gradient is allowed to run for an additional 200 min. Flow-rates are maintained at 30 ml/h in both the ninhydrin and sample lines. The retention time of the reaction coil is 11 min 15 sec (exactly) and the coil is maintained thermostatically at 100°C. Absorbance and height:width ratios of eluted peaks for a number of amines were published by Hatano e l al. A typical example of a separation can be seen in Fig. 30.2. Bio-Rex 70 (-400 mesh) appears to be the most suitable ion exchanger if polyamines have to be separated. The development of this technique was described in a series of TABLE 30.2 COMPOSITION AND CONDITIONS OF ELUTING BUFFERS (HATANO et al.) Benzyl alcohol was used for the elution system in order to prevent a tailing effect and to obtain well resolved chromatograms. When benzyl alcohol was added to the eluent, no difficulty was experienced with ninhydrin colour development. Buffer system
Concn. (N)
Benzyl alcohol concn. (%)
(1) Sodium n'trate (11) Sodium borate (111) Gradient ( 1 ) Sodium borate (2) Sodium salicylate (3) Sodium salicylate
0.1 16 0.025
0.5
5.28 8.02
0.35 0.60
0.05 0.20 0.20
0.4 -
10.00 11.50 12.50
0.60 0.65
*Adjusted with 6 N sodium hydroxide or hydrochloric acid. **Adjusted with sodium chloride solution.
References p.655
PH*
Ionic strength of sodium ion (M)**
0.70
640
AMINES PH r12
”‘“1
-11
-10 -9 -0
-1 -6
-5
30
60
90
120
150
180
210
EFFLUENT. rnl *BUFFER
I+
BUFFER II
BUFFER 111
240
-
Fig. 30.2. Chromatogram of an authentic mixture of 16 amines and eluting conditions of the buffer system (Hatano e t a [ . ) . Peaks: 1, methylamine; 2, ethylamine; 3, allylamine; 4, npropylamine; 5, isobutylamine; 6 , n-butylamine; 7, 1,2-propanediamine; 8, histamine; 9, isoamylamine; 10, n-amylamine; 11, tyramine; 12, putrescine; 13, phenethylamine; 14, cadaverine; 15, serotonin; 16, hexamethylenediamine. The amount of each amine was 0.4 pmole except for serotonin (1.0 pmole). All curves represent the peaks at 570 nm except for serotonin (440 nm). Monitored pH by a flow pH meter was recorded automatically.
papers by Morris et al., Rosenthal and Tabor, and Tabor et al. Columns (7 X 0.9 cm) of the above sorbent are attached to the usual equipment for automated amino acid analysis. Elution is carried out at 2 ml/min with the following series of buffers. The initial buffer is 0.438 M pyridinium acetate of pH 7.5. After 100 ml of the initial buffer have passed through the column, this buffer is immediately replaced with a second buffer, consisting of 0.5 M pyridinium acetate of pH 4.4. Elution times for polyamines in the above system were published by Dubin and Rosenthal. In the procedure reported recently by Morris, some slight changes in the elution buffer system were introduced. After application of the sample, the elution with the initial buffer was begun, which in this instance was 0.33 M pyridinium acetate of pH 5.7. After 100 ml of the initial buffer had passed through the column, the buffer was changed to 0.38 M pyridinium acetate buffer of pH 4.4 and elution continued at the same rate (ca. 15-30 ml/h). Various polyamines TABLE 30.3 ELUTION TIMES OF POLYAMINES AND RELATED COMPOUNDS ON BIO-REX 7 0 (MORRIS et al.) ~~~~
~~
Compound
Time (min)
Compound
Time (min)
Arginine Putrescine 1,3-Diaminopropane Cadaverine Acetylspermidine B Acetylspermidine A
4 25 21 32 34 42
Agm atine Spermidine Iminobispropylamine Ace tylspermine Spermine
59 64 64 68 18
ALIPHATIC MONO-, DI- AND POLYAMINES
64 1
and their acetyl derivatives were tested, and their elution volumes are summarized in Table 30.3. All of the commonly occurring polyamines can be quantitatively separated, with the exception of the pairs 1,3-diaminopropane and putrescine, and cadaverine and acetylspemidine, if they are present in a single sample. High salt concentrations disturb the above separations, and these techniques are therefore not directly suitable for analyzing such materials as tissue culture media, unless the sample is properly desalted by passing it through an ion-exchange desalting column prior to analysis or by a batch process with n-butanol. The choice of different ion exchangers suitable for the separation of polyamines was further extended in the work of Holder and Bremer, who used Amberlite IRP-64 and Dowex 50-X8 for this purpose. Elution of the Amberlite IRP-64 column is carried out first with a convex salt gradient obtained by continuous mixing of a potassium phosphate buffer of pH 7.1 (0.1 Mwith respect to phosphate) which is simultaneously 3.2 M with respect to potassium chloride with distilled water (column 0.9 X 30 cm; flow-rate 30 ml/h). The volume of distilled water used at the beginning of the separation is 200 ml. After 500 ml of the effluent have been collected, elution is continued with saturated potassium chloride solution in order to elute spermine (Fig. 30.3).
ml
Fig. 30.3. Separation of a mixture of diamines on Amberlite IRP-64 column with potassium chloride buffer (Holder and Bremer). Gradient elution is indicated in the figure; after 500 ml of the mobile phase had passed through the column, gradient elution was replaced with saturated potassium chloride solution in order to eluate spermine. Peaks: 1, 2,2'-dithiobis(ethylamine); 2, cadaverine; 3, putrescine; 4, 1,3-diaminopropane; 5, spermidine; 6 , spermine.
References p . 655
642
AMINES
TABLE 30.4 ELUTION VOLUMES OF SOME AMINES (BLAU) Amine
Elution volume (mll
Shape of peak
Recovery
Trimethylamine N-oxide Creatine Te tramethylammonium Die thy lamine n-Amylamine Isoamylamine Trime thylamine Piperidine n-Bu tylamine Pyrrolidine n-Propylamine Dimethy lamine Ethylamine Tyramine Glucosamine Me thylamine Canavanine Ethanolamine Adrenaline Arginine 3-Hydroxytyramine Noradrenaline SHydroxytry ptamine Ammonia pH 5.0 breakthrough
12 20 19 81 109 111 118 128 130 148 150 162 184 260 210 280 290 300 310 330 430 46 0 465 490 600
Very sharp Very sharp Sharp Sharp Sharp Sharp Sharp Sharp Sharp Sharp Sharp Sharp Sharp Broad Broad Broad Very broad Broad Very broad Broad Very broad Very broad Very broad Broad
Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative Quantitative 85% 85% Quantitative 80% 80% Not determined 70% 90% 70%
I
1
1
I
20
40
70
I
100
I
I
120
145
80% Quantitative
FRACTION NUMBEA
Fig. 30.4. Chromatographic separation of homologous series of volatile primary amines (Clayton and Strong). Solution containing cu. 0.05 mequiv. of each amine (C, to C,) was introduced on to a Celite 545 column, 38 X 100 mm, operated at a flow-rate of 0.5-1.0 ml/min. Fractions of 2-ml volume were extracted into 5 ml of water and titrated with dilute hydrochloric acid. The stationary phase consisted of methanol, ethanol and water; Iight petroleum, equilibrated with stationary phase, was used as the mobile phase.
643
AROMATIC AMINES
While the chromatographic separation on Amberlite IRP-64 is generally applicable, the separation of polyamines on Dowex 50-X8 is limited and is recommended preferably for the separation of simple mixtures such as 1,3-diaminopropane, putrescine and cadaverine mixtures. Aliphatic diamines and some other amino compounds with free amino groups, including histamine, are easy t o separate on Zeo-Karb 226 (Blau). Elution volumes found with this sorbent on a 0.8 X 112.5 cm column are given in Table 30.4. As with chromatography on Dowex 1-X2 (Holder and Bremer), Zeo-Karb 226 is also applicable mainly for concentrating dilute samples of mono- and polyamines. Partition chromatography on Celite 545 (Clayton and Strong) is one of the rare procedures used in the separation of amines in which the principle of ion exchange is not applied. The result of such a separation is shown in Fig. 30.4.
AROMATIC AMINES The chromatographic separation of primary aromatic amines has been studied using a wide variety of sorbents such as Celite, silica gel and Teflon 6. The ion-exchange chromatography of this type of compound is related t o both purifications and automated analytical separations, as described later (Tompsett). Recently, Lepri et al. described a highly efficient method involving the use of alginic acid and CM-cellulose. Separations on alginic acid were carried out in columns filled with alginic acid of 50- 150 mesh. Both columns had a cross-section of 0.94 cm2 and were filled with 4 g of ion exchanger. The columns were eluted with 0.1 M acetic acid or with 0.1 M hydrochloric acid (flow-rate 2 ml/min). Typical runs are shown in Figs. 30.5 and 30.6. With acetic acid as eluent, the expected differences were observed when comparing column chromatographic separations with thin-layer chromatography. The resolving power of the column separation is less than that of TLC for compounds with high RF values (above 0.5), while the column is more 2 10.
0
G t t
a
5’
8
0 0 VOLUME,
mi
Fig. 30.5. Elution curves for aromatic amines on an alginic acid column with 1 M acetic acid as eluent (Lepri er al. 1. The aromatic amines in the effluent were detected with p-dirnethylaminobenzaldehyde. The total quantity of amine eluted was dctermined spectrophotometrically after diazotization with nitrous acid followed by coupling with N-(1-naphthy1)ethylenediamine.The concentration is given in arbitrary units. Peaks: 1, sulphanilic, methanilic and orthanilic acids; 2, o-arsanilic acid; 3 , o-nitroaniline, 4, p-nitroaniline; 5, p-arsanilic acid; 6 , 4-aminosalicylic acid; 7 , o-aminobenzoic acid; 8, sulphanilamide; 9, p-aminoacetophenone; 10, 5-aminosalicylic acid; 1 1, p-aminobenzoic acid; 12, a-chloroaniline; 13, p-aminohippuric acid; 14, m-nitroaniline; 15, aniline; 16,o- and p-toluidine; 17, m-aminobenzoic acid; 18, o-anisidine and o-aniinophenol; 19, m- and p-aminophenol; 20, a-and p-naphthylamine.
References p . 655
644
AMINES 1
'O1
I
10
70
30 VOLUME. ml
Fig. 30.6. Elution curves for aromatic amines o n an alginic acid column with 0.1 M hydrochloric acid as eluent (Lepri et al.). The aromatic amines in the effluent were detected with p-dimethylaminobenzaldehyde. The total quantity of amine eluted was determined spectrophotometrically after diazotization with nitrous acid followed by coupling with N-(1-naphthy1)ethylenediamine.The concentration is given in arbitrary units. Peaks: 1, aniline; 2, p-aminodimethylaniline; 3,c-x- and pnapthylamide, o-phenylenediarnine and N-phenyl-p-phenylenedianiine;4, m- and p-phenylenediamine; 5, benzidine.
effective than the flat-bed arrangement for compounds with low R, values. In this case, separations not forseeable from the R, values are sometimes possible, such as the separation of o-chloroaniline and p-aminobenzoic acid (corresponding R , values 0.13 and 0.12) from isomeric toluidines, aminophenols, o-anisidine and p-aminobenzoic acid, which move with RF 0.10. On the other hand, the separation of amines that contain a sulphonic group from o-arsanilic acid and from o-nitroaniline is not possible. The possibilities of separating isomeric aromatic amines by these techniques were surveyed by Lepri et al. The great affinity of diamines can be reduced by using hydrochloric acid as the mobile phase. In this system, p-aminodimethylaniline is eluted together with o-phenylenediamine. The latter is also incompletely separated from its m -and p-isomers. The separation with hydrochloric acid as the mobile phase is particularly
VOLUME. ml
Fig. 30.7. Elution curvcs for aromatic amines on a carboxylmethylcellulose column with water as eluent (Lepri et al.). The aromatic arnines in the effluent were detected with p-dimethylaminobenzaldehyde. The total quantity of amine eluted was determined spectrophotometrically after diazotization with nitrous acid followed by coupling with N-(l-napthyl)ethylenediamine. The concentration is given in arbitrary units. Peaks: 1, sulphanilic, methanilic and orthanilic acids; 2, o- and p-arsanilic acids; 3, o-nitroaniline and o-aminobenzoic acid; 4,4-aminosalicylic acid and p-nitroaniline; 5 , sulphonilamide and p-aminohippuric acid; 6 , p-aminoacetophenone; 7 , p-aminobenzoic acid; 8, rn-nitroaniline; 9, m-aminobenzoic acid.
AROMATIC AMINES AND ALIPHATIC POLYAMINES IN MIXTURES
64 5
suitable for the separation of naphthylamines. Instead of the above solvents, 1 M monochloroacetic acid in water and in 50% isopropanol were used, but the separating power of these mobile phases is very much lower. Results achieved in the separation of aromatic amines on a CM-cellulose column are presented in Fig. 30.7 (identical column size as with alginic acid; CM-cellulose in the acid form obtained by treatment of the sodium salt of the ion exchanger with 1 M hydrochloric acid and subsequent washing with distilled water until the chloride ions disappeared; flow-rate 1 ml/min). As the affinity of CM-cellulose for aromatic amines is lower with respect to alginic acid, water was used as the mobile phase. This system is capable of separating n.1-aminobenzoic acid from other amines and in particular from p-aminobenzoic acid and rn-nitroaniline. In a similar manner t o alginic acid, it is also possible to separate all isomers of aminobenzoic acid. Amines that are strongly bound to CM-cellulose can be eluted with 1 M acetic acid. In this solvent, benzidine is separated from m- and p-phenylenediamine. Reversed-phase chromatography of aromatic amines can be carried out on cyclohexane-loaded Teflon 6 (Hedrick).
AROMATIC AMINES AND ALIPHATIC POLYAMINES IN MIXTURES Amberlite CG-50, Type 2 , can be recommended for the separation of complex mixtures of aliphatic polyamines and aromatic amines, since both of these types of compounds are eluted from this ion-exchange resin in the same pH range. The recommended procedure was described by Perry and Schroeder. Hirs' purification procedure has been recommended for the resin used. Before packing the column, the resin bed is suspended in a pyridine-acetate buffer of pH 4.32 (0.1 N) and rinsed several times before use in order to remove fines and to condition the resin. Most of the amines tested were detected by the conventional ninhydrin procedure. Some of the aniines, however, d o not give a positive reaction with ninhydrin and must be assayed by another suitable procedure after the effluent from the column has been split into two separate streams, one feeding the ninhydrin line and the other supplying a separate'fraction collector (Table 30.5).
TRYPTOPHAN METABOLITES Historically, the first liquid column separation of compounds related to tryptophan metabolism was carried out on a silica gel column eluted with a series of mobile phases based on n-heptane (Powell). Also, the separation of these compounds on a molecular sieve such as Sephadex G-25 or G-10 gives only a partial resolution, as reported by Iskric and Keglevic and Schlossberger er at. Nowadays, ion-exchange separations clearly predominate. Presumably the use of ionexchange celluloses in this field is now the fashion, but equally good results are obtained with QAE-Sephadex A-25 and Amberlite IR-120. Dowex chromatography is currently less developed. The method in which QAE-Sephadex A-25 is used has been developed by Bakri and Carlson; the sodium chloride concentrations at the sudden buffer changes are indicated in References p . 655
TABLE 30.5 ELUTION VOLUMES OF AMINES CHROMATOGRAPHED ON AMBERLITE CG-50 (PERRY AND SCHROEDER) Authentic compounds were chromatographed in mixtures on Amberlite CG-50 columns 45 crn in length and 0.9-1.0 crn in diameter at a flow-rate of 10 ml/h and a temperature of 40°C. Chromatograms were developed with 0.1 N pyridine acetate buffer (pH 6.32) for the first 250 rnl, and thereafter with 0.2 N pyridine acetate buffer (pH 6.12). ~~
Compound
Range of elution (ml)
Histidine 1-Methylhistidine Arginine Ethanolamine Ammonia Ethylamine F‘yrrolidhe N-Acetylhistamine Pyridoxamine N-Methylmetanephrine Metanephrine Epinephrine Norme tanephrine 1-Methylhistamine Norepinephrine Synephrine Isoam ylarnine Mescaline
25-3 1 25-31 31-42 32-40 33-42 3 8-46 49-58 49-65 68-79 78-89 102-115 101-124 112-130 112-130 116-132 118-135 119-136 124-142 127-142 132-147 134-150 135- 151 140-154 141-158
3,4-Dimethoxybenzylamine Octopamine 3-Me thoxy-4-hydroxybenzylamine Epinine 3,4-Dimethoxyphenylethylamine 3-Methoxytyramine
Elution peak* (mU 28 28 35 36 38 42 54 74 109 121 121
128
Compound
Range of elution (rnl)
3-Hydroxy4-rnethox y phenylethylamine Putrescine p-Hydrox ybenzylamine Cadaverine Dopamine Benzylamine p-Tyramine 3-Ethoxy-4-hydroxybenzylamine rn-Tyramine p-Methoxybenzylamine Histamine Bufotenin Phenylethylamine o-Tyramine p-Me thoxyphenylethylamine Kynuramine
145-163 140-171 151-171 163-1 83 163-190 169-189 170-196 177-193 183-202 192-215 202-225 205-235 228-254 218-262 228-261 229-261 277-297 282-303 365-321 310-335 328-352 380-4 15 408-466 465-505
2,2’-Dithiobis(ethylamine) Serotonin Agmatine N,N-Dimethyltryptamint: 5-Methoxytryptamine Tryptamine Spermidine 5-Methyltryptamine
*Elution peaks were not obtained for a number of m i n e s giving no colour or weak colours with ninhydrin.
Elution peak* (ml)
159 162
178 186
203 214 240 24 1 244 244 289 312 339 397 43 8 483
647
TRYPTOPHAN METABOLITES
1600
1800
2000
2200
2400
VOLUME. ml
Fig. 30.8. Elution pattern obtained for tryptophan metabolites from a 95 X 0.94 cm QAE-Sephadex
A-25 (CI-) column (solid line) and a 45 x 0.94 cm CM-cellulose (Na') column (broken line) (Bakri and Carlson). Buffer: 0.05 M Tris-hydrochloric acid, p H 7.9. Complex gradient for the QAE-Sephadex A-25 colulnn: 300 rnl each of 0,0.01,0, 0.076.0, 0.36, 0 and 0.60 M sodium chloride. Temperature: 4°C. The absorbance at 280 nm is plotted against elution volume.
Fig. 30.8, in which the overall pattern of separation can also be seen. The use of an additional CM-cellulose column allows the identification of tryptamine. According t o Arend et al., a mixture of tryptophan metabolites, including anthranilic acid glucuronide, o-aminohippuric acid, acetylkynurenine, kynurenine and indoxyl sulphate, can be analyzed in the following way. The metabolites are eluted from a Dowex 50 ion-exchange column with successive washes with hydrochloric acid of increasing concentration. Five acidic fractions are obtained as follows: 0.1 N , indoxyl sulphuric acid; 0.5 N , anthranilic acid glucuronide; 1.ON, o-aminohippuric acid; 2.4 N , acetylkynurenine; and 5 N , kynurenine. A procedure for separating this type of compounds was described recently by Chen and Gholson, in which DEAE-cellulose was used as an ion exchanger in either the amine or formate form. A mixture of tryptophan metabolites (300 pg each) was loaded on to the column (HCOO-; 1.2 X 30 cm) and the column was then eluted with 80 ml of 0.001 M triethylamine-formate (TEA-F) buffer of pH 4.0. Then elution was continued with a gradient formed by placing 250 ml of 0.001 M TEA-F o f pH 4.0 into a vessel into which 0.1 M TEA-F buffer of pH 4.0 was directed from an equal-sized (250 ml) beaker. The first two peaks escaping from the column (Fig. 30.9) were lyophilized and subjected t o the second separation step. The second column was packed with DEAEcellulose (amine form) and eluted with 140 ml of 0.001 M T E A - F buffer of pH 8; then elution was continued with a gradient obtained from equal volumes (200 ml) of 0.001 M TEA-F buffer of pH 8.0 and 0.05 M TEA-F buffer of pH 8.0. During the non-gradient elution 2-ml fractions were collected, while after the gradient has been introduced 4-ml fractions were taken. A typical run on the alkaline column (amine form) is presented in Fig. 30.10. References p . 655
648
AMINES
.
3 1001
45
80-
5F
0
60-
a
U L 1
0
=.
4020-
0
20
40
60
80
100
120
140
160
FRACTION NUMBER
Fig. 30.9. Separation of tryptophan metabolites on the first DEAE-cellulose column (HCOO- form) (Chen and Gholson). Peaks: 1, tryplophan, tryptamine, 5-hydroxytryptamine, 5-hydroxytryptophan, kynurenine, indole-3-acetonitrile and urea; 2, indole-3-carboxaldehyde; 3, 3-hydroxykynurenine; 4, indole-3-acetic acid; 5 , indole-3-accturic acid; 6 , 5-hydroxyindoleacetic acid; 7, kynurenic acid; 8, xanthurenic acid. Sonic peaks overlap slightly, as shown, beacuse different reagents were used to determine the various compounds.
'0°1
12 3
80 6
z 60
7
0
2 e
. 9
40 20
0
20
40
60
80 1 0 0 120 140
F R A C T I O N NUMBER
I5g. 30.10. Separation of tryptophan metabolites on the second DEAE-cellulose column (amine form) (Chen and Gholson). Peaks 1 and 2 from the first column were combined and applied to the second column. Peaks: 1, tryptamine and urea; 2, indole-3-acetonitrile; 3, indole-3-carboxaldehyde;4, kynurenine; 5,5-hydroxytryptamine; 6 , tryptophan; 7,s-hydroxytryptophan. Some peaks overlap slightly, as shown, because different reagents were used to determine the various compounds.
5-Hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid were separated on Dowex 1 (CH3COO-) by Nishino ef al. The column used was 7 X 1 cm in size; elution was carried out stepwise with 100 ml of water, 100 ml of 0.01 N acetic acid and 100 ml of 6 N acetic acid. Chromatography on Amberlite IR-120 is the oldest of the various ion-exchange techniques, and it still offers very good separations (Benassi e r a / . )(Fig. 30.1 1).
649
QUATERNARY AMMONIUM COMPOUNDS AND AMINO ALCOHOLS 1
+ 2 +-
3
+ 4 +5
+6
FRACTION
4
32-
. TIE
I
I
,,H 1 8 O I M PVRlDlNE
&
4-
EFFLUENT ml
p H 5 G 01M PVRlDlNE
I'ig. 30.1 1. Chromatographic lractionation pattern of tryptophan metabolites on a column of Ambcrlite The temperature was maintained at 3 3 i 0 . 1°C and the IK-I20 (pyridine), 28 X 0.9 cm (Benassi et d). flow-rate was adjusted to 1 2 nd/h with volatile formic acid-pyridine buffer. The concentration valucs, (pg/ml) were calculated from fluorimetric or colorimetric readings on the paper chromatographic spots eluted after column fractionation. The broken lines show the elution peaks of metabolites usually absent in the urine of normal subjects. Peaks: I , xanthurenic acid 8-methyl ether: 2, kynurenic acid; 3, xanthurenic acid; 4, N-acetylkynurenine; 5, N-acetyl-3-hydroxykynurenine; 6 , o-aminohippuric acid; 7, anthranilic acid; 8, kynurenine; 9, 3-hydroxyanthranilic acid; 10, 3-methoxy anthranilic acid; 1 1, 3-hydroxy kynurenine.
QUATERNARY AMMONIUM COMPOUNDS AND AMINO ALCOHOLS Since ion-exchange chromatography clearly predominates in the separation of different types of compounds that contain an amino group, it is not surprising that also betaine and quaternary ammonium compounds, which occur in the natural material associated with the oxidation of choline, have been subjected to separations of this type. Important contributions were made by Christianson et al., Courley et al. and Niemann. In the procedure reported by Speed and Richardson, Amberlite CC-50 is used. The flow-rate used ranged from 60 t o 80 ml/h and the column was operated at room temperature. A typical run is shown in Fig. 30.12. The pH 5 . 3 phosphate-citrate buffer can be also used for separation on Zeo-Karb 226 as published by Cromwell and Richardson.
CHoL"E
I00
BETAINE ALDEHYDE
300 Effluent buffer, rnl
Fig. 30.12. Separation of'bctaine, dimethylglycine, choline and betaincaldehyde on a 125 X 1.2 cm column of Amberlite CG-50 buffered at pH 7.3 and eluted with phosphate-citrate buffer of pH 5.3 (Speed and Richardson).
References p . 655
650
AMINES
For the separation of fatty alcoholamides, elution of silica gel with a stepwise gradient of ethanol-chloroform and methanol-chloroform (with an increasing concentration of the alcoholic component) was used by Hejna and Daly. In this manner, monoethanolamine and diethanolamine were separated. The separation of sphingosine, dhydrosphingosine and phytosphingosine was achieved on 30 X 2.2 cm column containing 50 g of silica gel (0.05-0.2 mm) which had been suspended in chloroform-methanol (1 : 1) packed by gravity and then washed with chloroform until transparent (Barenholz and Gatt). A gradient of increasing ammoniacal methanol in chloroform was used as the mobile phase. The lower mixing chamber of the gradient system contained 300 ml of chloroformmethanol-2 M ammonia solution (90: 10: I ) and the upper reservoir contained 600 ml of chloroform-methanol-2 M ammonia solution (50:50:5). When all of the 600 ml had been delivered from the upper reservoir, the separation was continued with 500 ml of chloroform-methanol-2 M ammonia solution (30:70:7). If fractions with a volume of 10 nil were collected, sphingosine appeared in fractions 43-47, fractions 48-58 contained dihydrosphingosine and fractions 63-67 were pure phytosphingosine. Lipids related to sphingosine (sphingosine esters) are dealt with in the chapter on lipids.
BIOCENIC AMINES In principle, the isolation of catecholamines from biological material is usually carried out by ion-exchange chromatography in a large series of columns suitable for many parallel samples. Quantitation after chromatography is a very complex operation which has been automated in two different ways. Both methods of evaluation (the ethylenediamine dihydrochloride method and trihydroxyindole method) have been subjected to a number of re-investigations and criticisms, the description of which is beyond the scope of this book. In this section we have limited ourselves to those methods in which individual catecholamines are chromatographically separated. Methods in which liquid column separations were used only for purification purposes have been omitted. There are not many methods that are suitable for the quantitative separation of the biogenic amines, noradrenaline, adrenaline, dopamine, 5-hydroxytryptamine, histamine and their metabolites, in a single small sample of tissue after a single extraction and purification procedure. Recently, a procedure was reported in which n-butanol is used in the organic extraction of catecholamines, 5-hydroxytryptamine and histamine from a small sample of tissue (Sadavongvidad). Atack and Magnusson developed a column chromatographic procedure which permits the total amount of each amine to be concentrated into small fractions. Noradrenaline and adrenaline have been separated from doparnine on a strong cation-exchange column 50 X 4.2 mm in size. The resin used was Dowex 50W-X4 (Na'), 200-400 mesh (Bertler ef al., Carlsson and Lindqvist). Noradrenaline, together with adrenaline, is eluted in the first 8 ml and dopamine in the following 12 ml of 1 N hydrochloric acid. Adopting the procedure of Green and Erickson and Kahlson et al., Atack and Magnusson were able to elute histamine with 5 ml of dilute hydrochloric acid after eluting both catecholamines. By using a large volume of the mobile phase (up to 20 ml), 5-hydroxytryptamine could be eluted with additional portions of 4-6 N aqueous hydrochloric acid or 0.01 N sodium hydroxide. The application of an alkaline
651
BIOCENIC AMINES
mobile phase was introduced by Wiegand and Scherfling. The volume of all the fractions could be minimized by using organic solvents; thus elution with 3 N ethanolic hydrochloric acid decreases the volumes of the fractions t o 4 ml (Schildkraut et al.). Except adrenaline and noradrenaline, all of the other amines are separated by the above methods. According t o Atack and Magnusson, the elution of noradrenaline, adrenaline and dopamine is also greatly facilitated by the use of other organic solvents, while the elution of 5-hydroxytryptamine is virtually unaffected. The procedure described by Atack and Magnusson can be briefly summarized as follows. The sample is loaded into the column at pH 2.5 and the corresponding amino acids are adsorbed together with catecholamines (Bertler et al., Kahlson et al., Wiegand and Scherfling). Elution is carried out with an organic mobile phase as indicated in Fig. 30.13. 5-Hydroxyindoleacetic acid may interfere in the assay of 5-hydroxytryptophan. Aqueous methanol (60%) elutes 5-hydroxyindoleacetic acid and 0.1 M phosphate buffer of pH 6.5 can be used for the elution of a mixed band of 5-hydroxytryptamine and dihydroxyphenylalanine. Histidine is usually eluted together with the other amino acids and the subsequent elution of amines is not affected b y the use of organic solvents and buffer mobile phases (Atack and Magnusson). The volume of the 5-hydroxytryptamine fraction can be reduced t o 3.5 ml by eluting with 1.8 N hydrochloric acid-ethylene glycol monoethyl ether (ethyl Cellosolve) (SO%), while still permitting the subsequent separation of histamine. Amberlite 1RC-50 gives a good separation of adrenaline, noradrenaline, hydroxytryptamine and dopa (Fig. 30.14, Kirshner and Goodall).
5- HI AA
SOLVENT
aqueous
HTP Hd
0.lM
NA A
1NHCI
DA
5-HT
1NHCI-
methanol
phosphate
ethanol
( 60 % 1
buffer PH6.5
(50%)
Hm
2.5 NHCl
Fig. 30.1 3. Order of elution of noradrenaline (NA) (together with adrenaline, A), dopamine (DA), 5-hydroxytryptamine (5-HT) and histamine (Hm) and their respective precursors (dihydroxyphenylalanine, DOPA; 5-hydroxytryptophan, HTP; and histidine, Hd) and 5-hydroxyindoleacetic acid (5-HIAA), from a strong cationexchange resin column (dimensions 7 2 mm in buffer by 4.0 mm I.D. Dowex 50W-X4 (Na'), 2 0 0 4 0 0 mesh) (Atack and Magnusson). The volume of 5-HT eluate can be reduced to 3.5 ml by eluting with 1.8 N hydrochloric acid-ethylene glycol monoethyl ether (ethyl Cellosolve) (SO%!), while still permitting the subsequent, separate clution of Hm.
References p . 655
652
AMINES
FRACTION NUMBER
Fig. 30.14.Separation of adrenaline, noradrenaline, hydroxytyramine and dopa on Amberlite IRC-SO (Krishner and Goodall). A 1-ml volume of a solution containing 400 pg of each of the compounds was chromatographed. Column 30 X 0.9 cm, equilibrated with 0.2 M ammonium acetate buffer at pH 6.1. Elution with 0.4 M ammonium acetate buffer at pH 5.0, starting at fraction 1. Fraction size 1.5 ml. The compounds in order t o emergence are dopa, adrenaline, noradrenaline and hydroxytyramine.
Gradient elution chromatography on ion-exchange cellulose (Whatman P-1 1 , cellulose phosphate) also proved to be a very satisfactory method for the separation of sympatomimetic amines. According to the procedure described by Merrills and Farrier, columns 30 X 0.5 cm in size are used. The column is equilibrated with 0.05 M ammonium acetate
A
t
START
Fig. 30.15. Recorder tracings obtained during chromatographic separation of various amounts of dopac (3,4-dihydroxyphenylaceticacid), L-tyrosine, dopa (3,4-dihydroxyphenylalanine),tyramine, adrenaline, dopamine and noradrenaline (1,2.5 or 5 p g of each) (Merrills and Farrier).
BIOGENIC AMINES
653
of pH 6.0 and an exponential salt gradient, formed by pumping a solution of 0.5 M ammonium acetate in 20% propanol adjusted to pH 6.0 into a mixing vessel containing 25 ml of 0.05 M ammonium acetate at the rate of 0.23 ml/min, is introduced. The mixture is pumped into the column at the same rate. The main advantage of this type of chromatography is that the effluent is suitable for direct scintillation counting, a procedure which, with low concentrations of catecholamines originating in tissues from labelled precursors, can hardly be avoided. In order t o identify the substances eluted, it is always necessary to add appropriate carriers, which are detected either by their optical density at 280 nm or by a suitable colorimetric reaction (Merrills). In a later paper Merrills and Offerman reported another detection procedure based on the possibility of recording anodic decomposition potentials of catechol or phenolic substances. Fig. 30.1 5 gives an example of a separation recorded by this procedure. In a later modification of this procedure, Deyl et af. succeeded in separating adrenaline, noradrenaline, isopropylnoradrenaline and its 0-methyl derivative. For the separation the synthetic catecholamine and its 0-methyl derivative from epinephrine and norepinephrine, two columns (40 X 0.6 cm) combined in series were used. These columns were filled up to 35 cm with Whatman CP-11 cellulose phosphate (column No. I) and Whatman CM-cellulose CM-32 (column No. 11). The columns were eluted with a linear gradient of ammonium acetate buffer with a concentration change from 0.05 to 0.25 M.The pH of the buffer was adjusted previously to 6.1; the flow-rate in both columns was less than 0.8 ml/min. Fractions of 2 ml were collected. The columns were loaded with 0.5 ml of a sample obtained after either purification procedure. The elution of the column was stopped after 100 ml of the eluent had passed through the system. Under these conditions, 0-methylisopropylnoradrenaline is eluted in fractions 3-7 and isopropylnoradrenaline in fractions 10- 15 (see Fig. 30.16). Some catechol derivatives can be effectively separated by adsorption on alumina (Drell). Recently, Routh ef ~ l introduced . a complex separation technique which combines alumina and two ion exchangers and allows the separation of not only epinephrine and norepinephrine, but also a number of compounds related to catecholamine metabolism such as dopamine, dihydroxyphenylalanine, 4-hydroxy-3methoxyphenylacetic acid (honiovanillic acid), 3,4-dihydroxyphenylaceticacid, 4-hydroxy-3-methoxymandelic acid (vanilmandelic acid) and 3,4-dihydroxymandelic acid. The separation is carried out on a series of three columns, of which the first is packed with Bio-Rad test column packing (catecholamine test kit), the second with Amberlite CG-400 (Cl-), 100-200 mesh, and the third with neutral alumina, Brockman activity I, 80-200 mesh. The dimensions of each column is 5.0 X 0.7 cm. Flow-rates in this separation are not important and flow is effected by gravity. The procedure is as follows. A 5-ml volume of urine is mixed with 14 ml of Na2 EDTA of concentration 1 g/100 ml, adjusted to pH 6.5 with 1 M sodium hydroxide solution, and chromatographed on the first column. The column is eluted with distilled water (5 ml) and the combined effluents are used for the following separation step. The first column is further eluted with 10 ml of boric acid of concentration 4 g/100 ml, which brings metanephrine, epinephrine and norepinephrine into solution. The combined effluents from the first column before the elution with boric acid are passedthrough the second column of References p . 655
AMINES
654
,
I
1.75
--E N
'
MIP
1.50 1.25
u
9a!
1.00
c \
0
3
0.75
I 0 rl
3
0.50 0.25
1
1.75
II!
IP
A
NA
(a)
I
MIP
A
NA
1.25
4.
1.00
g
0.75
I
1
0.25
20
Fig. 30.16. Chromatographc profile of the mixture of 0-rnethylisopropylnoradrenaline(MIP), isopropylnoradrenaline (IP), adrenaline (A) and n oradrenaline (NA) on cellulose phosphate (a) and on the confined system of cellulose phosphate and CM-cellulose (b) (Deyl et ul.).
Amberlite CG-400, which, after draining, is eluted with 5 ml of distilled water. All of the effluent is combined and used for dihydroxyphenylalanine quantitation. Then the second column is eluted with 25 ml of 1 Msodium chloride solution, the first 5 ml of the eluate being discarded and the remainder saved for further fractionation. A 5-ml aliquot of the eluate from the second column is mixed with 0.5 ml of 1 M sodium acetate of pH 6.5 and 1 rnl2.0 M sodium acetate and applied to the third column. This column is eluted with 15 ml of distilled water and the effluent used for homovanillic acid quantitation.
RE1:ERENCES
655
REFERENCES Arend, R. A., Leklem, J . E. and Brown, R. R., Advan. Autom. Anal., Technicon Int. Congr., 1969,2 (1970) 195;C.A.,73 (1970) 32189s. Alack, C. V. and Magnusson, T., J. Pharm. Pharmacol., 22 (1970) 625. Bakri, M. and Carlson, J . R., Anal. Biochem., 34 (1970) 46. Barenholz, Y . and Gatt, S., Biochim. Biophys. Acta, 152 (1968) 790. Benassi, C. A,, Veronese, F. M. and De Antoni, A., Clin. Chim. Acta, 17 (1967) 383. Bertler, A., Carlsson, A. and Rosengren, E., Clin. Chim. Acta,44 (1958) 273. Blau, K., Biochem. J . , 80 (1961) 193. Carlsson, A. and Lindqvist, M., Biochem. J., 54 (1962) 87. Chen, N. C. and Gholson, R. K.,Anal. Biochem., 47 (1972) 139. Christianson, D. D., Wall, J. S., Cavins, J. F. and Dimler, R . J., J. Chromafogr., 10 (1963) 43. Clayton, R. A. and Strong, F. F., Anal. Chem., 26 (1954) 579. Cromwell, B. T. and Richardson, M., Phyrochemistry, 5 (1966) 735. Deyl, Z., Pilny, J. and Rosmus, J . , J. Chrornatogr., 53 (1970) 575. Drell, W., Anal. Biochem., 34 (1970) 142. Dubin, D. T. and Rosenthal, S . M., J. Biol. Chem., 235 (1960) 776. Gourley, W. K., Haas, C . D. and Bakennan, A., Anal. Biochem., 19 (1967) 197. Green, H. and Erickson, R. W., Int. J. Neuropharmacol., 3 (1964) 315. Hatano, H., Sumizu, K., Rokushika, S. and Murakami, F., Anal. Biochem., 35 (1970) 377. Hedrick, C. E., Anal. Chem., 37 (1965) 1044. Hejna, J . J . and Daly, D., J. SOC.Cosmet. Chem., 21 (1970) 107. Holder, S. and Bremer, H. J . , J. Chromatogr., 25 (1966) 48. Iskric, S. and Keglevic, D., Anal. Biochem., 7 (1964) 297. Kahlson, C., Rosengren, E. and Thunberg, R., J. Physiol. (LondonJ,169 (1963) 467. Kirshner, N. and Goodall, M e . , J. Biol. Chem., 226 (1957) 207. Lepri, L., Desideri, P. G., Coas, V. and Cozzi, D., J. Chromatogr., 49 (1970) 239. Merrills, R. J., Aurom. Anal. Chem., Technicon Symp., 1965, Technicon Instruments, Chertsey, Great Britain, 1965. Merrills, R. J . and Farrier, J . P., Anal. Biochem., 21 (1967) 475. Merrills, R. J. and Offerman, J . , Biochem. J., 99 (1966) 538. Miyagi, T . and Ando, S., Annu. Rep. Inst. Food Microbiol., Chiba Univ., 6 (1953) 93. Morris, D. R.,MethodsEnzymol., 17 (1971) 850. Morris, D. R., Koffron, K. A. and Okstein, Ch., Anal. Biochem., 30 (1969) 449. Niemann, A., J. Chrornatogr., 9 (1962) 117. Nishino, M., Moguchi, T. and Kido, R.,Anal. Biochem., 45 (1972) 314. Perry, T. L. and Schroeder, W. A., J. Chromatogr., 12 (1963) 358. Powell, L. E., Nature (LondonJ,200 (1963) 79. Rosenthal, S. M. and Tabor, C. W., J. Pharmacol. Exp. Ther., 116 (1956) 131. Routh, J. I., Bannov, R. E., Fincham, R. W. and Stoll, J. L., Clin. Chem., 17 (1971) 867. Sadavongvidad, C., Brit. J. Pharmacol., 38 (1970) 353. Schildkraut, J . , Schanberg, S. M., Breese, G. R. and Kopin, I. J., Biochem. Pharmucol., 18 (1969) 1971. Schlossberger, H. G., Kuch, H. and Buhrow, I., Hoppe-SeylerSZ. Physiol. Chem., 333 (1963) 152. Speed, D. and Richardson, M., J. Chrornatogr., 35 (1968) 497. Tabor, H., Rosenthal, S. M. and Tabor, C. W., J. Biol. Chem., 233 (1958) 907. Tompsett, S. L., Anal. Chim. Acta, 2 1 (1959) 535. Wiegand, R. G. and Scherfling, E., J. Neurochem., 9 (1962) 113. Yoshioka, M., Ohara, A , , Kondo, H. and Kanazawa, H., Chem. Pharm. Bull., 17 (1969) 1276.
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Chapter 31
Other non- heterocyclic nitrogen compounds J . CHURACVEK
CONTENTS
...................................................................657 ............................................................... 657 ....................................................................... 659 ..................................................... 661 .................................................................... 664
Introduction Nitrocompounds Amides Cuanidineandureaderivatives References
INTRODUCTION In t h s chapter, the liquid chromatography of nitro compounds, urea and its derivatives, guanidines, nitrosamines, amides, azo compounds and aromatic hydrazo compounds is described. The chromatography of amides is mentioned only briefly because from the practical point of view the chromatographic separation of peptides and their mixtures is most important and therefore a special chapter is devoted to this aspect (Chapter 34). For the other compounds mentioned, the main aim of liquid column chromatography is their separation from other types of compounds. All modern chromatographic techniques have good prospects of being applied successfully in this field, but little attention has been devoted to them so far. An interesting technique was used for nitro compounds; some of them can be synthesized directly on the column on which they are then separated chromatographically from other components of the reaction mixture.
NITRO COMPOUNDS A typical example of the isolation of nitro compounds from industrial mixtures was described by Landram ef al. Chromatographic separation was used instead of the usual extraction procedures, which represents an important advance in work with explosives. This separation procedure was used satisfactorily for a number of double base propellants containing nitroglycerine, triacetin, 2-nitrodiphenylamine, resorcinol, ammonium perchlorate, aluminium, 2.4,6,8-cyclotetramethylenetriamine and nitrocellulose. The procedure is generally applicable to different types of propellants and it is not necessarily restricted to those which bear a large number of nitro groups. Both Chromosorb T (PTFE) and silica gel can be used for column packing in these instances. The principle of dry column chromatographic extraction should prove useful also with other non-propellant polymers. The main advantage of this procedure is the decrease in the operating time: in the example described above, the Soxhlet extraction took 1-4 days References p.664
657
658
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
while the chromatographic procedure took 1 h. In large-scale operations, the reduced consumption of solvents in chromatographic procedures is also important. I n the analytical field, ion-exchange chromatography is the prevailing method for the separation of nitro compounds. This method permits the separation of a series of nitroalkanes and nitro-aromatic compounds. Polarography is mainly used for detection (Kemula); a special chromatopolarographic apparatus was developed by Kcmula and Brzozowski for this purpose. The limiting diffusion current produced by organic substances is measured on a mercury drop electrode at a constant input potential (-1 V). Kemula and Brzozowski also described the separation of some nitroalcohols and nitrobenzoic acids by salting-out chromatography on a thermostatted cation-exchange column at 56,82,25 and 71°C. On Wofatit KPS-200, a mixture of four nitroalcohols was separated by elution with 1 M ammonium sulphate solution on a 37 X 0.7 cm column. The flow-rate was not an important factor in the separation. The sequence of the eluted compounds was 2-methyl-2-hydroxy-1,3-propanediol, 2-methyl-2-nitropropano1, 2-nitrobutanol and 2-hydroxymethyl-2-nitropentanol. Kemula and Brzozowski also separated a three-component mixture of nitroalkanes on a Dowex 50 column using ammonium sulphate solution as the mobile phase. The elution sequence observed was nitromethane, 2-nitropropane and 1-nitrobutane. The column dimensions were 1 1.5 X 0.7 cm and the flow-rate of the mobile phase did not exceed 9 ml/h. On the same ion exchanger, separations at various temperatures can also be effected: at 25°C with 1 Mammonium sulphate solution as eluent, nitromethane, 2-nitropropane and 1,3-dinitropropane are eluted first. After increasing the temperature in the column jacket to 71°C and using 0.5 M ammonium sulphate solution for elution, 1-nitrobutane and 1-nitropropane appear in the effluent. Under the same conditions, all three isomeric nitrobenzoic acids can also be separated, using 0.1 M ammonium sulphate solution in 0.019 M hydrochloric acid as the mobile phase. o-Nitrobenzoic acid is eluted at room temperature with a minimum retention time, p-nitrobenzoic acid is eluted at 56°C and m-nitrobenzoic acid at 82°C. The time required for the separation is about 5 h. During the analysis of the radiolytic products in the aqueous nitrate-ethylene system, ion-exchange chromatography on Dowex 50W-X8 was also employed. Ammonium sulphate solution of various concentrations served as the mobile phase. Nitromethane, hydrogen peroxide and sodium nitrite, present in the mixture, were detected polarographically. The curves were recorded a t a constant potential corresponding to the complete reduction of the dissolved oxygen and lower than the reduction potential of nitrate (Broszkiewicz and Przybylowicz). Kemula and Sybilska described a method for the separation and determination of 0.05-0.3 ing of a mixture of 0-,in- and p-nitroethylbenzene on a column packed with the clathrate nickel y-picoline thiocyanate. A solution of ammonium thiocyanate and y-picoline in acetone were used for elution. This separation, based on the formation of clathrates, may be considered to be a special case of ligand exchange. The specificity of the chromatography of nitro compounds consists in the possibility of preparing alkyl nitrates from the corresponding alkyl bromides on a silica gel column impregnated with silver nitrate, while the chromatographic separation of the mixture takes place simultaneously (Kuemmel). The reaction is unusual in that the breaking and forma-
659
AMIDES
tion of the covalent bonds occur on the adsorbent surface on reaction with the anion from the adsorbent, becoming covalently bound to the product. In addition, olefins are also produced from secondary bromides. The nitrates are readily separated from the olefins, which are strongly adsorbed on the column owing to the formation of silver complexes. A high ratio of adsorbent to sample is required so as to ensure the complete reaction of all the alkyl bromides.
AMIDES The possibility of applying liquid chromatography to the separation of coloured homologous N,N-dimethyl-p-aminobenzeneazobenzamides was demonstrated by ChuraEek and Jandera using an apparatus consisting of a pulse-free plunger feeding pump, a narrow bore column, a spectrophotometer with a flow-through measuring cell of their own design and a recorder. A strongly sulphonated styrene-divinylbenzene cation-exchange resin, Dowex 50W-X2 (H'), was used for the separation of the coloured compounds. Fig. 3 1.1 illustrates the chromatographic separation of some lower secondary amides. Their protonated forms are distributed between the external solution (mobile phase) and the solution in the resin particles in accordance with the basicities of the non-ionic forms. The equilibrium depends on the activity of H' in the external solution and in the resin particles.
0
4
8
12
16
20
Volume. rn 1
Fig. 3 1.1. Separation of secondary aliphatic amides of N,N-dimethyl-p-aminobenzeneazobenzoic acid (ChuriEek and Jandera). Column: 240 X 2.7 nim. Ion exchanger: Dowex 50W-X2 (H+) (200-400 mesh). Eluent: 0.925 M hydrochloric acid in 80.5%' ethanol. Flow-rate: 0.132 ml/min. Detection: optical density at 520 nm. Peaks: 1 = 1.5 p g of di-ti-butylamide; 2 = 1.5 p g of di+propylamide; 3 = 1.5 p g of diethylamide; 4 = 1.5 p g of dimethylamide; 5 = inert compound (I'onceau 6 R ) .
Because of the negligible solubility of these compounds in aqueous acid solutions, it is necessary to use mixed aqueous-organic media. The amount of the organic solvent present obviously affects the distribution equilibrium by its solubility and solvation effects, and also by the dielectric constant effect. References p.664
660
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
A cation-exchange resin with a low degree of cross-linking (Dowex 50W-X2) was used for the separation in order to improve the accessibility of the ion-exchange phase to the large molecules of the derivatives and to accelerate the diffusion rate in the resin. Coloured amides are sorbed quantitatively on Dowex 50W-X2 (H'), 200-400 mesh, from mixed aqueous-organic solutions (80%ethanol; 80%methanol). The sorbed compounds can be eluted with an aqueous ethanolic or aqueous methanolic solution of hydrochloric acid. The effect of the eluent composition on the chromatographic behaviour of some homologous amides was studied by ChurGCek and Jandera. Homologous amides are eluted in order of increasing basicities, i.e., in order of decreasing molecular weights. Amides with a higher basicity have a higher distribution coefficient than esters, the basicity of which is lower. Secondary amides are sorbed more strongly than the less basic primary amides. TABLE 31 .I VOLUME DISTRIBUTION COEFFICIENTS (DJOF SOME AMIDES OF N,N-DIMETHYL-pAMINOBENZENEAZOBENZOIC ACID ON THE CATION EXCHANGER DOJ'EX 50W-X2 IN 0.925 M HYDROCHLORIC ACID SOLUTION IN 80.5% ETHANOL (CHURACEK AND JANDERA)
D, is defined as the ratio of the amount of compound in unit volume of the ion-exchanger phase t o the same volume of external solution. Amide derivative
D,
Methylamide Ethylamide n-Propybdmide n-Bu tylamide n-Hexylamide Allylamide Dime thylamide Diethylamide Di-(n-propy1)amide Di-(n-buty1)arnide
8.6 7.4 6.5 5.6 4.7 6.4 9.9 6.6 4.7 3.5
TABLE 3 1 . 2 GEL CHROMATOGRAPHY OF AMIDES ON POLYACRYLAMIDES (STREULI) Column: 97 X 0.5 cm. Gels: 1 = Bio-Gel P-2, 100-200 mesh (polyamide gel),exclusion limit 2002600; 2 = Bio-Gel P d , 100-200 mesh, exclusion limit 1000-5000. Mobile phase: 0.01 Msodium chloride solution. Flow-rate: 1 ml/min. Detection: RI. Compound*
Urea Biuret Acetamide N-Me thylacetamide N,N-Dimethylacetamide Acrylamide N-revt. -Butylacrylamide N-Vinyl-2-p yrrolidone *Samples of 50 gl.
Kd
Gel 1
Gel 2
1.37 1.88 1.06 0.94 0.86 1.17 0.97 1.23
1.17 1.29 -
0.89 -
66 1
GUANIDINE AND UREA DERIVATIVES
The contribution of the CH2 group to the logarithm of the distribution coefficient proved t o be fairly constant for homologous primary aliphatic amides, increasing to some extent with decreasing hydrocarbon chain length. Secondary amides showed greater changes corresponding to the same molecular-weight contribution. Volume distribution coefficients (D,) of iso-derivatives are slightly lower in comparison with those of normal derivatives. The multiple bond contribution to D, does not seem to be significant (Table 3 1.1). The hydrochloric acid concentration in the mobile phase influences the equilibrium between the protonated and non-ionic forms of these compounds. Gel chromatography was also used for the separation of substituted amides. Bio-Gel P-2 and P-6 were used as the stationary phase and 0.01 M sodium chloride solution as the mobile phase. Distribution coefficients and other separation conditions are given in Table 3 1.2 (Streuli).
GUANIDINE AND UREA DERIVATIVES Kirkland separated urea derivatives, applied as antidiabetic agents, by means of highspeed liquid chromatography, using a 1 m X 2.1 mm column packed with Permaphase ETH and a 1% solution of dioxane in n-hexane as the mobile phase. Fig. 3 1.2 shows the
CI
0-
N H -CO -N ( CH, )2
Fenuron
Mon uron 4
Cl
Diuron
15
7.5 T I M E . MIN
References p.664
Fig. 31.2. Separation of substituted ureas (Kirkland). Column: 1000 x 2.1 mm. Sorbent: Permaphase ETH. Eluent: 1% solution of dioxane in n-hexane. Operating conditions: flowrate, 1 ml/min; temperature, 27" C; column pressure, 340 p.s.i.g. Detection: UV detector at 254 nm. Peaks: 1 = solvent; 2 = Neburon; 3 = Fenuron; 4 = Monuron; 5 = Diuron. Sample: 1.5 ~1 of a 0.25 mg/ml solution of each compound in methanol.
662
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
separation of a synthetic mixture of substituted aromatic ureas usinga relatively non-polar organic carrier. A similar mixture can also be resolved with the same packing using the reversed-phase technique with alcohol-containing water as the mobile phase. The order of elution of these substituted ureas is different in the two systems. Liquid chromatography was also used for the analysis of gaseous fluorinated organic nitrogen compounds of the guanidine type (Rebertus et al.). Silica gel is a satisfactory adsorbent for this type of compound. Alumina and molecular sieves tend to decompose the fluorinated unsaturated compounds. The separation of tris(difluoroamino)fluoromethane and pentafluoroguanidine is illustrated in Fig. 3 1.3. Either an inert fluoro-compound or a hydrocarbon such as n-heptane can be used as the solvent and mobile phase; however, the former is preferred because of its much greater stability toward oxidation. Tris(difluoroamino)fluoromethane is readily eluted with either of these solvents, but pentafluoroguanidine is removed completely only if large bed volumes are used. The separation of bis(difluoroamino)difluoromethane-tetrafluoroformamidine mixtures was also effected. The relative affinities of the compounds for silica gel increase in the order ( F ~ N ) J C F
&
8 2 0
a
Y
B I0
Fig. 34.15. The chromatographic separation of peptides produced by chymotrypsin digestion of carboxymethylated lysozyme (Canfield and Anfinsen). The solid line represents the optical density at 570 nm of aliquots subjected to ninhydrin analysis following alkaline hydrolysis. The shaded areas represent the optical density at 280 nm of the column effluent. The gradient used for the cellulose phosphate column is illustrated at the top of the figure.
767
ION-EXCHANGE CHROMATOGRAPHY TABLE 34.1 BUFFERS USED FOR ELUTION O F CELLULOSE PHOSPHATE COLUMNS BY CANFIELD AND ANFINSEN
A four-chamber Technicon Varigrad containing ammonium acetate buffer was used for fractionation on a 25 X 2.4 cm cellulose phosphate column. An eight-chamber Varigrad containing pyridinium acetate buffer was used with the same adsorbent and a column of the same dimensions. ~~
BuffeI
Ammonium acetate
Chamber
1 2 3 4
Pyridinium acetate
1 2 3 4 5
6 7 8
Acetate Volume (ml)
Concentration (M)
PH
1100 1100 1100 1100
0.02 0.07 0.13 0.20
3.95 4.03 4.30 5.06
500 500 500 500 5 00 500 500 500
0.05 0.10
3.91 4.02 4.21 4.42 4.63 4.80 5.01 5.04
0.15 0.20 0.25 0.25 0.30 0.45
SE-Sephadex The strong cation exchanger SE-Sephadex is awlphoethyl derivative of dextran gel, available in two porosity grades, designated C-25 and C-50. For the separation of peptides, the low porosity type C-25 is preferred, with a total capacity of 2.3 mequiv./g. This ion exchanger can serve advantageously in place of Dowex 50, owing to the similarity of the exchange groups and the lower non-specific adsorption. The swelling of the C-25 type of exchanger is essentially independent of pH over the pH range 2-12, thus allowing regeneration to be performed in the column. Konigsberg and Hill recommended this exchanger particularly for the fractionation of basic peptides. In some instances Dowex 50-X2 is not adequate either alone or in combination with Dowex 1, as some basic peptides are eluted at the void volume of Dowex 1 and are completely retained on Dowex 50. Two peptides from the peptic digest of haemoglobin could not be eluted from Dowex 50 without using sodium hydroxide, but they were eluted without difficulty from SE-Sephadex C-25 with the use of the same gradient of pyridine-acetate buffers as for Dowex 50. The amino acid sequence of these and Tyr-Pro-Try-Thr-Glu(NH2)-Arg-Phe. peptides are Leu- Ma-His-Lys-Tyr-His The fractionation of peptides obtained by tryptic digestion of Kazal-type trypsin inhibitor from porcine pancreas was reported by Tschesche and Wachter. For equilibration of the SE-Sephadex C-25 column, 0.05 M ammonium formate buffer of pH 4 was used, followed by stepwise or gradient elution with 0.05 M ammonium acetate solution of pH 7.0. References p . 771
768
PEPTIDES
The chromatography of peptides on SE-Sephadex C-25 in 8 M urea-containing buffers was used for the fractionation of tryptic hydrolyzates of the chain of pig immunoglobulin by Frankk and Novotny. A buffer containing 0.005 M potassium formate, adjusted with formic acid to pH 3.0, was used for equilibration of the column. The column was developed with the same buffer, which was superimposed by a linear ionic strength gradient of potassium chloride solution. General conclusions about the chromatography, based on experimental work with the described type of gradient, were drawn by Novotny. The relationships derived were expressed as a simple rule permitting the determination of the column volume and the slope of the gradient most suitable for a particular mixture of pep tides.
AFFlNITY CHROMATOGRAPHY* There are many advantages in using affinity chromatography for the purification and isolation of peptides, which can be separated by highly specific and reversible bonding to the adsorbent. The column operation is very fast, peptides can be isolated from a large volume and the column can be used several times. The disadvantage that the investigator must prepare the adsorbent himself is compensated by the high specificity and ease of handling once the adsorbent has been prepared. The preparation of adsorbents has been discussed in Chapter 13. Applications are illustrated here with a few examples. The isolation of nitrotyrosine-containing peptides from nitrated lysozyme by means of antibodies to nitrotyrosine attached to Sepharose was proposed by Helman and Givol (Fig. 34.16). The antibodies were prepared ingoats or rabbits by injection of a nitrotyrosineprotein conjugate. Purification of antibodies from antisera was achieved by affinity chromatography on a column of nitro-y-globulin-Sepharose conjugate by Wilchek et al. The adsorbed antibodies were eluted with 0.1 M acetic acid and used for coupling to Sepharose in order to prepare the antinitrotyrosine-Sepharose adsorbent. This adsorbent was used in the one-step column isolation of tryptic peptides containing nitrotyrosine residues from nitrated, reduced and carboxymethylated lysozyme. The same useful technique was applied to the isolation of nitrotyrosyl-containing peptides from porcine carboxypeptidase B by Sokolovsky. Affinity chromatography made possible the isolation of synthetic peptides on the basis of selective association with proteins attached to the solid matrix. This technique was used by Kato and Anfinsen for the isolation of RNase-S-peptide from the crude preparation resulting from the solid-phase synthesis. Another use of affinity chromatography is for the one-step isolation of affinity-labelled peptides. Affinity-labelled peptides were isolated from the active sites of Staphylococcus nuclease and pancreatic ribonuclease by Wilchek. The general method for the isolation of tryptophan-containing peptides is of special interest owing to the difficulties connected with the isolation of these peptides by ionexchange chromatography. The method proposed by Wilchek and Miron was tested on the isolation of tryptophan-containing peptides from human serum albumin and horse cytochrome c. The tryptophan residues of these proteins were quantitatively modified with the lllghly specific reagent 2,4-dinitrophenylsulphenylchloride, and the peptides *For a more detailed description of this technique, see Chapters 7 and 14.
769
AFFINITY CHROMATOGRAPHY
TUBE NUMBER
Fig. 34.16. Isolation of nitrotyrosyl peptides from nitrotyrosyl-lysozyme (Helman and Givol). A tryptic digest of 2 mg of reduced and alkylated nitrolysozyme was applied t o a 6 X 1 cm column of antinitrotyrosyl antibody-Sepharose conjugate that contained 30 mg of antibodies. The column was washed with 0.1 M ammonium hydrogen carbonate solution and the yellow nitrotyrosyl peptides 0, were eluted with 1 Mammonia solution (arrow). 0 , E z s o ,;
,,.
containing the modified tryptophan residues were liberated by tryptic hydrolysis and selectively adsorbed and purified on an anti-DNP antibody column. The preparation of DNP-antibody adsorbent was described by Wilchek et al. The use of this method can be extended to the isolation of peptides containing methionine, cystine, cysteine, tyrosine or lysine, provided that the residues are specifically modified. Eluents for these strongly bound peptides must be carefully selected so as not t o release the antibody from the solid matrix. It has been found that elution with 6 M guanidine hydrochloride released 2-3% of the antibody which was covalently bound to Sepharose.
Isolation of cysteine-containing peptides The isolation of cysteine-containing peptides by means of organomercurial adsorbents does not fall within the scope of the section on affinity chromatography as it does not exploit the unique property of macromolecules, namely their biological function. This method can be considered as an application of adsorption chromatography. The preparation of organomercurial derivatives of Sepharose was described by Cuatrecasas, Sluyterman and Wijdenes, of cellulose by Shainoff, of cross-linked dextran by Eldjarn and Jellum, and of maleic acid-ethylene copolymer by Liener. References p . 771
770
PEPTIDES
The last adsorbent was used by Liener and Chao to bind the SH-peptides of the peptic digest of insulin in which the disulphide bridges had been reduced with sodium borohydride. The release of peptides by elution with mercaptoethanol was followed by carboxymethylation of SH-peptides and their isolation by other methods.
PARTITION CHROMATOGRAPHY In spite of the fact that for a very long time paper chromatography was the method of choice for the fractionation of peptides, there were few attempts to transfer the experience with paper to chromatographic columns. The reason was probably the difficulty in finding a suitable material as the support for the anchored phase with good flow characteristics and low adsorption. Commercially available gels of different types and cellulose powder are nowadays most often used as the stationary phase support in the partition chromatography of peptides. Another advantage of these materials is that the suitability of solvent systems for the separation of the particular peptidic mixture can easily be tested by paper chromatography with the organic phase of the proposed solvent system being used as the developer. The most promising systems are those in which the RF values of peptides are different, and preferably around 0.5. Solvents systems with very low or very high RF values are not useful. The complete cycle of column operation consists in: (1) equilibration with the aqueous phase of the solvent system; (2) equilibration with the organic phase of the solvent system; (3) chromatography; (4) discharge of the two-phase system and the material not eluted; ( 5 ) washing of the column. After the completion of the last stage, the column is ready to enter the first equilibration stage of the next cycle. A complete cycle of a column packed with Sephadex C-25 usually requires 5 or 6 days. The procedure for the purification of oxytocin by column partition chromatography was developed by Yamashiro using a two-phase system similar to that used in the purification of oxytocin and vasopressin by counter-current distribution. The column was packed with Sephadex C-25 in 0.2 N acetic acid, and the volumes of solvents needed for equilibration were as shown in Table 34.2. TABLE 34.2 SOLVENTS FOR EQUILIBRATION Stage
Influent solvent
Total volume
Flow-rate (ml/h’ cm’)
1.3 X bed volume 0.3 X bed volume 7-10 X bed volume 1.5 X bed volume
10-15 5-10 5 5
5
Aqueous phase Organic phase Organic phase Pyridine-0.2 N acetic acid (X: Y) 0.2 N acetic acid
1.3 X bed volume
3-15
77 1
REFERENCES
The void volume is measured during stage 2 after the emergence of the organic phase of the solvent system at the column exit. The washing solvent used in the fourth stage is expressed in volume terms. The ratio X : Y was determined according to the solvent systems used in stages 1, 2 and 3. A washing system was found to be effective if not more than four volumes of it were required to obtain miscibility with one volume of the organic phase of the solvent system to be discharged. Solvents for the partition chromatography of oxytocin on Sephadex (3-25 column are listed in Table 34.3. TABLE 34.3 SOLVENT SYSTEMS FOR THE PARTITION CHROMATOGRAPHY OF OXYTOCIN ON A SEPHADEX G-25 COLUMN (YAMASHIRO) Solvent system
(I) (11) (111)
ri-Butanol-n-propanol-0.2 N acetic acid (2:1 :3) n-Bu tanol-benzene-pyridine0.2 N acetic acid (6:1:1 3 9 ) n-Butanol-benzene-pyridine0.1% acetic acid (6:2:1 :9)
PH
RF of oxytocin
X: Y (stage 4)
3.0
0.16-0.18
1:4
5.5
0.24-0.27
3:s
6.2
0.20-0.25
3:s
The use of partition chromatography with Sephadex G-25 as the supporting phase was particularly useful in the isolation of S-DNP labelled peptides from ATP-creatine phosphotransferase (Mahovald). The technique used was the same as described above but with the following solvent systems: (I) n-Butanol-n-propanol-3% pyridine in 3%aqueous acetic acid (2: 1:3); (11) n-Butanol-n-propanol-benzene-3% pyridine in 3% aqueous acetic acid (4: 1:1 : 6 ) ; (111) n-Butanol-n-propanol-benzene-3% pyridine in 3% aqueous acetic acid(8:1:3:12); (IV) n-Butanol-benzene- 3%pyridine in 3% aqueous acetic acid (1 :1:2). The effluent from the column was followed by measuring the absorbance at 330 nm, fractions being evaporated on a rotary evaporator and subjected to sequence analysis.
REFERENCES Azegami, M., Ishi, S. and Ando, T., J. Biochem., 67 (1970)523. Bock, M. R., Ling Nan-Sing, Anal. Chem., 26 (1954) 1543. Bornstein, P., Kang, A. H. and Piez, K., Proc. Nut. Acad. Sci. US.,55 (1966)417. Callaham, P. X.,McDonald, J. K. and Ellis, S., Merhods Enzymol., 25 (1972)282. Canfield, R. E. and Anfinsen, C. B., J. Biol. Chem., 238 (1963)2684. Canfield, R. E. and Liu, A. K., J. Biol. Chem., 240 (1965) 1997. Catravas, G. N.,Anal. Chem., 36 (1964)1146. Clarke, H. T.,J. Biol. Chem., 97 (1932)235. Cuatrecasas, P.,J. Biol. Chem., 245 (1970)3059. Delanay, R., Anal. Biochem., 46 (1972)413. Edmundson, A. B.,Methods Enzymol., 11 (1958)369. Eldjarn, L. and JeIlum, E., Acta Chem. Scand., 17 (1963)2610.
772
PEPTl DE S
Fohn, O.,J. Biol. Chem., 106 (1934) 311. FranBk, F. and Novotn?, J., Eur. J. Biochem., 11 (1968) 5591. Frankland, B. T., Hollenberg, M. D., Hope, D. B. and Schachter, B. J., Brit. J. Pharmacol., 26 (1966) 502. Goldfarb, A. R., Saidel, L. J. and Mosovich, E.,J. Biol. Chem., 193 (1951) 397. Helman, M. and Givol, D., Biochem. J., 125 (1971) 971. Hill, R. L. and Delanay, R., Methods Enzymol., 11 (1958) 339. Hirs, C. H. W., Methods Enzymol., 11 (1958) 325. Hirs, C. H. W., Moore, S. and Stein, W . H.,J. Biol. Chem., 211 (1954) 907. Holmquist, W. R. and Schroeder, W. A.,J. Chromatogr., 26 (1967) 465. Jansen, J . C.,J. Chromutogr., 28 (1967) 12. Kassel, B. and Brand, E., J. Biol. C h e m , 125 (1938a) 115. Kassel, B. and Brand, E.,J. Biol. C h e m , 125 (1938b) 131. Kato, I. and Anfinsen, C. B., J. Biol. Chem., 244 (1969) 1004. Konigsberg, W. and Hill, R. J., J. Biol. Chem., 237 (1962) 2547. Kostka, V., Morivek, L., Kluh, I. and Keil, B., Biochem. Biophys. Acta, 175 (1969) 459. KuSnir, J. and Meloun, B., Collect. Czech. Chem. Commun., 38 (1973) 143. Liener, I. E., Arch. Biochem. Biophys., 52 (1967) 67. Liener, I. E., and Chao Li-Pen, Anal. Biochem., 25 (1968) 317. Lugg,J. W. H., Biochem. J., 26 (1932) 2144. McDowall, M. A. and Smith, E. L.,J. Biol. Chem., 240 (1965) 4635. Machleidt, W., Kerner, W. and Otto, J., 2. Anal. Chem., 252 (1970) 151. Mahovald, T. A., Biochemistry, 4 (1965) 732. Miranda, F., Rochat, H. and Lissitzky, S . , J . Chromutogr., 7 (1962) 142. Moore, S. and Stein, W. H., J. Biol. Chem., 211 (1954) 907. Novotn?, J., FEBS Lett., 14 (1971) 7. Nyman, P. O., Strid, L. and Westermark, G., Eur. J. Biochem., 6 (1968) 172. Okuyma, T. and Satake, K., J. Biochem., 47 (1960) 454. Paar, C. W., Proc. Biochem. Soc., 324th Meeting, XXVII. Padieu, P. and Maleknia, N., Bull. SOC.Chim. Biol., 47 (1965) 493. Peterson, E. A. and Sober, H. A.,J. Amer. Chem. Soc., 78 (1956) 751. Plapp, B. V., Raftery, M. A. and Cole, R. D., J. Biol. Chem., 242 (1967) 265. Rochat, H., Rochat, C., Lissitzky, S. and Edman, P., Eur. J. Biochem., 17 (1970) 262. Samejima, K., Dairman, W., Stone, J. and Udenfriend, S., Anal. Biochem, 42 (1971) 237. Satake, K., Take, T., Matsuo, A,, Tazaki, K. and Hiraga, Y . ,J. Biochem., 60 (1966) 12. Schroeder, W. A., Methods Enzymol., 25 (1972a) 203. Schroeder, W. A., Methods Enzymol., 25 (1972b) 214. Shainoff, J. R., J. Immunol., 100 (1968) 187. Sluyterman, L. A. and Wijdenes, J., Biochim. Biophys. Acta, 200 (1970) 593. Sokolovsky, M., Eur. J. Biochem., 25 (1972) 267. Spackman, D. H. and Stein, W . H.,J. Biol. Chem., 235 (1960) 648. Titani, K., Shinoda, T. and Putnam, F. W., J. Biol. Chem., 244 (1969a) 3550. Titani, K., Whitley, E. J. and Putnam, F., J. Biol. Chem., 244 (1969b) 3521. Tschesche, H. and Wachter, E., Hoppe-Seyler’s Z. Physiol. Chem., 351 (1970) 1449. Wdchek, M., FEBS Lett., 7 (1970) 161. Wilchek, M. and Bocchini, V., Becker, M. and Givol, D., Biochemistry, 10 (1971) 2828. Wilchek, M. and Miron, T., Biochim. Biophys. Acta, 278 (1972) 1 . Yamashiro, D., Nature (London), 201 (1964) 76.
Chapter 35
Proteins
z. PRUS~K CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773 General rules for the separation of proteins. . . . . . . . . . . . Selection of the separation procedure Choice of temperature in column separ Separation according to molecular size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775 Gel permeation chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of proteins by distribution constants . . . Group separations - desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776 Fine separations of protein mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777 .. ...777 Determination of molecular weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778 Choiceofeluent ............................... Gel permeation chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778 ......... Gel permeation chromatography with recycling . . . . . . . . . Gel permeation chromatography of proteins in dissocia
................................. Chromatography on glass with controlled pore size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Ion-exchange chromatography ..................... Sorption and the choice of ion exchanger according to Range of pH used in the ion-exchange chromatography Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786 Selective elution from ion exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787 Chromatography on hydroxyapatite and on calcium phosphate . . . . . . . . . . . . . . . . . . . . . . .788 Solubility chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789 Technique of gel permeation chromatography in a detergcnt gradient. ...................... 798 Affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . ......... Detection of proteins in the effluent . . . . . . . . . . . . . . . . . Colorimetric detection . . . . . . . . . . . . . . . . . . .................... Spectrophotometric detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801 .............. 803 Detection by ultraviolet fluorescence . . . . . . . . . . . . . . Automation of spectrophotometric detection. . . . . . . . . . .8 0 4 805 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION Proteins are essential components of living matter and the range of their concentrations and molecular weights is wide. In contrast to other components of macromolecular character, which are present in every organism, proteins are extremely variable in their electrochemical properties. The many possible combinations of occurrence, and the sequence of amino acids in the polypeptide chain and of polypeptide chains in the protein also cause considerable variations in their solubilities in water and other strongly References p.805
773
774
PROTEINS
polar solvents. The many structural variations in proteins also mean that proteins may occur together in mixtures that have very different structures but are still similar with respect to their physico-chemical properties. The amphoteric character of proteins and the large variations in their molecular weights are the main factors that permit an effective separation by means of column processes, mainly in aqueous media. The specific binding properties of proteins became a factor that extended the availability of highly effective and specific separation processes by the method of affinity chromatography. On the other hand, the protease and peptidase activity of proteins that are regularly present in complex mixtures of starting material impose certain limitations on separation processes from the point of view of temperature and/or acidity of the eluent. An important factor in the separation is also the tendency of proteins to become aggregated or denatured. In the study of living matter, poorly soluble proteins that form the structure of cell membranes also cannot be ignored. Recently, requirements for efficient separations of lipoproteins were laid down, and attention was also devoted to the proteins of cell nucleus, where the main stress was laid not only on the resolving power of column methods but also on rapid procedures accompanied by small losses during the separation process. The aim of this chapter is to give a review of separation and detection methods, and t o emphasize, as far as possible, some general principles that enable an experimenter provided with basic information on the starting material t o suggest a rational separation procedure, although the trial-and-error method cannot be excluded completely during the search for the most suitable procedure for the isolation of proteins. GENERAL RULES FOR THE SEPARATION OF PROTEINS
Selection of the separation procedure The first requirement for the use of column separation methods is perfect solubilization of the proteins, which can be achieved during the extraction of biological preparations by a suitable choice of salt concentration in the solution, by detergents and by pH adjustment. After the elimination of mechanical impurities, the required protein is concentrated, either by precipitation procedures or by sorption on ion exchangers and subsequent desorption at a suitable ionic strength and pH of the solution. The elimination of salts and the transfer of the proteins into a buffer of the required composition can then be carried out by CPC;the same method can also be used for the basic separation according to the size and shape of the protein molecules. As the next step, the sorption method is usually applied, during which use is made of the charge or specific binding properties of proteins; see also the chapters on Enzymes (Chapter 36) and Affinity chromatography (Chapter 7). In the final part of the separation, GPC is again often applied in view of the fact that most proteins are usually transformed into a dry state by freeze-drying. In favourable instances, for example if the proteins possess extreme properties of solubility, charge or binding strength, some parts of the procedure can be omitted. Although this chapter deals with column operations, it should be mentioned that operations on columns are often controlled or even completed by electrophoretic procedures, for example electrophoresis in a polyacrylamide gel.
GENERAL RULES FOR THE SEPARATION OF PROTEINS
775
Choice of temperature in column separations of proteins With increasing temperature, the viscosity of the mobile phase decreases and the hydrodynamic resistance of the column and the time necessary for equilibration in the separation process are correspondingly shortened. Theoretically, the upper limit of the temperatures that can be used in column separations of proteins is that above which thermal denaturation of proteins occurs. This temperature depends on the individual properties of the proteins, whether they are more resistent to the formation of polymeric forms at higher or at lower temperatures. In practice, the temperature limit is often shifted downwards, especially if hydrolases of the protease type or other destructively acting enzymes are present in the separated mixture, which often occurs in work with tissue extracts. In such instances, the work must be carried out at temperatures close to or even below O'C, with the addition of several per cent of isopropanol. The necessity for this procedure can be avoided only if a medium is chosen the pH of which is far from the optimum pH of the interfering enzymatic activities, or a medium that contains, for example, inhibitors of proteolytic activities; if the activity is eliminated specifically, for example by affinity chromatography (Carey and Wells); or if a medium is selected that blocks the enzyme activity by its random-coil forming effect, e.g., highly concentrated solutions of guanidine hydrochloride or urea.
Separation according to molecular size
Gel permeation chromatography The principle of the GPC of proteins is the differing retardation of the molecules during the flow of their solution through a column consisting of gel particles, smaller molecules that are capable of penetrating easily into the gel pores being retarded more than larger molecules. The molecules that are unable to penetrate into the gel are eluted in the retained volume, G,of a given column. In addition to this prevailing process, ion exchange also may be involved to a small extent, as may the sorption properties of the gel matrix and also the partition effect of the mobile phase (eluent) provided that it is immiscible with the phase fixed on the gel. The principles of the GPC method are explained in Chapter 5. As proteins especially have a vast range of molecular weights, (103-106 daltons and above), the GPC method is used very extensively for the characterization and separation of protein mixtures. A detailed treatment of GPC techniques with a review of gels that are suitable for protein separations was given by Reiland.
Characterizationof proteins by distribution constants On the supposition that no interactions take place between the gel and the substances passing the gel, column GPC can be considered to be an example of liquid-liquid chromatography. During the passage through the column of a total volume V,, the substances in solution are distributed between the liquid volume, V,, around the gel References p.805
776
PROTEINS
particles and the volume, 6, which is the volume of the liquid inside the gel particles. After passing through the column, the substances are washed out in a volume V,. The properties of proteins can be characterized either by the distribution constant, Kd, according to the relationship
or by means of the distribution constant, Kav,which is more useful in practice, defined as
Both Kd and Kav are independent of the column geometry and can be used in the characterization of the molecular weights of proteins. As 5 is determined less easily than V,, the characterization of proteins by Kav is more advantageous. Both & and Kav have values from 0 to 1 unless an interaction of the gel matrix with the protein passing through takes place.
Group separations - desalting
GPC is used for group separations when a fine separation is not imperative; however, the separation usually requires a large amount of preparation. For example, a common
?, I
0
80
100
FRACTION NUMBER
Fig. 35.1. Last step in the purification of luteinizing hormone by gel permeation chromatography on Column: 96 X 2.5 cm. Sorbent: Sephadex G-75. Buffer: 0.05 M Sephadex G 7 5 (Hennen et d.). ammonium hydrogen carbonate solution. Operating conditions: flow-rate, 14 ml/h; fraction volume, 3.4 ml; sample A, crude porcine luteinizing hormone, 160 mg in 3 ml of buffer; sample B, material from A after selection, 100 mg in 3 ml of buffer. The GPC pattern of the a-sub-unit of luteinizing hormone is indicated by the broken line.
777
GENERAL RULES FOR THE SEPARATION OF PROTEINS
problem consists in the separation of protein components from the components of low molecular weight in the solution, ie., desalting. For such separations gels with a low exclusion limit are suitable, for example Sephadex G-25 and Bio-Gel P-6. The proteins are excluded (Kav = 0), while the substances of low molecular weight are strongly retarded and their Kav values are close to unity. The sample volume, V,, should not exceed one quarter of the column volume, 6 (V, > 0.25 V,).
Fine separations of protein mixtures GPC is used for finer separations of proteins, where the distribution constant K,, is usually between 0.1 and 0.3. Therefore, a suitable type of gel should be chosen for separation which would not cause either protein exclusion or an unnecessarily weak differentiation of the components of a mixture if a gel was chosen with an excessive exclusion limit. Agarose gels, polydextran and polyacrylamide gels are now available for a large range of molecular weights of proteins that occur in nature. During the separation of protein mixtures, attention must be paid to the small difference in viscosity between the sample solution and the eluent; the quality of the resolution is influenced by the sample volume, V,, which should be at least 25-100 times less than the column volume, (V, 2 25-100 V,). An example is shown in Fig. 35.1.
2.105 or mixture
Sephadex ion exchanger Cross-linking type"
lonogenic group
A-25 C-25
DEAE, QAE CM, SE, SP
A-50 C-50
DEAE, QAE CM, SE, SP
A-25 c-25
DEAE, QAE CM, SE, SP
*A = Anion exchanger, C = cation exchanger
785
ION-EXCHANGE CHROMATOGRAPHY
size, and microgranular celluloses give better resolutions than fibrous types, especially if they are kept humid. The matrix is formed by particles that are impenetrable t o proteins. For steric reasons, the capacity of cellulose ion exchangers is several times less for macromolecular proteins in comparison with the titratable capacity of ion exchangers for small ions. In a similar manner, the capacity is also changed in ion exchangers with a gel matrix, but the dependence of the capacity on the dimensions of the macromolecules is much more distinct because larger protein molecules can be bound only t o those ion exchanger groups which are on the surface of the gel particles. Examples of recommended types of more or less cross-linked polydextran gel ion exchangers in relation t o the molecular weights of the separated proteins are given in Table 35.3. From Table 35.3, it follows that polydextran ion exchangers with a denser matrix have a larger field of applications. The advantage of the higher capacity of a looser matrix of a polydextran ion exchanger for proteins with medium molecular weights is most striking if the capacities of the dry weight of ion exchangers are compared. As the initial ionic strength of the elution buffers is low, the same weight of ion exchangers with a looser matrix (Sephadex A-50 and C-50) of polydextran gels assumes a substantially larger volume. In fact, this decreases t o a certain extent the differences in capacity between ion exchangers with a matrix of the Sephadex G-25 and G-50 type. The ratios of the capacities for haemoglobin and ion exchangers derived from the polydextran gels Sephadex G-25 and Sephadex G-50 are given in Tables 35.4 and 35.5. TABLE 35.4 BINDING CAPACITY OF ION EXCHANGERS OF THE SEPHADEX TYPE FOR PROTEINS IN BUFFERS OF IONIC STRENGTH 0.01 (PHARMACIA, 1970) ~
Type of' exchanger
Capacity o f haemoglobin (w/win grams)
pH of buffer
QAE-Sephadex A-25 QAE-Sephadex A-50
0.3 6
8.0
DEAE-Sephadex A-25 DEAE-Sephadex A-50
0.5 5
8.0
CM-Sephadex C-25 CM-Sephadex C-50
0.4 9
5.0
SP-Sephadex C-25 SP-Sephadex C-50
0.2 7
5.0
The capacity of an ion exchanger cannot be utilized t o its full extent in separations on columns; in the sorption of proteins, 5 - 10%of the ion exchanger capacity at most is utilized. The sample should be introduced on t o the ion-exchange column in a small volume only when the stabilizing buffer is simultaneously the solvent for the sample and the eluent, without subsequent elution with a gradient of ionic strength and/or pH. As the samples are usually complex mixtures, which are most suitably separated by gradient elution, dilute protein solutions may be applied in a large volume. If the flow-rate is not References p.805
786
PROTEINS
TABLE 35.5 CAPACITY OF DEAE- AND CM-CELLULOSE (WHATMAN) FOR INSULIN AND LY SOZYME (REEVE ANGEL AND CO.) Type of ion exchanger
Designation
Capacity (w/w)
Remarks
Protein
Amount (g)
pH
DEAE-cellulose
DE-22 DE-23 DE-3 2 DE-5 2
Insulin
0.75 0.75 0.85 0.85
8.5
free base
CM-cellulose
CM-22 CM-23 CM-32 CM-52
Lysozyme
0.60 0.60 1.26 1.26
5.0
Na+
excessive, the sample remains sorbed on the upper part of the column, independent of the extent of dilution of the sample.
Elution Buffers with a sufficient buffering capacity are used for elution so that proteins will not affect the pH of the eluent by their own buffering capacity. The buffering ion in the eluent should, if possible, have the same charge sign as the functional group of the exchanger. Elution of proteins is carried out either by increasing the ionic strength of the buffer stepwise, or by a stepwise change in the pH of the buffer in the direction of the isoelectric point of the protein; continuous gradient elution can also be used with advantage, because the risk of the formation of artificial peaks is less than with a stepwise change in the eluent properties. Elution without a gradient, where the eluent is identical with the buffer that stabilizes the exchanger and also serves for the dissolution of the sample, can be used for very fine separations of closely related proteins. The separation of various types of haemoglobins, described by Dozy and Hujsman, is an example. The establishment of suitable conditions for elution without a gradient is a lengthy task and the requirement of the proximity of the isoelectric point and the pH of the eluent can serve as the only guideline. Optimum ionic strength and pH values for particular separations must be sought empirically. A further disadvantage is that the sample volume decides the peak widths obtained after elution. For gradient elution, the proteins are either bound strongly to the ion exchanger or they do not form any bonds. In gradient elution, only the values 0 or 1 for the R, value can be taken into consideration (Porath and Fryklund). Lampson and Tytell observed that a series of proteins is desorbed at a pH that differs from the isoelectric point by 0.40.6 pH units at an ionic strength of approximately 0.1 on an exchanger containing cationic carboxymethyl groups, and this relationship may help in the selection of optimum conditions for desorption.
787
ION-EXCHANGE CHROMATOGRAPHY
E
z
0
m
z 0
N
8Z 0.5a
m r
2
Y , ' -0.5 r t w
, , ,
, ,
1
sm
-0.3
a
-
Y 8
+m z
-0.1
0-
I
I
I
I
1
I
I
I
I
Fig. 35.3. Separation of sub-units of porcine luteinizing hormone by ionexchange chromatography on SE-Sephadex C-25 in 8 M urea (Hennen et d.). Column: 10 X 0.9 cm. Buffer: equilibrating buffer0.0025 M sodium acetate buffer, pH 4.9, in 8 M urea; two linear gradients of 0.025-0.10 M (150 ml; start: arrow 1) and 0.10-0.50 M (100 ml; start: arrow 2) of sodium acetate buffer, both in 8 M urea. Operating conditions: flow-rate, 11 ml/h; fraction volume, 3.7 ml; sample, 80 rng of porcine luteinizing hormone. Detection: absorbance at 280 nm (solid line). The gradient was applied after collection of the non-adsorbed fraction, the slope of the Na' concentration being indicated by the broken line.
An ionic strength gradient or the concentration of ions capable of binding with the functional groups of the exchanger with an opposite charge should always be increasing (Fig. 35.3). For an anion exchanger, the pH gradient decreases, but for a cation exchanger it increases. Most often ionic strength gradients are used. Within broad concentration limits, sodium chloride or potassium chloride gradients are used in the presence of buffer. The optimum resolution of proteins and peptides can be achieved by decreasing the steepness of the gradient only if the concentration of the eluted protein or peptide is equal to, or greater than, approximately M.However, in practice, the concentration of the eluted protein is more commonly M or less. In such instances, the quality of the separation apparently improves when a steeper gradient is applied; this was shown by Novotnf for the separation of light immunoglobulin chains o n SE-Sephadex and QAE-Sephadex.
Selective elution from ion exchangers Sometimes, if the proteins are distinguished in mixtures by their extreme charge, the ionic strength and the p H of the eluting buffer may be set so that the required substance is eluted individually, without being sorbed on the exchanger, while all others are retained by the exchanger. An example of such a procedure, which is also applicable t o larger scale work, is the purification of human immunoglobulin, IgG, on a QAE-Sephadex A-50 column, described by Joustra and Lundgren and outlined below. The method References p.805
788
PROTEINS
involves the use of buffers with a constant ionic strength during the elution and the regeneration. Regeneration is achieved only by a pH change, which, in the case of a strong anion exchanger and under the conditions mentioned, does not cause a change in gel volume. This enables the separation and regeneration process to be repeated many times in the same column. The serum is freed from 0-lipoproteins by the addition of Aerosil (Degussa, Frankfurt am Main, G.F.R.). Two grams of Aerosil380 are added to 100 ml of serum and the mixture is stirred at room temperature for 4 h. After centrifugation at 12,OOOg for 30 min, the supernatant is equilibrated by GPC on a Sephadex G-25 column with the elution buffer A (specified below). The serum is then diluted in a 1 : 2 ratio with elution buffer A and applied on to an ion-exchange column. If the P-lipoproteins are eliminated, a volume can be applied which may be up to the bed volume (V,) of the column. If the sample volume is further increased, transferring appears in the eluate. After the elution of IgG with buffer A, the ion-exchange column is regenerated with buffer B. The eluate of IgG is concentrated 10-fold by ultrafiltration and immediately lyophilized. The yield is approximately 70% of IgG, depending on the type of serum. The two buffers have the following compositions: Buffer A : ethylenediamine-acetic acid, pH 7.0, ionic strength I = 0.1. Ethylenediamine (2.88 g; distilled under reduced pressure) is dissolved in 73 ml of 1 M acetic acid and the volume is made up to 1 litre with distilled water. Buffer B: sodium acetate-acetic acid, pH 4.0, ionic strength I = 0.1. A 435-m1 volume of 0.6 M acetic acid plus 130 ml of 0.6 M sodium acetate are diluted to 1 litre with distilled water. An example of the separation is as follows. On a 11 X 1.5 cm column of QAE-Sephadex A-50 equilibrated with buffer A, a 10-ml sample of human serum was applied, equilibrated and diluted with buffer A. At a flow-rate of 8 ml/h . cm2, elution of IgG with 65 ml of buffer A was carried out and the column was regenerated with buffer B. The regeneration and the elution of IgG were controlled by UV absorption of the eluate at 254 nm. The first peak contained approximately 0.3% of IgC.
CHROMATOGRAPHY ON HYDROXYAPATITE AND ON CALCIUM PHOSPHATE The principle of the separation of proteins on hydroxyapatite (HA) (introduced by Swingle and Tiselius in 195 1 and developed by Tiselius et al.) and calcium phosphate gel (according to Price and Greenfield) consists in the interaction of negatively and positively charged groups of protein molecules with phosphate and calcium ions in HA, and it is therefore a special case of ion-exchange chromatography of proteins. Either microparticular crystalline HA of composition Ca10(P04)6(OH)2or calcium phosphate gel anchored on an inert carrier, for example cellulose, serves as the support. HA is amphoteric and the isoelectric point of the carrier is strongly affected by the method of preparation; its value changes in the range from 6.5 to 10.2. The binding of positively charged groups on HA is strongly influenced by salts, for example sodium and potassium chlorides, while with negatively charged groups in proteins the salt concentration does not affect the bond strength significantly. In addition, the effect of salt concentration on the sorptionof proteins is also a function of their molecular weight..The
SOLUBILITY CHROMATOGRAPHY
789
mechanism of binding, the application of HA chromatography to the separation of proteins and the operating technique for use with HA columns were described in a review by Bernardi. The use of HA chromatography seems to be particularly suitable for basic proteins and polypeptides, among which histones can be easily released by higher salt concentrations. The separation of proteins, especially enzymes, including a newer method for coating cellulose with calcium phosphate gel, was described by Koike and Hamada. It seems that the separation properties of HA and calcium phosphate are not yet sufficiently appreciated, owing to the polyfunctional character of the bond, originating from the amphoteric properties of the carrier, and from the few results available for the complete characterization of the effect of the molecular weight of the separated proteins on the course of the separation process. A survey of selected protein separations is presented in Table 35.f
SOLUBILITY CHROMATOGRAPHY The principle of solubility chromatography consists in the gradual precipitation of proteins and their elution from a column usually formed by a suitable inert carrier. Single proteins differ substantially in their solubilities in relation to various salt concentrations or ionic strengths, concentrations of organic solvents and detergents. The wide range of protein solubilities permits their separation on the basis of gradual solubilization of the protein precipitate by changes in salt concentration, organic solvents and detergents in suitable solutions; this method is analogous to classical fractional precipitation procedures used for the isolation of proteins. In contrast to batch fractional procedures, the use of columns permits a better separation of fractions because the application of a continuous gradient of the eluent permits the attainment of the optimum eluent concentration, which just suffices for elution but does not cause occlusion of the proteins from the solution. Keil er al. proposed a method which they called column gradient extraction of proteins, in which the different solubilities of proteins in the presence of ammonium sulphate are made use of. First, proteins are precipitated from a solution in concentric layers, on to a Kieselguhr carrier (Hyflo Supercel) using an increasing concentration of ammonium sulphate. When the proteins have been precipitated, the carrier with the layer of protein is introduced into a tube and in the second phase of the operation a solution of ammonium sulphate of decreasing concentration is pumped into the column. Elution of proteins takes place gradually down to zero concentration of the salt. Keil et af. demonstrated the efficiency of the method with several examples of the isolation of proteins. Fig. 35.4 illustrates the separation of the proteins of equine serum. Saffran er al. extracted hypophysis tissue on a column with a concentration gradient of solvents of increasing polarity, in the system ethanol-water-acetic acid, and achieved an appreciable enrichment of the fraction with corticotropic activity (ACTH). Diatomaceous earth was used as the carrier because it prevents an excessive increase in the hydrodynamic resistance of the column during gradual swelling of the tissue. The method of Saffran er al. is particularly suitable when a minor component of tissue preparations, such as acetone-dried residues, has to be isolated. Its advantage is that the eluent can be eliminated completely by evaporation. References p.805
4
TABLE 35.6 SELECTED PROTEIN SEPARATIONS ON ION EXCHANGERS Protein
\o
0
Source
Ion exchanger
Equilibration buffer
Sorption-desorption region
Bovine pituitary gland
CM-cellulose
0.01 M.Ammonium acetate, pH 4.6
Grif et al.
SE-Sephadex C-25
0.01 M Ammonium formate, pH 3.5
(a) Equilibration buffer Gradient (b) 0.2 M Ammonium acetate, RechromapH 6.7 tography (a) 0.01 M Ammonium format? Gradient pH 3.5 (b) 0.05 M Ammonium acetate, pH 5.5 (a) Equilibration buffer (b) 0.2 M Ammonium acetate, pH 6.7 Elution order a-MSH, 0-MSH, ACTH
Hussa et al.
Note
Reference
Hormones YLipotropic hormone
a-MSH (melanocyte stimulating hormone) 0-MSH ACTH (adrenocor ticotropic hormone)
Porcine and bovine pituitary gland
CM-cellulose
0.01 M Ammonium acetate, pH 4.6
TCT (thyrocalcitonin)
Porcine thyroid gland
CMcellulose
0.2 M Pyridine acetate, (a) Equilibration buffer pH 4.0 (b) 1.1 M Pyridine acetate, pH 4.0
Linear gradient
TCT
Porcine
CM-cellulose
0.062 M Pyridine acetate, pH 4.4
(a) Equilibration buffer (b) 1.86 MPyridine acetate, pH 5.7
Linear gradient
Bell et al.
FSH (follicle stimulating hormone)
Human pituitary gland
DEAE-Sephadex A-50
0.1 M phosphate, pH 6.8
Equilibration buffer
Without gradient
Peckham and Parlow
Continuous gradient
Hawker et al.
?I ;FI
;2;;. 3 0
2
Pa0
FSH LH + TSH (luteinizing and thyroid stimulating hormone)
Human pituitary gland
CM-Sephadex c-50
0.004 M Ammonium acetate, pH 5.5
(a) Equilibration buffer (b) 0.1 M Ammonium acetate, pH 6.7 (elution of FSH) (c) 0.1 M Ammonium acetate, pH 9.5
TSH
Bovine pituitary gland
DEAE-cellulose
0.005 M Sodium glycinate, pH 9.5
(a) Equilibration buffer (b) 0.005 M Sodium glycinate, pH 9.5, containing 0.02 M NaCl (c) As (b) containing 0.5 M NaCl
PLipotropic hormone
Bovine
CMcellulose
0.01 M Ammonium acetate, pH 4.6
(a) Equilibration buffer (b) 0.1 M Ammonium acetate, pH 6.8 (c) 0.2 M Ammonium acetate, pH 6.8
Light-adap ting hormone
Crustacean eyestalks, Pandalus borealis
0.075 M Ammonium acetate, pH 4.92, containing 2 . 1 0 P volumes of thiodigiycol
(a) Equilibration buffer (b) 0.1 M Ammonium acetate, pH 9.51
0.02 M NH, HCO,
Equilibration buffer
0.1 M Triethylamine formate equilibrated to pH 3.1
(a) Equilibration buffer (b) 0.1 M Triethylamine acetate, pH 5.5 (c) 0.1 M Triethylamine, pH 11.4
Rathnam and Saxena
0 bl
Red pigment concentrating hormone
Pandalus borealis
CM-Sephadex C-25
Dowex 50W-X2
Linear gradient
Lindsay et al.
Continuous gradient
Lohmar and Li
Fernlund
Re-chromatography without gradient Josefsson Stepwise
4
(Continued on p. 792)
5
4 v)
TABLE 35.6 (mntinued)
h)
Protein
Source ~~~~~~~~
Prolac tin
Blood proteins Fibrinogen
Fibrinogen
Prothrombin
Plasminogen
Plasminogen
Human pituitary gland
Ion exchanger
Equilibration buffer
Sorption-desorption region
0.012 M Tris-HCI, pH 1.6
(a) Equilibration buffer (b) As (a), in 0.05 M NaCl (c) As (a), in 0.5 M NaCl (a) Equilibration buffer (b) As (a), in 0.2 M NaCl (c) As (a), in 0.5 M NaCl
Prolactin eluted at 0.2 M NaCl
Linear gradient
0.01 M Ammonium acetate, pH 5.6
Human blood platelets
DEAE-cellulose
0.05 M Tns-HC1, pH 1.2
(a) Equilibration buffeF (b) Equilibration buffer containing 0.5 M NaCl
Human
DEAE-cellulose
0.005 M Trisphosphate, pH 8.6
(a) Equilibration buffer (b) 0.02 M Tris-phosphate, pH 4.3 (c)-(h) Increasing molarity to 0.5 M Tris-phosphate, decreasing pH to 4.1
Human
Bovine
Reference
Stepwise
Hwang et al.
~
DEAEcellulose
CM-cellulose
Bovine
Note
DEAE-Sephadex A-50
0.1 M sodium phosphate, pH 6.0
(a) Equilibration buffer (b) Equilibration buffer in 1.O M NaCl
DEAE-Sephadex A-50
0.02 M Sodium phosphate, pH 8.0, containing 0.04 M NaCl
(a) Equilibration buffer (b) Equilibration buffer in 0.05 M e-aminocaproic acid
0.1 M Sodium acetate, pH 5.0, containing 0.3 M NaCl
(a) Equilibration buffer (b) Equilibration buffer in 0.6 M NaCl
CM-Sephadex c-50
Ganguly
Mosesson et al. Sevenchamber mixer, Trisphosphate and pH gradient
Linear gradient
Ingwall and Scheraga
Wallen and Wimm -0
a Linear gradient of NaCl
Nagasawa and Suzuki
XI
%
Metalloproteins Haemocyanin
8 0 2
P
Decapod crustaceans: Collinectes
DEAE-cellulose
0.05 M Ammonium hydrogen carbonate, pH 1.8
sapidus, Libinia emarginota, Gecarcinus lateralis
0 co b
Stellacyanin
Umecyanin
Myoglobin
Cytochrome csss
Rhus vernicifera Horse radish
Mollusc, Busycon caricum
Czithidiu fasciculata
(a) Equihbration buffer (b) Equilibration buffer containing 0.5 M NaCI, 0.002 M MgCI,
Concave gradient
Kerr
0
z P
0
5 Amberlite CG-50
0.05 M potassium phosphate, pH 5.5
(a) Equilibration buffer (b) 0.2 M KH,PO,
Stepwise
Peisaca et al.
(a) Equilibration buffer (b) 0.03 M Sodium acetate, pH 5.1
Rechromatographed stepw ise
Paul and Stigbrand
8GI w
%
CM-cellulose
0.004 M Sodium acetate, pH 4.7
DEAE-cellulose
0.05 M Sodium phosphate, pH 5.5
DE AE-Sephadex A-50
0.04 M NH,HCO,
SE-Sephadex C-50
Equilibration buffer Tris-cacodylate, pH 6.5,ionic strength I = 0.1
Amberlite CG-50
0.01 M Sodium phosphate, pH 8.0
(a) 0.01 M Sodium phosphate, pH 8.0 (b) As (a), containing 0.4 M NaCl
0.01 M Sodium phosphate, pH 8.0, containing 0.01 M K, Fe(CN),
(a) 0.01 M Sodium phosphate, pH 8.0
Rechromatographed Linear gradient Na'
(b) As (a), containing 0.4 M NaCl
Cytochrome eluted at 0.19 M Na'
Read
Equilibration buffer Re-chromatographed
Kusel et al.
(Continued on p. 794)
3: 4
4 \o
TABLE 35.6 (continued) Protein Cytochrome c
Cytochrome c
P
Source
Ion exchanger
Equilibration buffer
Sorption-desorption region
Note
Reference
Spirillum itersonii
DE AE-cellulose
0.003 M Tris-HCI, pH 8.7
Equilibration buffer
Cytochrome not adsorbed
Clark-Walker and Lascelles
CM-cellulose
0.002 M Potassium phosphate, pH 6.2
(a) Equilibration buffer (b) 0.05 M Potassium phosphate, pH 6.2
Re-chromatographed stepwise
CM-cellulose
0.05 M Na,HPO, containing 0.05 M
Equilibration buffer
Without gradient
Dixon and Thompson
Linear gradient cytochrome b5 eluted at 0.25 M KCl
Ito and Sato
Horse heart
NaH,PO,, pH 6.75 Cytochrome b ,
Rabbit liver
DEAE-Sephadex A-50
0.02 M Tris-HCl, containing 0.5% Triton X-100, 0.002 M EDTA, 0.1 MKCI
(a) Equilibration buffer (b) Equilibration buffer in 0.3 M KC1
Transfenin
Human
DEAE-Sephadex A-50
0.05 M Tris-HC1, pH 8.0
(a) Equilibration buffer (b) 0.07 M Tris-HCI, pH 8.0 (c) 0.15 MTris-HCI, pH 8.0
Haemoglobin
Human
DEAE-cellulose 01 DEAESephadex
0.05 M Tris-HC1, pH 8.4-7.2 for different Hb types
0.05 MTris-HCI, pH 8.3-7.1
Haemoglobm
Ascaris lurnbricoides body walls
DEAE-cellulose
0.001 M Potassium phosphate, pH 7.0
(a) Equilibration buffer (b) 0.03 M Potassium phosphate, pH 7.0 (c) 0.3 M Potassium phosphate, pH 7.0
Aisen et aL
Stepwise pH gradients
Horton and Chemoff
Okazaki et al. Two linear gradients
p
Histones Histones
Calf thymus
CM-cellulose
2 0
a
0.04 M Sodium acetate, pH 4.6, in 3.1 M urea
(a) Equilibration buffer (b)-(i) Increasing molarity of sodium acetate, 0.1-0.6 M and increasing pH, 5.7-8.6
0.005 M Potassium phosphate, pH 6.8, containing 3.1 M urea
(a) Equilibration buffer (b)-(h) Equilibration buffer containing NaCl of increasing molarity, 0.150.60 M (i) 0.05 N HC1 (i) 0.10N HC1
P
3 CI,
Stepwise
Yang
f:
2
cl 3: ia
0
F
8n P
%
3:
Histone
Histone
HeLa cells
Calf thymocytes
Amberlite CG-50
Amberlite CG-50
0.1 M Sodium phosphate, pH 6.8, in 8% guanidine hydrochloride
0.1 M Sodium phosphate, pH 6.8, in 8% guanidine hydrochloride
(a) Equilibration buffer (b) 0.1 M Sodium phosphate, pH 6.8, in 8.5% guanidine hydrochloride (c) 0.1 M Sodium phosphate, pH 6.8, in 13% guanidine hydrochloride (elution of lysine-rich histones) (d) 0.1 M Sodium phosphate, pH 6.8, in 40% guanidine hydrochloride (elution of arginine-rich histones) (a) Equilibration buffer (b) 0.1 M Sodium phosphate, pH 6.8, containing 8% guanidine hydrochloride (c) 0.1 M Sodium phosphate, pH 6.8, containing 13% guanidine hydrochloride (d) 0.1 M Sodium phosphate, pH 6.8, containing 40% guanidine hydrochloride
Sadgopal and Bonner
.e
Non-linear gradient
Linear gradient, Pallotta and 8-13% Berlowitz guanidine hydrochloride Linear gradient, 13-40% guanidine hydrochloride 4
a
v1
(Continued on p. 796)
TABLE 35.6 (continued)
4 \D Q\
Reference
Protein
Source
Ion exchanger
Equilibration buffer
Sorption-desorption region
Note
Inhibitors Trypsin inhibitor
Potato
DEAE-cellulose
0.02 M Sodium borate, pH 8.3
(a) Equilibration buffer (b) Equilibration buffer in 0.2 M NaCl
Linear gradient
Cellulose phosphate
0.02 M Sodium borate, pH 7.0 or 6.1
Equilibration buffer
Re-chromatographed
Gradient produced by rectangular Varigrad
Frattali and Steiner
Two linear gradients
Cechovi et al.
Linear gradient of NaCl
Iwamoto and Abiko
Linear gradient
Samuelsson and Pettersson
Trypsin inhibitor
Soya bean
DEAEcellulose
0.05 M Ammonium acetate, pH 5.0
(a) Equilibration buffer (b) 0.5 M Ammonium acetate, pH 6.5
Trypsin inhibitor B
cow
DEAEcellulose
0.02 M Sodium phosphate, pH 7.2
(a) Equilibration buffer (b) Equilibration buffer in 0.01 M NaCl ( c ) Equilibration buffer in 0.02 M NaCl
An tiplasmin
Human plasma a2-macroglobulin
DE AE-Sephadex A-50
0.02 M Tris-HC1 in 0.1 MNaCI, pH 7.7
(a) Equilibration buffer (b) 0.02 M Tris-HC1, pH 7.7, in NaCl of increasing molarity
Mistletoe, Viscum album L.
SE-Sephadex C-25
0.067 MPhosphate, pH 5.0
(a) 0 . 0 3 3 M KH,PO, (sample) (b) 0.067 MPhosphate, pH 5.0 (c) 0.067 M Phosphate, pH 8.0, NaCl to 0.125 M "a+)
Toxins Viscotoxins
colostrum
Hochstrasser et al.
Cb
w
0
Neurotoxin
b 3 0
2
P
03 0 b
Anaphylatoxin
Scorpion venoms Andronoctus australis
DEAE-Sephadex A-25
0.1 M Ammonium acetate, pH 8.5
Equilibration buffer
Buthus occitanus
Amberlite CG-50
0.2 M Ammonium acetate, pH 6.7,6.3 or 6.15
0.2 M Ammonium acetate, pH 6.7, 6.3 or 6.15
CM-Sephadex C-50
0.2 M Ammonium acetate, pH 6.7
0.02 M Ammonium acetate, pH 6.7
CM-Sephadex C-50
0.02 M Sodium acetate, pH 5.6
(a) 0.02 M Ammonium formate, pH 5.5 (b) 0.5 M Ammonium formate, pH 6.5
Leiurus quinquestriatus Hog serum
Without gradient
Miranda et al.
Linear gradient of ammonium formate
Vogt
A- toxin
Clostridiurn botulinurn
DEAE-Sephadex A-50
0.15 M Tris-HCI, pH 8.0
(a) Equilibration buffer Linear (b) 0.15 M Tns-HCI, pH 8.0, in gradient of 0.3 M NaCl C1concentration
Dasgupta et al.
B-toxin
Clostridiurn botulinurn
DEAE-cellulose
0.07 M Tris-HCI, pH 8.0
(a) Equilibration buffer (b) 0.07 M Tris-HCI, pH 8.0, in 0.5 M NaCl
Linear gradient, toxin eluted at 0.14 M C1-
Beers and Reich
E-toxin
Clostridiurn botulinurn
CM-Sephadex C-50
0.02 M Sodium acetate, pH 6.0
(a) Equilibration buffer (b) 0.02 M Sodium acetate, pH 6.0, in 0.5 M NaCl
Linear gradient
Kitamura et al.
798
PROTEINS
I
0.9~
E
8
? 0.6 w
0
z a m
8 Cn 0.3-
z
0
I VOLUME. ml
Fig. 35.4. Separation of horse serum by solubility chromatography (Keil e l d). Column: 22 X 2.7 cm. Sorbent: Kieselguhr (Hyflo Supercel). Eluent: gradient of ammonium sulphate in water. Operating conditions: flow-rate, 44 ml/h; fraction volume, 22 ml; sample, 5 0 ml of horse serum; mixer diameter, 10 cm; height of solution, 12.5 cm, containing ammonium sulphate of 85% saturation; reservoir diameter, 10 cm; height, 15.5 cm, containing water. Solid line, absorbance at 280 nm; broken line, percentage of saturation of ammonium sulphate gradient.
TECHNIQUE OF GEL PERMEATION CHROMATOGRAPHYIN A DETERGENT GRADIENT If a constant concentration of detergent in the eluent is used for the GPC of proteins solubilized by the detergent, then the ratio of detergent to protein is usually higher than necessary. The effect of the detergent manifests itself in reversible and often also irreversible changes. The ratio of detergent to protein is critical from the point of view of the preservation of the original protein properties and therefore it is advisable to keep the lowest practical detergent concentration during the separation. The condition of minimum detergent concentration is fulfilled if the proteins are separated on a column in a concentration gradient of the detergent. The procedure according to Swanljung was used for the purification of the crude ATPase concentrate. The separation has four steps: in the first, a Sepharose 6B column is stabilised with a detergent-free buffer, in the second the column is washed with a buffer in which the detergent concentration is gradually increased, in the third the sample is applied on to the column dissolved in the buffer the concentration of which corresponds to the highest detergent concentration in the column, and in the fourth the column is washed with a buffer of constant (highest) detergent concentration. If water-insoluble proteins attain the lowest critical value of detergent concentration in the column, i.e., if the volume of the buffer with the detergent gradient is not much less than that of the column, then precipitation occurs and the proteins move further with the lowest detergent concentration at which they enter the solution. In a similar manner to ATPase, a series of flavoproteins and the b-c, cytochrome complex were also separated. This method of separation is an inverted variant of zone precipitation gel chromatography described by Porath. In this case also a concentration gradient is formed, which moves slowly down the column. Faster moving proteins begin to precipitate at the critical ammonium sulphate concentration and then move with the same speed as the gradient.
AFFINITY CHROMATOGRAPHY
799
AFFINITY CHROMATOGRAPHY The principle of affinity chromatography was defined by Cuatrecasas (1972) as a method by which a substance or a group of substances is separated specifically from a mixture on the basis of the affinity given by the biological bonding function of macromolecules, and the technique was rapidly accepted for use in protein separations. This promising selective separation method is described in general terms in the chapter on affinity chromatography (Chapter 7). In this chapter, the possibilities for the separation of proteins with a non-enzymatic character will only be briefly mentioned as the isolation of enzymes is described in Chapter 36. In addition to the purification of a series of enzymes and their inhibitors or substrates, which may be alternatively bound on the matrix, the purification of antibodies and antigens, hormone-transport and vitamin-binding proteins, and group purifications of proteins that contain a glycidic component, also belong to the group of separations by affinity chromatography. On the border of the definition of affinity chromatography is the method of isolation of SH-proteins on agarose-organomercury derivatives. The fixation of antibodies offers wide possibilities, and of the carriers available agarose was found to be the most suitable. In contrast to the commonly used method of the reaction of activated agarose with proteins, it is advantageous if the pH value is shifted to lower values during the preparation of the carrier with bound antibodies; the optimum pH is 6-7. Cuatrecasas (1970) found that at this pH the capacity of the adsorbent is one order of magnitude higher than when the reaction takes place in a very alkaline medium, although the amount of the bound antibodies remains unchanged. Agarose with bonded antibodies against human somato-mammotropin (HCS) was used by Weintraub for the preparation of HCS, labelled and non-labelled, for radioimmunoassays. The same sorbent was used by Guyda and Friesen for the elimination of 99% of growth hormone activity from the homogenate of simian hypophyses. An example of group isolation is the isolation of glycoproteins according to Aspberg and Porath. Glycoproteins undergo binding with concanavalin A and agarose is used as the support, and today, commercial preparations of agarose-bound concanavalin A are available for affinity chromatography. The binding site of concanavalin A is specific for a-D-glucosyl, a-D-mannosyl and other sterically similar residues that are usually present in glycoproteins. Allan et al. isolated, by affinity chromatography on concanavalin A-sorbent, glycoprotein receptors of concanavalin from the membranes of porcine lymphocytes of plasma in a medium containing sodium deoxycholate. Instead of the preparation of adsorbent according to Allan et al., it is also possible to use commercially available preparations, for example Con A-Sepharose, (Pharmacia, Uppsala, Sweden) and Glycosylex A (Miles-Seravac, Lausanne, Switzerland). The separation of glycoproteinic receptors of concanavalin A according to Allan et al. is carried out as follows. Porcine lymphocyte plasma membranes (20 mg of protein) are extracted with 5 ml of 1% sodium deoxycholate and the soluble fraction, forming approximately 85% of the membrane protein, is washed through the adsorbent column and eluted with 1% deoxycholate until the extinction at 280 nm reassumes the value of the blank. Approximately 75%of the added proteins are eluted. The receptor glycoproteins (5% of the content of membrane proteins) are eluted with a solution of methyla-D-glucoReferences p.805
800
PROTEINS
pyranoside (20 mg/ml in 1% deoxycholate). The eluted material is precipitated by addition of one tenth of the volume of 2% acetic acid and the precipitate is extracted three times with 10-ml portions of 95% ethanol in order to eliminate the sugar and deoxycholic acid. The preparation, dried in a current of air, contains five components of glycoproteins that can be resolved by polyacrylamide gel electrophoresis in 0.1% sodium dodecylsulphate.
DETECTION OF PROTEINS IN THE EFFLUENT Colorimetric detection The modification by Lowry er al. of the colorimetric determination of proteins with the phenolic reagent described by Folin and Ciocalteau and the biuret reaction is a sensitive and universal colorimetric detection method that is very suitable for following the protein concentration in the effluent; it permits the determination of down to 5-100 pg of protein in 1 ml. The blue-violet coloration formed is sufficiently stable and the reaction can be carried out not only on single fractions but also in a continuous flowthrough system with adjusted reagent concentrations. The detection method of Lowry et al. is about 100 times more sensitive than the biuret reaction, and the ninhydrin colorimetric method also does not attain the sensitivity of the method of Lowry er al. without previous alkaline hydrolysis of the sample. Thus, in a 0.05-ml micro-cell an amount of less than 0.02 pg can be determined. The colour intensity depends on the tyrosine content, as in the original Folin reaction. The method is empirical and for quantitative determinations calibration with a protein, the concentration of which is determined by spme other method, is necessary. The calibration is carried out in the same system as that used for the actual determination. The colorimetry of soluble proteins according to Lowry et al. is carried out as follows. A 1-ml volume of protein solution and 5 ml of solution C are mixed and allowed to stand at room temperature for at least 10 min. Solution D (0.5 ml) is added rapidly and the mixture stirred immediately (within 1 to 2 sec). After standing for 30 min, colorimetry is carried out either at a wavelength of 500 nm if the protein Concentration is high, or at 750 nm if the concentration is low. Bovine or human serum albumin at concentrations from 0.02 to 0.5 rng/ml can be used as a standard. Solution A is 2% of sodium carbonate in 0.1 N sodium hydroxide; B is 0.5% of copper(I1) sulphate (CuS04. 5 HzO) in 1%sodium tartrate (or 1% sodium potassium tartrate); C is 50 ml of A plus 1 ml of B; and D is 1 ml of concentrated Folin-Ciocalteau reagent (diluted to a concentration corresponding t o 1 N acid). The phenol reagent is prepared according to Folin and Ciocalteau as follows. Ammonium molybdate ((NH4)z Moo4 * 2H20; 25 g) and 100 g of sodium tungstate (Naz W 0 4 .2Hz0) are mixed with 700 ml of water in a 1500-ml flask, 50 ml of 85% phosphoric acid and 100 ml of concentrated hydrochloric acid are added and the mixture is refluxed for 10 h. Then 150 g of lithium sulphate are added to the boiling solution, followed by a few drops of bromine solution and 50 ml of water. The mixture is boiled for a further 15 min without a reflux condenser. When all of the excess of bromine has been boiled off, the reaction mixture is cooled, made up to 1 1 and filtered. The filtrate should not be greenish, as interference in the colorimetry will result.
DETECTION OF PROTEINS IN THE EFFLUENT
80 1
In the biuret reaction, on reaction of Cu2+with the -NH-CO- group, which is characteristic of proteins and peptides, a complex is formed the absorption maximum of which is about 555 nm. The colour intensity is not affected by ammonium salts and for all proteins it is dependent only on the number of peptide groups. The biuret method is suitable for the determination of higher concentrations of proteins (0.25-25 mglml). The colour yield is standardized with pure protein. The biuret reaction can also be carried out in a continuous flow-through system. The procedure is as follows: 0.1-4 ml protein solution containing 1-5 mg of protein is made up to 5 ml with solution A, then 5 ml of solution B are added and the mixture is heated on a water-bath at 32°C for 30 min, the resulting blue-violet coloration being measured at 5 5 5 nm. Solution A is 0.85%sodium chloride solution. Solution B comprises 45 g of sodium potassium tartrate, 15 g of copper(I1) sulphate (CuSO, . 5H20), 5 g of potassium iodide, and 0.2 M sodium hydroxide solution (without carbonate), prepared as follows. After the dissolution of the sodium potassium tartrate, copper(I1) sulphate is added while stirring until it is dissolved. Finally, potassium iodide is added and the mixture is made up to 1 1 with 0.2 M sodium hydroxide solution. Sodium hydroxide solution without carbonate can be prepared by heating 50% sodium hydroxide solution at 90°C for 24 h and, after sedimention of sodium carbonate, diluting the clear solution with boiled water.
Spectrophot ometric detection The principle of the spectrophotometric detection of protein and peptide solutions consists in the measurement of the absorbance of the solutions in the region of the absorption maximum. Most proteins contain tyrosine, phenylalanine and, at lower concentrations, also tryptophan, in the form of amino acid residues. The absorption maxima of these residues appear in the short-wave region of the W spectrum, and absorptions characteristic of the peptide bond, the helical structure of the peptide chain, disulphide bonds of cystine residues, etc., also appear in the same part of the spectrum. For practical determinations of the concentration of proteins, the absorption maxima of tyrosine and tryptophan are the most important. The relatively broad maximum enables the determination of the absorbance at a wavelength of 275-280 nm. Above 280 nm, substantial changes in absorbance may take place with proteins as a result of the effect of pH, because the ionization of the tyrosine hydroxyl group in strongly alkaline conditions causes both a change in the extinction coefficient and a shift of the absorption maximum to higher wavelengths. The changes in the extinction coefficient of tyrosine as a function of pH are evident from Table 35.7. The shift in the absorption maximum of tryptophan in the same pH interval is substantially less and so is the change of the absolute molar extinction coefficient E . For 0.1 N hydrochloric acid, the Lax. of tryptophan is 278 nm ( E = 5450), while for 0.1 N sodium hydroxide solution A,,,. is 280.5 nm ( E = 5250). In the direction of shorter wavelengths, a minimum appears in the spectrum of proteins, followed by a steep increase in absorbance. If an eluent with a low absorbance is used, the sensitivity of detection may be increased several times when measuring below 235 nm in comparison with measurement at 280 nm. Measurements at these wavelengths can be made on spectrophotometers of average quality. An example is shown in Fig. 35.5. References p . 805
802
PROTEINS
TABLE 35.1 VARIATION OF THE MOLAR EXTINCTION COEFFICIENT pH of medium
E
1500 1300 2600
(E)
OF TYROSINE WITH pH
hmax.(nm)
1.09* 8.0 12
211.7 275 293
*O. 1N HCI W v)
--_- 230 nm
-4.0
280 nrn
2K
- 3.0 Mz - 2.0 ,2 o a
0
60
180
300 420 EFFLUENT VOLUME, ml
540
Fig. 35.5. Separation of thyrocalcitonin concentrate by partition chromatography on Sephadex (3-25 (Hawker e t al.). Example of differences in absorbance at 280 and 230 nm. Vertical lines are the bioassay responses. Eluent: n-butanol-acetic acid-water (7: 1:9).
At 210-220 nm, the detection of proteins is very specific and sensitive. However, buffers in which the components contain carbonyl groups are unsuitable for use in the 210-220 nm region and the choice of components for the buffered eluents is limited. For the 192-194 nm region, in which the presence of substances that contain peptide bonds can be determined specifically, only aqueous solutions of alkali metal fluorides can be used. The importance of the spectrophotometric determination of proteins at wavelengths below 210 nm is limited to special cases of GPC. The sensitivity of the spectrophotometric determination at 210 nm is comparable with the sensitivity of the colorimetric detection of Lowry et al. Tombs et al. mentioned a sensitivity of 2 pg/ml for serum proteins. The absorbance is due mainly to the peptide bond and therefore proteins have a similar extinction coefficient (see Table 35.8). In the presence of UV-absorbing impurities which interfere at 260-280 nm, i.e., substances with a typical spectrum of nucleic acids, the absorbance at 220 nm is almost identical with that in the colorimetric determination of proteins according to Lowry et af. Wrigley and Webster have shown that at 220 nm, succinate, phthalate and barbiturate buffers cannot be used, while sodium hydroxide solution, acetate, glycine and Tris buffers can be used up to a concentration of 0.01 M.Sodium chloride solution, cacodylate, borate, phosphate and ammonium sulphate can be used even at concentrations above 0.1 M. For proteins that contain, in addition to aromatic amino acids, further groups with characteristic absorption maximum bands in the long-wave UV region or bands in the
803
DETECTION OF PROTEINS IN THE EFFLUENT
TABLE 35.8 COMPARISON OF EXTINCTION COEFFICIENTS (E.;?,,) OF; PROTEINS AT WAVELENGTHS OF 280 AND 210 nm (TOMBS e t a [ . ) Protein
Human serum albumin Human immunoglobulin Bovine serum albumin Human siderophilin
El%
cm 280 nni 1
6 15 6.8
210 nm 203 213 204 200
14
visible region are utilized for detection. The requirement is that the groups with distinct light absorption should remain firmly bound to apoprotein in the course of the separation process. The measurement of absorbance in the long-wave UV region is used for the sensitive detection of some enzymes with firmly bound prosthetic groups. In the visible region, some types of metalloproteins may be detected by absorption spectrophotometry, especially those which contain complexed iron, copper, chromium, vanadium, etc. Characteristic absorption maxima of some metalloproteins are listed in Table 35.9. TABLE 35.9 CHARACTERISTIC ABSORPTION MAXIMA O F SOME METALLOPROTEINS IN THE VISIBLE REGION OF THE SPECTRUM Metalloproteins Haemoproteins Haemoglobin (human) Haemovanadin Cy tochromes Haemocyanins Caeruloplasrnin Plastocyanin Ery throcuprein
hm,,,(nm) 412 415
425 550-560 563-580 605-610 591 655
Extinction coefficients and wavelengths of maximum absorption are dependent o n the oxidation state of the metal. The ratio of absorbance in the visible and the U V regions, i.e., Avieble/Azso,is used as a criterion for the purity of metalloproteins. If the solutions are turbid, the true value of the optical density at 280 nm may be determined by means of optical density values in the visible region of the spectrum, by extrapolating the plot of log O.D. versus log h. The contribution of light scattering may represent 50-60% of the effective optical density at 280 nm.
Detection by ultraviolet fluorescence The principle of this method consists in the measurement of the ultraviolet radiation emitted at 340-350 nm due to the excitation of tryptophan or tyrosine in the protein References p.805
804
PROTEINS
molecule by radiation of a shorter wavelength (about 280-290 nm). The greatest part of the emission is due to the tryptophan residue, in relation to the molecular structure and the ionization state of the protein. The fluorescence of the phenylalanine residue is less important, especially in the presence of tyrosine and tryptophan in the protein molecule. The sensitivity of the detection by fluorescence is approximately 1000 times higher than that based on absorption at 280 nm; however, the method is much more demanding with respect to the purity and properties of the eluent. A complication in the application of the method arises as a result of individual changes in the dependence of the fluorescence intensity on pH in various types of proteins. Another method of making use of the fluorescence measurements and thus increasing the sensitivity of detection independently of the presence of tryptophan or tyrosine and phenylalanine in the protein consists in labelling the proteins with fluorescent reagents, such as fluorescein isothiocyanate or 5-dimethylaminonaphthalene-1-sulphonyl chloride (Dns chloride). This method was used for labelling antibodies, for example, by Coldstein ef al. and Rinderknecht. Antibodies labelled with a fluorescent group can be further utilized for the detection of antigens. In all methods in which UV light or UV fluorescence is used for measurements, it should be borne in mind that short-wave UV radiation, if sufficiently intense. may damage the protein molecule, which may also undergo photo-oxidation. The energy of the absorbed light should therefore be as low as possible and the time 01- -.-osure as short as possible.
Automation of spectrophotometric detection Automation of the detection process can be based either on the less often used fractional principle as proposed, for example, by Croulade e t a/. , or on the flowthrough principle. The most commonly used apparatus for continuous control of the process of protein separation is the absorptiometer for the UV region of the spectrum. Its function is based mainly on the presence of tyrosine, tryptophan and phenylalanine in proteins. Currently used apparatus is of simpler single-beam construction and transmittance recording, serving as semi-quantitative indicators of the presence of proteins, while less commonly used are the more efficient double-beam absorptiometers, which permit higher amplification with lower noise at the zero line. Current types of the UV absorptiometers are provided with low-pressure mercury lamps emitting at 253.7 nm. The dose-of the UV radiation energy is about lo-" Einstein/ min, i.e.,approximately 0.09 pW.For specific protein detection, the radiation of 280 nm obtained by means of a fluorescent transformer (lead-glass or crystal) is most convenient. If the sensitivity requirements, and especially the requirements placed on the specificity of detection, are lower, absorptiometers for 253.7 nm can also be used. If the recording is carried out simultaneously at 253.7 and 280 nm, it is possible to estimate, according to Thacker et al., the purity of the protein eluates on the basis of the ratio of absorbances, Azso/Az53.7, from the point of view of contamination with the nucleic acid components that often accompany complex mixtures of proteins. A simple apparatus for the flow-through detection of proteins at 280 nm, fitted with a magnesium spectral lamp, was proposed by Bennett ef al. More efficient types of UV absorptiometers, provided with a deuterium lamp that affords a continuous spectrum in the UV region, or with a monochromator and a logarithmic amplifier of the transmittance
805
REFERENCES
signal (Hoffmann), are almost as useful as UV spectrophotometers, because they give a direct record of the optical density. Although the capacity of flow-through cells represents a volume of only 10- 10- ml, the volume of the quartz flow-through cell and the necessary hydraulic system always causes broadening (tailing) of the protein peak. In the region of the maximum protein peak, especially at higher protein concentrations, the density of the eluent changes abruptly. Hence, flow-through detection on the effluent is suitable in instances when a direct fractional determination of the protein concentration is not possible for reasons of volume or time. Flow-through detection is indispensable in the recycling process of GPC.
'-
REFERENCES Aisen, P., Leibman, A. and Reich, H. A., J. Biol. Chem., 241 (1966) 1666. Allan, D., Auger, J. and Crumpton, M . J., Nature (London), 236 (1972) 23. Andrews, P., Biochem. J., 91 (1964) 222. Andrcws, P., Biochem. J., 96 (1965) 595. Andrews, P., Methods Biochem. Anal., 18 (1970) 1. Aspberg, K. and Porath, J., Acta Chem. Scand., 24 (1970) 1839. Beers, W. H. and Reich. E., J. Biol. Chem., 244 (1969) 4473. Bell, P. H., Colucci, D. F., Dziobkowski, C., Snedeker, E. H., Barg, Jr., W . F. and Paul, R., Brochemistry, 9 (1970) 1665. Bennett, P. A., Sullivan, J. V. and Walsh, A., Anal. Biochem., 36 (1970) 123. Bernardi. G.,Methods Enzymol., 22 (1971) 325. Bidlingmayer, B. A. and Rogers, L. B., Anal. Chem., 43 (1971) 1882. Bryce, C. F. A. and Crichton, R. R., J. Chromatogr., 63 (1971) 267. Carey, W. F. and Wells, J. R., Biochem. B i y h y s . Res. Commun., 4 1 (1970) 574. Cechovi, D., Jonikovi-Svestkovi, V. and Sorm, F., Collect. Czech. Chem. Commun., 35 (1970) 3085. Clark-Walker, G. D. and Lascelles, J., Arch. Biochem. Biophys., 136 (1970) 153. Cuatrecasas, P., J. Biol. Chem., 245 (1970) 3059. Cuatrecasas, P., Aduan. Enzymol., 36 (1972) 30. Dasgupta, B. R., Berry, L. J . and Borroff, D. A., Biochim. Biophys. Acta, 214 (1970) 343. Dixon, H. B. F. and Thompson, C. M., Biochem. J . , 107 (1968) 427. Dozy, A. M. and Huisman, H. J., J. Cbromutogr., 40 (1969) 62. Fernlund, P., Biochim. Biophys. Acra, 237 (1971) 519. Folin, 0. and Ciocalteau, V., J. Biol. Chem., 73 (1927) 627. Frattali, V. and Steiner, R. F., Biochemistry, 7 (1968) 521. Canguly, P., J. Biol. Clzem., 247 (1972) 1809. Goldstein, G., Slizyo, 1. S. and Chase, M. W., J. Exp. Med., 114 (1961) 89. Grif, L., Cseh, G. and Medzihradszky-Schweiger, H., Biochim. Biophys. Acta, 175 (1969) 444. Groulade, J., Chicault, M. and Waltzinger, W., Bull. SOC. Chim. Biol., 49 (1967) 1609. Guyda, H. and Friesen, H., Biochem. Biophys. Res. Commun., 41 (1971) 1068. Haller, W., Tympner, K. D. and Hannig, K., Anal. Biochem., 35 (1970) 23. Hawker, C. D., Rasmussen, H. and Glass, J. D., Proc. Nut. Acad. Sci. US.,58 (1967) 1535. Hennen, G., Prusik, Z. and Maghuin-Rogister, G., Eur. J. Biochem., 18 (1971) 376. Hochstrasser, K., Werle, E., Siegelmann, R. and Schwarz, S., Hoppe-Seyler's Z. Physiol. Chem., 350 (1969) 897. Hoffmann, L. G., J. Chromatogr., 40 (1969) 39. Horton, B. F. and Chernoff, A. I., J. Chromatogr., 47 (1970) 493. Hussa, R. O., Landon, J. and Winnick, T., Biochem. J . , 114 (1969) 519. Hwang, P., Guyda, H. and Friesen, H., J. Biol. Chem., 247 (1972) 1955. Ingwall, J . S. and Scheraga, H. A., Biochemistry, 8 (1969) 1860. Ito, A. and Sato, R., J. Biol. Chem., 243 (1968) 4922.
806
PROTEINS
Iwamoto, M. and Abiko, Y., Biochim. Biophys. Acta, 214 (1970) 402. Josefsson, L., Biochim. Biophys. Acra, 148 (1967) 300. Joustra, M. and Lundgren, H., Annual Colloquium, Protides of Biological Fluids, Brugge, 17 (1969) 5 11 Keil, B., Keilovi, H. and HartoEk, I., Collect. Czech. Chem. Commun., 27 (1962) 2940. Kerr, M. S., Comp. Biochem. Physiol., 34 (1970) 301. Kitamura, M., Sakaguchi, S. and Sakaguchi, G., Biochim. Biophys. Acta, 168 (1968) 207. Koike, M. and Hamada, M., Methods Enzymol. 22 (1971) 339. Kusel, J . P., Suriano, J . R. and Webcr, M. M., Arch. Biochem. Biophys., 133 (1969) 293. Lampson, G. P. and Tytell, A. A., Anal. Biochem., 11 (1965) 374. Lindsay, K. H., Starnes, W. K., Hershman, J. M. and Pittmann, J. A., Proc. Soc. Exp. Biol. Med., 131 (1969) 875. Lohmar, P. and Chom Hao Li, Biochim. Biophys. Acta, 147 (1967) 381. Lowry, H. O., Rosebrough, N. J . , Farr, A. L. and Randall, R. J., J. Biol. Chem., 193 (1951) 265. Miranda, F., Kupeyan, Ch., Rochat, H., Rochat, C. and Lissitzky, S., Eur. J. Biochem., 16 (1970) 514. Morivek, L.,J. Chromatogr., 59 (1971) 343. Morgan, R. G . H., Barrowman, J . and Borgstrom, B., Biochim. Biophys. Acra, 175 (1969) 65. Mosesson, M. W., Alkjaersig, N . , Sweet, B. and Sherry, S., Biochemistry, 6 (1967) 3279. Nagasawa, S. and Suzuki, T., J. Biochem. (Tokyo), 66 (1969) 273. Novotny, J., FEBS Lett., 14 (1971) 7. Okazaki, T., Wittenberg, B. A., Briehl, R. W. and Wittenberg, J. B., Biochim. Biophys. Acta, 140 (1967) 258. Pallotta, D. and Berlowitz, L., Biochim. Biophys. Acta, 200 (1970) 538. Paul, K. G . and Stigbrand, T., Biochim. Biophys. Acta, 221 (1970) 255. Peckham, W. D. and Parlow, A. F., Endocrinology, 84 (1969) 953. Peisaca, I., Levine, W. G . and Blumberg, W. E., J. Biol. Chem., 242 (1967) 2847. Pharmacia, Supplement to Sephadex Ion Exchanger Booklet, Pharmacia, Uppsala, Sweden, 1969, pp. 2-3. Pharmacia, Sephadex-Ionenaustauscher (Leitfaden zur Ionenaustausch- Chromatographie] Pharmacia, Uppsala, Sweden, 1970, p. 12. Porath, J . , Nature (London), 196 (1962) 47. Porath, J . and Bennich, H., Arch. Biochem. Biophys., Suppl., 1 (1962) 152. Porath, J. and Fryklund, L., Nature (London), 226 (1970) 1169. Price, V. E. and Greenfield, R. E., J. Biol. Chem., 209 (1954) 365. Rathnam, P. and Saxena, B. B.,J. Biol. Chem., 245 (1970) 3725. Read, K. R. H., Comp. Biochem. Physiol., 22 (1967) 1. Reeve Angel & Co., Technical Bulletin 1032, 2000 and 15 M, H. Reeve Angel & Co., Clifton, N.J., U.S.A. Reiland, J . , Methods Enzymol. 22 (1971) 287. Rinderknecht, H., Nature (London), 193 (1962) 167. Sadgopal, A. and Bonner, J., Biochim. Biophys. Acta, 207 (1970) 206. Saffran, M., Muhlstock, B. and Caplan, B., Can. J. Biochem. Physiol., 39 (1961) 653. Samuelsson, G. and Pettersson, B.,Acta Chim. Scand., 24 (1970) 2751. Shipman, W. H. and Cole, L. J., Anal. Biochem., 29 (1969) 490. Swanljung, P., Anal. Biochem., 43 (1971) 382. Swingle, F. M. and Tiselius, A., Biochem. J., 48 (1951) 171. Thacker, L. H., Scott, C. D. and Pitt, W. W., J. Chromatogr., 5 1 (1970) 175. Tiselius, A., Hjertkn, S. and Levin, O., Arch. Biochem. Biophys., 65 (1956) 132. Tombs, M. P., Souter, F. and Macbdgan, N. F., Biochem. J., 73 (1959) 167. Vogt, W., Biochem Pharmacol., 17 (1968) 727. Wallen, P. and Wiman, B., Biochim. Biophys. Acta, 221 (1970) 20. Weintraub, B., Biochem. Biophys. Res. Commun., 39 (1970) 83. Wrigley, C. V. and Webster, H. L., J. C;promatogr., 33 (1968) 534. Yang, H., J. Biol. Chem., 245 (1970) 6404. Zahler, P. H. and Wallach, D. F. H., Biochim. Biophys. Acta, 135 (1967) 371. '
I
Chapter 36
Enzymes 0. MIKES
CONTENTS Special requirements for the chromatography of enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801 Techniques and automated analyses. . . . . . . . . . . . . . . . . . ............... . . . . . . . . . . . . .809 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813 Oxidoreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816 Hydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818 823 Lyases ....................................................................... Isomerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825 Ligases . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
SPECIAL REQUIREMENTS FOR THE CHROMATOGRAPHY OF ENZYMES Enzymes differ from many other natural substances in their fragile niacromolecular polyvalent amphoteric nature and from other proteins in their specific topography, reflecting their particular activities . The special, very susceptible and complicated structure of enzymes must be kept in mind when methods are selected for their isolation and fractionation. Some types of enzymes very easily change their tertiary structure, which is accompanied by a loss in activity, often irreversible. The ever-present risk of the denaturation of enzymes is generally known. The surface denaturation of enzymes in contact with unsuitable chromatographic sorbents that allow strong hydrophobic contacts has often been observed; adsorbents of a hydrophilic nature are preferably used. The researcher will certainly not expose enzyme preparations t o extreme conditions of hcat, pH, oxidizing or reducing agents, detergents or unsuitable organic solvents. However, what may be neglected are slight changes in conformation of enzymes which are not connected with a loss of activity, but which modify the enzymes so that they then differ from the products of proteosynthesis. For example, Sipos and Merkel found that exposure of protease from marine bacteria t o an increase in temperature in the presence of Ca2+ changes the optimum temperature of this enzyme, and the same was later found t o be true of bovine trypsin and chymotrypsin. A similar influence of some ions on amylase activity is also known. Mike; et al. found changes in the optimum pH of digestion of haemoglobin with alkaline protease from Aspergillus f l a w s exposed for a certain time to an elevated temperature in the presence of various additives. These transformations, which lead t o polymorphism of certain enzymes, also take place t o a certain extent at ambient temperature but not at 4°C. In most instances, it is important when treating enzyme preparations to work in a laboratory at a temperature of just above O"C, as the risks of References p. 829
807
ENZYMES
Fig. 36.1. Principle of the anaerobic column system (Repaske). A, plastic bottle as a reservoir of gas; B, buffer reservoir for stepwise elution or a gradient device; C, chromatographic column; D, serum bottle with hypodermic needles for collecting fractions; e, calibrated end unit; g, stream of oxygenfree gas; m,magnetic stirrer; s, stopcocks.
enzyme autolysis, of proteolytic attack on raw enzymic preparations or of microbial contamination are minimized at this temperature. Another general rule is to work as rapidly as possible. Other risks arise from the sensitivity of many enzymes to the ions of heavy metals. Therefore deionized water or water distilled from glass should be used for all extractions and in the preparation of all buffers. Special ion-exchange treatment is necessary in some instances. Ions extracted from the walls of the vessels used sometimes influence the results and bottles coated with silicone films should be used in such instances. Some enzymes are sensitive to the method of concentration and cannot even be lyophilized. The stability of the enzyme studied should certainly be examined at the beginning of any work. Specific operating procedures must be followed in special instances, for example, contact with oxygen in the air must be avoided with some enzymic systems (hydrogenases, enzymes of methane bacteria, for the study of enzymic reduction and oxidation of various substrates, enzymic systems of anaerobes and of anaerobic mutants, experiments involving
TECHNIQUES AND AUTOMATED ANALYSES
809
the reduction of disulphide bridges in enzymes, etc.). All of these procedures require special conditions, e.g., anaerobic glove-boxes. Column chromatography of oxygen-sensitive enzymes requires a strict anaerobic technique. Repaske described eqxpment for the chromatography of hydrogenases with a 90% recovery (Fig. 36.1). All spaces and lines are carefully washed with a suitable oxygenfree gas, introduced into the system through bottle A, the same gas is bubbled through the buffers used before application to the column and the slurry of the sorbent is carefully introduced into the column using an anaerobic atmosphere. Also, the collection and preservation of fractions is performed under oxygen-free conditions. This or similar equipment cannot be replaced by the usual chromatographic apparatus in which reducing agents in elution buffers are used, as very low recoveries result. For longer systematic experiments with oxygen-sensitive enzymes, an anaerobic laboratory has many advantages, allowing greater versatility and the easy utilization of a long sequence of different methods without contact with the outer atmosphere. The construction of such an anaerobic chamber with all of the necessary equipment was described by Poston et al., who discussed the anaerobic facilities at the National Institute of Health, Bethesda, Md., U.S.A. The laboratory is systematically washed with nitrogen and the Last traces of oxygen are removed by reaction with hydrogen on a catalyst bed. Persons who work in the laboratory, of course, must wear masks supplying them with air and are connected to a vacuum line for removing exhaled air. Normal laboratory equipment and methods can be used in such a chamber.
TECHNIQUES AND AUTOMATED ANALYSES Many effective methods have been developed for the separation of enzymes from various components of living matter and for their fractionation and concentration. These methods include the following: crystallization (Davies and Segal, Jakoby, Zeppezauer); dialysis, ultrafiltration and hollow-fibre techniques (Blatt, McPhie, Rony); electrophoretic methods (Prusi’k, Shuster, Vesterberg, and Chapter 35); extraction (Penefsky and Tzagoloff, Tzagoloff and Penefsky); lyophilization (Everse and Stolzenbach); solvent precipitation and methods involving the use of soluble non-ionic polymers (Fried and Chun, Kaufman); and zonal centrifugation (Cline and Ryel). The most important and most often used fractionation method for the preparation and analysis of enzymes is chromatography. A bibliography on column chromatography by Deyl et al. covers the period from 1967 to 1970 and contains 1687 references to papers dealing with enzymes; this was extended to 1972 by 131 references to papers using affinity chromatography for the isolation of enzymes by Turkovi in Chapters 7 and 14, and elsewhere. In papers cited by both authors, 3288 different column chromatographic experiments were described. It may be interesting for the reader to compare the survey of the different methods in Table 36.1, but it should be noted that in fact the proportion of individual methods changes with time. In recent years. great progress has been made in the use of affinity chromatography. Recent references to particular methods are as follows: affinity chromatography (Cuatrecasas, Cuatrecasas and Anfinsen (197 1a, b), Feinstein, Porath, Turkovi, and Chapters 7 and 14); chromatography on hydroxyapatite References p.829
810
ENZYMES
TABLE 36.1 SURVEY OF USE O F DIFFERENT CHROMATOGRAPHIC METHODS FOR THE FRACTIONATION OF ENZYMES Calculated from the bibhagraphic data collected by Deyl e t al., Turkova, and those given in Chapters 7 and 14. Method Liquid-sotid chromatography (aluminium oxide, brushite, calcium phosphate modifications, hydroxyapatite) Liquid-liquid chromatography (Celite, cellulose, silica gel, starch) Gel permeation chromatography (agarose, polyacrylarnide gel, polydex tran) Ionexchanging derivatives of cellulose Ionexchanging derivatives of polydextran Ion-exchange resins Affinity Chromatography Other methods
Percentage 7.2 0.4 41.5
30.5 11.8 4.3 4.0 0.3
(Bernardi, and Chapter 35); chromatography on calcium phosphate gel (Koike and Hamada, and Chapter 35); detergent gradient gel chromatography - technique for the purification of membrane-bound enzymes (Swanljung, Swanljung and Frigeri); gel permeation chromatography (Reiland, and Chapters 5 and 12); electrochromatographic methods (Prusi’k); ion-exchange chromatography (Himmelhoch, Mikes, and Chapters 6 and 13); and substrate-specific elution (Pogel and Sarngadharan). The last method, specific elution with substrate, is a combination of classic chromatographic sorption with specific desorption, which has the benefit of specific enzymesubstrate, enzyme-inhibitor or enzyme-effector interactions. The enzyme is bound, for example, on an ion exchanger, in the usual way. The desorption is effected by a gradient of the substrate concentration in addition to the usual ionic strength gradient. Only the particular enzyme that is capable of specific interaction with the substrate changes its tertiary structure a little, while other proteins and enzymes are not influenced. The specific interaction modifies topographic parameters (e.g., the number of charged groups on the surface of the enzyme and the number of hydrophobic contacts), which is reflected in changes in binding forces to the ion exchanger or other sorbent. The final effect is specific desorption. Examples of this modern method for the specific separation of enzymes are the purification of chicken pancreatic ribonuclease by elution from cellulose phosphate with ribonucleic acid (Eley) and the purification of rabbit liver fructose-l,6-diphosphataseon CM-cellulose (Sarngadharan et d.).The efficiency of the method can be seen from the next example: in the purification of glucose-6-phosphate dehydrogenase by elution of the enzyme from CM-Sephadex with 2 mM glucose-6-phosphate (Rattazzi), a 5 1-fold increase in specific activity was achieved with 91% recovery of the enzyme activity. The need to repeat individual enzyme experiments using standard procedures led to the development of the automation of enzyme analysis, which has great importance primarily for clinical biochemists. However, research biochemists often must repeat large
81 1
TECHNIQUES AND AUTOMATED ANALYSES
REFRIGERANT
COOLER A N D
MASTER SAMPLER
WXER
WffiTE
Fig. 36.2. Schematic diagram of single flow path autoanalytical system for enzymes based on Technicon AutoAnalyzer components and used for the monitoring of column chromatographeffluents (example compiled according t o Beck and Tappel, Eveleigh el al. and Tappel). Solid Lines, Liquid flow; broken Lines, refrigerant flow; dotted Lines, electrical control. The slaved sampler can be replaced with a substrate refrigerator, programmed multichannel valve and distributor, w h c h allow increased substrate capacity.
numbers of experiments, which can hardly be realized without automation. The first approaches to this problem were made several years ago (cf , Moss, Schwartz et al., VeEerek et al., and others). The present apparatus (still being developed) allows the complete automation of processes, including calculation and presentation of results and feedback control of instruments by computer. A recent survey of automated instruments, allowing continuous flow analysis, discrete sample analysis and continuous kinetic experiments, was given by Schwartz (197 1). A survey of instruments allowing multiple enzyme analyses was presented by Tappel. Instruments available for automated analysis of discrete samples have been reviewed by Alpert. In most automated enzyme experiments that have been described, the Technicon AutoAnalyzer (Schwartz, 1968) was used, which can be adapted for the analysis of all types of enzymes (cf ,Wacker and Coombs) and, by References p . 829
812
ENZYMES
using a sample splitter, also for the simultaneous analysis of several enzymes in the same sample (Smythe et d.). The systems for automated multiple enzyme analysis are applicable to continuous monitoring of column chromatograph effluents (c$, Beck and Tappel) in addition to other functions. The principle or such apparatus (c$ , Bradley and Tappel, Eveleigh et a/.) is illustrated by Fig. 36.2. The master sampler acts as a column fraction collector. The on-off timer distributes the appropriate amounts of enzymes and substrates to the mixer. The air bubbles split the stream of solution into well mixed segments, which are then heated in the heating bath. After mixing with a suitable reagent, which terminates the enzyme reaction, and developing the chromatotropic product, and after deaeration, the streams flow through colorimeters. In order to measure peak absorbances accurately, part of the concentrated stream effluent is then diluted with water (or with a suitable diluent) in a fixed proportion and measured again. This system was used for the simultaneous determination of six enzyme activities (acid phosphatase, a-D-gdactosidase, 0-D-galactosidase, 0-D-glucuronidase, aryl sulphatase and N-acetyl-0-D-glucosaminidase) in the soluble enzyme fraction of rat liver lysozymes chromatographed on a CM-cellulose column. Only about one quarter of each 7.7-ml fraction was consumed in this determination (Beck and Tappel). The method was improved (Tappel) in order to allow further simultaneous determinations (additional activities: a-L-fucosidase, 5'-phosphodiesterase I, 5'-phosphodiesterase IV, a-D-glucosidase, 0-D-glucosidase and a-D -mannosidase) directly in the soluble fraction of rat liver lysozomes, omitting chromatography. Fig. 36.3 illustrates the results obtained. Examples ot purification or tractionation of particular enzymes using the various methods mentioned above are described below in greater detail. As an introduction
03
-
05 -
09 -
! a H9
0.3
I
a2-
I
I Ik I
e
MINUTES
Fig. 36.3. Automatic determination of the activities of 1 1 hydrolytic enzymes from rat liver lysozymes (Tappel). Solid line, absorbance at 420 nm; broken line, absorbance at 505 nm. a, a-L-fucosidase; b, 5'-phosphodiesterase 1; c, 5'-phosphodiesterase IV; d, a-D-glucosidase; e , a-D-galactosidase; f, p-Dgalactosidase; g, 0-D-glucosidase; h, a-D-mannosidase;i, p-D-glucuronidase;j, N-acetyl-p-D-glucosaminidase; k, arylsulphatase.
813
OXIDOREDUCTASES TABLE 36.2 SURVEY O F DIFFERENT TYPES O F ENZYMES FRACTIONATED BY COLUMN CHROMATOGRAPHIC METHODS (DEY L e t al.) Enzyme type
Proportion of published papers (70)
E.C.1: oxidoreductases E.C.2: transferases E.C.3: hydrolases E.C.4: lyases E.C.5: isomerases E.C.6: ligases Other enzymic preparations
19.0 22.4 36.8 1.3 1.4 5.6 1.5
Table 36.2 surveys the proportions of published papers that describe column chromatographic methods for the treatment of the main classes of enzymes.
OXIDOREDUCTASES The preparation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pseudomonas using a column of hydroxyapatite gel and stepwise elution was described by Bensch and Rodwell. Their procedure is an example of a successful fractionation using reducing additives in the preparation of a reductase. The cells of Pseudomonas were grown aerobically at 30°C and pH 7 in a medium containing 12 mh4 ammonium DL-mevalonate and collected from the stationary phase. All other manipulations were carried out at 0-5°C. The washed, centrifuged and frozen cells retained full activity for several months. The turbid supernatant of sonically disrupted cells (50 mM Tris-hydrochloric acid, pH 7.1 ; 1.2 mM EDTA) was centrifuged at 60,OOOg and fractionated using ammonium sulphate (25-35% of ammonium sulphate precipitate contains the enzyme). This preparation can be stored frozen for several weeks. After dissolving the preparation in 10 mM potassium phosphate solution (pH 6.8), 10 /.IM in dithiothreitol, the solution was dialyzed overnight against this buffer (the tubing was previously boiled in 1 mM EDTA) and subjected to chromatography. A 6.0 X 2.5 cm column of Bio-Gel HT hydroxyapatite gel was equilibrated with 10 mh4 potassium phosphate solution (pH 6.8), 10 /.IM in dithiothreitol. After the application of the sample, the column was washed with 30 ml of the above buffer (one column volume) and eluted successively with 60-ml portions of potassium phosphate solution (pH 6.8), 10 pM in dithiothreitol and 20, 28 and 40 mh4 in total phosphate. Fractions of 3 ml were collected and combined according to the scheme illustrated in Table 36.3. The combined fractions 42-48 were made 2.5 mM in EDTA and brought to 40% saturation in ammonium sulphate. The precipitate was dissolved in 100 mM Tris-hydrochloric acid (pH 7.1), 2.5 mM in EDTA and 1 O f l in dithiothreitol and dialyzed against the same buffer. This purified oxidoreductase loses activity rapidly in the absence of EDTA or dithiothreitol. Fractions 49-70 can be dialyzed and re-chromatographed on hydroxyapatite. Gradient elution gave an improved yield but only half of the final specific activity. References p.829
814
ENZYMES
TABLE 36.3 SURVEY OF PURIFICATION OF Pseudomonas 3-HYDROXY-3-METHYLGLUTARY L COENZYME A REDUCTASE ON A HYDROXYAPATITE COLUMN (BENSCH AND RODWELL) Equivalent of 760 mg dry weight cells = 100%. The activity was tested using mevalonate oxidation. ~~~~
Fraction
Volume (ml)
Total activity (1.U.)
Total protein (mg)
Crude extract after sonic treatment Fraction after ammonium sulphate precipitation and dialysis Chromatographic fractions 49-70 Chromatographic fractions 42-48
12.8
1070
575
2.0
570
34.8
61.1
436
23.0
32.9
77
1.98
Specific activity (I.U./mg)
~
~~
Enrichment
~
Yield (%)
1.o
100
16.4
8.8
53
18.9
10.2
40
38.9
21.0
1.86
7.2
33 I t
0
200
400 VOLUME,
600
mi
Fig. 36.4. Chromatography of milk xanthine oxidase preparation on DEAE-Sephadex using gradient elution (Roussos and Morrow). Column dimensions: 1.6 cm X 4.9 cm2. Load: 201 mg of protein. The ion exchanger was equilibrated with 10 mMpotassium phosphate buffer (pH 7.4), 1 mM in EDTA (starting buffer). After washing off all enzyme activities and proteins, the linear gradient elution (400 ml of 100 mM potassium phosphate buffer (pH 7.41, 10 f l i n EDTA, t o 400 ml of 10 mMpotassium phosphate) was started. Protein was determined by the method of Lowry ef al. Enzymic activity was tested at 25°C and the unit was defined according to Fridovich (cf:also Avis et ul.).
81 5
OXIDOREDUCTASES
The purification of milk xanthine oxidase by a combination of chromatography on anion-exchange polydextran and chelating resin was described by Roussos and Morrow. Trace amounts of some heavy metals often play an important role when present in the purified enzyme preparation because they can be considered to be an integral part of the enzyme. Bovine milk xanthine oxidase serves as an example. Therefore Roussos and Morrow made an effort to prepare enzyme preparations free from molybdenum and very low in iron, and favoured the conclusion that molybdenum and probably also iron are not essential in the xanthine-oxygen oxidoreductase activity. All procedures were carried out at 0-4°C. Commercial xanthine oxidase (Worthington Biochemicals Corp., Freehold, N.J., U.S.A.) was dialyzed twice against 10 mil4 potassium phosphate solution (pH 7.4), 1 mil4 in EDTA. The supernatant was applied to a preequilibrated DEAE-Sephadex column (Fig. 36.4). The eluates with the highest specific activitv (fraction 111) were pooled and applied on to a chelating resin column. The Chelex 1UU column ( 2 cm X 4.2 cm’) was pre-equilibrated with 20 mil4 phosphate buffer (pH 7.4), 1 mil4 in EDTA. After the application of the sample, the column was washed with the same buffer (5 ml) and the combined effluent was dialyzed against 100 pA4 phosphate buffer (pH 7.4) 10 pM in EDTA. Electrophoretic examination indicated the presence of homogeneous protein. The enzyme prepared in this manner possessed a specific activity that was significantly higher than those hitherto reported (about 3-fold), TABLE 36.4 RESULTS O F THE PURIFICATION O F TRIPHOSPHOPY RIDINE NUCLEOTIDE ISOCITRATE DEHYDROGENASE FROM Bacillus stearothermophilus USING A COMBINATION O F METHODS (HOWARD AND BECKER) Fraction
(1)
(2)
(3) (4)
(5)
(6) (7)
Combined supernatant fractions after sonic disruption of cell suspension Supernatant of the mixture of the preceding fraction and sodium DL-isocitrate after the pH was adjusted to 4.9-5.0 Solution of the pellet after ammonium sulphate precipitation at pH 7.5 Desalted protein (Sephadex G-25) treated with ammonium sulphate and chromatographed on an agarose (Bio-Gel A-0.5 m) column in the presence of a small amount of ammonium sulphate Solution of combined and reprecipitated fractions chromatographed on hydroxyapatite (Bio-Gel HTP) Active pooled fractions after chromatography on ECTEOLA-cellulose (Cellex E) Combined homogeneous fractions after chromatography of concentrated solution (ultrafiltration; Amicon UM-2 membrane) on a Sephadex G-75 column
References p.829
Total activity (1.U.)
Total A,,,
A,,,/A,,,
1488
40,000
0.55
0.037
1300
11,400
0.56
0.11
1270
3280
0.745
0.39
968
34 1
1.6
2.84
645
52.4
Specific activity
1.61
12.3
248
8.16
1.66
30.4
128
3.73
1.70
34.2
816
ENZYMES
it was active in the absence of molybdenum and the content of iron was 3-4-fold lower than that in previous preparations. The minimum molecular weight calculated on the basis of the FAD content was 354,000 t 94,000, i.e., 2.3-fold higher than the value published previously for the crystalline preparation. In many instances, the application of one or two chromatographic or other purification steps is not sufficient to obtain a homogeneous enzyme from a natural source and a combination of several methods must be used. The isolation of isocitrate dehydrogenase using successive chromatographic and other methods (Howard and Becker) is an example. The efficiency of the procedure is illustrated by the survey in Table 36.4 (in which the details are omitted). All solutions used in the preparation of isocitrate dehydrogenase contained 1 mM EDTA and no antioxidants were used. A 1000-fold purification (based on the absorbance at 280 rlm) was achieved. The yield of total activity was 8.6%. On the other hand, the method of affinity chromatography does not require as many operations. Newbold and Harding described a single-step procedure that gave a 4000-fold purification of dihydrofolate reductase from mammalian skin. This comparison illustrates the efficiency of the latter method.
TRANSFERASES The purification of ornithine carbamoyl-transferase from Halobacterium salinarium using gel filtration, sucrose gradient centrifugation and chromatography on calcium phosphate gels was described by Dundas. This is an example of the processing of a typical extremely halophilic enzyme, which shows a high activity in 4 M sodium chloride solution and is rapidly and irreversibly inactivated in a salt-free environment. Therefore, all purification procedures were carried out on 4.3 M sodium chloride solutions. A 0.1 M solution of L-ornithine stabilizes the enzyme and was included throughout the purification. Owing to the necessarily high ionic strength, the use of ion-exchange purification techniques is impossible in this instance. The broken cells of Halobacterium salinarium were resuspended in 4 M sodium chloride-0.1 M ornithine solution and the supernatant was fractionally precipitated with acetone at 0°C. The dissolved precipitate was dialyzed against 4.3 Msodium chloride-0.1 M ornithine and stored at -30°C. The specific activity increased 5-1 5-fold. Gel filtration was carried out on a Sephadex G-200 column previously equilibrated with 4.3 M sodium chloride-0.1 M ornithine solution. The same solution was used for elution. This procedure resulted in an approximately 2-fold increase in specific activity. Volumes of 1 ml of acetone-purified fractions were centrifuged using 30-ml gradients made with 25% (w/v) sodium chloride and 0.1 M ornithine and a decreasing sucrose concentration from 30% (w/w) to 10% (w/w). After centrifugation (Beckman 60 Ti angle rotor, 5"C, 60,000 rpm for 6-12 h), the tubes were collected, giving 0.7-ml fractions. The pooled runs gave a 2-fold increase in specific activity. The best result (a 6-fold increase in activity) was obtained by calcium phosphate gel chromatography (Fig. 36.5) or by its combination with prior gel filtration. The sorbent was prepared according to Tiselius et al. as described by Levin. The combined active fractions were dialyzed in the presence of urea and mercaptoethanol and the electrophoretic and sedimentation pattern obtained showed the homogeneity of the preparation.
817
TRANSFERASES
. cn
r2O0
?.
z
w
100
a
0
ELUATE.ml
Fig. 36.5, Calcium phosphate gel chromatography of ornithine carbamoyl transferase (Dundas). Column dimensions: 20 X 2.5 cm. Washing: 500 ml of 5 mM phosphate buffer (pH 6.8'1, 500 ml of 4.5 M sodium chloride, and 100 ml of4.3 M sodium chloride, 0.1 M in ornithine. Then the sample (60 mg of protein) was applied. Eluent: 4.3 M sodium chloride, 0.1 M in ornithine solution, with stepwise increasing phosphate concentration, 5-300 mM (input phosphate concentration to 50, 100, 150, 200, 250 and 300 d a f t e r 300, 550, 750, 850, 950 and 1150 ml, respectively). Fractions: lOml (or 25 ml).
The isolation of three multiple forms of aminoacyl transferase I of rat liver using hydroxyapatite and polydextran chromatography was described by Schneir and Moldave. This method permitted the preparation of three forms of aminoacyl transferase I (differing in molecular weight) from rat liver mitochondria and proved the transformation of highmolecular-weight species to lower-molecular-weight enzyme. This fractionation is briefly described below. Homogenized excised livers were centrifuged at 10,000g and the supernatant, after precipitation by adjustment to pH 5.0, was freed from low-molecular-weight components by passing it through Sephadex (3-25 (0.05 M Tris-hydrochloric acid, pH 8). This and all other treatments were performed at +4"C. The effluent (500 ml), containing transferases I and 11, was made 1 mM in dithiothreitol (all other solutions used for washing also contained this sulphydryl-activating agent) and mixed with 166 ml of well suspended hydroxyapatite (Clarkson Chemical Co., Inc.) at 4°C for 1 h. The supernatant after centrifugation (lOO,OOOg, 10 min, 4°C) with washings of the sediment was discarded. The residue was eluted successively with three 300-ml volumes of potassium phosphate solutions at pH 6.8 to obtain fractions extracted with 0.125, 0.15, 0.175,0.25 and 0.50M potassium phosphate solution. The active fraction (0.25 M) was precipitated by ammonium sulphate to a final concentration of 70% (pH 7) and concentrated by dialysis under vacuum against Tris-hydrochloric acid buffer. Gel filtration of this sample is illustrated by Fig. 36.6a. The combined fractions 31-37, 42-48 and 56-72 were precipitated with ammonium sulphate and, after vacuum dialysis, applied individually to the same column. The results are illustrated in Fig. 36.6b. Form A of transferase I can be converted into form B when exposed briefly to the influence of 1 M ammonium chloride solution; prolonged incubation leads to loss of activity. Form B seems References p.829
81 8
ENZYMES
FRACTION NUMBER
Fig. 36.6. Chromatography of transferase 1 on Sephadex G-200 (Schneir and Moldave). Column dimensions: 90 X 1.5 cm. Eluent: 0.05 M Tris-hydrochloric acid pH 8, 1 mM in dithiothreitol. Flowrate: 8 nil/h. Fractions: 1.1 ml. Temperature: 4°C. Activity tests are described in the original paper. (a) Load: 1 ml of solution after hydroxyapatite treatment (see text) containing 25 mg of protein. (b) Survey of re-chromatography of pooled and dialyzed fractions 31 -37,42-48 and 56-72 from the preceding chromatography. These three samples were applied individually. (b) represents a composite of three columns. A, B and C: three forms of transferase I.
to be a natural enzyme, while form C could represent active sub-units, and the existence of form A can be explained by the binding of B to large molecular weight materials that have no enzymic activity. The purification of tyrosine aminotransferase by affinity chromatography was described by Miller et al. They purified ~-tyrosine-2-oxoglutarateaminotransferase using affinity adsorbents containing pyridoxamine phosphate covalently bound to agarose (Sepharose 4B). The sorbed enzyme can be released by changing the buffer, with 125-fold purification. Further purification can be achieved by gel permeation chromatography on Sephadex G-200 columns. A mixture of different isozymes is obtained by this method, because affinity chromatography binds and releases all molecules with the same specific activity.
HYDROLASES During the isolation of enzymes, sometimes multiple forms are prepared with the same type of activity but differing in their chromatographic or electrophoretic properties. There may be several causes of this phenomenon, and some examples are given below.
819
HY DROLASES TABLE 36.5 PURIFICATION OF ARYLSULPHATASE FROM P. aeruginosu (DELISLE AND MILAZZO) Enzyme assays after Dodgson and Spencer. Fraction
Acetone powder extract DEAE-cellulose DEAE-Sephadex (NH,),SO, precipitate DE AE-Sephadex (PH 8.5) Acrylamide gel electrophoresis
Total protein (mg)
1539 432.8 199.2 112.0 26.5 6.6
Specific activity (u/mg)
Recovery (%)
p-Nitrophenol sulphate
Nitrocatechol sulphate
p-Nitrophenol sulphate
Nitrocatechol sulphate
0.0224
0.045 I
100
100
0.0941 0.137 0.1862 0.7592
0.1 249 0.239 0.3686 1.444
118* 79 60 58
78 68 59 55
2.49
4.464
48
42
*Endogenous inhibitor was present in the initial extract.
The isolation of arylsulphatase isoenzymes from Pseudomonas aeruginosa using cellulose or polydextran ion-exchange chromatography was described by Delisle and Milazzo. This is an example of a simple method leading to the isolation of an identical pair (charge-isomers) of microbial arylsulphate sulphohydrolases, formed by the microorganism in duplicate. The enzyme was extracted from an acetone powder of the micro-organism in 0.05 M Tris-hydrochloric acid buffer (pH 7.5). Sorption on DEAE-cellulose or DEAE-Sephadex and elution using 0.01 M Tris-hydrochloric acid buffer (pH 7.5) and linear sodium chloride gradients 0-1 M and 0.1-0.6 M, respectively, led to the first purification shown in Table 36.5. Further purification was achieved by precipitation with ammonium sulphate. The enzyme was present in the fraction salted out between 35-75% of saturation. The fraction after preparative disc electrophoresis had the highest enrichment (about 100-fold) with an approximately 45% yield. The enzyme, when examined by gel electrophoresis, indicated a high purity; only two zones were present, both hydrolyzing both substrates. The presence of two isoenzymes in the microorganism was proved by repeated cross-experiments. The method of Hedrick and Smith was used to establish whether these two enzymes were size- or charge-isomers or perhaps both. The results are shown in Fig. 36.7. Parallel lines demonstrate that the two enzymes are similar in size but that they differ in charge; they are therefore charge-isomers. The isolation of two active forms of lipase from Rhizopus arrhizus using a cation exchanger was described by Semkriva et al. This is an example of the isolation of two forms of an enzyme, the first being a direct product of the microorganism and the second being formed from the first by a slow conversion of an unknown type. The lyophilized powder (5 g; activity 40 units/mg = specific activity 400), prepared according to Laboureur and Labrousse, was dissolved in 800 ml of water at 2°C and the solution was pumped (300 ml/h) into a 27 X 1.7 cm column of Amberlite IRC-50 (Type References p.829
820
ENZYMES
190
-
*
g
'80-
X
h Y
8 170-
s
160
-
. I -
0
4 6 8 GEL CONCENTRATION, "/.
2
10
Fig. 36.7. Application of Hedrick and Smith's method for distinguishing types of arylsulphatase enzyme isomers (Delisle and Milazzo). The effect of different polyacrylamide gel concentrations on electrophoretic mobility of the sample was examined. Points represent the average of triplicate determinations on each gel.
T
I 9
T 0.6 -
I I
u
1.0 -
E
8
0.4 -
8 z a k 8 0.29
';i
1 8
so00
-I mi
I: 0.05 2
rt
I
WOOW
P
3
E
4
J
50
75
100
125
ELUATE, ml
Fig. 36.8. Chromatography of a concentrate of a lipase from Rhizopus arrhizus on a weakly acidic cationexchange resin, Amberlite IRC-50 (Simdriva eta!.). Column dimensions: 20 x 0.9 cm. Buffer: the column was equilibrated with buffer B (see text). Sample: solution after Sephadex filtration (lo5 units). Washing: 30 ml of buffer B (10 ml/h). Desorption: a linear gradient of 50 ml of buffer B to 50 ml of buffer A. Solid line, lipase activity; broken line, total proteins; dotted line, buffer concentration in the eluate. I and 11: different lipase forms. For the measurement of the lipase activity and definition of the unit, see original paper.
82 1
HYDROLASES
IRF-97, Rohm and Haas, Philadelphia, Pa., U.S.A.) equilibrated with 20 mM calcium acetate solution at pH 4.7. The column was washed (300 ml/h) with 400 ml of 5 mil4 calcium acetate solution and eluted (20 ml/h) with 1 M ammonium acetate buffer (pH 5.7) 5 mM in Ca" (buffer A). The yield was 95% and the specific activity about 5000. The pooled active fractions were concentrated to 6.5 ml by vacuum dialysis against 50 mM ammonium acetate buffer (pH 5.7), 5 mMin calcium acetate (buffer B). The solution was freed from non-active contaminants on a 32 X 3.2 cm Sephadex G-75 column equilibrated and eluted with buffer B. Lipase emerged as a symmetrical peak (in 30 ml; 1.6 V o ;average specific activity 8000). This fraction was re-chromatographed on an Amberlite IRC-50 column (Fig. 36.8). Two active peaks (I and 11) were obtained with a specific activity of 9200 and 8000, respectively. The yield was 95%. The re-chromatography and disc electrophoresis of peak I immediately and after storage for 16 or 92 days in the cold indicated the conversion of form I into 11, the latter being stable. The chromatographic resolution of two forms of alkaline proteinase from Aspergillus ~ Z Q V U S arising by conversion of the native enzyme when exposed to conditions near the transition state was described by Mike: ef al. The polymorphism of alkaline aspergillopeptidase, caused by transformations of the native enzyme when exposed either to high temperatures (near the transition temperature) or to treatment with 8 M urea at room temperature, was described. Various substances (Ca", EDTA or eaminocaproic acid) added at a concentration of 0.02 M specifically influenced the forms that arise. The new forms of enzyme differed in the optimum pH of cleavage of haemoglobin, and in the specificity of cleavage of the B-chain of oxidized insulin. In the example given in Fig. 36.9, an aqueous solution of the native enzyme was heated for 20 min at 45°C without any additives. The figure illustrates the influence of pH on the separation of the forms that arise. The best results were obtained only at pH 5.9, because at lower and higher pH peaks I and I1 coalesced.
0 20
8
016
012
&!om
$ $!
004
m 4
0
0
5
10
15
FRACTIONS
Fig. 36.9. Establishment of the optimum pH for thc chromatographic separation of two forms of enzyme formed by thermal treatment of alkaline proteinase from Aspergillus fluvus (Mike's et al. ). Sorbent: DEAE-Sephadex. Column dimensions: 20 X 1 cm. Buffers for equilibration and elution: the same concentration of phosphate was used (0.01 M), but the buffers differed in the pH indicated for particular runs. Simple elution was used. Fractions: 1.5 ml/h. Temperature: 4°C.
References p.829
822
ENZYMES
The purification of acetylcholinesterase using affinity chromatography was described by Kalderon er al. Affinity chromatography of enzymes can utilize their specific interaction with fixed antibodies, substrates, effectors or inhbitors. The last case is illustrated by the example given below. The phenyltrimethylammonium ion is a good competitive inhibitor of acetylcholinesterase (Wilson and Alexander) and its affinity for the enzyme decreases with increase in ionic strength (Changeux). Hence there are good conditions for the possibility of selective desorption of the enzyme by changing the salt concentration in the eluent. Kalderon e l al. prepared this inhibitor in the fory of [N(e-aminocaproy1)-p-aminophenyl]trimethylammonium bromide hydrobromide, [HJ\I(CHJ,C0.NHC,H4.N(CH3),] .2 Br (abbreviation: e-aminocaproyl-PTA), the €-amino group of which served to link the inhibitor to the BrCN-activated Sepharose 2B (Axen et al.). Also, e-aminocaproyl-PTA was shown t o be a good inhibitor of the enzyme (the inhibition constant Kiis 6 llM). A crude preparation of the enzyme was prepared from toluene-treated tissue from the electrical organ of the electric eel by extraction and fractionation with ammonium sulphate according to Leuzinger and Baker. The fraction between 15 and 35% ammonium sulphate was dissolved in 0.1 M sodium chloride-0.01 M phosphate (pH 7) and dialyzed against the same buffer for 12 h at 4°C. The enzyme prepared as described had a specific activity of 240 units per milligram of protein (1 unit being the amount of enzyme that hydrolyzes 1 pmole of acetylcholine per minute) and was applied on the column (cf:, Fig. 36.10). The first peak represents the unsorbed enzyme accompanied by other proteins in the washings. The second peak was found to have a specific activity of 4100 units per milligram of protein, i.e., 17 times greater than the specific activity of the enzyme applied to the column. Higher concentrations of the inhibitor bound to Sepharose sorbed a larger
VOLUME. ml
Fig. 36.10. Elution pattern of acetylcholinesterase from an affinity chromatography column (Kalderon et al. ). Adsorbents: c-aminocaproyl-PTA-Sepharose (0.16 pmole/ml of inhibitor). Column dimensions: 40 X 1.1 cm. Load applied on the column: 10 ml of a solution of crude enzyme preparation containing 15,000 units of esterase activity in 0.1 M sodium chloride-0.01 M phosphate (pH 7). Time of equilibration: 1 h. Washing: 0.1 Msodium chloride-0.01 Mphosphate (pH 7). Elution: 1 Msodium chloride-0.01 Mphosphate (pH 7 ) . Fractions: 5 ml. Evaluation of fractions: Absorbance at 280 nm and determination of activity according to Kremzner and Wilson (pH-stat method at pH 7 and 25°C). Full line, protein (A ); dashed Line, enzymic activity.
823
LYASES
amount of enzyme but the desorbate had a lower specific activity (e.g., about 2000 units per milligram of protein with Sepharose containing 1.4 pmole/ml of inhibitor). The probable explanation is the non-specific sorption of proteins on ion-exchange quaternary ammonium groups which cannot be suppressed by the effect of higher ionic strength during the sorption. The isolated enzyme displayed only one band on disc electrophoresis but contained aggregates owing to the known lability of acetylcholineesterase to lower ionic strength. They decreased the specific activity to 40% in comparison with the purest enzyme.
LYASES A simple method for the purification of L-glutamate 1-decarboxylase from Escherichia coli using a DEAE-Sephadex column and crystallization of the enzyme was described by Strausbauch e l al. The starting material was a by-product after purification of pyruvate oxidase from E. coli described by Williams and Hager. The procedure of the latter authors was interrupted before the last protamine sulphate fractionation step, which was replaced with DEAE-Sephadex column chromatography, as illustrated in Fig. 36.1 I . The second peak was found to contain glutamate decarboxylase (60% purity). The active fractions were collected, concentrated by precipitation in ammonium sulphate and frozen.
600
8 P
500
3 2 >
4000
P
2
3003 200
100
0
20
40
60
80
100
120
140
160
180
200
FRACTION NUMBER
Fig. 36.1 1 . Chromatographic purification of glutamate decarboxylase (Escherichia coli) on a DEAESephadex A-50 column (Strausbauch et al.). Elution: linear gradient 0.02 M potassium phosphate buffer (pH 5.7)-0.3 M phosphate (pH 5.3). The pooled fractions of the second peak were further processed by crystallization. For the determinations of the enzyme activities and the definition of the units, see the original paper.
References p. 829
824
ENZYMES
For crystallization, the thawed fraction (40 mg/ml of protein) was adjusted to pH 6.5 with sodium phosphate buffer (0.05 M final concentration) and solid ammonium sulphate was added in small portions in the cold over a period of 5 days. Thin needles or flat plates appeared at a 15% (w/v) concentration of ammonium sulphate, which was then added up to 20% (w/v). Crystallization was repeated twice after dissolving the crystals in a minimum amount of 0.05 M phosphate buffer of pH 6.5. When examined by various methods, the crystals represent a homogeneous enzyme. The isolation of chicken breast muscle aldolase by a combination of ion-exchange chromatography on cellulose and ammonium sulphate precipitation was described by Marquardt. Pure homogeneous aldolase (fructose 1,6-diphosphate D-glyceraldehyde-3phosphate lyase) was prepared in six steps. All operations were carried out at 0-5°C. (I) Extraction. Breast muscles of freshly killed mature female chickens were chilled on ice and frozen in 200-g amounts. The thawed preparations were homogenized with 600 ml of 1 mM EDTA-5 mM 2-mercaptoethanol (pH 7.6) and the supernatant and the filtrate were used for further processing. (11) First precipitation. Solid ammonium sulphate was added to 50% saturation over 1 h and the supernatant after centrifugation (25,00Og, 30 min) was saturated to 63% over 1 h. After 3 h, the suspension was centrifuged again and the precipitate dissolved in the minimum volume of 0.1 M Tris-50 mM EDTA- 10 mM 2-mercaptoethanol, pH 7.5. (111) 1b'AE-cellulose chromatography. The enzyme wab
-NO
KCW -40mM
KCI
-
-500mM
KCI-
Fig. 36.1 2. Chromatography of chicken muscle aldolase on a CM-cellulose column (Marquardt). Ion exchanger: Cellex CM. Column dimensions: 2 2 X 4 cm. The column was equilibrated with pH 6.5 buffer (see text) and the unsorbed protein of the sample was flushed with 150 ml of the pH 6.5 buffer. Elution: stepwise, 40 mM potassium chloride in a pH 6.5 buffer and 500 mM r otassium chloride in the same buffer. Flow-rate: 2.6 ml/min. Second peak: aldolase. Third peak: lactate dehydrogenase. Enzyme tests for these two enzymes were those of Rajmakur er al. and Komberg, respectively.
ISOMERASES
825
dialyzed for 16 h against 12 I of 50 mM Tris- 10 mM sodium phosphate- 10 mM EDTA-5 mM 2-mercaptoethanol, pH 9.2. After adjustment to pH 9.3, the centrifuged solution (50,00Og, 20 min) was chromatographed on a 33 X 4.5 cm TEAE-cellulose (Cellex T, Bio-Rad Labs, Richmond, Calif., U.S.A.) column equilibrated with pH 9.2 dialyzing buffer. The column was washed with the same buffer (5 ml/min) and the void volume (identified by the coloured solution) emerged slightly before aldolase (approximately 30 ml). (IV) Second precipitation. The active pooled fractions were saturated with ammonium sulphate to 70%over 1 h and centrifuged (25,00Og, 30 min). The sediment was dissolved in 75 ml of 10 mM sodium phosphate-5 mM EDTA-5 mM 2-mercaptoethanol (pH 6.5) and dialyzed against 12 1 of the pH 6.5 buffer. (V) Chromatography on CM-cellulose.The centrifuged supernatant (50,000 g , 30 min) of dialyzed enzyme was sorbed on to a CM-cellulose column (Cellex CM, Bio-Rad Labs.) and chromatographed (cc, Fig. 36.12). All fractions with a specific activity of 20 and better were combined. (VI) Crystallization. The combined fractions were saturated with solid ammonium sulphate to 50% and centrifuged at 50,OOOg for 30 min. Solid ammonium sulphate was added until the first turbidity appeared. After 12 days at 4"C, the enzyme began to crystallize and the process was completed by adding 4% of ammonium sulphate. The crystals were collected after 1 day by centrifugation. Recrystallization was carried out as described above except that the buffer used was 0.1 M sodium phosphate- 1 mM EDTA, pH 7.6. The purification was 4.4-fold and the yield 50%. Various methods proved the homogeneity of the preparation.
ISOMERASES Only 24 papers describing the use of column chromatographic methods for the fractionation of isomerases were found in the bibliography by Deyl et al. Therefore, only one example of this class of enzymes will be given here as an illustration of an in uitro hybridization of mouse phosphoglucose isomerase variants (Carter and Yoshida). D-Glucose-6-phosphate ketol-isomerase has been found to exist in three genetically determined electrophoretic phenotypes in various mouse tissues: Phenotype I: associated with F type (fast cathode-migrating enzyme); Phenotype 11: associated with S type (slow migrating enzyme); Phenotype 111: possesses three enzyme components, F, FS and S (FS being an enzyme of intermediate mobility). When F and S types of mice were cross-bred, the offspring showed three enzyme bands; two of them were identical with the F and S bands of the parents, but the third seemed to be a hybrid of F and S. To verify this possibility, Carter and Yoshida tried to prove that the recombination occurred also in vitro. Hybridization with the crude extract was not possible and therefore the purification of enzymes was necessary. The muscle tissue from three phenotypes of laboratory mouse was selected as a source of enzymes and was pooled. After homogenization in 0.01 M potassium chloride solution and fractionation with 0.03 M zinc acetate and ammonium sulphate (Noltmann), the enzyme was dialyzed against 0.005 M sodium phosphate buffer (pH 6.8) and purified on a 35 X 1.5 cm column of calcium phosphate gel equilibrated with the same buffer. The enzyme was eluted with a linear gradient comprising 300 ml of 0.005 M sodium phosphate References p.829
826
ENZYMES
6
I3O 120
EFFLUENT. ml
r:
1"
Fig. 36.1 3. Chromatography of mouse phosphoglucose isomerase on a CM-Sephadex column (Carter and Yoshida). Column dimensions: 35 X 1.5 cm. Equilibration: 0.005 M sodium phosphate buffer, pH 6.8. Elution: linear gradient, 300 ml of the same buffer-300 ml of 0.025 M sodium phosphate buffer, pH 6.8. For enzyme assay and definition of the unit, see the original paper. S, FS and F are enzymes of the three phenotypes (see text), distinct in starch gel electrophoresis.
to 300 ml of 0.025 M sodium phosphate (pH 6.8). The active fractions were pooled and concentrated by vacuum dialysis and the enzyme preparation was then dialyzed against the more diluted buffer of pH 6.8 and chromatographed on CM-Sephadex (Fig. 36.13). The material of the active enzyme peak was used for further experiments. For hybridization, equal volumes (0.1 ml) of solution of peaks F and S of the same activity were mixed, and a similar aliquot of the FS peak was prepared. These two solutions were made 2 M in guanidine hydrochloride, 50 mM in 2-mercaptoethanol, 35 mM in EDTA and 25 mM in sodium phosphate buffer, pH 6.8. The solutions were kept on ice for 3 min and then dialyzed against 2-1 volumes of 0.005 M sodium phosphate, pH 6.8. Then the solution was concentrated to the initial volume by vacuum dialysis. A control sample without the guanidine hydrochloride treatment was also prepared. The samples were examined electrophoretically. The results can be summarized as follows: F i- S + guanidine hydrochloride FS + guanidine hydrochloride ____5___* F + F S + S Offspring from the mating of F and S phenotypes Control samples were without effect on the hybridization. The three enzymes in the three phenotypes are composed of peptide chains (sub-units) f and s, controlled by two different alleles and associated in the enzymes in question, ff, fs and ss.
LIGASES The isolation of two methionyl-tWA synthetases from Escherichia coli and the evidence for their specific interaction with particular tRNA using chromatography on
LIGASES
827
methylated albumin was described by Cerhova and Rychlik. A problem in proteosynthesis has been to decide whether the amino acid is attached to the respective tRNAs by only one enzyme or whether there are as many activating enzymes as there are tRNAs for the same amino acid. CerhovP and Rychlik isolated two methionyl tRNAs and studied their specific interaction. The crude extract, after sonic disintegration of E. coli cells (100 g in 500 ml of 0.025 M Tris-hydrochloric acid buffer, pH S.O), was incubated at 37°C for 2 h. The autolyzate was precipitated with ammonium sulphate, the enzyme being separated between 35 and 55% saturation. The sediment, after centrifugation at 30,OOOg for 10 min, was dissolved in 0.02 M potassium phosphate, pH 7.2 (100 mg/ml of protein) and chromatographed on a Sephadex G-75 column (50 X 4 cm), equilibrated by and eluted with the same buffer. The active 5-ml fractions were pooled and stirred for 10 min with alumina Cy gel (1 2 mg/ml) at pH 6.5. The centrifugation gel was washed with 200 ml of water. The enzyme was eluted with 100 ml of 0.1 Mpotassium phosphate solution of pH 7.0 and dialyzed against 0.02 M phosphate buffer of pH 7.5. Chromatography on a 20 X 2.5 cm column of DEAE-cellulose (gradient: 0.07 to 0.2 M phosphate buffer; 5-ml fractions resolved the preparation into two peaks, I and 11, both charging tRNA with methionine. On re-chromatography, the positions of the peaks remained unchanged. Methionyl-tRNA synthetase I differs from 11: the double-labelling experiments showed that each of them acylates with methionine a different species of methonine-tRNA. tRNA was charged with [U-'4C] rnethionine (4.7 pCi/pmole) by methionyl-tRNA synthetase I or with [U3H]methionine (153 pCi/pmole) by methionyl-tRNA synthetase 11. Both samples were mixed and chromatographed on a methylated albumin-Kieselguhr coluTn (Fig. 36.14). Methionyl-tRNA of E. coli was resolved into two components (cf. also Cerna er a/.);3H was found to be attached to the first and 14C to the second component. Methionyl-tRNA synthetase I selectively acylated the second component of methionine-tRNA, and methionyl-tRNA synthetase I1 the first component. Hence the two different methionyl-tRNA synthetases correspond to two different species of methionyl-tRNA. The mechanism of pyruvate carboxylase formation from apoenzyme and biotin in a thermophilic bacillus was described by Cazzulo er al. (1969, 1970). This is another example of the use of chromatographic methods for the solution of biochemical problems. F'yruvate carboxylase is formed by the attachment of biotin to its inactive protein precursor, apopyruvate carboxylase. This reaction is catalyzed by holoenzyrne synthetase. The reconstruction of the pyruvate carboxylase activity in cell-free extract requires acetyl-CoA, Mg2+and ATP in addition to biotin. An experiment was designed to demonstrate the effect of acetyl-CoA on the incorporation of labelled biotin into the apoenzyme. First the apoenzyme was purified from a culture of Bacillus coagulans (a variant of B. stearothermophilus). The washed cells were digested with lysozyme and the extract was fractionated with ammonium sulphate. Apoenzyme and holoenzyme synthetase can be resolved from this preparation by chromatography on Sephadex G-200 (cf., Fig. 36.15a). the apoenzyme being eluted well before holoenzyme synthetase. The following incubation mixture was prepared: 2.2 mg of apoprotein and 3.1 mg of holoenzyme synthetase obtained after chromatography on Sephadex G-200, plus 12.5 pmoles of References p.829
828
ENZYMES 0.7
c
0.5
T
8 w
0
y
:
'
m
FRACTIONS
3 c o,5'[
,
u
y2 y2+ 8 Q 1.5 3 9 0
n:
21
25
30
35
40 45 50 FRACTIONS
55
60
Fig. 36.14. Chromatography of methionyl-tRNA on methylated albumin-Kieselguhr using specific double labelling (Cerhovi and Rychik). The 20 X 2.5 cm column of methylated albumin-Kieselguhr was prepared according to Yamane and Sueoka. Load: 10 mg of [U-'"C] methionyl-tRNA (specific activity 1.4 pCi/pmole) and 3 mg of [U-3H]methionyl-tRNA (specific activity 4.9 pCi/pmole). 1, gradient 0.25 M-0.35 M sodium chloride solution; 2, values; 3, number of impulses from methionyl-t RNA linked through methionyl-tRNA synthetase 11; 4, number of impulses from methionyl-tRNA linked through methionyl-tRNA synthetase I.
Fig. 36.15. Chromatographic evidence of the role of acetyl-CoA on the synthesis of pyruvate carboxylase using a Sephadex G-200 column (Cazzulo etal., 1970). Column dimensions: 30 X 1.7 cm. Equilibration: 50 mM Tris-hydrochloric acid (pH 7.6), 1 mM in EDTA and 0.4 M in ammonium sulphate. The solution of precipitated incubation mixture (see text) was applied to the column. The proteins were eluted with the same buffer as above. Fractions: 1 ml. Assays: 0 , protein spectrophotometrically; A, radioactivity; A, holopyruvate carboxylase spectrophotometrically (Cazzulo et ai., 1969). Apopyruvate carboxylase (0)or holoenzyme synthetase ( 0 ) were tested after incubation with other additive substances (details of the procedures are given in the original paper). a, Experiment without acetyl-CoA; b, experiment with acetyl-Co A.
magnesium chloride, 3.1 pmoles of ATP. 0.17 pmoles of C+)-["C] biotin (10 pCi) and 37.5 pmoles of Tris-hydrochloric acid (pH 7.6), in a final volume of 7 ml. After incubation for 30 min at 45°C and cooling to O'C, 20 pmoles of EDTA were added and the proteins precipitated with 20 ml of saturated ammonium sulphate solution. The precipitate was dissolved in 0.5 ml of 50 mM Tris-hydrochloric acid (pH 7.6, 1 mM in EDTA and 0.4 M in ammonium sulphate, and the solution was applied t o the column (Fig. 36.15b).
REFERENCES
829
When acetyl-CoA was not included in the incubatioh mixture (Fig. 36.15a), there was little incorporation of (+)-[“C] biotin into apoprotein and no formation of pyruvate carboxylase activity was observed. When 2.5 pmoles of acetyl-CoA were included in the incubation mixture in a duplicate experiment (Fig. 36.15b), a large peak of radioactivity was associated with a protein that possessed pyruvate carboxylase activity. This peak was eluted at the same place as that occupied in the above experiment by the apoprotein. On the basis of these and other experiments, Cazzulo et al. (1970) considered the acetyl-CoA to be an allosteric effector of the reconstitution process.
REFERENCES Alpert, N. L., Clin. Chem., 15 (1969) 1 1 98. Avis, P. G . , Bergel, F. and Bray, R. C., J. Chem. Soc., London, (1955) 1100. Axin, R., Porath, 1. and Ernback, S . , Nature (London), 214 (1967) 1302. Beck, C. and Tappel, A. L., Anal. Biochem., 21 (1967) 208. Bensch, W. R. and Rodwell, V. W., J. Biol. Chem., 245 (1970) 3755. Bernardi, G.,MethodsEnzymol.,22 (1971) 325. Blatt, W. F.,MethodsEnzymol., 22 (1971) 39. Bradley, D. W. and Tappel, A. L., Anal. Biochem., 33 (1970) 400. Carter, N. D. and Yoshida, A., Biochim. Biophys. Acta, 181 (1969) 468. Cazzulo, J . J., Sundaram, T. K. and Kornberg, H. L., Nature (London/, 223 (1969) 1137. Cazzulo, J . J . , Sundaram, T. K. and Kornberg, H. L., Nature (London/,227 (1970) 1103. Cerhovi, M. and Rychlik,J., Collect. Czech. Chem. Cornmun., 32 (1967) 3808. Cerni, J . , Rychli’k, I . and Sorm, F., Collect. Czech. Chern. Cornmun.,31 (1966) 336. Changeux, J . P., Mot. Pharmacol., 2 (1966) 369. Cline, G . B. and Ryel, R. B.,Methods Enzyrnol., 22 (1971) 39. Cuatrecasas, P., Aduan. Enzymol., 36 (1972) 29. Cuatrecasas, P. and Anfinsen, C. B., Annu. Rev. Biochem., 40 (1971a) 259. Cuatrecasas, P. and Anfinsen, C. B.,Methods Enzymol., 22 (1971b) 345. Davies, D. R. and Segal, D. M., Methods Enzymol., 22 (197 1) 266. Delisle, G. and Milazzo, F. H., Biochim. Biophys. Acta, 21 2 (1 970) 505. Deyl, Z., Rosmus, J . , Juiicovi, M. and Kopeck$, J., Bibliography of Column Chromatography 196770, Elsevier, Amsterdam, London, New York, 1973. Dodgson, K. S. and Spencer, B., Methods Biochem. Anal., 4 (1957) 246. Dundas, 1. E . D.,Eur. J. Biochern., 16 (1970) 393. Eley, J . , Biochernisrry, 8 (1969) 1502. Eveleigh, J . W., Adler, H. J . and Reichler, A. S . , Automat. Anal. Chem., Technicon Symp., 1967, Vol. 1, Mediad Inc., White Plains, N.Y., 1 9 6 8 , ~ 311. . Everse, J. and Stolzenbach, F. E.,MethodsEmymol., 22 (1971) 33. Feinstein, G . , Naturwissenschaften, 5 8 (1971) 389. Fridovich, I., J. Biol. Chem., 237 (1962) 584. Fried, M . and Chun, P. W., Methods Enzymol., 22 (1971) 238. Hedrick, J . L. and Smith, A. J.,Arch. Biochem. Biophys., 126 (1968) 155. Himmelhoch, S. R., Methods Enzymol., 22 (1971) 273. Howard, R. L.and Becker, R. R., J. Biol. Chem., 245 (1970) 3186. Jakoby, W. B.,Methods Enzymol., 22 (1971) 248. Kalderon, N., Silman, I., Blumberg, S. and Dudai, Y., Biochim. Biophys. Acta, 207 (1970) 560. Kaufman, S.,Methods Enzymol., 22 (1971) 233. Koike, M. and Hamada, M.,Methods Enzymol., 22 (1971) 339. Kornberg, A., Methods Enzymol., 1 (1955) 491. Kremzner, L. T. and Wilson, I. B., J. Biol. Chem., 238 (1963) 1714.
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ENZYMES
Laboureur, P. and Labrousse, M., C.R. A.cad. S c i , Paris, 259 (1964) 4394;Bull. SOC.Chim. Biol., 4 8 (1966) 747. Leuzinger, W. and Baker, A. L., Proc. Nat. Acad. Sci. U.S., 57 (1967) 446. Levin, O., Methods Enzymol., 5 (1962) 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.,J. Biol. Chem., 193 (1951) 265. McPhie, P.,Methods€hzymol., 22 (1971) 23. Marquardt, R. R., Can. J. Biochem., 47 (1969) 517. Mke$ 0. (Editor), Laboratory Chromatographicand Other Separation Methods, Ellis Horwood Publ. Co., Chichester, and SNTL, Prague (Czech version), 1975, in press. Mike;, O., Worowski, K. and Turkovi, J., Collect. Czech. Chem. Commun., 38 (1973) 3339. Miller, J., Cuatrecasas, P. and Thompson, E. B., Biochim. Biophys. Acta, 276 (1972) 407. Moss, D. W., Med. Electron. Biol. Eng., 3 (1965) 327. Newbold, P. C. H. and Harding, N. G. L., Biochem. J . , 124 (1971) 1. Noltrnann, E. A.,J. Biol. Chem., 239 (1964) 1545. Pencfsky, H. and Tzagoloff, A., Methods Enzyrnol., 22 (1971) 204. Pogel, B. M. and Sarngadharan, M. G.,Merhods Enzymol., 22 (1971) 379. Porath, J . , Biotechnol. Bioeng. Symp., No. 3, 1972, p. 145. Poston, J . M., Stadtman, T. C. and Stadtman, E. R., Methods Enzymol., 22 (1971) 49. Prusik, Z., in Mike:, 0. (Editor), Laboratory Chromatographicand Other Separation Methods, Ellis Horwood Publ. Co., Chichester, and SNTL, Prague (Czech version), 1975, in press. Rdjmakur, T. V., Woodtin, B. M. and Rutter, W. J.,Methods Enzymol., 9 (1966) 491. Rattazzi, M. C., Biochim. Biophys. Acta, 181 (1969) 1. Reiland, J., Methods Enzymol., 22 (1971) 287. Repaske, R., Methods Enzymol., 22 (1971) 322. Rony, P. P., Biotechnol. Bioeng., 1 3 (1971) 431. Roussos, G. G. and Morrow, B. H., Biochem. Biophys. Rex Commun., 29 (1967) 388. Sarngadharan, M. G., Watanabe, A. and Pogell, B. M.,J. Biol. Chem., 245 (1970) 1926. Schneir, M. and Moldave, K., Biochim. Biophys. Acta, 166 (1968) 58. Schwartz, M. K., Automat. Anal. Chem., Technicon Symp., 1967, Vol. 1, Mediad Inc., White Plains, N.Y., 1968, p. 587. Schwartz, M. K.,Methods Enzymol., 22 (1971) 5. Schwartz, M. K., Kessler, G. and Bodansky, O.,Ann. N. Y, Acad. Sci., 87 (1960) 616. S h i r i v a , M., Benzonana, G. and Desnuelle, P., Biochim. Biophys. Acta, 144 (1967) 703. Shuster, L., Methods Enzymol., 22 (1971) 412 and 434. Sipos, T. and Merkel, J., Biochem. Biophys. Rex Commun., 31 (1968) 522; Biochemistry, 9 (1970) 2766. Smythe, W. J . , Shamos, M. M., Morgesern, S. and Skeggs, L. T., in Automat. Anal. Chem., Technicon Symp., 1967, Vol. 1, Mediad Inc., White Plains, N.Y., 1968, p. 105. Strausbauch, P. H., Fischer, E. H., Cunningham, C. and Hager, L. P., Biochem. Biophys. Res. Commun., 28 (1967) 525. Swanljung, P., Anal. Biochem., 4 3 (1971) 382. Swanljung, P. and Frigeri, L., Biochim. Biophys. Acta, 283 (1972) 391. Tappel, A. L.,MethodsEnzymol., 22 (1971) 219. Tiselius, A., Hjert6n. S. and Levin, O., Arch. Biochem. Biophys., 6 5 (1956) 132. Turkovi, J., in Mike's, O., (Editor), Laboratory Chromatographicand Other Separation Methods, Ellis Horwood h b l . Co., Chichester, and SNTL,Prague (Czech version), 1975, in press. Tzagoloff, A. and Penefsky, H. S.,Merhods Enzymol., 22 (1971) 219. Vezerek, B., Kicl, K., Kolaiik, L., Chundela, B. and VeEerkovi, J., Chem. Listy, 5 3 (1959) 279. Vesterberg, O.,Methods Enzymol., 22 (1971) 389. Wacker, W. E. C. and Coombs, T. C., Annu. Rev. Biochem., 38 (1969) 539. Williams, F. R. and Hager, L. P., Biochim. Biophys. Acta, 116 (1966) 168. Wilson, I. B. and Alexander, J.,J. Biol. Chem., 237 (1962) 1323. Yamane, T. and Sueoka, N., Proc. Nat. Acad. Sci. US.,50 (1963) 1093. Zeppezauer, M., Methods Enzymol., 22 (1971) 253.
Chapter 37
Low-molecular-weight constituents of nucleic acids Nucleosides, nucleotides and their analogues
s. Z A D R A ~ I L CONTENTS Introduction ................................................................... 831 General techniques in the separation of low-molecular-weight components of nucleic acids ...... 832 Automated procedures for the analysis of nucleic acid components ........................ 836 839 Individual types of nucleic acid constituents .......................................... 839 Purine and pyrimidine bases and their analogues .................................... Nucleosides ................................................................ 842 Nucleotides and oligonucleotides ............................................... .847 851 Complexmixtures ........................................................... References .................................................................... 855
INTRODUCTION Nucleic acids are composed of phosphoric acid, a sugar component (deoxyribose or ribose) and purine and pyrimidine bases (adenine, guanine and cytosine, thymine or uracil). These basic components can be isolated from total hydrolyzates of polymers, while partial hydrolysis of the polynucleotide chain leads to fragments in the form of nucleosides, nucleotides and oligonucleotides (Table 37.1). These components can also be isolated from the cell pool, the composition of which is the result of an equilibrium between biosynthetic and catabolic cell processes (Table 37.2). In addition to the above substances, cellular pools also contain more highly phosphorylated nucleoside derivatives (diphosphates and triphosphates) and some nucleotide-type coenzymes (DPN, TPN, NAD, FAD, UDPG, coenzyme A, etc.) (Hutchinson). In addition to the four fundamental nucleosides for each type of nucleic acid, six so-called minor components were found in DNA (mostly from bacteriophages) and about 35 in RNA (mainly tRNA). A survey of these components, which can be isolated only as cleavage products of natural polymers because they are formed by modification of the fundamental components on the macromolecular level, is shown in Table 37.3. Obviously, when studying the structure and function of nucleic acids, many synthetic intermediates are encountered, e.g., nucleosides with protective groups in organic synthesis, and analogues of bases and nucleosides, e.g., azapyrimidines (Skoda), the separation of which must therefore also be taken into account in experimental work (Zadratil, 1972). References p.855
83 1
83 2
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
TABLE 37.1 HYDROLYTIC PROCEDURES FOR THE ISOLATION OF NUCLEIC ACID COMPONENTS ~~
~
Conditions
Substrate(s)
Final products
Reference
72%HClO, (12N), 1OO"C, 1-2 h
DNA, RNA, oligonucleotides
Purine and pyrimidine bases
98- 100% HCOOH, 175"C, 1 h 0.3 M KOH, 37°C 16 h Crotalus adamanteus venom (1:lO enzyme: substrate) and bacterial alkaline phosphatase (1:30), 37"C, 20-24 h DNase I (1 :loo), 37"C, 2 h, pH 6-7, and Naja naja venom (1:50), 37"C, 5 h, pH 8-9 Specific nucleases (various conditions of complete and partial hydrolyses)
DNA and oligonucleotides RNA and oligonucleotides tRNA and RNA in buffer of pH 8.6 with 5 mM M a ,
Purine and pyrimidine bases 3'( 23-Mononucleotides (96%) Nucleosides
Littlefield and Dunn, Marshak and Vogel, Sluyser and Bosch Wyat, Wyat and Cohen Bock, Singh and Lane Hall (1965)
Denatured DNA in a buffer with 30 mM MgSO,
Nucleosides
Pifhovi et al.
DNA, RNA and oligonucleotides
Mono- and oligonucleotides
Zadratil (1973)
TABLE 37.2 EXTRACTION OF. CELL NUCLEOTIDE POOL (HUTCHISON AND MUNRO) Extraction agent
Conditions
Removal of extraction agent
Trichloroacetic acid
5-10% at 4"C, repeated four times 1.2-6%at 4"C, repeated 3-4 times
Extraction with diethyl ether (several times) AS KClO, by centrifugation for 10 min at 2000 g below 4" c
Perchloric acid
GENERAL TECHNIQUES IN THE SEPARATION OF LOW-MOLECULAR-WEIGHT COMPONENTS OF NUCLEIC ACIDS In order to separate purine and pyrimidine bases, nucleosides and nucleotides, all types of chromatographic techniques are employed in practical laboratory work, including paper chromatography (paper electrophoresis and fingerprinting techniques), thin-layer chromatography (Randerath) and all types of column chromatography (adsorption, partition, ion exchange and gel permeation). It is usually stated, from the quantitative point of view, that the optimum amounts of nucleotides for separation by means of the above techniques are 0.2-30 pg for TLC, 10-200 pg for paper chroniatography and 100-500 pg for paper electrophoresis, while for columns the optimum amount is between 50 pg and several hundred milligrams. Evidently, these ranges may be considerably
833
GENERAL TECHNIQUES TABLE 37.3 LIST OF NUCLEOSIDES OCCURRING IN NATURAL NUCLEIC ACIDS (HALL, 1971) Compound
Compound
Adenosine 1-Methyl2-MethylN6 -MethylN 6 , N"Dimethy12'-O-MethylN6-( A'-Isopentenyl)N6-(~is-4-Hydroxy-3-methyIbu t-2-eny1)N6 -(Aa-Isopentenyl)-2-methylthio2' (3')-GRibosylN-[ 9-(~-D-Ribofuranosy1-9H-purind-yl) carbamoyl] -L-threonine-[N-(nebularin6-ylcarbamoyl)] -L-threonine
Uridine 3-Methyl5-Methyl2'-O-Methyl2-Thio-5-carboxymethyl(methyl ester) 5-Hydroxy5-Carboxymethyl5,6-Dihydro4-Thio2-Thio-5-( N-me thy laminome thy])Pseudo[ 5-(p-D-Ribofuranosyl)uracil] 2'-O-Methylpseudo[ 54 2'-O-Methylribosyl)uracil]
Inosine 1-Methyl-
Deoxyadenosine N6 -Methyl-
Guanosine 1-Methyl7-MethylN2-Methyl2'-0MethylN', N2-Dimethyl-
Deoxyguanosine
Cytidine 3-Methyl5-Methyl2'-O-MethylN 4 , 02-DimethylN4-Acetyl2-Thio-
Deox y u r idi ne 5-Methyl(thymidine) 5-Hydroxy methyl5-(4', 5'-dihydroxypenty1)-
Deoxycy tidine 5-Methyl5-Hydroxymethyl-
exceeded in special cases of analytical or preparative separations. In order to prepare samples for chromatography and further treatment, it is usually necessary t o remove excessive concentrations of salts and low-molecular-weight substances (e.g., urea), which would have an unfavourable influence on later separation and analysis. As mixtures of low-molecular-weight substances are involved, the use of dialysis and normal exclusion in gel filtration (the main methods of desalting nucleic acids) is greatly limited. In general, chemical and adsorption methods can be recommended, e.g., extraction with an acetone-alcohol mixture (Blumson and Baddiley, Christianson e t a / . ) and adsorption on activated carbon (Rudner et al.; Zadraiil, 1972), which can also be used in a column arrangement (adsorption at pH 4-5 and elution,with ammonia-containing alcohol). With column techniques, anion-exchange carriers on a cellulose base (DEAEReferences p.855
834
LOW-MOLECULAR-WEIGHT CONSTITUENTS OF NUCLEIC ACIDS
cellulose with elution with carbonate and hydrogen carbonate buffers; Cohn and Bollum; Rushizky and Sober, 1962) are mainly used for nucleotides and their polyphosphates. Most salts used in chromatographic gradients can be separated from nucleic acid components, particularly nucleosides and bases, by means of gel filtration on Bio-Gel P-2 (Table 37.4; Uziel; Uziel and Cohn, 1965a, b) and Sephadex columns (Flodin). Hence the two main types of gel carriers employed can also, in general, be used in order to desalt bases, particularly due to adsorption of the bases on the column material. Most salts (chlorides, iodides, acetates, formates, phosphates, etc.) are eluted earlier in the form of sharp, distinct peaks (Khym and Uziel, 1970; Simkin). TABLE 31.4 DESALTING OF NUCLEIC ACID COMPONENTS ON A COLUMN OF BIO-GEL P-2 (UZIEL; UZIEL AND COHN, 1965b) Compound
Kd (at room temperature)
Oligonucleotides (RNA included) at pH 8 Adenosine-5'-monophospha te Cytidine- and uridine-5'-monophosphates Guanosine-5'-monophosphate Acetate and Tris
0 0.24 0.3 0.7 0.8 0.82 0.84 0.85 1.o 1.1 1.4 1.6 1.8 2.1 3.1
HP0;Formate H,PO; and ammonium hydrogen carbonate Tris H' (as C1-) and CIFormic and acetic acids Urea and BrCytidine, uridine and thiouridine Cytosine, thymine and uracil Adenosine and guanosine Adenine and guanine Oligonucleotides (RNA included) at pH 3
8
Insoluble poly-N-vinylpyrrolidone is a recent material for desalting all nucleic acid components; when used in a column it separates nucleotides, nucleosides, pyrimidines and purines in that order (Lerner er al.) and can be eluted with water (Dougherty and Schepartz, 1969a). In this instance also, as with Bio-Gels, salts pass through the column unimpeded while nucleoside and bases are delayed (interaction by hydrogen bonds; Dougherty and Schepartz, 1969b). Lithium chloride and sodium chloride can easily be separated from all components, while ammonium sulphate is eluted together with the nucleotide fraction. For nucleotides, it is therefore advantageous to use the above procedures with substituted cellulose or gels for desalting. With respect to the wide variations in the chemical compositions of the substances involved, the differences in the dissociation of the various substituents (for pK values, see Table 3 7 . 9 , distribution coefficients, electron density, size and shape of molecules, etc., may all be utilized in separation processes. The sorbents most widely used in separating bases, nucleosides and nucleotides are synthetic ion exchangers, which were introduced by Cohn (1949a, b), and which together with gradient elution (Hurlbert er al., Schmitz er al.) are the most generally applicable separation techniques. For oligonucleotides,
835
GENERAL TECHNIQUES
classical ion-exchange resins were replaced mostly with substituted celluloses and dextran gels (Rushizky and Sober, 1968; Staehelin), which, used with urea-containing gradient solutions (Tomlinson and Tener), are the main tool for use in column separations of enzyme hydrolyzates of nucleic acids in sequence analysis.
TABLE 37.5 pKH VALUES OF PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES* (SMITH) Compound
Adenine 6-Methy laminopurine 6-Dimethylaminopurine Adenosine 2’-phosp ha te 3’-phosphate S’-phosphate 5’-pyrophosphate 5‘-triphosphate Guanine Guanosine 2‘-phosphate 3’-phosphate 5’-phosphate 5’-pyrophosphate 5’-triphosphate Hypoxanthine Inosine 5’-phosphate Xanthine Xan th osine Cytosine 5-Methylcytosine 5-Methylcytidine Cytidine 2’-phospha te 3’-phosphate 5’-phosphate 5’-pyrophosphate 5‘-triphosphate Uracil Uridine 2‘-phosphate 3’-phosphate 5’-phosphate 5’-pyrophosphate 5’-triphosphate Thymine Thymidine-5’-phosphate
Primary phosphate
-
0.89 0.89
-
0.7 0.7
-
1.54
Amino (basic) 4.22 4.18 3.87 3.45 3.80 3.65 3.74 3.95 4.0 3.3 1.6 2.3 2.3 2.4 2.9 3.3 1.98 ~
-
-
-
-
-
4.45 4.6 4.28 4.22 4.36 4.28 4.5 4.6 4.8
~
-
0.8 0.8
Secondary phosphate
9.8 9.99 10.5 -
6.15 5.88 6.05 6.26 6.48
5.9 5.9 6.1 6.3 6.5 ~
6.0 ~
-
-
-
-
-
-
5.9 5.9 6.4 6.5 6.6 6.5
.o
-
-
-
1.6
-
*The deoxyribonucleotides have pK values similar to those of the ribonucleotides.
References p.8SS
-
9.2, 12.3 9.16 9.7 9.7 9.4
8.9, 12.1 8.75 8.9 7.4, 11.1 5.75 12.2 12.4
6.17 6.0 6.3 6.4 6.6
-
1 .o
-
-
-
1
Enol (acidic)
9.5 9.17 9.4 9.4 9.5 9.4 9.5 9.8 10.0
836
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
Partition and adsorption column chromatography are mainly carried out with Kieselguhr (Celite; Hall, 1962, 1964), cellulose (Randerath and Struck), silica gel (zemlitka et al.), hydrophilic gels (Kull and Soodak) and aluminium oxide (Prystas and Sorm, 1964a, b), usually of commercial purity grade. Before column preparation, Kieselguhr is generally purified by washing with acid (2-3 M) and water to remove UV-absorbing impurities, as UV absorbance measurements at different wavelengths is the only practical detection method available. Eluents applied in this field are organic solvents (their purity with respect to polar admixtures is very important) and their mixtures, the elution capacity of which increases with the polarity of the mixture and also depends to some extent on the carrier employed (Trappe). Binary and ternary elution mixtures, similar to those used in paper chromatography, are used in partition chromatography, particularly for anomalous and minor components of nucleic acids and for synthetic intermediates. Sorbents available for the gel permeation chromatography of low-molecular-weight substances include cross-linked dextran gels (Sephadex, Pharmacia, Uppsala, Sweden) and polyacrylamide gels (Bio-Gel, Bio-Rad Labs. Richmond, Calif., U.S.A.). The gels mostly work on the molecularsieve principle (group-wise separation of nucleotides, nucleosides and bases), but they also have some adsorption and ion-exchange properties (greater affinity to aromatic and heterocyclic compounds). For this reason, dilute neutral buffer solutions are used for elution instead of water (De Bersaques, ZadraGl et d.).pH changes of the elution solution can obviously be utilized to separate those substances which are more strongly bound to the column bed (Khym and Uziel, 1970). AUTOMATED PROCEDURES FOR THE ANALYSIS OF NUCLEIC ACID COMPONENTS Most column-type fractionation techniques are nowadays carried out with the use of automatic fraction collectors and instruments recording the substance analyzed. In an attempt to shorten as much as possible the time needed for separation, to enhance the sensitivity and resolution of the column (nanomoles of substances separated), etc., special chromatographic instruments and systems were developed even for the separation of nucleic acid components (in analogy with amino acid analyzers). One of the systems for the analysis of nucleoprotein components (ninhydrin-positive and UV-absorbing substances) is based on the principle of the amino acid analyzer with a column of Amberlite IR-120, amplified with an LKB UvicordvModel I1 instrument with recording facilities for absorbance measurements at 254 nm (Zeniiek eta].). The entire process is shown diagrammatically in Fig. 3 7.1. High-pressure cation-exchange chromatography was employed in a Varian Aerograph Model 41 00 liquid chromatograph system with a dynamically filled column of the cation exchanger VC-10 (lox;giving a high degree of homogeneity of the column formed; Scott and Lee) for automatic analysis of the nucleotide composition of RNA and DNA (Burtis, 1970a). This fully automated system with recording facilities separates on the column hydrolyzate corresponding to 0.25 pg of RNA within 2-4 min (Fig. 37.2). Uziel et al. recommend a similar microanalytical method for separating UV-absorbing substances on a Dowex 50 column and related cation-exchange resins, applying it to the separation of a mixture of bases obtained by the gradual degradation of the polynucleotide chain by periodate oxidation (Khym
837
AUTOMATED PROCEDURES
P O S I T I O N OF COMPONENTS
1
I IIII
+
TIME
VOLUME
I
COLUMN :
45‘
TEMPERATURE
120
180
I
,
150
60
3.80
2.785
1
i
240
100
50
REGENERATION
ELUTION wiin BUFFER pH
I I
1 1
II 60
1
MIN
I
ML
I T
’
w
5.00
H
H I
I
Fig. 37.1. Diagrammatic representation of a separation of ninhydrin-positive and UV-absorbing substances in the amino acid analyzer with a column of Amberlite IR-120 (eenf3ek et al.).
0.08 -
8
N
or” -
T
I
I I
@
*A
0.48
-
016
-
0
, 0
I
d
2 3 ELUTION TIME. MIN
1
Fig. 37.2. Separation of ribonucleosides and deoxyribonucleosides by high-pressure cation-exchange chromatography on a Varian Aerograph Model 4100 liquid chromatograph with a column of VC-10 cation exchanger (Burtis, 1970a). Column: 25 X 0.24 cm. Elution: 0.4 Mammonium formate solution, pH 4.0. Flow-rate: 50 ml/h. Pressure: 3000 p.s.i. Temperature: 75°C. Sample: (a), mixture containing uridine ( l ) , guanosine (2), adenosine (3), and cytidine (4), 0.1 pg of each; and (b), mixture containing thymidine (A), deoxyguanosine (B), deoxyadenosine(C), and deoxycytidine (D), 0.4 gg of each.
References p. 855
838
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
and Uziel, 1968). An ion-exchange technique of high efficiency (a column of the cationexchange resin Aminex A-4 eluted with a citrate complex gradient made in a Technicon Autograd instrument) was used in the analysis of a mixture of nucleotides, nucleosides and bases (Murakami et al.) and polyphosphates, nicotinamide and flavine nucleotides (Drobishev et al.) in an automatic system with a Hitachi Model 034 liquid chromatograph (with detection at 260, 270 and 280 nm). Thirteen components (0.1-0.8 pmoles) of the mixture were separated with good resolution in quantitative (k 3%) and qualitative analyses (Murakami et al.). A similar separation of deoxyribonucleotides on a Zipax SAX column was used for determinations of the oligomers nucleotide composition (Gabriel and Michalewskyh A fully automated system with a Dowex 2 column and two continuous-flow measuring I
1
I
1
I
I
I
1
w1
UDP
VOLUME, rnl
Fig. 37.3. Separation of a Mycoplasmn acidic extract and 100 nmole of UMP, UDP, UDPAG and ADP (broken lines) added to, by an automated anion-exchange column chromatography (Virkola). Column: Dowex 2-X8 (200-400 mesh), 12 X 0.8 cm. Elution: a gradient prepared in a Varigrad mixer with nine compartments containing 200 ml each of the following solutions: compartment 1, water; 2,0.5 M formic acid; 3 and 4 , 4 M formic acid; 5-7,4 M formic acid with 1 Mammoniurn formate. Flow-rate: 29.2 ml/h. Temperature: 22°C.
Fig.37.4. Schematic representation of an automated liquid chromatograph based on the Varian Aerograph model (Burtis, 1970a).
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
839
cells (ensuring a stable background line) was employed by Virkola to separate an extract of acid-soluble substances in Mycoplasma laidlawii A. The applicability of this analytical system is shown in Fig. 37.3. Burtis (1970b) employed the same principle in the analysis of urine, using a Varian Aerograph Model LCS-4010 urine analyzer. Dinucleotides (Kennedy and Lee) and adenosine polyphosphates (Schmukler) can also be analyzed with a commercial nucleic acid analyzer (e.g., the Picker-Nuclear LCS-1000 analyzer) with a column of anion-exchange resin. In all of these instances, the analytical application of ion-exchange columns combinei with control and detection systems is involved, and precise preparation of homogeneoils column fillings, buffer solutions and elution gradients is also required, together with, in many instances, special instrumentation for corresponding results to be achieved (Anderson, Thacker et al., Uziel et al.). A general diagram of a liquid chromatograph based on the Varian Aerograph system is shown in Fig. 37.4.
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS Purine and pyrimidine bases and their analogues In laboratory practice, these substances are encountered as components of whole nucleic acid hydrolyzates or as synthetic substances. The main separation technique is paper chromatography, the main advantage of which is sifiiplicity. Most of the bases involved contain at least one substituent that is capable of ionization, which thus gives the molecule a positive or negative charge (Table 37.5). These properties permit the use of both ion-exchange resins and hydrophilic gels containing a certain proportion of ionizable groups (dextran). On the other hand, even slight differences in the structure of the substituents have a substantial influence on the behaviour of bases in the course of elution in partition column chromatography.(analogous to paper chromatography). A good example of the most common ion-exchange separation process is the use of a Dowex 50 (H’) column, which, when eluted with 2 N hydrochloric acid, gives distinct p e a b for all fundamental bases of RNA (Cohn, 1949b) in the order uracil, cytosine, guanine and adenine. The same column eluted with a linear gradient of 1-4 M hydrochloric acid has been used for the isolation of methylated bases, mainly guanine derivatives, from the perchloric acid hydrolyzate of the total RNA (Craddock et al.). Similar ion-exchange resins can be used to analyze bases split off in the gradual periodate oxidation of polyribonucleotides (Khym and Uziel, 1968). An anion-exchange column of Dowex 1, which also serves as an example of the first application of ion-exchange columns to nucleic acid components, combines the advantages of elution at constant solution concentration with good resolution of the sorbent (Fig. 37.5). Synthetic anion exchangers can be replaced with DEAE-cellulose (Weith and Gilham), which, however, is mainly employed to separate higher components (mono- and oligonucleotides) and rather serves in the instances mentioned to separate groups of bases for preliminary purification. The method of partition or adsorption chromatography with Kieselguhr as sorbent in the field of nucleic acid components has achieved the widest application in separating nucleosides (see p. 843), for which it has been worked out in great detail (Hall, 1971). This “nucleoside” procedure was also used by Hall (1967) to separate the main bases of References p . 855
840
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
VOLUME, ml
Fig. 37.5. Separation of purines and pyrimidines on a Dowex 1 column (Cohn, 1949b). Column: 8.5 X 0.49 cm. Elution: 0.2 M ammonia solution with 0.025 Mammonium chloride (pH 10.6) changed, as indicated by the arrow, for 0.1 Mammonium chloride (pH 10.0). Flow-rate: 60 ml/h. Sample: cytosine (11, uracil (2), thymine (3), guanine (4), and adenine (5),1-2 mg of each base. 6
8
4
0
0
Fig. 37.6. Separation of purine and pyrimidine bases by partition chromatography on Kieselguhr (Hall, 1967). Column: 42 x 1.9 cm column of 50 g of Celite 545-Microcel E (9:l) mixture in a lower phase of the solvent system ethyl acetate-2-ethoxyethanol-l0% formic acid (4:1:2). Elution: 400-ml linear gradient of upper phase with a concentration of formic acid decreasing to zero, followed by an upper phase of ethyl acetate-1-butanol-water (1 :1 :1). Flow-rate: 60 ml/h. Sample: 2 ml of lower phase of the starting solvent system containing 5 mg of each of the bases thymine (I), uracil (2), adenine (3), guanine (4), and cytosine (S), applied in the form of a suspension with 4 g of the carrier mixture.
nucleic acids, as shown in Fig. 37.6. The separation of bases on poly-N-vinylpyrrolidone, based on hydrogen bonding of the bases with the sorbent, likewise belongs to this group of methods (Dougherty and Schepartz, 1969b). Adsorption of purine bases on the sorbent material during gel permeation chromatography has already been mentioned in the section on desalting. This interaction of bases, which is actually an ion-exchange process, allows the mutual separation of substances within this group to take place together with separation of the group of bases from higher components (Table 37.6). Sweetman and Nyhan (1968, 1971) have shown that the
84 1
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
TABLE 37.6 DISTRIBUTION CONSTANTS (Kd VALUES) OF SOME PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES ON VARIOUS SEPHADEX COLUMNS Procedures: A, column 25 X 1.5 cm eluted with 130 mM ammonium formate of pH 6 (De Bersaques); B, column 100 X 1 cm eluted with 50 mM sodium dihydrogen phosphate of pH 7 (Sweetman and Nyhan, 1968); C, column 150 X 1.3 cm eluted with 10 n M ammonium carbonate of pH 9 (Gorbach and Henke); D, column 35 X 3.5 cm eluted with water (Gelotte); E, column 90 X 2.2 cm eluted with 0.005% aqueous ammonium carbonate of pH 7.6 (ZadraZil et d); P, column 180 X 3.5 cm eluted with 10 d a m m o n i u m carbonate of pH 9 (Hohn and Pollman). Compound
K , value on Sephadex column C-15
G-10
Adenine Adenosine 2'-Deoxy5'-phosphate 5'-pyrophospha te 5'-triphospha te Guanine Guanosine 2'-Deoxy5'-phosphate Hypoxanthine Inosine 2'- De o xyXanthine Xan thosine Cytosine Cytidine 2'-Deoxy5'-phosphate Uracil Uridine 2'-Deoxy5'-phosphate Thymine 1-p-D-RibofuranosylThymidine 5'-phospha te 5'-pyrophosphate 5'-triphosphate
G-25
A
B
A
C
D
E
F
6.00 3.36 3.23 0.9 1 0.4 1 0.29 3.35 2.43 2.66 0.8 2.17 1.26 1.26 3.15 1.83 1.43 0.99 1.09 0.4 1.56 1.07 1.09 0.45 1.86 1.oo 1.24 0.52 0.28 0.19
7.66 4.23 4.23 0.93
4.26 2.92 2.81 1.37 0.74 0.50 2.80 2.29 2.40 1.07 1.89 1.31 1.30 2.89 1.95 1'.26 1.06 1.16 0.54 1.50 1.21 1.15 0.74 1.63 1.15 1.29 0.73 0.44 0.32
4.62 2.79
2.2 1.7
3.38 2.59
3.62 2.50
-
5.84 3.14 3.28 0.82 2.83 1.56 1.60 4.34 2.51 1.84 1.32 -
0.40 1.91 1.28 -
0.44 2.23 -
2.23 ~
-
-
-
0.58
0.1
0.85
-
-
-
-
-
-
-
3.22 2.09
-
3.40 2.60
3.23 2.30
-
-
-
-
0.43 2.20 1.50
0.4 1.6 1.2
0.9 1
1.04
-
2.20 1.50 1.50 1.18
1.6
~
-
1.27 (3') -
-
-
.-
-
-
-
-
1.8
-
-
-
-
-
1.6 1.2
1. s o 1.60
1.69 1.42
-
-
-
-
0.37 1.18 0.85
0.1 1.1 1.o
0.66 1.82 1.66
0.69 1.54 1.27
-
-
-
-
0.10 1.45
0.1
0.66
-
-
0.69 1.54
-
-
-
-
-
-
-
1.23 0.89
-
-
-
-
-
-
-
-
~
1.02 ~
adsorption of purines, the mechanism of which they investigated, may serve not only for separation but also to predict the elution volume of a molecule of a given structure on Sephadex G-10, and vice versa. As the elution volume of purines are very high when elution is carried out with water, dilute phosphate or volatile buffers (ammonium carbonate and hydrogen carbonate) are used when separating bases, for practical reasons. Khym References p , 855
842
LOW-MOLECULAR-WEIGHT CONSTITUENTS OF NUCLEIC ACIDS
and Uziel(l970) used columns of Sephadex G-10, equilibrated with 0.01 M hydrochloric acid or ammonia solution and ammonium chloride buffer of pH 9.7 to separate purine and pyrimidine bases in the order uracil, cytosine, adenine, guanine and uracil, cytosine, guanine, adenine, respectively. The variation of the pH between 9 and 10 considerably altered the elution profile of the mixture (a dependence on the ionization of the individual bases).
Nucleosides As the sugar component of a nucleoside does not contribute greatly to changes in the formation of the charge of the molecule (with the exception of the use of borate buffers with ribose derivatives; Khym and Cohn, Khym and Zill), the same applies to the separation of nucleosides as to that of bases. The main separation method in this field is adsorption and partition chromatography, although ion-exchange and gel permeation chromatography are also utilized. Natural nucleosides of both types can be separated by a similar ion-exchange method to that described for bases [Dowex 1 in the formate cycle with elution by ammonium formate at pH 10.2 (Andersen et al.; Cohn, 1950) or Dowex 50 used in the “nucleoside
..
‘c
0.6
0.4
0.2
I
I
4
8 VOLUME.ml 12
Fig. 37.7. Separation of ribonucleosides on a sulphonated polystyrene cationexchange column (Singhal and Cohn). Column: Bio-Rad Aminex A-6, 50 X 0.5 cm with void and inner volumes of 3 and 5 ml, respectively. Elution: 0.02 Mammonium carbonate with ammonia solution of (A) pH 9.3 or (B) 9.85. Flow-rate: 0.98 ml/cm2 and 0.194 ml/min. Temperature: 50°C. Sample: 10 pl of about 100 nmole mixture of pseudouridine (l),uridine (2), thymidine (ribo-, 3), cytidine (4), guanosine (S), adenosine (6) and 4-thiouridine (7).
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
843
analyzer” according t o Uziel et al. ] . Using the ion-exclusion or ion-repulsion techniques on a column of the cation exchanger Bio-Rad Aminex A-6, Singhal and Cohn achieved one of the best nucleoside separations known (Fig. 37.7). When sodium or potassium borate to 10-5M) in an elution solution of pH 8-10 is used, substances containing the cis-diol group obtain another negative charge (borate diols are formed), which facilitates ion-exchange separation. Jaenicke and Von Dahl used the borate form of a strong anion-exchange resin t o separate ribonucleosides (in the order cytidine, adenosine, uridine and guanosine), eluting with increasing concentrations of borate buffer of pH 9.2 and 0.01-0.1 M sodium chloride solution. Ion-exchange chromatography, however, is more important for the separation and purification of nucleoside products from reaction mixtures of organic synthesis. Ziff and Fresco isolated uridine and cytidine derivatives from the mixture obtained by oxidation of 4-thiouridine (Dowex 1 (Cl- or CH3COO-) and Dowex 50 (H+), both eluted by salt gradients); Bobek ef al. (1969a, b) isolated 6-azauridine and 6-azapseudouridine derivatives and Bobek and Sorm isolated homouridine and homocytidine on the same types of ion exchangers. The separation of different nucleoside mixtures, differing also in their sugar components, was effected on a Dowex 1 column by Dekker and by Gin and Dekker. Metabolic conversions of isopentenyladenosine were studied using a DEAE-cellulose column (Hall et al.). Partition chromatography on a Celite 545 column, when combined with paper chromatography (Fig. 37.8), allows nearly all of the nucleoside components known so far t o occur in tRNA enzyme hydrolyzates t o be separated (Table 37.3). Because of the general importance of this technique, a detailed description of the column preparation and the sample application is given below (Hall, 1962, 1967, 1971). A suspension of Celite 545 in 3 N hydrochloric acid is washed on a suction funnel with the same acid until a clear filtrate is obtained. After washing with distilled water until the filtrate is neutral, the Celite is washed with ethanol and dried in a thin layer for 16 h at 100°C. Microcel E is washed in the same manner. The column is best prepared by means of the “dry pack” technique. To 160 g of a dry mixture of Celite 545-Microcel E (9: I ) , roughly two parts of the aqueous (lower) phase of the solvent system employed (see p. 845) are added so as to obtain a free-floating powder (the bonding capacity of the sorbent for the liquid is just saturated); in this form, the powder is suitable for filling a thick-walled glass tube (height 80 cm, I.D. 2.54 cm, fitted with a ground-glass joint) by means of a rod with a flat end cut at a right-angle, moving closely in the column (in a similar manner t o the piston of a syringe). Small portions of the above moist Celite are compressed into compact layers by means of the rod, the height of these layers being equal t o the column diameter. In this way, a very compact carrier column is obtained with no chanelling effect on elution. A column of this type is suitable for separating 1-1.5 g of RNA hydrolyzed t o nucleosides. The best method of sample application is t o dissolve the lyophilized enzyme RNA hydrolyzate in 9 ml of the aqueous phase of the solvent system and, after centrifuging for 15 min at 15,000 g, and mixing with 1 8 g of dry Celite 545-Microcel E (9: I ) , to fill the moist suspension obtained on t o the top of the column prepared as above. After application of the sample, the column can be eluted with the appropriate solvent system (see Fig. 37.8). References p. 855
tRNA hydrolyzate Fig. 37.9 Column Solvent F and G
p"
1
P
Fig 37 1Oc Solvent Column H
m
m n Fig 37 10b Solvent Column E
N6- Methy Iadenosine 3- Methy Iuridine
Guanosine N6_Met hy ladenosine
2'- 0-Methy I$-Methylguanosine guanosrne Cytidine $-DimethUridine 2'-O-Methyl- ylguanosine pseudouri2I-O-Methdine ylguanosine
5*
Fig 37 10d Solvent Column J
Guanosine Guanosine DeoxyCytidine
2I - 0 - Methy Icytidine
Gwnosine 2-Amino-4hydroxy-5Pseudouridine methylforCytidine mam~do-6ribosylaminopurine Cytidine I-Methyladenosine
P
Pseudouridine Cytidine N -(Purin6-ylcarbamoyl )amino no acid 1- Methyladenosine
Pseudouridine Cytidine I-Methyladenosine
r
Fig. 37.8. General procedure for the complete separation of nucleosides from a tRNA enzymic hydroiyzate (Hall. 1967). A-J: solvent systems used (see p. 845). Numbers in parentheses represent the developing time of paper chromatography in the given solvent system.
E*
2
E
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
84 5
Figs. 37.9 and 37.10 show the distrioution of the total hydrolyzate into individual fractions and their re-chromatography on smaller columns of the same sorbent. The peaks obtained after re-chromatography were divided into individual components by chromatography on Whatman No. 3MM paper (Fig. 37.8). The solvent mixtures used for chromatography were: (A) 1-butanol-water-concentrated ammonia solution (86: 14:s); (B) ethyl acetate-2-ethoxyethanol-2% aqueous formic acid (4: 1 :2); (C) 2-propanol-concentrated hydrochloric acid-water (680: 176: 144); (D) 2-propanol-water-concentrated ammonia solution (7:2:1); (E) ethyl acetate-1-propanol-water (4: 1 : 2 ) ;(F) ethyl acetate2-ethoxyethanol-water (4:1:2); (C) ethyl acetate-1 -butanol-ligroin (b.p. 66-75°C)water (1 :2: 1: 1); (H) 1-butanol-water-concentrated ammonia solution (3: 1 :O.OS); and (J) ethyl acetate- 1-butanol-water (1 : 1 : 1). Hal1 (1962) separated deoxyribonucleosides on a Celite 545 column in solvent system B in the order thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine.
40
3
P
N T
20
0
Fig. 37.9. Fractionation of an enzymic hydrolyzate of tRNA by partition chromatography on a column of Celite 545-Microcel E (Hall, 1965, 1967). Column: 8 0 x 5.08 cm column made of 6 9 0 g of carrier mixture ( 9 : l ) in the aqueous phase of solvent system F. Elution: upper phase of solvent system F changed for an upper phase of solvent systeni G at the point marked by the arrow. Flow-rate: 600 ml/h. Sample: 35 ml of aqueous phase of F containing nucleosides of the enzymic hydrolyzate of tRNA (5.4 g), applied as a suspension with 8 0 g of carrier mixture; fractions 1-6, containing N6-methyladenosine ( I ) , adenosine ( 2 ) , uridinc (3), methylated guanosines (4),guanosine ( 5 ) , and cytidine ( 6 ) as their main components, were used for further separations (see Figs. 37.8 and 37.10).
Neutral aluminium oxide can be used with advantage as sorbent in the adsorption separation of protected nucleosides, e.g., anomeric ribofuranosyl derivatives of uracil (elution with a benzene-ethyl acetate mixture in various proportions; Prystag and Sorm, 1964a, b, 1965, 1966). The Kd values in Table 37.6 characterize the properties of the main nucleosides on Sephadex columns and the possibility of their mutual separation by gel permeation chromatography with the use of eluents of different concentrations and pH values (De References p.855
846
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
16
I II!
I
I
0
1500
8
3000
VOLUME. ml
Fig. 37.10. Re-chromatography of fractions from Fig. 37.9 on a column of Celite 545-Microcel E (Hall, 1965, 1967). Column: 80 X 2.54 cm column made of 150 g of carrier mixture. Flow-rate: 150 ml/h. (a) fraction 1 of Fig. 37.9 eluted with solvent system H; (b) fraction 4 of Fig. 37.9 eluted with solvent system E; (c) fraction 5 of Fig. 37.9 eluted with solvent system H; (d) fraction 6 of Fig. 37.9 eluted with solvent system J. Numbers in each figure indicate regions the fractions of which are used for further separation by paper chromatography (see Fig. 37.8).
4
r- 0.10
0.05
0
30
60
90
ELUTION TIME.MIN
Fig. 37.11. Separation of deoxyribonucleosides on a column of fractionated Sephadex G-10 (Ehrlich et nl., 1971a). Column: 25 X 0.5 cm. Elution: 0.025 M ammonium carbonate of pH 10.4. Flow-rate: 3 ml/h. Sample: 50 &I containing 0.25 A zbo unit of each deoxyribonucleoside: thymidine (11, deoxycytidine (2), deoxyguanosine (3) and deoxyadenosine (4).
Bersaques; Gelotte; Gorbach and Henke; Hohn and Pollman; Sweetman and Nyhan, 1968; Zadraiil et al.). The most successful separation of nucleosides was carried out by Bernardi and co-workers on commercial and fractionated gels (to obtain a more homogeneous column filling), viz. Bio-Gel P-2 (Carrara and Bernardi, Piperno and Bernardi) and Sephadex G-10 (Ehrlich et al., 1971a). It was found that both types of gel can be used
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
847
for precise determinations of the base composition of nucleic acids even with a different elution order of the components to be separated (Fig. 37.1 1). Bio-Gel P-2 can also be used t o separate nucleosides in the presence of a larger amount of nucleotides, which are eluted considerably earlier than on Sephadex G-10. As a microanalytical method, this procedure has been applied in many instances t o determine the nucleoside composition of enzyme hydrolyzates of DNAs (Bernardi et al., 1968, 1970; Corneo et al.) and of terminal nucleotides (Ehrlich er al., 1971b). Both ribonucleosides and deoxyribonucleosides can be separated on the same column of fractionated Bio-Gel P-2 in a single operation in the presence of sodium tetraborate in the elution solution at pH 10.1 (Fig. 37.12; Piperno and Bernardi). A Sephadex G-10 column eluted with a citric acid-phosphate buffer of pH 3.5 can be recommended for the separation of thymidine from its analogues 5-bromo- and 5-iododeoxyuridine (Braun, Visser).
VOLUME.ml
Fig. 37.12. Separation of ribonucleosides and deoxyribonucleosides on a column of fractionated Bio-Gel P-2 (Piperno and Bernardi). Column: 60 x 0.8 cm. Elution: 0.1 mM borate in 2 mMammonium carbonate of pH 10.2. Flow-rate: 2.8 ml/h. Sample: 50 pl containing 1 A,,, unit of each ribunoclcoside: (1) uridine, (2) guanosine, (5) cytidine and (6) adenosine; and 2 A,,, units of each deoxyribonucleoside: (3) deoxyguanosine, (4) thymidine, (7) deoxycytidine and (8) deoxyadenosine.
Nucleotides and oligonucleotides In contrast to bases and nucleosides, nucleotides bear a strongly acidic phosphate group, so that they are mainly ionized as anions, although their behaviour t o some extent also depends on the substituents of the base present (Table 37.4). They cannot be separated by means of paper chromatography, but with respect to their charge, paper electrophoresis is frequently employed (Markham and Smith, Sanger and Brownlee, Smith). For the same reasons, the main column separation technique is ion-exchange chromatography on polystyrene resins (Dowex), as well as on substituted celluloses and dextrans. In order t o separate oligonucleotide mixtures on the basis of the molecular sizes of the components, DEAE-substituted carriers eluted with urea-containing gradients (Tener) can be recommended in addition t o gel permeation chromatography (Stanley). A column of anion-exchange resin in the chloride cycle (Dowex l), successively eluted with water and diluted hydrochloric acid, was first employed by Cohn (1950) and was found to be an ideal tool for separating RNA alkaline hydrolyzates (elution order: bases and nucleosides, CMP, AMP, UMP and GMP). For a routine method of investigating the base composition of RNA Katz and Comb recommend a Dowex 50 (H') column for separating UMP (0.05 M hydrochloric acid), GMP and mixtures of AMP and CMP, which can be further separated, if necessary (e.g., in order t o measure their specific radioactivity), on a Dowex 1 column. Dowex 1 with stepwise concentration elution with acetate at pH References p.855
848
LOW-MOLECULAR-WIGHTCONSTITUENTS OF NUCLEIC ACIDS
4.7 also served to separate the nucleotides in a DNA enzyme hydrolyzate (Sinsheimer and Koerner) and, with ammonium acetate of pH 4.3, to resolve 5-hydroxymethylcytidylic acid from its mono- and diglycosylated derivatives (DNA from T-even phages; Lehman and Pratt). Similarly, 5,6-dihydrouridylic acid was isolated from the total RNA hydrolyzate after treatment with RNase T1 on a Dowex 1 column with a complex ammonium formate gradient (pH 3 . 9 , and re-chromatography of the minor peak between cytidylic and pseudouridylic acid was carried out on a DEAE-Sephadex column (linear gradient of 0.1-0.7 M ammonium carbonate; Madison, Madison and Holley). Anion-exchange resins of the Dowex 1 type were also used in the separation of oligonucleotides, serving as sorbents in the first attempts to characterize the primary structure of nucleic acids (Volkin and Cohn, RNA enzyme hydrolyzate after treatment with pancreatic RNase; Fig. 37.13). Using gradient elution with formic acid and formate, oligonucleotides from a similar hydrolyzate were isolated on Dowex 1-X2columns (Zadraiil and Sormovi). Oligonucleotides from a DNA enzyme hydrolyzate were separated in similar manner by Sinsheimer. The separation of isomeric 3 ’ 5 ’ -and 2‘3’-dinucleoside monophosphates was carried out by Taylor and Hall. A widely used anion-exchange sorbent is DEAE-cellulose or its Sephadex analogue, which, in addition to being used for the fractionation of reaction mixtures in a synthetic laboratory (HolL !nd korm, separation of isomeric monophosphites on a column in borate buffer; HolL and ZemliEka, fractionation of a mixture of nucleoside and mono- and
Fig. 37.13. Preparative fractionation of a pancreatic RNase digest of calf liver RNA on a Dowex 1 column (Voikin and Cohn). Column: 15 x 1.09 cm, Dowex 1-X2 (400 mesh). Elution: for fractions I to X, the following solutions were used in a stepwise manner: 0.005 N hydrochloric acid; 0.01 N hydrochloric acid; then 0.0125 M ; 0.025 M ; 0.05M, 0.1 M , 0.2 M 0.3 M, 1 .OM, and 2.0 M sodium chloride added to 0.01 N hydrochloric acid in each instance. Sample: 700 mg of RNA hydrolyzed with 10 mg of RNase. Parentheses indicate an unknown sequence or a mixture of sequences. Brackets indicate empirical composition.
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
849 1.0
0.5
> !z n
4
P 3
Fig. 37.14. Separation of oligoadenylic acids from an Azatobacter nuclease digest of poly-A on a column of DEAE-cellulose (Staehelin et al., 1959). Column: 30 X 0.9 cm. Elution: a gradient prepared in a Varigrad mixer with six compartments containing ammonium hydrogen carbonatecarbonate buffer of pH 8.6 with the following concentrations: 1, 0.01 M ;2, 0.24 M ;3, 0.01 M ;4 and 5, 0.20 M ;6 , 1 M.
dinucleotide by a linear trietliylammonium hydrogen carbonate gradient of pH 7.5; Tener ef al., fractionation of oligomers in synthetic polymerization of thymidylate) also find
a particularly important field of application in separating oligonucleotides in enzyme hydrolyzates of nucleic acids and polynucleotides (Staehelin er al., 1959, separation of homologous oligoadenylic acids from poly-A enzyme hydrolyzate, Fig. 37.14; and a similar fractionation of RNA hydrolyzate obtained with pancreatic RNase, Fig. 37.1 5). It can be seen from the latter-two examples, that the form of the gradients to which this column fractionation is always bound is decisive for the degree of separation. In general, for DEAE-substituted cellulose and dextran, the homologous oligonucleotides are separated in accordance with their chain lengths (Fig. 37.14), while the prevailing base of the oligonucleotide increases the elution volume in the series C, U, A and G (Fig. 37.15). The introduction of buffers containing 7 M urea (Tener, Tomlinson and Tener) simplifies the situation in such a way that at a neutral pH (5.4-8.0) the oligonucleotide mixture is separated in accordance with the magnitude of the charge only, i.e., according to the number of phosphate groups or degree of polymerization (Fig. 37.16), even when a simple linear gradient is applied. Subfractionation according to the composition of the bases in fragments bearing the same charge can be carried out on the same column simply by omitting the urea (the above-mentioned order of influence of bases) or by elution at lower pH (2.7-4.0) in the presence of urea (protonization of adenine and cytosine), and also by elution at higher pH (8.5-10.0 with carbonate or hydrogen carbonate buffers) with no urea (ionization of uracil and guanine). Urea-contajning buffers have been found to be very useful, particularly for separating purine and pyrimidine “isopliths” after specific chemical degradation of nucleic acids, apyrimidinic and apurinic acids being References p.855
850
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS I
I
AU
0.4
>
ca
4
0
z 0.2
1
400
I
VOLUME, ml
600
Fig. 37.1 5. Separation of a pancreatic RNase digest of yeast RNA o n a column of DEAE-cellulose (Staehelin et al., 1959). Conditions as in Fig. 37.14, except for the last Varigrad compartment, which contained 0.4 M buffer.
I '
"
40
1
1
120
I
1
200
1
I
280
I
1
360
FRACTION NO.
Fig. 37.16. Fractionation of an RNase T1 digest of yeast RNA on a column of DEAE-Sephadex in the presence of 7 M urea (Tener). Column: 50 X 4 cm. Elution: a linear gradient of sodium chloride in 0.02 M Tris-hydrochloric acid buffer of pH 7.6 with 7 M urea. Flow-rate: 100 ml/h. Fractions: 20 ml. The peaks are numbered according t o the chain-length of the compounds present.
intermediates in the process (Habermann; Petersen and Reeves; Vanyushin and Bur'yanov, 1969a, b). Mononucleotides are not usually separated in the course of fractionation on molecular sieves and, therefore, Sephadex G-10 and Bio-Gel P-2 are used particularly to separate adenosine and thymidine polyphosphates, which fractionate according to their molecular weights when eluted with formate at pH 6 (De Bersaques), and to study, for example,
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
85 1
nucleotide-metal interactions (Colman). The separation of oligothymidylic acids and a study of the influence of their terminal phosphate group on the separation was likewise successful (Haynes er al. ; Hohn and Pollman). Stanley verified the possibility of using columns of Sephadex G-25, G-50, G-75 and GI00 to separate substances by their degree of polymerization, finding that 0.5 M ammonium hydrogen carbonate of pH 8.6 with 8 M urea is needed for an elution process that is independent of the purine bases content, and that the elution volume o f oligonucleotides may also be influenced by their secondary structure. Combinations of different sorbent columns, such as Sephadex, Bio-Gel, DEAE-cellulose and hydroxyapatite, were used during a study of oligodeoxyribonucleotide biosynthesis in pulse-labelled mammalian cells (Schandl and Taylor).
Complex mixtures In order to separate complex mixtures containing components of different types (bases, nucleosides, nucleotides and their polyphosphates, or oligonucleotides together with their sequence isomers, etc.), encountered mainly when isolating the acid-soluble pool of cells and tissues (Table 37.2) or fractionating enzyme hydrolyzates of nucleic
c-----
E L U T I O N TIME,HOURS
Fig. 37.17. Separation of RNA components and the position of some bases, nucleosides and nucleotides on a column of Dowex 1 (Hori). Column: Dowex 1-X8 (200-400 mesh), 150 x 0.9 cm. Elution: a gradient prepared in a Varigrad mixer with nine compartments containing 135 ml of acetate buffer of pH 4.4 of the following concentrations: l , O . l M ; 2, water; 3 , 1 M ; 4 , 1.2 M; 5 , 2 M; 6,0.4 M ; 7, 3 M ; 8 and 9, 2.5 M. Flow-rates: 0.445 ml/min (A) and 0.89 ml/min (B). Temperature: 35°C (A) and 45°C (B). Components: 1 = 5-methylcytosine; 2 = pyrimidine; 3 = pseudouridine; 4 = 2-aminopyrimidine; 5 = thymidine; 6 = 5-hydroxymethyluracil; 7 = purine; 8 = hypoxanthine; 9 = xanthine; 1 0 = cytidineS'-phosphate; 11 = xanthosine; 12 = pseudouridine-monophosphate; 1 3 = thymidylic acid; 14 = uridine5'-phosphate; 15 = uridine-2'-phosphate; 16 = inosinc-S'-phosphatc; 17 = adenosine-S'-phosphate; 1 8 = guanosine-5'-phosphate.
References p.855
852
LOW-MOLECULAR-WEIGHTCONSTITUENTS OF NUCLEIC ACIDS
L
A
-
B
-
C
~
D
+
Fig. 37.18. Separation of a yeast nucleotide extract on a Dowex 1 column (Schmitz). Column: 19 x 0.8 cm, Dowex 1-X8. Elution: linear gradients of 0-4 M formic acid (A) and 0-0.2 N (B), 0.2-0.4 N (C) and 0.4-0.8 N (D) ammonium formate in 4 N formic acid. Flow-rate: 60 ml/h. Fractions: 4 ml. Sample: a neutralized perchloric acid extract of 40 g of yeast. 1 = Adenosine; 2 = cytidine-5'-phosphate; 3 = diphosphopyridinenucleotide; 4 = adenosine-5'-phosphate; 5 = guanosine-5'-phosphate and triphosphopyridinenucleotide;6 = cytidine-5'-pyrophosphate; 7 = inosine-S'-phosphate; 8 = uridine-5'phosphate; 9 = adenosine-5'-pyrophosphate; 10 = uridinediphosphoaminosugar peptide; 11 = uridinediphosphoglucose or -galactose; 12 = guanosine-5'-pyrophosphate;13 = cytidine-triphosphate; 14 = uridine-5'-pyrophosphate and uridinediphosphoglucuronicacid; 15 = adenosine-S'-triphosphate; 16 = guanosine-5'-triphosphate;17 = uridine-S'-triphosphate.
6 0.3
m 0.2
VOLUME, ml
Fig. 37.19. Separation of oligonucleotides from an RNase T1 digest of tRNALhzorj on a column of DEAE-cellulose (Uziel and Gassen). Column: Whatman DE-32, 120 X 0.5 cm. Elution: a linear gradient of 0.05-0.75 Mammonium acetate of pH 8.3 (total volume 760 ml). Flow-rate: 12 ml/h. Sample: RNase T1 hydrolyzate of 47 A,,, units in 0.2 ml of 0.2 M Tris (pH 7.3). Letters A, G, C and U correspond to basic nucleotides; 9 = pseudouridylic acid; T = ribothymidylic acid; S = 4-thiouridylic acid; D = 5,6-dihydrouridylic acid (all with 3'-phosphate end in oligonucleotide).
853
INDIVIDUAL TYPES OF NUCLEIC ACID CONSTITUENTS
acids, ion-exchange chromatography is used exclusively, and, if necessary, subfractionation is carried out on several columns under different conditions, as stated in the preceding section on nucleotide fractionation. Thus the same rules apply for the separation of these mixtures and, therefore, the discussion here is limited to a few examples. The possibilities of separating acid extracts containing UV-absorbing substances of different origins have already been mentioned in conjunction with the description of automatic “nucleoside and nucleotide analyzers”, in which use is made of Dowex-type anion and cation exchangers (see p. 836). Fig. 37.17 shows one such example of the separation of a synthetic mixture of low-molecular-weight RNA components on a Dowex 1-X8 column (Hori) with an acetate buffer gradient, prepared in a Varigrad instrument (Peterson and Sober). Lesser demands on the separation of yeast nucleotide extract, with good resolution, are illustrated in Fig. 37.18 (Schmitz), where the separation of polyphosphates and nucleotide coenzymes is also involved. The successful separation of complicated mixtures of polynucleotides and oligonucleotides is an essential condition for elucidating the primary structure of polynucleotides (Brownlee). Ion-exchange chromatography, particularly on DEAE-cellulose and DEAESephadex, using gradient elution in the presence of 7 M urea (Rushizky et al., Tomlinson and Tener) as well as at elevated temperatures (Penswick and Holley), which decrease the secondary interaction with the carrier, completely satisfies the demands of successive separation (see p. 849) of the components of total and partial hydrolyzates of nucleic acids. Uziel and Gassen, who carried out sequence studies of tRNALhEoh.,mentioned several illustrative examples (Figs. 37.19-37.2 1). Table 37.7 shows several completed tRNA structures, in the investigation of which various column techniques were employed. For
TABLE 31.1 SOME PRIMARY STRUCTURES OF t RNAs ELUCIDATED USING COLUMN FRACTIONATION TECHNIQUES tRNA
Source
Number of bases
Reference
Alanine I Serine 1 and I1 Serine
Yeast Yeast Rat liver
77 85 85
Tyrosine wrosine Phenylalanine Phenylalanine Phenylalanine Valine Valine 1 Lsoleucine Aspartic acid Tryptophane Glutamic acid 11 Arginine 111
Yeast Tomlopsis urilis Yeast Escherichia coli Wheat germ Yeast Torulopsis utilis Tomlopsis utilis Yeast Yeast Escherichia coli Yeast
78 78 76
Holley et al., Merrill Zachauetal. (1966a.b) Delihas and Staehelin, Staehelin e t al. (1968) Madison e t al. Hashimoto e t al. Raj-Bhandary er al. Uziel and Gassen Dudock e t al. Bayev e t al. Mizutani et al. Takemura e t al. Gangloff e t al. Keith e t al. Ohashi e t al. Kuntzel et al.
References p.8SS
76 76 77 75 17 75 75 76 75
854
0.5
LOW-MOLECULAR-WEIGHT CONSTITUENTS OF NUCLEiC ACIDS
1
i
Fig. 37.20. Re-chromatography of tetranucleotide mixture from Fig. 37.19 on a column of DEAEcellulose at pH 3.7 (Uziel and Gassen). Column: Whatman DE-32, 140 x 0.6 cm. Elution: a linear gradient of 0.1-0.75 Marnmonium formate of pH 3.7. Flow-rate: 39.6 rnl/h. Sample: mixture of C,G, UC,G and T W G from Fig. 37.19.
I
I
GCD
:
t
1.2
I ui z
CCGC
I.U.C
, 0 ’
-
0.8
m
p 0
‘
0.4
0
I so
300 VOLUME, ml
Fig. 37.21. Separation of oligonucleotides from an RNase A digest of tRNALh& on a column of DEAE-cellulose (Uziel and Gassen). Column: Whatman DE-32, 100 X 0.5 cm. Elution: a linear gradient of 0.02-0.3 M sodium chloride in 0.02 MTris-hydrochloric acid (pH 7.8) with 7 M urea (total volume 500 ml). Flow-rate: 12 ml/h. Sample: RNase A hydrolyzate of 75 A , , , units of tRNA. Symbols as in Figs. 37.19 and 37.20.
similar examples of oligonucleotide separations, the comprehensive work of Professor Khorana’s laboratory, published in well known biochemical journals under the over-all title of “Studies on polynucleotides” (about 100 papers have been published so far) is to be recommended; these papers contain a large amount of information of a similar type in the fields of both natural and synthetic polymers.
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Chapter 38
Nucleic acids
s. Z A D R A ~ I L CONTENTS Introduction and general techniques in nucleic acid separations ........................... 859 Deoxyribonucleic acids .......................................................... 862 Ribonucleic acids. ............................................................. .873 Polynucleotides and large oligonucleotides ........................................... 878 Automated procedures and polynucleotide sequence analysis ............................. 880 References .................................................................... 883
INTRODUCTION AND GENERAL TECHNIQUES IN NUCLEIC ACID SEPARATIONS Nucleic acids are the fundamental genetic material of all living organisms. The study of their structure and of the molecular mechanism of their functions necessitates a wide range of methodical approaches, by means of which nucleic acids are isolated (liberated from cells, subcellular particles and bonds to other cell components), purified (to remove low- and high-molecular-weight admixtures, both natural and those added in the isolation process) and fractionated (to decrease the heterogeneity of the final preparation with respect to chemical composition, molecular weight and higher structures). In these processes, the various techniques of modern column chromatography play a substantial role. With regard to the complex chemical composition of the macromolecule (nitrogenous bases and the sugar-phosphate chain, giving the polymer a negative charge at neutral pH) and the widely varied arrangements of individual types of nucleic acids (native and singlestranded DNA, circular and supercoiled DNA, viral and phage RNAs, rRNA, mRNA, tRNA, DNA-RNA hybrids, natural and synthetic mono- and polythematic polymers, etc.), ionexchange, adsorption and partition chromatographic techniques and gel permeation chromatography can be used to separate nucleic acids. The procedures used for isolating nucleic acids are generally complex processes that differ according to the source of the material to be isolated. Some general steps are illustrated in Table 38.1. Reviews by Cantoni and Davies and Kirby (1964) serve as introduction to the detailed study of isolation methods adapted to individual nucleic acid sources. The technique of gel filtration on a column of Sephadex G-150 can, however, also be used for the direct isolation of high-molecular-weight DNA obtained from nuclei lysate in guanidine hydrochloride (Pivec and Stokrova). Similarly, Sepharose 4 B can be used with animal DNA (Loeb and Chauveau) and for transforming DNA from bacterial lysate (Satava et aZ.), elution of the column in both instances being carried out with citrate containing 2 M sodium chloride. An example of such an elution is shown in Fig. 38.1. References p.883
859
860
NUCLEIC ACIDS
TABLE 38.1 BASIC STEPS OF ISOLATION PROCEDURES FOR NUCLEIC ACIDS _
_
~
Isolation step
Methods used
References
Tissue and cell disruption
Mechanical (alumina, pressure, glass beads) Chemical - detergents Enzymic - lysozyme, pronase
Isolation of subcellular particles Nuclease inhibitors
Differential centrifugation, density gradients, zonal Centrifugation EDTA, citrate, polyvinyl sulphate, bentonite, sodium dodecylsulphate, phenol
FraenkelConrat and Singer (1954a, b), Kay e t a l . , Nomoto e t a l . , Marmur, Berns and Thomas Brakke, Reid
Deproteinization
Phenol, organic solvents (chloroform), sodium dodecylsulphate Specific nucleases (DNase, RNase)
Enzymic degradation Precipitation Further separation and fractionation
Ethanol, methoxy- and ethoxyethanol, isopropanol Density gradients (sucrose, CsC1)
Counter-current distribution
Aqueous polymer two-phase system Chemical methods
Brownhill et al., Singer and FraenkelConrat, Littauer and Sela, Keller e t al. Kay er QL, Marmur, Kirby (1964) Marmur, Fleissner and Borek Kirby (1956), Kay e t al., Marmur Vinograd and Hearst, Zadrd~iland Mach, Schildkraut et al., Williamson Kirby et al., Kidson and Kirby (1963, 1964), Doctor etal., Zachau erat. 11961) Albertsson Stephenson and Zamecnik, Katchalsky et al.
Chromatography
Most of the known types of chromatographic sorbents can be used for the separation of nucleic acids (mixtures of polyanions). A brief review of these sorbents, together with their general separation mechanisms, is given in Table 38.2. However, all sorbents are not equally effective with different types of nucleic acids. The method most generally used to detect the separated macromolecules is UV absorbance measurement a t 260 nm. Differences in types of higher structures are most easily studied by observing the hyperchromic effect after thermal or chemical denaturation, or chemical or enzymic hydrolysis. The spectrophotometric method may be accompanied by the more sensitive measurement of radioactivity (incorporation of [32P] phosphate or 3Hand ''C-specific precursors, e.g., thymidine or uridine). Qualitative and quantitative analysis of the fractions obtained can be carried out in most instances by means of colour reactions with the sugar component, such as the orcinol reaction for RNA (Ceriotti) and the diphenylamine reaction for DNA (Burton), which are used most
86 1
INTRODUCTION AND GENERAL TECHNIQUES
FRACTION ho. Y
Fig. 38.1, Isolation of a Bacillus subtilis DNA on a Sepharose 4 8 column (Satava el a[.). Column: 80 x 3 cm. Elution: 2 M sodium chloride in 0.01 M sodium citrate solution. Flow-rate: 24 ml/h. Fractions: 10 ml. Sample: 15 ml of a lysozyme lysate of 2 g of wet bacterial cells labelled with [’HI thymidine. I, DNA; Ha, cellular proteins; IIb, mainly lysozyme. 1, Radioactivity; 2, absorbance at 260 nm. TABLE 38.2 SOME CHROMATOGRAPHIC MATERIALS USED IN NUCLEIC ACID FRACTIONATION Type of chromatography
Separation forces involved
Materials
Ion exchange
Ion exchange and weak secondary interactions (aromatic residues, etc.)
Adsorption
Electrostatic and complex interactions, charge density in molecules (nucleic acid-protein interactions) Hydrophilic and hydrophobic affinity (differences in distribution coefficients)
ECTEOLA-, DEAE-, BD-, BNDcelluloses; DEAE- and QAESephadexes MAK, PLK and histone-Kieselguhr (Celite); hydroxyapatite; nitrocellulose; Amberlite (with Mg” or A13*) Sephadex (3-25; Sephadex LH-20; diatomaceous earth (Chromosorb W); Kieselguhr (Ceiite); aluminium oxide Dextran gels (Sephadexes); polyacrylamide gels (BioCel P); agarose gels (Sepharoses; Bio-Gel A); agar; porous glass
Partition
Gel permeation
Sieving effects (differences in molecule size and conformation)
frequently to determine nucleic acids in tissue extracts and in isolated preparations (Hutchison and Munro). Specific nucleases (DNase, RNase, Neurospora nuclease, Lehman enzyme, RNase H, etc.) can be used in a similar manner. Important information may also be obtained from other analytical methods, e.g., velocity and equilibrium sedimentation in an ultracentrifuge (molecular-weight heterogeneity, chemical composition and molecular shapes; Schildkraut et al, , Smith and Levine), hybridization (sequential relationships; Kennell, Walker), electron microscopy (molecular length and shape; Lang References p . 883
862
NUCLEIC ACIDS
et al., Lang and Mitani), etc. Of biological and biochemical methods, bacterial transformation (Spizizen) and determination of the acceptor activity for amino acids in tRNA (Cherayil and Bock, Von Ehrenstein) deserve mention.
DEOXYRI BONUCLEIC ACIDS The primary structure of DNA consists of a long, non-branching deoxyribosophosphate chain with four fundamental nitrogenous bases (adenine, guanine, cytosine and thymine). The elementary higher structure of DNA is the Watson-Crick right-handed double helix, consisting of two polynucleotide chains winding round the same axis and held together by their base interaction A-T and G-C (Watson and Crick). The molecular weight of different samples varies within a wide range, from lo6 to 3 . lo9 daltons, being generally the result of degradation of the molecule during isolation, because extremely large molecules (e.g., circular DNA of Escherichia coli, 2.8. lo9 daltons; Bleeken el al., Cairns), of the order of lo6 nucleotide pairs, are very sensitive to mechanical degradation. Therefore, most DNA samples are mixtures of fragments of different sizes, the mean molecular weight of which is about lo7 daltons. In addition t o the linear polymer, the structure of a covalent closed circle was also found in many instances, e.g., single-stranded circular DNA of the (PX-174phage (Kleinschmidt er al.) and its double-stranded replicative form (Rush and Warner), which was also found in the h phage (Caro, MacHattie and Thomas, Radloff et al., Weissbach et al.) and mitochondria1 DNA. All these properties of DNA molecules may affect their behaviour during column fractionation and can be utilized for their separation. Adsorption chromatography on hydroxyapatite, methylated albumin-Kieselguhr and polylysine-Kieselguhr columns or nitrocellulose is the best method for separating DNA from RNA and higher oligonucleotides, and for removing proteins. These sorbents are also used with advantage for separating native (double-stranded) and denatured (single-stranded) DNA. Of the many methods available for preparing hydroxyapatite, the method according to Tiselius et al. has found the widest use, being preferred even over commercial preparations: Hydroxyapatite is prepared as a precipitate by gradual mixing of equal volumes (2 1) of 0.5 M calcium chloride solution and 0.5 M disodium hydrogen orthophosphate solution dropwise (120 drops/min) from separating funnels in a beaker. The precipitate obtained is decanted four times by washing with distilled water (4 1 each time). Finally, distilled water is added so as to give a final volume of 4 1, and 100 mI 40% sodium hydroxide solution are added. The mixture is boiled for .1 h with agitation. The precipitate is again decanted four times by washing with water, then 0.01 M phosphate buffer of pH 6.8 is added, the suspension is heated to boiling and immediately cooled, then boiled for a further 5 min after decanting and addition of the same buffer. This operation is repeated, with boiling for 15 min, and then twice more with 0.001 Mphosphate buffer, boiling for 15 min each time. The hydroxyapatite thus obtained can be stored in 0.001 M phosphate buffer of pH 6.8. In order to prepare the column, the adsorbent is introduced into the column, the lower end of which is stopped with glass-wool, at atmospheric pressure. When the column has been filled, the adsorbent is washed from the walls and the uppermost
DEOXYRIBONUCLEIC ACIDS
863
adsorbent layer is stirred in order t o create an even surface for sample application. The water content of the column is about 70%. Methylated albumin-coated Kieselguhr (MAK) is usually prepared in a similar manner in the laboratory (Mandell and Hershey). As it can be used universally to fractionate nucleic acids, a detailed description of the preparation of this column is given below. A 4.2-ml volume of 12 N hydrochloric acid is added in small portions t o 5 g of bovine serum albumin (fraction V) in 500 ml of absolute methanol, by which process the protein is dissolved and re-precipitated. The suspension is allowed t o stand at room temperature in the dark for 3 days with occasional stirring and, after centrifugation at 4000 g for 10 min, the sediment is washed at least three times with absolute methanol and twice with diethyl ether. It is then dried in vacm over potassium hydroxide, and likewise storeu in the form of an easily water-soluble powder. Hyflo Supercel (Kieselguhr) is washed before preparation of the MAK column with 200 ml of 1 N sodium hydroxide solution per 100 g of carrier, filtered and washed on the filter with another 300 ml of 1 N sodiur hydroxide solution. Further washing is carried out with 500 ml of 1 N hydrochloric acid and then a large volume of distilled water so as t o remove the acidic reaction of the filtrate. The washed Kieselguhr is then dried in an oven. A 20-g amount of washed Kieselguhr is suspended in 100 ml of 0.1 M sodium chloride solution in 0.05 M phosphate buffer of pH 6.7 and boiled briefly in order t o remove air. After cooling, 5 ml of 1% aqueous methylated albumin solution is added with stirring, the mixture is stirred for a further 15 min, then 2 0 ml of the same buffer with 0.1 M sodium chloride solution are added and stirring is continued for 5 min. The suspension is centrifuged for 10 min at 2000 g and washed with 0.4 M sodium chloride solution in the same buffer. The final sediment is suspended in 125 ml of buffered 0.4 M sodium chloride solution, and can be stored in a cool place for several weeks. In order t o prepare a 400 X 2 0 mm column with three layers, 8 g of Kieselguhr and 2 ml of 1% methylated albumin solution are taken for the first layer, 6 g of Kieselguhr in 40 ml of buffered 0.4 M sodium chloride solution and 10 ml of MAK suspension from the first layer (lower protein content) for the second, and 1 g of Kieselguhr in 10 ml of buffered 0.4 M sodium chloride solution (protein-free - covering layer) for the third layer. The three suspensions described are introduced into the column in the above order on to a 2-cm layer of cotton-wool or cellulose, and the column is washed wiih 150 ml of buffered 0.1 M sodium chloride solution. Amounts of 1.2-7 mg of nucleic acids in 1050 ml of buffered 0.1 M sodium chloride solution can be applied t o such a column. The initial buffer concentration depends on the composition of the nucleic acid mixture (lower concentration in the presence of tRNA, which is gradually increased for native DNA, rRNA and denatured DNA). A typical example of the results obtained with this column is shown later in Fig. 38.5. Poly-L-lysine-coated Kieselguhr (PLK), prepared in a similar manner t o MAK, with a ratio of 1 mg of polylysine t o 1 g of Kieselguhr can also be used in a similar manner (Ayad and Blamire, 1968, 1969). Separation on a nitrocellulose column (prepared from a washed carrier, which, being gradually compacted in a chromatographic tube with a glass rod, makes a homogeneous column; Armstrong and Boezi), analogous t o the use of nitrocellulose membrane filters in hybridization (Gillespie and Spiegelman, Nygaard and Hall), is a very rapid method for separating nucleic acids. References p.883
864
NUCLEIC ACIDS
1
I
I
50
I
I
70
1
90
FRACTION No.
Fig. 38.2. Separation of a mixture of native and heat-denatured transforming DNA from H . influenzae on a hydroxyapatite column (Chevalier and Bernardi). Column: 15 X 2 cm. Elution: a linear gradient of 0.001 -0.5 M potassium phosphate, pH 6.8 (100 ml of each). Fractions: 2.7 ml. Sample: 36 ml of a mixture of 775 pgof heat-denatured DNAand 375 pgof native DNA. 1, Denatured DNA; 2,native DNA.
TABLE 38.3 PROPERTIES AND CHROMATOGRAPHIC BEHAVIOUR OF NUCLEIC ACIDS ON A HYDROXYAPATITE COLUMN Nucleic acid
DNA Calf thymus
Saccharornyces cerevisiae Mitochondria Nuclei Polyoma virus
Phage T2 Phage qX-174 RNA E. coli tRNA Plant rRNA Ehrlich ascites Tumour Plant virus Polynucleotides
*Stepwise elution.
Structural characteristics
Buffer molarity at elution peak
Reference
Native
Bernardi (196 1, 1969b3
Denatured
0.20-0.22 0.20, 0.25* 0.10,0.15*
Circular Linear Linear and circular Superhelix
0.27 0.25 0.29 0.26
Bernardi el al.
Unglucosy lated Glucosylated Single-stranded circle
0.22 0.25-0.27 0.10,0.15*
Clover-leaf structure High-molecular
0.13 0.15 0.15,0.20*
Replicative form Random-coiled Double-stranded Triple-stranded
0.20 0.10-0.15 0.20-0.22 0.45
Bourgaux-Rdmoisy et al:, Bourgaux and BourgauxRamoisy Bernardi (1969a, b) Bernardi (1969b, c ) Bernardi (1969c) Pinck et al. Bernardi and Tirnasheff Pinck et al. Bernardi ( 1 9 6 9 ~ )
865
DEOXYRIBONUCLEIC ACIDS
The principle of separation and fractionation on hydroxyapatite consists in electrostatic interaction between the negative phosphate groups of the polymer and the positive charges of calcium ions in the sorbent crystals (Bernardi, 1965). Thus, an elution can be carried out with a concentration gradient of phosphate buffer solution or by increasing the column temperature with a constant concentration and pH of the eluent (Miyazawa and Thomas). This mechanism is clearly demonstrated by the different behaviour o f native and denatured DNA (Fig. 38.2) and RNA (Table 38.3, Bernadi, 1965, 1969a, b , c ; Chevalier and Bernardi). No changes in the DNA molecule were observed during the chromatographic process and fractionation with respect t o base composition o r molecular weight did not occur. The different behaviour of some nucleic acids, evidently caused by conformational differences in the molecules from the form of native double-stranded DNA (dependence on molecule volumes: Bernardi, 1969a) is summarized in Table 38.3. The hydroxyapatite column was also used t o obtain evidence of the fact that the residual transforming activity of heat-denatured DNA is related to the native-like (eluted in the same position, Fig. 38.3), probably cross-linked, sample fraction (Chevalier and Bernardi). The determination of the number of sex factors on E. coli chromosomes has been carried out by the same method (Frame and Bishop). When stepwise elution is used, however, artefact peaks may be formed (Fig. 38.4); this formation evidently depends on unsuitable DNA:carrier ratios or on the length of the column (Bernardi, 1961). Elution a t a constant but elevated temperature (60- 70°C) is used to isolate rapidly denaturing DNA fractions (satellite DNA, Votavova et al. ; renaturation studies, McGallum and Walker). When variable temperatures are used, with a phosphate buffer of constant concentration, DNA can be fractionated by the base composition, denatured fractions being obtained (Miyazawa and Thomas). The method can be modified for use with large amounts of material and larger DNA sample series by using a heated centrifuge, which
30
50
70
90
FRACTION No.
Fig. 38.3. Chromatography of an alkali-denatured DNA from H. influenzoe on a hydroxyapatite column and its residual transforming activity (Chevalier and Bernardi). Column: 20 x 1.2 cm. Elution: a Linear gradient of 0.001 -0.5 Mpotassium phosphate, pH 6.8 (150 ml of each). Fractions: 2.4 ml. Sample: 1.8 mg of alkalidenatured transforming DNA. Circles indicate. the number of transformed cells (cathomycin marker). The specific biological activity of fraction 76 was 43% of untreated DNA.
References p.883
866
NUCLEIC ACIDS
FRACTION No.
Fig, 38.4. Chromatography of native calf thymus DNA on il column of hydroxyapatite with stepwise elution (Bernardi, 1961). Column: A, 5 X 1.3 cm; Band C, 3 X 1.3 cm. Elution: stepwise with phosphate buffer of indicated concentrations. Fractions: 3 nil. Sample: 1.28 mg of DNA. The first arrow indicates the point of DNA application. A, separation of the original DNA sample; B and C, re-chromatography of the material from peaks 1 and 2, respectively, of A. The whole procedure shows the artifact origin of the second peak.
speeds up the process considerably (Brenner et ul., Flamm et ul.). In most instances the yield obtained by chromatography on a hydroxyapatite column is 90- 100%. Native DNA can be separated by stepwise elution from denatured DNA and DNA-RNA complex on nitrocellulose (Armstrong and Boezi, Klamerth), being eluted in the opposite order to that on hydroxyapatite. Riggsby, who modified the nitrocellulose column technique used by Bautz and Reilly , achieved approximately 100-fold purification of genetically labelled DNA. Differing from the preceding sorbents, the MAK column, which operates mainly on the basis of electrostatic interaction between DNA and the basic protein, is able not only t o separate DNA from RNA and the native form from the denatured form of DNA, but also to fractionate double-stranded DNA by its composition and molecular weight. However, with insufficiently deproteinated samples, the fractionation may be influenced to a large extent by the presence of residual proteins. A three-layer column (Mandell and Hershey), the preparation of which is described above, can be used to separate at room temperature the entire extract of nucleic acids from E. cofi (Fig. 38.5) into four fractions, which are successively eluted at a sodium chloride concentration of about 0.4M for tRNA, 0.6 M for DNA, 0.75 M for 16s rRNA and 0.85 M for 2 3 s rRNA, in 0.05 M phosphate buffer of pH 6.7. It is generally advanta-
867
DEOXYRIBONUCLEIC ACIDS
geous to separate about 1 mg of DNA on 10 ml of MAK suspension, the column height being irrelevant. Transformation DNA from B. subtilis (Pivec et al., Zadra%ilet a!.) was repeatedly fractionated on a similar column (Sponar et al.), the fractions obtained differing in terms of mean base composition and molecular weight, and also in biological activity for various markers (Fig. 38.6). For elution from the MAK column, native DNA with a mean molecular weight of lo7 to 2 . lo7 is eluted in the sodium chloride concentration range 0.60-0.75 M and denatured DNA at 0.80-0.95 M . A decrease in the molecular weight leads to a decrease in the concentration of the eluting salt, while the G-C content is indirectly proportional to the concentration of the eluting solution (Cheng and Sueoka, Mandell and Hershey). The recovery, particularly of denatured DNA, is highest with an elution temperature of less than 10°C, decreasing with increase in temperature and becoming practically zero at about 40°C (Roger). Stepwise elution gives a better recovery of the sample applied, while gradient elution is better for analytical purposes as it is more sensitive to differences in the desorption of individual molecules.
“ “ “ “ “ I
23 S
Fig. 38.5. Chromatography of ”P-labelled nucleic acids extracted from E. coli on a methylated serum albumin column (Takai et ul.). Column: according to Mandell and Hershey (see text). Elution: a linear gradient of 0.2-1.OM sodium chloride in phosphate buffer. Flow-rate: 24 ml/h. Fractions: 4 ml. Sample: a phenol-extracted and alcohol-precipitated nucleic acid mixture from 200 ml of a bacterial culture labelled with 32P.Closed circles, absorbance at 260 nm; open circles, four fractions of labelled mRNA (1 -4).
The different cytosine contents of individual DNA strands from bacteria have enabled the MAK column with an intermittent gradient (Rudner et al., 1968a, b, 1969) to be used to separate the complementary strands of thermally or alkaline-denatured DNA (Fig. 38.7). Poly-L-lysine is a further protein sorbent used with Kieselguhr (Ayad and Blamire, 1968). An extract of whole nucleic acid from B. subtilis (Fig. 38.8) and a DNA preparation from the same source (Fig. 38.9) was reproducibly fractionated, particularly by the base composition (Ayad and Blamire, 1968, 1969), as sonication of the DNA sample or thermal denaturation did not markedly influence the elution profile (Table 38.4). In spite of some advantages (reproducibility, affinity for the base composition used to separate plasmide References p.883
NUCLEIC ACIDS
868
I\ '0.52 056
0.63 0.71 NaCl MOLARITY
i 0.79
Fig. 38.6. Repeated fractionation oPB. subtilis DNA on an MAK column (Pivec et ul.). Top curve Column: 25 X 3.5 cm prepared according to Mandell and Hershey (see text). Elution: a linear gradient of 0.5-1 M sodium chloride in 0.013 Mphosphate buffer of pH 7 . Flow-rate: 40 ml/h. Fractions: 7 ml. Sample: 20 mg of transforming DNA. I, oligonucleotides and residual protein; 11, DNA distribution peak. Fractions A to E were used for rechromatography. Curves A to E - Column: 15 X 2 ern, prepared and eluted as mentioned above. Flow-rate: 20 ml/h. Fractions: 5 ml. Samples: material of fractions A to E, each about 2.5 mg.
from chromosomal DNA (Cannon and Dunican), known fractionation mechanism, etc.), PLK chromatography is not yet being widely used. This applies to a far greater extent to other carriers of a similar nature, such as histone (Ayad and Wilkinson, Brown and Martin, Brown and Watson, Tichonenko), protamine (Ligault-DBmare et a/.) etc., and sometimes even chemically bound to cellulose instead of Kieselguhr. In order to fractionate bacterial and animal nucleic acids, a method of interaction between the mixture to be separated and the hexamine cobalt(I1) salt of synthetic or natural polynucleotides bound to Kieselguhr has been developed (Lin). The sorbents used are denatured DNA, poly-A, poly-I, poly-C and poly-U, and the mutual bond depends on the presence of dioxane, so that there is no need for the polymers involved to be sequentially complementary. Elution is carried out with a linear dioxane gradient mixed with a buffer. On the column with bound poly-I, complementary strands of denatured DNA from E. coli were separated, while partial fractionation of tRNA and rRNA was achieved on bound animal DNA (fin). Experiments with DNA fractionation on Amberlite IRC-50 in the presence of Mg2+ (Frankel and Crampton) and on IR-120 with A13+(Kothari, 1970a, b, 1972) appear to lie
869
DEOXYRIBONUCLEIC ACIDS
FRACTION
No
Fig. 38.7. Denatured B. subfilis DNA eluted from an MAK column with the use of linear gradient (A) and by the intermittent gradient technique (B) for the separation of complementary strands (Rudner et al., 1968a). Column: 15.5 X 1.9 cm. Elution: a linear sodium chloride gradient in 0.05 M sodium phosphate of pH 6.7 ( A ) or the same used with the intermittent technique (B). Fractions: 5 ml. Sample: 2 mg of denatured DNA. A, heat-denatured DNA with a total gradient volume of 4 0 0 ml (0.6-1.2 M sodium chloride); recovery 64%. B, alkali-denatured DNA with a total gradient volume of 450 nil (0.7-1.4 M sodium chloride); recovery 80%. As indicated by arrows, the gradient was cut at tube 44 and reconnected at tube 53.
,
0.2
2.0
-
OLlGO NUCLEOTIDES
cif
8
N P
4
I
1.0
0.1
I
0
I
'
I
10
20
p
10
30
FRACTION No.
Fig. 38.8. Separation of a nucleic acid mixture extracted from B. subtilis on a PLK column (Ayad and Blamire, 1969). Column: 5 g of PLK material. Elution: a linear gradient of 0.4-4 M sodium chloride in 0.02 M phosphate buffer of pH 6.7 (150 ml). Flow-rate: 20 ml/h. Fractions: 4 ml.
on the borderline with ion-exchange chromatography. As weak cation exchangers are involved, DNA fractionation is a matter of adsorption (formation of chelate complexes between the carboxyl groups of the resin, metal ions and the phosphate groups of DNA). This means that the sorbent used fractionates by base composition, A T-rich molecules being more firmly bound to the carrier (Mindich and Hotchkiss, 1964b; Pullman and Pullman). Transforming DNA from H. influenzae was fractionated in this way in order to
+
References p.883
870
NUCLEIC ACIDS
r 1.5
1
1
I
I
, /’
I
-
0
8
, , , ,
P
/
/
0
20 FRACTION No.40
Fig. 38.9. Fractionation of B. subtilis native DNA on a PLK column (Ayad and Blamire, 1968). Column: 10 g of PLK material. Elution: a linear gradient of 0.4-4 Msodium chloride in 0.02 M potassium phosphate buffer of pH 6.7 (150 ml). Flow-rate: 20 ml/h. Fractions: 4 ml.Sample: 1.5 mg of DNA in 15 ml of 0.4 M buffered sodium chloride. 1, Low-molecular-weight DNA admixtures; 2, heterogeneous fraction of DNA of no definite conformation; 3, high-molecular-weight native DNA (each fraction of different G t C content). TABLE 38.4 BEHAVIOUR O F Bacillus subfilis DNA ON A PLK COLUMN (AYAD AND BLAMIRE, 1968) Structural characteristics
Molarity of elution buffer ~
Nd t ive Sonicated Heatdenatured
Peak I
Peak I1
Peak Ill
0.5 0.6 0.6
1.20 1.25 1.35
2 .o 1.84 2.07
cumulate biological activity to certain markers (Mindich and Hotchkiss, 1964a). Initially, there was an intensive study of the ion-exchange fractionation of DNA, particularly on substituted cellulose types (Bendich et al., 1955, 1958; Davila et al., 1965a, b; Kit, 1960a, b; Otaka et al.; Rosenkranz and Bendich), which are more suitable than synthetic ion exchangers because of their hydrophilic nature, better permeability and enormous surface area and, therefore, greater capacity for macromolecules. It was found, however, that this field is more suitable for fractionating complex mixtures of oligonucleotides and tRNA than for high-molecular-weight DNA. Recently, benzoylated and naphthoylated DEAE-cellulose, functioning on the basis of differentiation of the secondary structure of molecules, was used to separate an extract of nucleic acids from E. coli infected with MS2 phage. The separation, however, was only partial, viz., of rRNA from the mixture of DNA and tRNA (Sedat et al.). This type of carrier was also used in order to fractionate replicating DNA of h phage (Kiger and Sinsheimer). The physical basis of separation has not yet been fully clarified, but these columns have been used successfully for the separation of the native and partially denatured DNAs (Iyer and Rupp, Pyeritz et aL) and, being treated with deoxycholate, also for the purification of the bacterial genes for rRNAs (Udvardy and Venetianer).
87 1
DEOXYRIBONUCLEIC ACIDS
DNA itself can be covalently bound to a cellulose and serves as an adsorbent for isolating complementary DNA strands and RNA (according t o the sequence) from a mixture of different nucleic acid molecules (Bautz and Hall). The ideal conditions for adsorption are comparable with those which favour renaturation of nucleic acid strands. By decreasing the ionic strength and increasing the column temperature the adsorbed polynucleotide can be eluted. As with ion-exchange resins, the preparation of a gel column for nucleic acid fractionation is the same as for the chromatography of low-molecular-weight substances (the manufacturers supply detailed instructions for column preparation with all gel types). Originally, gel columns (sorbents with higher degrees of cross-linking, see Fig. 38.10) were used in order t o replace and speed up dialysis (desalting of high-molecular-weight samples and removal of phenol after isolation, Shepferd and Petersen), which means purification of the macromolecule in the course of its isolation (Bauer and Johanson) or removal of superfluous components in studies of interactions with DNA (Attardi et al., Hanson, Sekine et al.). The actual separation of macromolecules was achieved by separating DNA and RNA on more porous gels of the dextran type, such as Sephadex G-200 (Fig. 38.1 1, Bartoli
mobocular weight operating
ranges
mol. ~ p l c a l Qnpoum
p ’ ? “18;’
d j
Tobaaa Mosalc Vlrus lnlluetua Vlrus POllovtus RNA
Catalase Human FGlobu
Nucleosldes
[
SEPHADEX
rmq
810-GEL
P
810-GEL P
Fig. 38.10. Relationship between operating range and molecular weights for gel filtration materials (Pharmacia and Calbiochem leaflets).
References p.883
87 2
NUCLEIC ACIDS
and Rossi), or of the agarose type, such as Sepharose (Fig. 38.12,C)berg and Philipson), the particles of which are capable of absorbing and delaying the flow of macromolecules. The above examples show that separations on gels of both types are based on differences in molecular weight and shape. As mentioned already (p. 861), Sepharose is also used in order to separate DNA from admixtures that are difficult to remove (Cozzarelli et al., Young and Jackson) and this is therefore one of the mildest separation methods (Loeb and Chauveau). The relatively recent introduction of agarose carriers into laboratory use and the possibility of simple elution with gradient-free solutions indicate that these gels should find wider application in the future. The resolution of the column depends, as with low-molecular-weight substances, on the ratio between the volume of the sample and that of the column filling. Gels are also used as stationary phases, serving to anchor DNA as the fractionation
i
0.7
DNA
0.7
m
a
DENATURATED
0.3
I
DNA RNA
50
100 VOLUME, ml
150
4 I
200
Fig. 38.1 1. Separation of liver DNA and RNA by gel filtration on a Sephadex G-200 column (Bartoli and Rossi). Column: 25 X 2.5 em. Elution: 1 Msodium chloride in 0.1 M Tris-hydrochloric acid buffer of pH 7.2. Flow-rate: 30 ml/h.
g 0.4
16
Q
T
P
ti
3 H
X
o.2
m 4 0
30
50 70 BED VOLUME.%
0
Fig. 38.12. Gel filtration of K B cell nucleic acid mixture and ["PI RNA of poliovirus on a column of 1%agarose (Oberg and Philipson). Column: 60 X 2.1 cm. Elution: 2.1OW M sodium phosphate buffer of pH 6.0 with 10-3Mmagnesium chloride. Flow-rate: 2 ml/h.cma. Sample volume: 1.5 ml.
RIBONUCLEIC ACIDS
873
agent (hybridization on an agar column, Bendich and Bolton) where it served, for example, to isolate the DNA anticodon strand (DoskoCil and Hochmannova). When bound to various gel carriers, DNA can also be used for the purification of DNases (Naber et al., Poonian et al., Schabort) or of other enzymes that take part in DNA metabolism and biosynthesis (polymerases, ligase, etc.). The use of partition chromatography (a continuation of the now little used technique of counter-current distribution, Kidson and Kirby, 1963, 1964) is limited in the case of DNA to a modified gel carrier, such as hydrophobic Sephadex LH-20 (Kidson, 1969). A column prepared in the organic phase of a multicomponent solvent system (amyl alcohol, 2-methoxyethanol, 2-butoxyethanol, tripentylamine, acetic acid and trilithium citrate) could distinguish the native and denatured DNAs by elution with a linear gradient of citrate in the aqueous phase (used as the mobile phase). This method, the separation mechanism of which is due to a stronger interaction of the denatured DNA phosphate groups with the amine, with exchange of Li', was used successfully to study the structure of E. coli DNA in the vicinity of the replicating point (Kidson, 1968).
RIBONUCLEIC ACIDS In comparison with DNA, RNAs are a far more heterogeneous group with macromolecules that differ distinctly in structure and function. RNAs are found in all living cells: rRNA with a molecular weight between 0.5. lo6 and 2 . lo6 daltons (80% of total RNA), tRNA ( 2 . 5 . lo4 daltons; 15% of total DNA) and mRNA (very heterogeneous and labile, mean molecular weight lo5 daltons; 5% of total DNA). In the primary structure, a large number of minor modified components are added to the four fundamental bases, particularly in the case of tRNA (Hall). The secondary structure is likewise very varied (Cox), from totally double-stranded viral RNA (Gomatos and Tamm), through molecules with extensive hydrogen bonding (rRNA and tRNA, Cox, Levitt) up to linear singlestranded structures of mRNA (in a translation process, Matthaei and Nirenberg). As most of the techniques that are capable of separating RNA from DNA have already been mentioned in the preceding section on DNA, we shall now limit the discussion to fractionations that differentiate between individual types of RNA molecules. Owing to the great variations in the structures of the molecules all column chromatographic techniques are used. From the field of the adsorption chromatography, the MAK column separates all RNA types (Fig. 38.5) including mRNA, the heterogeneity of which can be studied in this way (Monier et al., Oravec). It can also be used in order to fractionate infectious RNA from poliomyelitis virus (Cocito et al.). Most attention, however, has been devoted to the most complex group of tRNA. Using ''C-labelled amino acids, Sueoka and Yamane found that sub-fractionation takes place in the tRNA peak (Fig. 38.13), even allowing a search for modified tRNA after infection of bacteria by a phage (Kano-Sueoka and Sueoka), after transformation of animal cells by oncornaviruses (TrBvniCek and Riman) or after incorporation of fluorouracil (Lowrie and Bergquist). When Kieselguhr is replaced with silicic acid, the capacity of the methylated albumin column for fractionation of tRNA from E. coli increases up to 100-fold (Stern and Littauer, Ziv et a1.j. A situation similar to that References p . 883
874
NUCLEIC ACIDS I
-
i
0
N u) * W
0.8
I
I
I
I II
-
FRACTION NO.
Fig. 38.13. Chromatography of a mixture of 16 aminoacyl tRNAs from E. coli on an MAK column (Sueoka and Yamane). Column: 8 X 1.8 cm. Elution: a linear gradient of 0.2-1.1 M sodium chloride in 0.05 M phosphate buffer of pH 6.7 ( I 10 ml of each solution). Plow-rate: 60 rnl/h. Fractions: 2 ml. Sample: 2.5 mg of tRNA incubated with a mixture of one radioactively labelled amino acid and the 19 remaining non-radioactive amino acids. The vertical lines indicate the positions of the main radioactive peaks of the individual aminoacyl tRNAs (multiform not being marked).
for MAK also applies to the use of PLK (Fig. 38.8). Gradient elution successively liberates fractions according to their increasing molecular weight, but for double-stranded RNA and 16s and 23s rRNA the secondary structure and conformation may also be important (Ayad and Blamire, 1970). Differing from its widespread application with DNA, the hydroxyapatite column cannot be used with high-molecular-weight rRNA because it is degraded during chromatography (Bernardi and Timasheff). Owing to their structural analogy with DNA, however, RNA replicative forms and intermediates can be fractionated (Pinck et al.). In spite of a detailed study of the fractionation of different tRNAs, viral RNA and polynucleotides (Bernardi, 1969c), chromatography of RNA on hydroxyapatite has not been widely used. Of greater importance for tRNA are ion-exchange procedures on substituted celluloses and Sephadex, which permit the isolation of pure specific tRNAs that can be used for sequential analysis and for protein biosynthesis. The existence of weak bonding forces (secondary interactions dependent on RNA base composition) is of decisive importance in fractionation owing to the similarity of the separated molecules in terms of size and charge. Control of these forces, together with changes in pH and the concentration gradient, may be decisive for satisfactory separations (urea, temperature, BD and BND substitution, etc.). Detailed studies of these problems were made by Bock and Cherayil and Cherayil and Bock, who used DEAE-cellulose and DEAE-Sephadex under different conditions so as to
875
RIBONLCLEIC ACIDS
~
: 0 N
30
40
50
60 FRACTION NO
Fig, 38.14. Fractionation of tRNA on a DEAE-cellulose column with a urea gradient (Cherayil and Bock). Column: 100 X 1.5 cm. Elution: an exponential gradient of 0-7 M urea in 0.02 A I Trishydrochhric acid buffer of pI1 7.5 with 0.34 M sodium chloride prepared in a constant-volume mixer containing 300 ml of a starting solution. Flow-rate: 15 nil/h. Fractions: 7.5 ml. Sample: 200 mg of yeast tRNA. Arg, Val, Leu and His reprcsent the acceptor activity of tRNA fractions for thc given amino acids. Material of regions A, B and C was used for further re-chromatography (Fig. 38.15). Solid line, absorbance at 280 nm.
permit the isolation or enrichment of most specific tRNAs (Figs. 38.14 and 38.15). General conclusions concerning this fractionation process can be drawn: (a) secondary interaction of the polynucleotide with cellulose is considerably greater than with dextran (mainly purine bases) and can be eliminated with the aid of urea; (b) separation by length of the polyanion chain can be achieved on both cellulose and dextran ion exchangers in the presence of urea; (c) re-chromatography of the peaks at lower pH values facilitates the further separation of tRNA by the base composition (partial protonation of adenine and cytosine residues, Fig. 38.15) and (d) the use of a urea gradient considerably increases the resolution of the column for tRNA compared with a sodium chloride gradient Similarly, a change of column temperature in the 20-65°C range (Baguley et al.) causes, on elution, the tRNA peak to widen and t o shift towards a higher concentration of the eluting gradient (a change in molecular conformation takes place). Secondary interactions, particularly of the lipophilic part of the chain, may be enhanced by the introduction of benzoyl (BD), and benzoyl and naphthoyl (BND) groups into DEAE-cellulose (Gillam et al., 1967). This leads to stronger binding of the polynucleotide, in spite of the simultaneous decrease in capacity of the ion exchanger. tRNAs specific for the aromatic amino acids phenylalanine, tyrosine and tryptophan can therefore be separated more easily from other tRNAs in the form of aminoacyl tRNA (Gillam et al., 1968; Fink et al. ; Maxwell et a/.), the affinity of which t o the column is greater (they are eluted with 1 M sodium chloride solution only after the addition of ethanol). In this instance, however, the decisive factor for successful separation is the purity of the amino References p.883
876
NUCLEIC ACIDS
FRACTION NUMBER
Fig. 38.15. Re-chromatography of fractions A, B and C of tRNA from Fig. 38.14 on a DEAESephadex column at pH 4.5 (Cherayil and Bock). Column: 100 X 1.2 cm. Elution: a linear gradient of 0.52-0.7 Msodium chloride (the total volume 500 ml) in the presence of 0.03 Macetate buffer of pH 4.5 and 7 M urea. Flow-rate: 8 ml/h. Fractions: 4 ml. Sample: fractions A, B and C from the previous column (Fig. 38.14) adjusted t o 7 M urea and pH 4.5. Val, Phe, Arg, Pro, Gly, Tyr, Leu and His represent the acceptor activity of tRNA fractions for a given labelled amino acid. Solid Line, absorbance a t 260 nm I
A
'3
I
1
f f '
3 dire $3 h r E
Fig. 38.16. Chromatography of aminoacyl tRNA on a BD-cellulose column (according t o Dejesus and Gray). Column: 90 X 1.5 cm. Elution: a linear gradient of 0.55-0.9 M sodium chloride (A) or 0.75-1.2 M sodium chloride with 10%ethanol (B), both in 0.01 M sodium acetate of pH 5 with O.O02P/I magnesium chloride. Fractions: 10 ml. Sample: 100 Mg in 10 ml of aminoacyl tRNA; each radioactively labelled aminoacyl tRNA was prepared separately.
877
RIBONUCLEIC ACIDS
acid and of the synthetase preparation used for aminoacylation. An example of an aminoacyl tRNA separation and a BD-cellulose column (Dejesus and Gray) is given in Fig. 39.16. The separation of both free and aminoacylated tRNAvet and tRNAlet can be achieved and the effect of formylation can be studied by this technique (Samuel and Rabinowitz, Stanley, White and Bayley). Partition chromatography is used almost exclusively to fractionate tRNA and is a continuation of experience gained with counter-current distribution. The best known variant is reversed-phase chromatography (Kelmers et al.), in which the carrier of the stationary phase (4% dimethyllaurylammonium chloride in isoamyl acetate) was Chromosorb W (diatomaceous earth) and elution was effected with a salt gradient in the presence of 0.01 M magnesium chloride. The column served to differentiate 16 tRNAs, multiforms being observed with tRNASer and tRNAArg. Further modifications have been made by the same group (Pearson et al., Weiss and Kelmers) and widely used by others (Gallagher ef al., Muller et d.).Muench and Berg achieved a 24-fold enrichment of the specific activity of tRNA in single-stage partition chromatography on Sephadex G-25 in which a column with an aqueous phase of a solvent system containing potassium phosphate buffer of pH 6.88, ethoxyethanol, butoxyethanol, mercaptoethanol and triethylamine was eluted with a linear gradient of triethylamine in the organic phase. Differing from DNA, RNAs of lower molecular weight can also be separated on gels
-----rRNA
tRNA
6
2
8
a
1
VOLUME. r n l
FRACTION NO.
Fig. 38.17. Gel permeation Chromatography of [ I4C] methyl and ['HI uracil-labelled tRNA of E. coli on a Sephadex G-100 column (Schleich and Goldstein). Column: 150 X 2 cm. Elution: 1 M sodium chloride. Sample: tRNA isolated on a DEAE-cellulose column in a volume of 0.5 ml. - - - - -, Absorbance at 260 nm; -.-.-, 3H; , I4C. The tRNA peak represents 75% of the total material. ~
Fig. 38.18. Fractionation of rat liver ribosomal RNA by Sepharose 2B gel filtration (Petrovii e t a / . ) . Column: 200-ml bed. Elution: gradually with 0.5 and 0.1 M sodium chloride (arrow) in 0.02 M Tris-hydrochloric acid buffer of pH 7.5 containing 0.1% of sodium dodecylsulphate and 0.0025 M EDTA. Flow-rate: 5 ml/h. Temperature: 21°C. Sample: 6.3 mg of rRNA.
References p . 883
87 8
NUCLEIC ACIDS
with a higher degree of cross-linking by gel permeation chromatography. A preparation of whole tRNA from E. coli, isolated on DEAE-cellulose, was fractionated on a Sephadex (2-100 column (Fig. 38.17), on which residues of rRNA, mRNA and even of 5 s rRNA were found to be well separated (Schleich and Goldstein). Agarose gels can also be used in order to separate and purify high-molecular-weight rRNAs (Fig. 38.18, NovakoviC and PetroviC, PetroviC et al.), and viral RNA (e.g., poliomyelitis virus, Bberg et al.). In the latter instance, with 2% agarose, the elution volume of RNA with a molecular weight of 2 . lo6 depends greatly on conformation changes caused by the presence of Mg2': M lithium sulphate solution elutes RNA at the void volume, while lo-' M lithium sulphate solution with Mmagnesium sulphate elutes RNA at 60% of the bed volume. A tRNA1le covalently bound to Sephadex may be mentioned as an example of affinity chromatography using RNA for the isolation and purification of specific t RNA synthetases (Bartkowiak and Pawelkiewicz).
POLYNUCLEOTIDES AND LARGE OLIGONUCLEOTIDES Chemically, polynucleotides do not differ from natural nucleic acids and they can therefore be fractionated by a similar or even identical procedure. Homologous oligo- or polynucleotides of synthetic origin, derived from both the ribo- and deoxyribo- types, can be fractionated relatively easily by size on columns of DEAE-cellulose and DEAE-
1.o
0.5
t
dI 0
800
400
VOLUME, ML
Fig. 38.19. Separation of oligonucleotides from a n Azotobacter nuclease digest of poly-A on a DEAE-cellulose column (Stevens and Hilmoe). Column and elution gradient as in Fig. 37.14. Sample: 17.5 Mmole of poly-A hydrolyzed for 30 min at pH 7.7.
879
POLYNUCLEOTIDES AND LARGE OLIGONUCLEOTIDES
Sephadex (Hall and Sinsheimer, Narang et al., Tener et al.). Fig. 38.19 shows the elution profile of the partial enzyme hydrolyzate poly-A obtained from a DEAE-cellulose column by using a complex gradient of volatile buffers (Stevens and Hilmoe). In order t o resolve the oligonucleotide mixture in the enzyme hydrolyzate of natural polynucleotides, however, buffers containing 7 M urea must be used (see the separation of nucleic acid components, p. 831 ), which eliminates secondary interactions with the carrier. Gel permeation chromatography with dextran and agarose has been widely applied in this field, having now become the main fractionation technique. In the synthetic preparation of polythymidylic acid containing up t o 24 monomer units, gel filtration on Sephadex G-15, G-25 and G-75 was used to isolate the intermediates and final product; this technique resulted in complete separation on elution with 0.1 M triethylammonium hydrogen carbonate of pH 7.5. The products can be desalted by direct lyophilization (Narang et al.). The separation of oligonucleotides on these gels can also be used in the study of the influence of terminal groups of oligonucleotides on elution (Haynes et al., Hohn and Pollman, Hohn and Schaller). A combination of chromatography on an MAK column with thermal chromatography on a hydroxyapatite column (Fig. 38.20) was used in order to isolate the natural polymers poly d(A-T) in an extract of r.ucleic acids from the testes of Cancer borealis (Brzezinski et ul.). This isolation is an example of the possibility of using the resolution of the MAK column according to base composition and polymer size in order t o fractionate polynucleotides, combined with the different re-naturation rates of the fractions on a hydroxyapatite column at elevated temperature.
6.0 d [A-T)
2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 F R X T I O N NO.
TEMPERATURE;
oc
Fig. 38.20. Isolation of poly d(A-T) from purified crab DNA on an MAK column and by thermal (A) Column: MAK, 9.6 X 2.5 cm. Elution: chromatography on hydroxyapatite (Brzezinski et d.). stepwise with the indicated concentrations of sodium chloride in 0.05 M phosphate buffcr of pH 6.7. Flow-rate: 6.5 ml/min. Fractions: 19.6 ml. Sample: 2800 A,,, units of crab DNA in 0.4 M buffered sodium chloride. Each peak contains both poly d(A-T) and the major DNA component in different proportions. (B) Column: hydroxyapatite, 42 X 2.5 cm. Elution: 0.14 M phosphate buffer of pH 6.7 with varying temperature (40°C; 60°C; 65°C; from 68°C t o 95°C in 3°C intervals; 100°C) and 5-min equilibrating time. Flow-rate: 3 ml/min. Fractions: 15 nil. Sample: peak malerial from (A) eluted with 0.7 M sodium chloride, 81% of xhich is poly d(A-T). Only the fractions collected at 7 lo k
\
all-trans- RETINENE (“381
)
(aa264)
D2
k 7 ”
Fig. 44.2. Separation of a synthetic mixture of various fat-soluble vitamins (Zile and De Luca). Column: 60-cm long consisting of four 15-cm sections, adjacent sections decreasing in diameter by a factor of approximately 1.4; the uppermost section had an I.D. of 13 mm, the lowest 4.5 mrn. Sorbent: silicic acid. Eluent: gradient of a mixture of diethy1 ether in Skelly-Solve B (0-ca. 30%). Detection: UV spectrophotornetrically according to the spectral characteristics of the individual components. Fractions of 10.6 ml collected.
Calciferols Calciferols are substances with antirachitic efficiency, in terms of chemical structure they are the steroids and from the analytical point of view one must note their instability towards light and in the presence of oxygen. For the separation of calciferols, their different adsorption activities towards various adsorbents are utilized. Silicic acid is the most commonly used polar adsorbent (Bell and Kodicek, De Luca, Haussler et al., Myrtle e t al., Suda et al.), often in combination with subsequent rechromatography on Celite with methanol-water as stationary phase (De Luca, Haussler et al., Myrtle et al., Suda et al.). Partially deactivated alumina (Mariani and Mariani-Vicari, Takahashi and Yamamoto) and silica gel (Lawson et al.) have also been used for the separation of the vitamin D group. Dollwet and Norman used Factise (a polymerized and vulcanized vegetable oil) as the stationary phase. References p . 9 76
958
VITAMINS
For the elution of substances separated on the polar adsorbants silica1 gel, silicic acid and alumina, gradient elution with the mixtures light petroleum (b.p. 40-60°C or 60-80°C) -diethy1 ether-methanol (Bell and Kodicek, Haussler et al., Lawson et al., Myrtle et al.), diethyl ether-I ,2-dichloroethane-methanol (Haussler et al., Myrtle et a/.), diethyl ether -Skelly-Solve B (Suda et al.) and a non-gradient system using 16.5% of diethyl ether in light petroleum (b.p. 40-60°C or 60-80°C) (Takahashi and Yamamoto) or 20% of 1,2dichloroethane in diethyl ether (Haussler et al.) have been used. From the stationary phase system of methanol-water on Celite, calciferols were eluted with a gradient of the mixture 1,2-dichloroethaneinlight petroleum-l,2-dichloroethane (Haussler etal.) or Skelly-Solve B (De Luca, Suda et al.). With the reversed phase of Factise, a mixture of water and acetone was used for the elution. Calciferols can be detected in eluents by utilizing their absorption maxima in UV light (300-220 run) (Dollwet and Norman), spectrophotometrically after reaction with antimony trichloride (Mariani and Mariani-Vicari, Takahashi and Yamamoto) or radiometrically (Dollwet and Norman).
Chromatographic separation of vitamins Dzand D3 and related compounds using Factise For the preparation of the reversed phase (Dollwet and Norman), Factise is first triturated in acetone, and then forced with the bottom of an erlenmeyer flask through a 60-mesh sieve and collected below on a 100-mesh sieve. The Factise is kept moist with a small amount of acetone during this screening procedure, and the screened Factise is then stored in acetone. Later the Factise polymer is washed several times with glacial acetic
-
-
-
- 800 -
100 -
- 600 -
1.60
140
120
w
0
2LT
0 v) m
080-
-
-
7 DEHYDROCHOLESTEROL
- 400 E Q
ERGOSTEROL
U
060-
Q
040
020
--
-
-
190
200
210
220
230
240
250
260
270
280
290
200
300
Fig. 44.3. Separation of ['HI vitamin D, ,vitamin D, ,vitamin D,, ergosterol and 7dehydrocholesterol (Dollwet and Norman). Column: 214 X 0.8 cm. Stationary phase: Factise. Eluent: 5% of water in acetone. Detection: solid line, absorbance at 264 and 282 nm; broken line, radioactivity.
FAT-SOLUBLE VITAMINS
959
acid and acetone (Hirsch). Before filling the columns, the Factise is slurried several times in the solvent system to be used, 50 ml of the slurry are then forced into the columns, the Factise is allowed to settle and the eluent is then pumped through the column for 30 min. After completing the preparation of the column, the eluent is pumped out overnight at a flow-rate of ca. 10 ml/h so as to compact the column. The sample in 1.5-2 ml of acetone is placed on the top of the column, then rinsed into the column with at least four or five 0.5-ml portions of the eluent (5% of water in acetone) before the eluent is pumped through the column. Fractions of 1 ml are collected at a flow-rate of 10 ml/h and evaporated under vacuum. Then 5 ml of absolute ethanol are added, the contents stirred and the UV spectrum is scanned between 300 and 220 nm. This procedure was used for the separation of (a) vitamin Dz , D3 and ergosterol; (b) cholesterol and 7dehydrocholesterol; (c) dihydrotachysterol-2; (d) tritiated vitamin D3 and D2 ;(e) [4-14C]vitamin D3 ;and (0 [ 1,2-3H] cholesterol (Fig. 44.3) (Dollwet and Norman).
Isolation and identification of 2.5-hydroxyergocalcijerol in blood Blood is collected and mixed immediately with one tenth of its volume of 0.1 M sodium oxalate solution so as to prevent clotting. Plasma is separated from the cells by means of a De Lava1 blood separator and is made up to 70% saturation with ammonium sulphate and allowed t o stand at 4°C for 7 days. The precipitate of the protein is collected by centrifugation (25,000 rpm for 25 min; Sharples AS-16-P centrifuge) and extracted with methanol-chloroform (2: 1). After further addition of chloroform, the denatured proteins are removed by filtration through glass-wool and re-extracted with a further portion of methanol-chloroform (2: 1). The phases are allowed to separate and the aqueous phase is drawn off and re-extracted with chloroform. The combined chloroform layers are washed with tap water and allowed to stand for 24 h. The chloroform phase is concentrated to 50 ml, washed with saturated sodium chloride solution and dried over anhydrous magnesium sulphate. The solvent is then evaporated to dryness and the residue dissolved in 100 ml of Skelly-Solve B (a petroleum fraction, b.p. 65-67°C) (Suda et al.). The first chromatography is carried out on a column of silicic acid (60 X 1 cm). The column is eluted with a diethyl ether-Skelly-Solve B gradient, obtained by running 400 ml of 85%diethyl ether in Skelly-Solve B from a holding chamber into a 250-ml mixing chamber initially containing 250 ml of Skelly-Solve B. Then diethyl ether is placed in the holding chamber and finally methanol is applied directly on to the column. From the fractions obtained, the fraction with the greatest biological activity is collected, evaporated to dryness and rechromatographed on a multibore silicic acid column. The mixing chamber contains 250 ml of Skelly-Solve B and the holding chamber 400 ml of 85% of diethyl ether in Skelly-Solve B. As soon as the holding chamber has been emptied, it is fdled with 300 ml of diethyl ether. The eluted fractions of the main peak are collected, combined and chromatographed on a Celite partition column. For this purpose, 200 ml of Skelly-Solve B is equilibrated at 10°C with an equal volume of methanol-water (8:2), I 5 ml of the methanolic phase is mixed with 20 g of Celite and dry packed into a 60 X 1 cm column in 2 c m portions. The upper phase is used as the mobile phase. The residue obtained from the silicic acid column chromatography (after evaporation) is applied in a small volume (1 -3 ml) of mobile phase to the column and is developed with mobile phase, References p . 9 76
960
VITAMINS
5-ml fractions being collected. Tubes 11- 17 are combined, evaporated to dryness as before and re-chromatographed on another partition column. The separated substances are determined radiometrically or spectrophotometrically (UV region) or by GLC (Suda et al.).
Tocopherols The preparation of a sample of natural material includes extraction and saponification. Owing to the high sensitivity of tocopherols to oxidation, antioxidants (ascorbic acid, pyrogallol, etc.) are added before saponification. Otherwise, the saponification is carried out in the manner described, for example, for vitamin A. For the separation of tocopherols by column chromatography, different adsorbents are used. The chromatographic procedure utilizes the different adsorption affinities of the tocopherols and interfering substances to the adsorbent used. Silica gel (Cassagne and Baraud, Skinner et al.), silica gel with Celite (Williams), silicic acid (Dicks-Bushnell), silicic acid with diatomaceous earth (Cinquina), Florisil hydrated to different degrees (Dicks-Bushnell, Skinner et al.), magnesium hydrogen orthophosphate (Dicks-Bushnell) and a mixture of alumina, zinc carbonate and Celite (Millar and Caravaggi) were found t o be suitable for this purpose. The elution from the column of silica gel was carried out with a gradient of n-hexanebenzene-diethyl ether-methanol (Cassagne and Baraud) or by stepwise elution with 0.5% 3%, 10%and 20% of diethyl ether in n-hexane. Stepwise elution using a mixture of isooctane, 22% of chloroform in isooctane and 50% of chloroform in isooctane from the column of silicic acid (Cinquina) or a mixture of Skelly-Solve F and diethyl ether (98:2) (Dicks-Bushnell) has been used. Stepwise elution was applied to a Florisil column using Skelly-Solve F-diethyl ether (199: 1 , 9 9 :1 and 39: 1 ) (Dicks-Bushnell). With an aluminazinc carbonate-Celite column, a mixture of benzene and cyclohexane (1 :4) was used. The detection and determination of the separated substances is mostly carried out by W spectroscopy (246 and 292 nm) (Cinquina) before and after reduction with potassium borohydride (Williams) or using the iron(II1) chloride-dipyridyl reaction at 520 nm (Dicks-Bushnell, Williams).
Separation arid determination of quinones and a-tocopherol Approximately 30 g of leaves of the Viciafaba (broad bean) plant are frozen in liquid air and ground with 40-50 g of anhydrous disodium hydrogen orthophosphate. The finely ground leaves are lyophilized overnight at 4°C in the dark and the lyophilized leaves are re-ground, extracted with acetone and filtered. The residue is washed with acetone until colourless. The liquid extract is dried and the green residue is dissolved in chloroform, again evaporated, the residue re-dissolved in n-hexane (b.p. 67-70°C) and a known volume of the solution applied to the top of a Kieselgel G-Celite (1 : 1, w/w) column. The mixture of Celite and Kieselgel G is thoroughly washed with diethyl ether, which removes a yellow contaminant, and dried at 100°C for 1 h. The column material is prepared as a slurry with n-hexane, packed into the column (6 X 1.2 cm) and washed with a small volume (10 ml)
FAT-SOLUBLE VITAMINS
9 61
of n-hexane. The sample of quinones is eluted with 60 ml of OS%, 60 ml of 3 .O%, 60 ml of 10%and finally with 20% of diethyl ether in rzhexane. Fractions of approximately 3.5 ml are collected and all of the fractions are evaporated to dryness and the residues re-dissolved in 3 ml of 95% ethanol and identified (Williams). Determination of a-tocopherol in serum
To 4 ml of serum are added 2 ml of water and 6 ml of ethanol containing 1 % of pyrogallol. The mixture, containing 0.3 ml of 1 1 Npotassium hydroxide, is refuxed for 15 min at 90°C and, after cooling, is extracted with three 20-ml volumes of diethyl ether. The combined ethereal fractions are dried with 5 g of anhydrous sodium sulphate and evaporated to dryness at 40°C under a stream of nitrogen. The residue is dissolved in 0.1-0.5 ml of n-hexane and the solution is applied to a column of 0.6 g of alumina, 0.6 g of basic zinc carbonate and 0.3 g of Celite 545. The column is developed with benzenecyclohexane (1 :4). The first 3 ml of eluate contains @carotene,the next 10 ml a-tocopherol, and ubichromenol remains on the column (Millar and Caravaggi).
Vitamin K group There are many homologues of vitamin K , such as K 1 ,K2 and K 3 . Vitamins K I and Kz also have homologues with side-chains of various lengths at the 3-position. The K vitamins are extremely sensitive to light and special care must be taken t o avoid their photolysis during analytical operations. Cclumn chromatography has been used for the preliminary purification of analyzed material and for the isolation of the pure substances on a preparative scale. The most common adsorbents used were alumina, Decalso, Florisil, Permutit, zinc carbonate, silicic acid and occasionally magnesium oxide. Synthetic K vitamins were separated on alumina deactivated with dilute acetic acid. For the separation of homologues of vitamin K, a methylated Sephadex (Nystrom and Sj6vall) and solvent mixture comprising chlorofornmethanol-nheptane (1 : 1 :2) have been used. Column chromatography has in most instances been used especially for the preliminary purification of test material from interfering substances. Separation of vitamin K z on capillary columns of methylated Sephadex This method is suitable for the separation of the isoprenologue homologues of vitamin K2 (Kz (10) -Kz (a)). Columns with a diameter of 2 cm are prepared with about 25 g of methylated Sephadex G 2 5 superfine. The samples (0.2-1 mg) are applied to the columns in 0.5-1 ml of solvent. Capillary columns are prepared in the following w y . A small piece of glass-wool and a 2 c m length of stainless-steel capillary tube (O.D. 1/16 in., I.D. 0.25 mm, cut to a tip at the distal end) are inserted into the distal end of a PTFE tube about 2 m in length and this tube is filled with the solvent to be used for chromatography. The proximal end is References p . 9 76
962
VITAMINS
connected with a stainless-steel tube (O.D. 1/16 in., I.D. 0.6 mm, length 5 cm) silversoldered t o a stainless-steel cylindrical reservoir (O.D. 30 mm, length 100 mm) that contains a slurry of methylated Sephadex G-25 superfine in the same solvent. The upper end of the cylindrical reservoir is connected to a nitrogen tank and a pressure of about 1-2 kp/cm* is applied. The slurry passes slowly through the capillary into the PTFE tube. Clogging is prevented by vibrating the reservoir. When the PTFE tube is completely filled with the gel, the pressure is released and the tubing is disconnected from the reservoir. An injection port is attached t o the proximal end of the PTFE tubing and connected to another cylindrical reservoir (300 X 38 mm) that contains the solvent to be used (chloroform-methanol-n-heptane, 1 : I :2). A pressure of 1-3 kp/cm2 is applied. A 5-1.11 volume of a solution of the sample in the solvent is injected into the column. The elution of vitamin K2 derivatives with chloroform-methanol-n-heptane (1 :1 :2) is followed by measurement of the absorption at 270 nm or by using a platinum chain-flame ionization detector. The compounds are eluted in order of decreasing molecular weight and in order of increasing polarity (Nystrom and Sjovall).
WATER-SOLUBLE VITAMINS It is not possible to separate this large group of vitamins, which consist of different chemical constituents, in one universal procedure and the behaviour and separation of individual water-soluble vitamins will therefore be described. Of new techniques, high-speed liquid chromatography has been used for the separation of water-soluble vitamins. The chromatographic conditions required for single component samples are summarized in Table 44.2 (DuPont). TABLE 4 4 . 2 SEPARATION OF WATER-SOLUBLE VITAMINS USING HIGH-SPEED LIQUID CHROMATOGRAPHY (DUPONT) Vitamin
Column
Mobile phase
Niacinam ide Riboflavin Pyridoxine Corrinoide Thiamine mononitrate Ascorbic acid Niacin Folic acid
Zipax SCX Zipax SCX Zipax SCX Zipax SCX Zipax SCX Zipax SAX Zipax SAX Zipax SAX
pH 3 , 0 . 0 7 M NaCIO, pH 4 , 0 . 1 4 64 NaCIO, pH 4 , 0 . 1 4 M NaCIO, pH 9 , 0 . 2 M NaCIO, pH 9 , 0 . 6 M NaCIO, pH 7 , no modifier pH 7 , 0 . 0 0 2 M NaNO, pH I, 0.02 M NaNO,
Thiamine During all analytical operations with thiamine and its derivatives, their instability, especially in neutral and alkali media, must be kept in mind. For the separation of thiamine and its derivatives and metabolites, zeolite ion exchang-
WATER-SOLUBLE VITAMINS
963
ers have most often been used. If the concentration of thiamine in a sample is low, the best results can be achieved using Permutit T (Decalso). For higher concentrations of thiamine, some synthetic cation exchangers such as Amberlite GC-50 and Dowex have most often been applied (Amos and Neal; Diorio and Lewin; Matsuo and Suzuoki; Neal, 1968, 1969; Suzuoki et ul.). Dowex 1-X4 was found to be advisable for the separation of thiamine, monophosphothiamine, diphosphothiamine and triphosphothiamine (Koike et al.). Diorio and Lewin used Dowex AG 50W-X8for the separation of pyrimidine precursors of thiamine. Thiamine can be eluted from a Decalso column using an acidic solution of potassium chloride. Koike et al. eluted thiamine and its derivatives from Dowex 1-X4 with water followed by an acetate buffer. Neal (1968, 1969) eluted thiamine from Amberlite GC-50 with water followed by pyridine-acetic acid-water (7.5:1.5:91). Amos and Neal used a linear gradient of distilled water to 0.35 M pyridinium acetate for Amberlite GC-50 and 0.0 1 N hydrochloric acid for Sephadex G-10. Diorio and Lewin used 2 M ammonium hydroxide solution for Dowex AG 50W-X8.
Separation o f thiamine from interfering substances on Decalso The acidic extract, or the enzymatic hydrolyzate in the case of thiamine phosphate, is transferred by pipette into the column of Decalso, and thiamine in the acidic extract is bound on the ion exchanger. A 1-g amount of normally grained Decalso binds quantitatively 40 pg of thiamine. For isolation from natural materials, there is a maximum amount of about 10 pg, owing to the decreasing activity of Decalso in the presence of other compounds with the same binding activity. After the application of a sample, the column is washed with hot distilled water and thiamine is eluted with an acidic solution of potassium chloride.
Separation o f thiamine, moriophosphothiamine (MPT), diphosphothhmine (DPT)and triphosphothiamine (TPT) The sample ( 5 ml, pH adjusted to 4.5 with 0.1 N hydrochloric acid) is passed through a 190 X 6 mm column of Dowex 1 -X4 (CH3COO-), 200- 400 mesh. Thiamine and MPT are eluted with 14 ml of water, DPT with 24 ml of 0.1 M sodium acetate buffer of pH 4.5 and TPT with 24 ml of 1 M sodium acetate buffer of pH 4.5. The separation of thiamine plus MPT, DPT and TPT is sharp (Fig. 44.4), but thiamine and MPT must be distinguished by the thiochrome method, before and after acid phosphatase digestion (Koike et ul.).
Separation of thiamine from its metabolites in urine and cells Metabolites of thiamine from urine and cells are chromatographed on a column of Amberlite GC-50 (H'), eluted with water followed by pyridine-acetic acid-water (7.5: 1.5 :91). Thiamineacetic acid (4-methylthiazole-5-acetic acid) is the main metabolite, followed by the fraction corresponding to thiamine (Neal, 1968, 1969). Amos and Neal in addition separated 2-methyl4-amino-5-formylaminomethylpyrimidine on the same column. References p . 9 76
VITAMINS
964
60
w 0 2
w 0
4 0
v)
W
6J -1 LL
2.0
3
1
L
1.0
10
20
VOLUME, rnl
Fig. 44.4. Chromatography of thiamine and its phosphoric acid esters from biological materials (Koike d.). Column: Dowex 1-X4 (CH, COO-). Eluent: water followed by acetate buffer of pH 4.5. Peaks: 1 = thiamine + MPT; 2 = DPT; 3 = TPT.
er
0
30
60
120
90
150
180
FRACTION NUMBER I
0
I
0.5
I
I
I
I
1.0
1.5
2.0
2.5
I
3.0
I 3.5
CALCULATED MOLARITY OF HCL
Fig. 44.5. Chromatography of pyridine precursors on a Dowex A C 50W-X8 (H') column (Diorio and Lewin). Eluent: linear gradient of hydrochloric acid (0.4 N in 2 1). Peaks: 1 = 2-methyl-4-amino-53 = 2-methyl4-aminomethylhydroxyrnethylpyridine; 2 = 2-methyl4amino-5-formylpyrhidine; pyrimidine; 4 = 2-methy14-amino-5-methoxymethyIpyrimidine.
965
WATER-SOLUBLE VITAMINS
Separation of pyrimidine precursors of' thiamine
O M Dowex
AG 50 W-X8
This procedure has been used for the separation of precursors from Neurospora crassa (Diorio and Lewin). Dowex AG 50W-X8 (H') is mixed with broth on which Neurospora crassa has grown. The mixture is stirred for 1 h before filtering and discarding the filtrate. The resin is then washed with distilled water and eluted with 2 M ammonium hydroxide solution. The eluate containing the pyrimidine precursors is applied to a column of washed Dowex 50W-X8 (H') (70 X 1.5 cm) and the column is then washed with distilled water until the effluent is at pH 5-6. Elution of pyrimidine precursor compounds is accomplished with a linear gradient of hydrochloric acid (0-4 N in 2 1). With this procedure, four compounds can be separated in the above material (Fig. 44.5). It is possible to recover 90-95% of biologically active precursors in the analyzed medium.
Riboflavin and other flavins Substances of this type are generally not very stable when exposed to light, so that all analytical procedures, including the chromatographic separation, must be carried out in darkness or subdued red light. The determination of riboflavin and flavins is generally most often based on their fluorescense. In natural materials, however, many interfering fluorescent compounds occur, which must be removed prior to its determination. For this purpose, chromatographic procedures have most often been used. The separation of riboflavin from an interfering fluorescent compound was first made on several activated clays. Klatzkin et al. used Florid, while Strohecker and Henning used Permutit. Nowadays ion exchangers are most often used for these purposes (Kozioiowa and Koziol). Good results were obtained with Zeo-Karb 215, Wofatit P, F and KS, Lewatit PN and KSN, Staionit FN and F extra, MSF resin and phenolcarboxylic resins such as Lewatit CNS and Zeo-Karb 216. All types of phenolic resins gave completely quantitative sorption of riboflavin, lumiflavin and lumichrom from aqueous solutions, dilute acids (up to 0.5 N) and salt solutions (up to 2 IV)over a pH range from 1 to 8. The capacity of the resins was found to be virtually unaffected by the form of the resin (H+, Li', Na+, K+ or NH;) and closely related to the number of free phenolic OH groups. For the separation of riboflavin, Lammi and Lerner used poly-N-vinylpyrrolidone (Polyclar AT). Recently, Lerner substituted Polyclar AT for Bioclar G. Carletti et al. achieved the satisfactory separation of all pure flavins on cellulose. Gupta et al. (1967b) used DEAEcellulose for the same purpose. Riboflavin can be eluted from Permutit T (Decalso) with water and from phenolic resins with alkaline solutions such as sodium, potassium and ammonium hydroxide, sodium carbonate and sodium tetraborate. Riboflavine 5'-phosphate (FMN) and flavineadenine dinucleotide (FAD) in aqueous solutions are only partly bound by resorcinoltype resins and are not sorbed on sulphonic- and carboxylic-type resins. They can be eluted from resorcinol resins using a 10%aqueous solution of acetone, and riboflavin with a 1: 1 acetone-water mixture. For Polyclar AT, elution of riboflavin with water or salt solution is most suitable (Lammi and Lerner). tert -Butanol-0.01 N hydrochloric acidReferences p.976
966
VITAMINS
water (50:2.5240) followed by water is a suitable elution procedure for cellulose (Carletti et al.) and 0.01 M sodium carbonate solution for DEAE-cellulose (Gupta eral., 1967a, b).
Separation of riboflavinfrom thiamine and vitamin B6 on Permutit A neutral or weakly acidic sample solution (pH 4-6) containing 5-50 pg of riboflavin is applied on a column of Permutit T (Decalso). Riboflavin is not bound on Permutit and can be eluted easily with water. In this way, riboflavin can be separated from thiamine and vitamin B6,which remain bound on the column of Permutit (Strohecker and Henning).
Separation of riboflavin from vitamin B I 2and cytochrome c on Bioclar G Bioclar G is slurried in a minimal volume of deionized water and the slurry poured into a 40 X 0.8 cm column. The sample containing vitamin B12 (red), riboflavin (yellow) and horse cytochrome c (orange) is applied on the column, which is eluted with water. Vitamin BIZ begins t o appear in the effluent at 5 min and is completely removed after 7 min, riboflavin follows immediately after this and its elution is completed in 14 min. At this point, water remaining above the bed is removed and replaced with 1% sodium sulphate solution. After 3 min, cytochrome c begins to emerge from the column and is completely recovered in 8 min (Lerner).
Separation of flavins and lumazines on cellulose This method is suitable for the determination of flavins and lumazines in flavinogenic and 6-methylmicrobes. A good separation of FMN, FAD, 6,7-dimethyl-8-ribityllumazine 7-hydroxy-8-ribityllumazineis achieved on a 20 X 0.75 cm column packed with cellulose powder. The eluent is terr.-butanol-O.Ol N hydrochloric acid-water (50:2.5:40) followed, after elution of FMN, by water at a flow-rate of 15 ml/h (Carletti et al.).
Separation of riboflavin from flavin nucleotides The separation of riboflavin from other flavin nucleotides is successful with all types of phenolic resins (Zeo-Karb 215, Wofatit F, P and KS, Lewatit PN) using a two-step elution. FMN and FAD can be eluted with water and pure riboflavin in the next step with acetone-water (1 :1). With a resorcinol-type resin, the nucleotides can be eluted with a 5-10% aqueous solution of acetone and riboflavin with a 50%acetone-water mixture. For the determination of total flavins in biological materials, the crude extract obtained from samples by hydrolysis with 0.1 N sulphuric acid is neutralized with ammonia solution and then passed through the column of resorcinol resin. The column is washed with 0.1 N ammonium sulphate in order t o remove the non-flavin compounds.
WATER-SOLUBLE VITAMINS
967
Nicotinic acid and its derivatives No specific procedure is available for the determination of nicotinic acid and its amide and therefore their separation prior to determination is necessary. Nicotinic acid and its derivatives are stable under normal conditions and therefore none of the chromatographic procedures need special conditions. The methods originally used for the isolation of nicotinic acid from biological materials were based on its selective adsorption on aluminium silicate (Perlzweig et af.). Finholt and Higuchi separated nicotinic acid from nicotinamide on the ion exchanger Amberlite IRA400. McDonald and Stewart separated nicotinamide from nicotinamide mononucleotide using Dowex 2 (HCOO-) (anionic constituents were separated by this step) and nicotinamide from nicotin nucleotide using Amberlite IRC-50. Kahn and Blum used Dowex 1 (HCOO-) and Lee et af.Dowex 50 (H') in combination with Dowex 1 . Nicotinic acid formed by the degradation of several nicotinic acid esters could be satisfactorily separated from other degraded components using the anion-exchange resin Amberlite CG4B (Suzuki and Tanimura). The separation of small amounts of thionicotinamide and selenonicotinamide from the corresponding NADP analogues was achieved by chromatography on Sephadex G-10 and G-25 (Christ eta/.). The separation of nicotinic acid on Amberlite IRA400 is based on its binding on the resin from pH 4.5 to 5.0; nicotinamide can be eluted with water at this pH (Finholt and Higuchi). Kahn and Blum eluted nicotinic acid and its derivatives from Dowex 1 using water followed by a concave gradient of formic acid. Lee et al. separated metabolites of nicotinic acid on Dowex 50 with water as eluent followed by their separation on Dowex 1 again with water as eluent.
Separation o f tzicotinic acid f r o m its degradation products Amberlite CG-4B (50 g) is purified by soaking it in distilled water, washng it with 50 ml of 0.5 N hydrochloric acid and subsequently washing it with 500 ml of 0.5 N sodium hydroxide solution in a column after removal of the acidic solution with distilled water. The resin is converted from its hydroxide form into its chloride form again with 0.5 N hydrochloric acid. This product is washed with distilled water and poured into a chromatographic tube, making a column of dimensions 4 X 0.9 cm. A 5-ml volume of 1 M acetate buffer (pH 4.9) is passed through the resin before addition of the sample solution. A 5-ml volume of a sample solution of degraded nicotinic acid esters of polyhydric alcohols is adjusted to pH 2.0-2.3 by addition of 5 ml of sodium hydroxide solution at an appropriate concentration and then the mixture is shaken with an equal volume of chloroform in a glass-stoppered tube for 10 min so as to remove slightly soluble compounds, and then centrifuged for 5 min. Then 5 ml of the aqueous layer and 1 ml of 1 M acetate buffer (pH 4.9) are transferred by pipette into a reservoir connected to the column and passed slowly through the column at a rate of 0.7 ml/min. The partly solvolyzed nicotinic acid esters of polyhydric alcohols are eluted with distilled water into a 100-ml calibrated flask at a rate of 1.4 ml/min. The nicotinic acid absorbed to the resin is eluted with 0.3 N hydrochloric acid and exactly 50 ml of effluent are collected in a 50-ml calibrated flask at a rate of 1.4 ml/min (Suzuki and Tanimura) References p . 976
968
VITAMINS
Separation of nicotinic acid from its derivatives on Dowex 1 This method was used by Kahn and Blum for the separation of nicotinic acid and its derivatives from Astasia longa cultivated in a synthetic medium containing [7-I4C] nicotinic acid. A trichloroacetic acid extract is applied to a Dowex 1 (HCOO-) column (50 X 0.9 cm), which is washed with water. After about 100 fractions of 10 ml each have been collected, a concave gradient of formic acid is applied to the column and fractions of 5 ml are collected.
Pyridoxine group This group consists of pyridoxol, pyridoxal and pyridoxamine. Before their determination, it is necessary to extract these components using acidic or enzymatic hydrolysis and separate them using column, thin-layer or paper chromatography. The original simple method for the separation of substances that interfere in the determination of pyridoxol was adsorption on activated clay. At present, the use of ionexchange resins based on weakly acidic cation exchangers has been developed. Strohecker and Henning recommend the ion exchanger IV, while F'latzer and Roberts used Dowex AG W-X8. Takanashi et al. carried out the separation on Dowex followed by Amberlite, while Johansson and Lindstedt used Dowex AG 50W-X8. Contractor and Shane followed the metabolism of pyridoxol on rats using cellulose phosphate, charcoal and DEAEcellulose. From ion exchanger 1V (Merck), pyridoxol can be eluted with 1 N hydrochloric acid, from Dowex AG W-X8 with a hot mixture of 0.6M potassium chloride and 0.1 M potassium dihydrogen orthophosphate (Platzer and Roberts). Takanashi et al. eluted pyridoxol from a Dowex column with acetate buffer of pH 4 and from an Amberlite CG-120 column with 0.4 M phosphate buffer of pH 7.5. Johansson and Lindstedt used Dowex 50W-X8 with 0.05 M ammonium formate solution of pH 4.25 followed by gradient elution with I00 ml of 0.05 M ammonium formate of pH 4.25 in a closed chamber to which 0.5 M ammonium formate of pH 7.5 was added.
Separation of vitamin B6 from interfering materials on a Dowex column A 10-ml volume of wet settled Dowex AG W-X8 (K', 100-200 mesh), is placed in a 25.5 X I .O cm glass column and adjusted to pH 4.5 by washing with 50 ml of 0.01 M potassium acetate solution of pH 4.5. An acid extract of 500 mg of tissue or vitamin B, standards adjusted to pH 4.5 is placed on the column, which is then washed with 50 ml of 0.02 M potassium acetate solution of pH 5.45. The vitamin B6 components are eluted in one step with 30 ml of a boiling mixture of 0.6M potassium chloride and 0.1 M potassium dihydrogen orthophosphate of pH 8.0. Then 4.5 ml of 1 M calcium chloride solution are added to the eluate and the volume is adjusted to 35 ml with water. The mixture is centrifuged at 700 g for 15 min and the supernatant fluid is assayed for total vitamin B,. A convex gradient of increasing pH and molarity of potassium acetate, pH
a
969
WATER-SOLUBLE VITAMINS
0.4
0.3
I
PN
I
\
\
PM
\
U
0
0.7
0
0.3 0.2
I
n
I
H. d i m i n u t a
n
/
PN
TUBE
\
NUMBER
Fig. 44.6.Chromatography of vitamins B, (Platzer and Roberts). Column: Dowex AG W-X8 (K+). Eluent: boiling 0.6 M potassium chloride and 0.1 M potassium dihydrogen orthophosphate (pH 8.0) and convex gradient of increasing pH and molarity of potassium acetate, pH 5.45 and 0.02 M t o pH 7.0 and 0.1 M. Peaks: PL = pyridoxal; PN = pyridoxine; PM = pyridoxamine.
5.45 and 0.02M to pH 7.0 and 0.1 M ,is used for the separation of the vitamin B6 group (Fig. 44.6) (Platzer and Roberts).
Separation of vitamin B6 compounds on Dowex 50 W-X8 A perchloric acid extract of muscle tissue is prepared. A suitable portion is dissolved in 1-2 ml of 0.05 M ammonium formate buffer of pH 4.25 and placed on a 40 X 0.9 cm column of Dowex 50W-X8. The column is eluted first with 100 ml of 0.05 M ammonium formate of pH 4.25, then a gradient is started with 100 ml of 0.05 M ammonium formate of pH 4.25 in a closed chamber to which 0.5 M ammonium formate of pH 7.5 is added (Fig. 44.7) (Johansson and Lindstedt). Refereiices p . 9 76
VITAMINS
970
20/
A
4
sba VOLUME, ml
Fig. 44.7. Chromatography of vitamin B, from mouse liver (Johansson and Lindstedt). Column: Dowex 50W-X8.Eluent: ammonium formate, pH 4.25, and gradient of ammonium formate. A, unhydrolyzed extract; B, same extract after hydrolysis. Peaks: 1 = pyridoxine-5’-phosphate and pyridoxal-5‘-phosphate; 2 = pyridoxalamine-5’-phosphate; 3 = pyridoxal; 4 = pyridoxine; 5 = pyridoxamine.
Biotin Chemical and physicochemical methods for the determination of biotin are used only rarely; the biological activity of biotin is most often tested microbiologically. In special instances, column chromatography can also be used. Column chromatography has been applied to the study of metabolites during the cultivation of microorganisms on culture media containing biotin or during the biosynthesis of biotin or its vitamers. For the binding of biotin or biotincontaining peptides, Sepharoseavidin columns are efficient (Bodanszky and Bodanszky). Avidin can be coupled with Sepharose 4 B activated with cyanogen bromide. Enzymatically synthesized [“C] desthiobiotin was purified by anion-exchange chromatography on Bio-Rad AG 1-X8; the desthiobiotin-avidin complex was isolated on Sephadex G-25F (Eisenberg and Krell).
Separation o f biotin from interfering compounds on Dowex Iwahara et al. studied the products isolated from culture filtrates of Pseudomonades grown on a biotin-salts medium to which [“C] carbonyl-labelled biotin was added. The bacterial culture is centrifuged for 20 min at 16,000 g in order to remove the cells. Dowex 50W-X8(100-200 mesh) is stirred into a total of 9 1 of supernatant solution which has been filtered through paper. The resin is washed with 6 1 of 70% ethanol,
WATER-SOLUBLE VITAMINS
97 1
combined with the first filtrate and concentrated to 600 ml. After removal of the final precipitate, the solution (pH 7.0) is divided into three equal portions and each portion is poured over a 7 0 X 1.5 cm column of Dowex 1-X2 (HCOO-), 100-200 mesh. The radioactive materials are eluted from the column by successive treatments with water and 0.01 Marid 0.1 M formic acid. Yang et al., in studies on the changes to biotin due to Rhodotomla, Penicillium and Endomycopsis also investigated its separation on Dowex 1-X2. Eisenberg and Maseda followed the biotin derivatives from Penicillium chrysogenum on Dowex 5OW-X8.
Pantothenic acid and coenzyme A In addition to the separation of pantothenic acid and its derivatives from natural materials, column chromatography has also been applied to prepare some analogues of pantothenic acid and, especially, to prepare coenzyme A. Saccharides and riboflavin, during alkaline hydrolysis of analyzed materials, form a brown colour that interferes in the spectrophotometric determination. The colour can be removed on ion exchanger V (Merck) (Strohecker and Henning). Nagase et al., in the preparation of some pantothenic acid analogues, used different types of Amberlite. Yoshioka er al. separated D-pantothenic acid 4-phosphate from the reaction mixture on DEAE-Sephadex. Cha et al. and Zahler and Cleland used DEAE-cellulose for the separation of coenzyme A. DEAE-cellulose was also used for the separation of some analogues of coenzyme A (Shimizu et al., 1968) and this procedure was improved using DEAE-Sephadex A-25 (Shimizu et aZ., 1970a) and Dowex 50 (Shimizu et al., 1970b). Stearylcoenzyme A synthesized by Haeffner was isolated using Sephadex G-15. Pantothenol can be separated from pantothenic acid on an anion exchanger, the elution being carried out with water. Pantothenic acid is eluted with 0.1 N hydrochloric acid (Knobloch). Yoshioka et al. eluted pantothenic acid 4-phosphate from DEAE-Sephadex with an increasing gradient of ammonium carbonate (0-1 .O M). In order to separate coenzyme A, Cha et al. used DEAEcellulose and gradient elution with triethylamine hydrogen carbonate (pH about 7.5) from 0 to 0.8 M in a total volume of 1 1. For a similar purpose, DEAEcellulose and a linear gradient of lithium chloride from 0.05 to 0.5 M was used by Zahler and Cleland and by Shimizu et al. (1968). Stearylcoenzyme A can be eluted from Sephadex G-15 using 0.5% mercaptoethanol.
Separation of coenzyme A analogues Some coenzyme A analogues, such as a-rnethyl-, 0-methyl- and acarboxycoenzyme A, can be separated on a DEAEcellulose column (Shimizu et al., 1968). The reaction mixture is passed through a 30 X 2 cm column of DEAEcellulose (Cl-) and elution is carried out using a linear salt gradient. The reservoir contains 0.3 M lithium chloride in 0.003 N hydrochloric acid (1 1) and the mixing vessel contains 0.003 N hydrochloric acid (1 1). The same workers (Shimizu et aZ., 1970a) improved this method by using DEAE-Sephadex A-25 (Cl-) with the same eluent. References p . 9 76
972
VITAMINS
Folic acid and other pteridine derivatives During the preparation of samples, their instability in light and in the presence of air must be considered. Hence all preparative procedures, including the chromatographic separation, must be carried out in the dark (or subdued red light) and under an atmosphere of nitrogen. For the separation of a mixture of pteridine derivatives, columns of cellulosic ion exchangers are mostly used. Columns of cellulose phosphate are utilised mainly for the preparation of synthetic mixtures of pteridines (Chippel and Scrimgeour, Guroff and Rhoads, Jones and Brown, Mitsuda and Suzuki); cellulose phosphate was also used for the separation of substances of this type from natural material (Fukushima, Rembold et d , ) .For the isolation of pteridines from natural material, DEAE-cellulose (Chippel and Scrimgeour; Gupta and Huennekens; Cupta et al., 1967b; Ho and Jones; Zakrzewski etal.; Zakrzewski and Sansone) or Dowex 1-X2 (Kaufman) and Dowex 1-X8 (Rembold et nl., Sugiura and Goto) can also be used. For the differentiation of isomers of methyl tetrahydropteridines, Whiteley et al. applied the cation exchanger Dowex 50, while Curoff and Rhoads used CM-Sephadex. The modified celluloses used for this purpose are represented by ECTEOLA-cellulose (Fukushima, Rembold et al. ). For the separation of pteridines from natural material, it is possible t o use a molecular sieve of the Sephadex type. Sephadex (3-10 and G-25 (Dewey and Kidder, Fukushima, Sugiura and Goto), suitable for the separation of low-molecular-weight substances, were applied for this purpose. In some special instances, Florisil (Guroff and Rhoads), alumina (Hla-Pe and AungThan-Batu) and cellulose (Sugiura and Goto) were used for the separation of pteridine derivatives from other interfering ballast components. Combinations of the above methods have been often used for the separation of natural mixtures In a study of the biosynthesis of pteridines in the tadpole of the bullfrog, Fukushima used separations on ECTEOLA-cellulose, cellulose phosphate and Sephadex G25. Guroff and Rhoads applied Florisil and Sephadex in studies on the hydroxylation of phenylalanine by Pseudomonas species. To study the properties and metabolisms of pteridine derivatives in the rat liver, Rembold et al. applied Dowex I-X8, ECTEOLAcellulose and cellulose phosphate. When using these ion exchangers, various eluents have been used according to the procedure and the properties of the separated components and the ion exchanger used. The elution of pteridines from a column of CM-Sephadex was carried out with a gradient of a mixture of sodium acetate in Cleland's reagent (a solution of dithiothreitol) (Guroff and Rhoads). For the separation of synthetic standards of pteridines on cellulose phosphate, elution in two steps was used, acidic and neutral substances being eluted with water and basic substances with 5% formic acid and buffer solution (Rembold et d.).Using Dowex 1-X2 (CH3 COO-), the elution of reduced biopterine derivatives was carried out with 0.028 M 2-mercaptoethanol (Kaufman). For the elution of pteridines from a column of DEAE-cellulose, a gradient of ammonium acetate (Chippel and Scrimgeour, Gupta and Huennekens, Ho and Jones) in admixture with mercaptomethanol, or a phosphate gradient in admixture with potassium ascorbate (Rohringer et a[.), was used.
973
WATER-SOLUBLE VITAMINS
Separation of pteridines in the tadpole of the bullfrog The skin (10-1 5 g) is boiled in water (1 5 ml) for 5 min and homogenized in a Waring blender for 1 min. The homogenate is mixed with 80 ml of ethanol and centrifuged. This procedure is repeated and the combined supernatant fluid is concentrated by evaporation. A concentrated extract is applied to a pH 7 cellulose column (1 5 X 3 cm). Yellow fluorescent substances are eluted with water first, followed by blue fluorescent substances. As isoxanthopterin and pterin-6-carboxylic acid are not eluted readily with water, these compounds are eluted with 0.1 N hydrochloric acid. The yellow fluorescent fraction is concentrated and applied to a 20 X 1.8 cm Sephadex G-25F column and the yellow fluorescent fraction from this column is collected, concentrated and applied to a 20 X 1.8 cm cellulose phosphate column. The fraction is resolved into three zones; the first eluted material consists mainly of riboflavin and the second of sepiapterin. The concentrated blue fluorescent fraction from the first column is applied to a pH 7 cellulose column (35 X 1.8 cm), and biopterin and 2-amino4-hydroxypteridine are separated from each other in this step. The biopterin fraction is purified using a 20 X 1.8 cm cellulose phosphate column. Chromatographic data for the normal pteridines on the columns used are presented in Table 44.3. TABLE 44.3 COLUMN CHROMATOGRAPHY OF PTERIDINES (FUKUSHIMA) Compound
Biopterin Neopterin Dihydrobiopterin Sepiapterin 2-Amino4hydroxypteridine 6-Hydroxymethylp terin lsoxanthopterin Pterindcarboxylic acid Lumazine
Relative elution volume
Kd
pH 7 ECTEOLAcellulose column
Cellulose phosphate column
Sephadex G 2 5 F column
100 103 60 67 126 126 350** - ***
100 97
1.6 1.6 2.0 2.5 1.8 1.8 2.2
107
-
*
26 112 121 1s 10 13
1 .o
1.7
*The compound is not stable o n this column. **Rough estimation. ***Not eluted with water.
Corrinoids Corrinoids are substances derived from simple corrine with centrally bound cobalt within complexes of these compounds with proteins. For the separation of compounds of this type, ion exchangers and molecular sieves have been used, particularly Amberlite XAD-1 and XAD-2 (Kamikubo and Narahara), DEAEcellulose (Grasbeck et al., Wolff et al.) and DEAE-Sephadex A-50 (Ellenbogen and Highley , Grasbeck el al.). Cation exchangers used for the separation of corrinoids include References p . 9 76
974
VITAMINS
CM-cellulose (Finkler et al.; Tortolani et al., 1970a) Dowex 50W-X2 (Tortolani et al., 1970a), SP-Sephadex C-25 (Tortolani et al., 1970b) and CM-Sephadex C-50 in combination with other molecular sieves (Grasbeck et al., Simons and Weber). Molecular sieves of the Sephadex type play an important role in studies on the qualitative and quantitative relationships between cobdamins and their protein carriers. Using Sephadex G-50 (Gullberg, 1970a), G-25 (Hom), G-100 (Finkler et al., Garrido-Pinson et al., Simons and Weber), G-150 (Gullberg, 1970b) and G-200 (Grasbeck et al., Hom, Olesen et al., Simons and Weber), the protein carriers of cobalamins were isolated from different sources (human plasma, human gastric juice, human leukocytes, etc.). Yurkevich et al. used Sephadex G-15 for the separation of cobalamins in the form of triphenylphosphine complexes. Simons and Weber and Wolff et al. eluted corrinoids from DEAEcellulose using a gradient of pH and molarity of a phosphate buffer while Simons and Weber used a gradient of sodium chloride and phosphate buffer for elution from DEAE-Sephadex A-50. With Amberlite, Kamikubo and Narahara used ethanol and acetone for the elution. For molecular sieves, Tris-sodium chloride buffer (Gulberg, 1970a,b) and phosphate buffer (Garrido-Pinson et al.) were mostly used.
Separation and determination of corrinoids on an SP-Sephadex column SP-Sephadex is dispersed in a beaker with a 0.05 M sodium acetate buffer (pH 5.0) and poured into the chromatographic column (20 X 0.9 cm). The column is washed repeatedly with distilled water in order to remove excess ions and the eluate is checked for spectrophotometric purity in the wavelength range 260-400 nm. Then 2 ml of a solution containing 50 pg/ml of each corrinoid are applied on the column. The first elution is carried out with 40 ml of water, followed by 7 0 ml of 0.05 Macetate buffer (pH 5.0),
I-
H20
+CH3COONa
0.05 M 1 - C H ~ C O O HpH 5
n
DBC B12CH3
to
io
So
70
9’0
iio
EFFLUENT, m,
Fig. 44.8. Separation of cyanocobalamin (B12CN), methylcobalamin (B12CH, ), hydroxycobalamin ( B I Z O H )and cobamamide (DBC) (Tortolani el al., 1970b). Column: SP-Sephadex C-25 (Na+) Eluent: distilled water followed by 0.05 M sodium acetate buffer of pH 5.0.
975
WATER-SOLUBLE VITAMINS
and 2-ml fractions are collected. Cyanocobalamin and methylcobalamin are eluted separately with distilled water, while cobalamide and hydroxycobalamin remain fixed at the top of the column (Fig. 44.8); they can be separated by increasing the ionic strength of the eluent. Each 2-ml fraction is read at the corresponding wavelength. All operations must be conducted in subdued red light so as to prevent photolytic degradation of the cobalamins (Tortolani eC al., 1970b). L-Ascorbic and L-dehydroascorbic acids Column chromatography is not often used in analyses of vitamin C, paper chromatography more often being employed. Ascorbic and dehydroascorbic acids behave in a similar manner to sugars, and in many instances the chromatographic procedures for sugars can be applied to these substances. Friberg ea al. separated osazones of dehydroascorbic acid using chromatography on charcoal, Sephadex (3-50 and DEAE-Sephadex A-25 while Fiddick and Heath separated bound ascorbic acid from rat adrenals by gel filtration using Sephadex G-50 with 0.05 N sodium chloride solution as eluent (Fig. 44.9).
I
80
100
120
140
160
I 180
FRACTION. rnl
Fig. 44.9. Separation of ascorbic and dehydroascorbic acids from rat adrenals (Fiddick and Heath). Column: Sephadex (3-50. Eluent: 0.1 N sodium chloride solution. Peaks: H, -H, represent probably complexes of amino acids with polypeptides; L, and L, represent ascorbic acid and dehydroascorbic acid, respectively,
References p . 9 76
976
VITAMINS
In some instances, chromatographic techniques can be applied to separate ascorbic and dehydroascorbic acid from compounds that might interfere in their determination. Davidek et al. and Grundovi et al. separated anthocyanine pigments, which interfere in the polarographic determination of dehydroascorbic acid, on polyamide or Dowex SOW. Anthocyanine pigments are bound on the column and ascorbic and dehydroascorbic acids together with non-interfering substances can be eluted with water or 2% oxalic acid. Separation
o f L a s c o r b i c and L d e h y d r o a s c o r b i c acids on polyamide powder
Polyamide powder (54 mesh) is mixed with water and allowed to stand for about 2 h and then a 20 X 1 cm column is prepared. A 20-ml volume of analyzed extract (the extract of ascorbic and dehydroascorbic acids is prepared with 2% of oxalic acid) is added to the top of the column. Ascorbic and dehydroascorbic acids are eluted into a 50-ml calibrated flask with 2% oxalic acid and the eluate is used for analysis. Dowex 50W-X4 (Na') can be used instead of polyamide (Davidek el a l . ) .
REFERENCES Amos, W. H. and Neal, R. A., J. Biol. Chem., 245 (1970) 5643. Bell, P. A. and Kodicek, E., Biochem. J . , 1 1 5 (1969) 663. Bertram, S. and Krisch, K., Eur. J. Biochem., 1 1 (1969) 122. Bodanszky, A. and Bodanszky, M., Experientia, 26 (1970) 327. Carletti, P., Strom, R., Giovenco, S., Barra, D. and Giovenco, M. A., J. Chromatogr., 29 (1967) 182 Cassagne,C. and Baraud, J., Bull. SOC.Chim. Fr., (1968) 1470. Cha, S., Cha, M.,ChungJa. and Parks, Jr., R., J. Biol. Chem., 242 (1967) 2582. Cliippel, D. and Scrimgeour, K. G., Can. J. Biochem., 48 (1970) 999. Christ, W., Schmidt, D.and Coper, H., J. Chromatogr,, 51 (1970) 537. Cinquina, C. L., J. Bacteriob, 95 (1968) 2436. Contractor, S. F. and Shane, B., Biochem. Biophys. Res. Commun., 39 (1970) 1175. Crain, F. D., Lotspeich, F. J. and Krause, R. F.,J. Lipid Res., 8 (1967) 249. Davidek, J., Grundovi, K., Velitek, J. and JaniEek,G., Lebensm. -Wiss. Technol., 5 (1972) 213. De Luca, H. F.,J. Agr. Food Chem.,17 (1969) 778. Dewey, V. C. and Kidder, G. W.,J. Chromatogr., 31 (1967) 326. Dicks-Bushnell, M. W.,J. Chromatogr., 27 (1967) 96. Diorio, A. F. and Lewin, L. M., J. Biol. Chem., 243 (1968) 4006. Dollwet, H. H. A. and Norman, A. W., Anal. Biochem., 25 (1968) 297. DuPont, Liquid Chromatography Methods Bulletin, DuPont, Wilrnington, Del., March, 1972. Eisenberg, M. A. and Krell, K.,J. Biol. Chem., 244 (1969) 5503. Eisenberg, M. A. and Maseda, R., Biochemistry, 9 (1970) 108. Ellenbogen, L. and Highley, D. R.,J. Biol. Chem., 242 (1967) 1004. Eremina, G. V., Lab. Delo, (1968) 81. Fiddick, R . and Heath, H., Biochim.,Biophys. Acta, 136 (1967) 206. Finholt, P. and Higuchi, T., J. Pharm. Sci., 51 (1962) 655. Finkler, A. E., Green, P. D. and Hall, Ch. A., Biochim. Biophys. Acta, 200(1970) 151. Friberg, V., Lohmander, S. and Carlsson, G., Acta Chem. Scand., 221 (1968) 3037. Fukushima, T.,Arch. Biochem. Biophys., 139 (1970) 361. Garrido-Pinson, G. C., Turner, M. D., Miller, L. L. and Segal, H. L., Biochim. Biophys. Acta, 127 (1966) 478. Grasbeck, R., Simons, K. and Sinkkonen, I., Biochim. Biophys. Acta, 127 (1966) 47.
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Grundovi, K., Davidek, J., VeliSek, I. and JaniEek, G . , Lebensm. Wiss. Technol., 6 (1973) 11. Gullberg, R., Clin. Chim. Acta, 27 (1970a) 251. Gullbcrg, R., Clin. Chim. Acta, 29 (1970b) 97. Gupta, V. S. and Huennekens, F. M., Biochemistry, 6 (1967) 2168. Gupta, V. S., Kraft, S. C. and Samuelson, J. S.,J. Chromatogr., 26 (1967a) 158. Gupta, V . S., Ozols, J . G and Huennckens, F. M., Biochemistry, 6 (1967b) 2159. Guroff, G. and Rhoads, C. A., J. Biol. Chem., 244 ( 1 969) 142. Haeffner, E. W., J . Chromatogr., 50 (1970) 140. Haussler, M. R., Myrtle, J. F. and Norman, A. W., J. Biol. Chem., 243 (1968) 4055. Hirsch, J.,J. LipidRes., 4 (1963) 1. Hla-Pe, U.and Aung-Than-Batu, Clin. Chim. Acta, 24 (1969) 381. Ho, P. P. K. and Jones, L., Biochim. Biophys. Acta, 148 (1967) 662. Horn, B. L., Biochim. Biophys. Acta, 175 (1969) 20. Iwahara, S., McCorrnick, D. B., Wright, L. D.. and Lih, Ch., J. Biol. Chem., 244 (1969) 1393. Johansson, S. and Lindstedt, S., Biochemistry, 7 (1968) 2327. Jones, T. H. D. and Brown, G . M.,J. Biol. Chem., 242 (1967) 3989. Kahn, V. and Blurn, J. J.,J. Biol. Chem., 243 (1968) 1441. Kamikubo, T. and Narahara, H., Vitamins, 37 (1968) 225; C.A., 68 (1968) 9313131. Kaufman, S., J. Biol. Chem., 242 (1967) 3989. Klatzkin, Ch., Norris, F. W. and Wokcs, F.,J. Pharm. Pharmacol., 1 (1949) 915. Knobloch, E., Fyzikalni ChemickC Metody Stanoveni Vitaminb, Czechoslovak Academy of Sciences, Prague, 1956. Koikc, H., Wada, T. and Minakarni, H., J. Biochem., 62 (1967) 492. Kozidowa, A. and Koziol, J., J. Chrornatogr., 34 (1968) 216. Lammi, C. J. and Lerner, J . , J. Chromatogr., 43 (1969) 395. Lawson, D. E. M., Wilson, P. W. and Kodicek, E., Biochem. J . , 1 1 5 (1 969) 269. Lee, Y . C., Gholson, R. K. and Raica, N., J. Biol. Chem., 244 (1969) 3277. Lerner. J.,J.Chem. Educ., 47 (1970) 32. McDonald, J. W. D. and Stewart, H. B., Can. J. Biochem., 45 (1967) 363. McLaren, D. S., Read, W. W. C., Awdeh, Z. L. and Tchalian, M.. Methods Biochem Anal., 15 (1967) 1. Mariani, A. and Mariani-Vicari, C., Ann. Ist. Super. Sunita, 4 (1968) 90. Matsuo, T. and Suzuoki, Z., J. Biochem., 65 (1969) 953. Millar, K. R. and Caravaggi, C., N . Z . J. Sci., 13 (1970) 329. Mitsuda, H. and Suzuki, Y . , Biochem. Biophys. Res. Commun., 36 (1969) 1. Myrtle, J. F.. Haussler, M. R. and Norman, A. W., J. Biol. Chem., 245 (1970) 1190. Nagase, 0.. Tagawa, H. and Shimizu, M., Chem. Pharm. Bull., 16 (1968) 977. Neal, R . A,, J. Biol. Chem., 243 (1968) 4634. Neal, R. A,, J. Biol. Chem., 244 (1969) 5201. Nystrorn, E. and Sjovall, J.,J. Chromatogr., 24 (1966) 212. Olesen, H., Rehfeld, J., Horn, B. L. and Hippe, E., Biochim. Biophys. Acta, 194 (1969) 67. Perlzweig, W. A,, Levy, E. D. and Sarett, H. P., J. Biol. Chem., 136 (1940) 729. Peterson, P. A., J. EioI. Chem., 246 (1971a) 34. Peterson, P. A , , J. Biol. C!em., 246 (1971b) 44. Peterson, P. A. and Berggard, I., J. Biol. Chem., 246 (1971) 25. Platzer, E. C . and Roberts, L. S., Comp. Biochem Physiol., 35 (1970) 535. Rembold, It, Metzger, H., Sudershan, P. and Gutensohn, W., Biochim. Biophys. Acta, 184 (1969) 386. Roberts, A. B. and De Luca, H. F.,J. LipidRes., 9 (1968) 501. Rohringer, R., Kim, W. K. and Samborski, D. J., Can. J. Biochem., 47 (1969) 1161. Shichi, H., Lewis, M. S., Irreverre, F. and Stone, A. L., J. Biol. Chem., 244 (1969) 529. Shimizu, M., Nagase, O., Hosokawa, Y. and Tagawa, Y., Tetrahedron, 24 (1968) 5241. Shimizu, M., Nagase, O., Hosokawa, Y., Tagawa, H. and Yotsui, Y., Chem. Pharm. Bull., 18 (1970a) 838. Shimizu, M., Nagase, O., Okada, S. and Hosokawa, Y., Chem. Pharm. Bull., 18 (1970b) 3 13. Simons, K. and Weber, T., Biochim. Biophys. Acta, 117 (1966) 201.
978
VITAMINS
Skinner, W. A., Parkhurst, R. M., Scholler, J. and Schwarz, K., J. Med. Chem., 12 (1969) 64. Strohecker, R and Henning, H. M., Vitamin-Bestimmungen,Verlag Chemie, Weinheim, 1963. Suda, T., De Luca, H. T., Schnoes, H. K. and Blunt, J . W., Biochemisfry,8 (1969) 3515. Sugiura, K. and Goto, M., J. Biochem., 64 (1968) 657. Suzuki, T. and Tanimura, Y.,Chem. Pharm. Bull., 17 (1969) 1422. Suzuoki, N. Z., Matsuo, T. and Tominaga, F., J. Biochem., 63 (1968) 792. Takahashi, T. and Yamamoto, R, Yukugaku Zusshi. 89 (1969) 993; C A . , 71 (1969) 84510t. Takanashi, S., Matsunaga, J. and Tamura, Z., J. Vitamin., 16 (1970) 132. Tortolani, G., Bianchini, P. and Mantovani, V., Furmaco, Ed. Prut., 25 (1970a) 772. Tortolani, G., Bianchini, P. and Mantovani, V., J. Chromufogr.,53 (1970b) 577. Whiteley, J . M., Drais, J . H. and Huennekens, F. M., Arch. Biochem. Biophys., 133 (1969) 436. Williams, J. P.,J. Chromufogr.,36 (1968) 504. Wolff, R., Linden, G. and Nicolas, J. P., Bull. SOC.Chim. Biol., 51 (1969) 191. Yang, H. C., Kusumoto, M., Iwahara, S., Tochikura, T. and Ogata, K., Agr. Biol. Chem., 33 (1969) 1730. Yoshioka, M., Samejima, K. and Tamura, Z., Chem. Phurm. Bull., 17 (1969) 1265. Yurkevich, A. M., Rudakova, 1. P. and Pospelova, T. A., Zh. Obshch. Khim., 39 (1969) 425. Zahler, W. L. and Cleland, W. W., J. Biol. Chem., 243 (1968) 716. Zakrzewski, S. F., Evans, E. A. and Phillips, R. F., Anal. Biochem., 36 (1970) 197. Zakrzewski, S. F. and Sansone, A., J. Biol. Chem., 242 (1971) 5661. Zile, M. and De Luca, H. F., Anal. Biochem., 25 (1968) 307.
Chapter 45
Antibiotics V . BETINA
CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................................. Penicillins and cephalosporins PenicillinsandgAPA ......................................................... Cephalosporins and 7.ACA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... Carbohydrate antibiotics .............................. Streptomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... Neomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . bnamycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gentamicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ljncomycin group . . . . . . . . . . . . . . . . ........................................ Other aminoglycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotransformations of aminoglycosidic antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrocyclic antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrolides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other macrocyclic antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................... Tetracyclines and related antibiotics . . . . Tetracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anthracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleoside antibiotics including polyoxins . . . . . . . . . . . . . . . . . .................... peptides and related antibiotics ................................................... Analogues of amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Actinomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................................... References . . . . . . . . .
979 980 980 984 985 986 989 989 991 992 994 994 994 996 996 996 998 999 1000 1000 1000 1001 1003 1005
INTRODUCTION Antibiotics are microbial metabolites possessing antimicrobial. cytotoxic. antitumour. antinematodal or insecticidal properties. Within the three decades from 1937 t o 1966. discoveries of 1630 antibiotics were reported but 427 of them were rediscoveries (Neelameghan et al.,). Some tens of new antibiotics have been discovered since 1966. Antibiotics belong to very different groups of organic compounds but until now a classification from a chemical point of view has been virtually non.existent . The classification of antibiotics used in the “Bibliography of Column Chromatography”(Dey1 et al.) was accepted in writing the present chapter . Clssical and modern techniques of column chromatography are indispensable tools in the study and preparation of antibiotics. They are used mainly for the following purposes: References p .1005
979
980
ANTIBIOTICS
(i) laboratory and commercial isolation and purification of antibiotics; (ii) separation of mixtures of antibiotics; (iii) structural studies; (iv) studies of the biogenesis and preparation of labelled antibiotics; (v) studies of chemical modifications and biotransformations of antibiotics; and (vi) purity control. Samples of various qualities can be applied on to chromatographic columns. They include filtrates and extracts of fermentation broths, crude concentrates of antibiotics, partially purified substances and pharmaceutical preparations. Liquid-solid chromatography is widely used in separations of antibiotics belonging to different chemical groups. Applications of gel chromatography in the field of penicillins, tetracyclines and peptidic antibiotics are included in this chapter. Ion-exchange chromatography is of a great value for the isolation and separation of carbohydrate and peptidic antibiotics. It is also helpful in the separation of amino acids as building units of the latter group of compounds, which is important in structural studies. In addition to physical and chemical methods used in qualitative and quantitative determinations of compounds separated by means of CC, assays of antibiotics are also based on their biological activity. Aliquots of fractions can be tested for inhibitory effects on sensitive microorganisms, and the zones of inhibition and their diameters then indicate the presence and amounts of active components in eluates. As paper chromatography and thin-layer chromatography are usually combined with CC in order to check the purity of eluates, bioautography must be mentioned as a general method for the detection of antibiotics. The bioautography of antibiotics in PC and TLC is based on their inhibitory effects. Using sensitive microorganisms, the positions of antibiotics on paper or thin-layer chromatograms are rendered visible by the zones of inhibition. Technical details and references concerning the bioautography of antibiotics can be found elsewhere (Betina, 1972,1973). This chapter deals mainly with antibiotics which are or could be used pharmaceutically, in agriculture or in basic research. In some instances, chemically related antibiotics, semisynthetic and synthetic derivatives and analogues are also included.
PENICILLINS AND CEPHALOSPORINS Penicillins and 6-APA Penicillins are N-acyl derivatives of 6-aminopenicillanic acid (6-APA). Cephalosporin N is also a derivative of 6 - M A and is now named penicillin N. Its side-chain is identical with that of cephalosporin C, which is an N-acyl derivative of 7-aminocephalosporanic acid (7-ACA). Cephdosporin PI is a steroid compound. Natural, biosynthetic, semisynthetic and synthetic derivatives of 6 - M A and 7-ACA are often named as p-lactam antibiotics. Isolation of 6-APA at the end of the fifties made possible the preparation of large numbers of N-substituted derivatives, the “semisynthetic penicillins”. Since that time some 1800 different penicillins have been prepared and studied by a pharmaceutical
981
PENICILLINS AND CEPHALOSPORINS
company (Naylerj and the number examined throughout the world must amount to several thousands. In recent years, CC was used in three fields of studies of penicillins: a) in chemical transformations of penicillins and 6-APA; b) in studies of penicillin allergy in humans; c) in isolation of polymers formed during storage of aqueous solutions of penicillins and 6-APA. Attempts to convert penicillins into other p-lactam systems have been fruitful. Morin e l al. (1969a) described transformations of penicillin sulphoxide into cephalosporin compounds. A A3-cephem derivative was prepared from phenoxymethylpenicillin sulphoxide methyl ester and was purified by C C . The conversion was (schematically) as follows: 0
COOCH:,
Phenoxy methylpenicillin sulphoxide methyl ester
Methyl 3-methyl-7(2-phenoxyacctarnido)-3cephem-4-carbox ylate
Chromatography on columns of silica gel was used extensively in transformations of penicillins into new p-lactam systems by Heusler and by Fechtig ef al. A new intermediate, 7-aminocephalocillanic acid, was prepared by Scartazzini er al. and contains structural characteristics of both 6-APA and 7-ACA. Its N-acyl derivatives were named “cephalocillins”, and two of the cephalocillins prepared were purified on columns of silica gel using toluene-acetic acid ester (4: 1) as the eluent. High-speed LLC was used successfully for the analysis of the methyl benzyl esters of penicillins. A Zipax column was developed with an ethanol-5% n-hexane solution which served as mobile phase. The elution time of the methyl benzyl esters is governed by the percentage of n-hexane in the mobile phase. The separation of the methyl benzyl ester of penicillin G from its impurities was achieved within 4 min. As the penicillins vary widely in solubility and in the substitutions on the molecule, it is doubtful that any single chromatographic system will be applicable to all of the penicillin derivatives (Schmit j. 6-APA and benzylpenicillin were found to contain small but significant amounts of a high-molecular-weight protein that stimulated the formation of antibodies of penicilloyl specificity (Weston). Protein impurities were isolated by fractionation of the sodium salt of 6-APA on columns of Sephadex C-25 (Fig. 45.1 ). $-Lactam antibiotics form polymers during storage as aqueous solutions. 6-APA also forms a dimer, presumably through nucleophilic attack of the amino group of one molecule to the Elactam of another to form a penicillin. Dennen separated penicillins, 6-APA and a degradation mixture of 6-APA by gel chromatography (Fig. 45.2). References p . I005
982
ANTIBIOTICS
FRACTIONS
Fig. 45.1. Separation of protein impurities from the sodium salt of 6-aminopenicillanic acid (Weston). Sorbent: Sephadex (2-25. Columns equilibrated and eluted with 0.5% sodium chloride solution. Detection: eluates were scanned automatically at 254 nm with a n LKB Uvicord 01 manually at 280 nm with a Uvispek 700 spectrophotometer. A = impurities; 6-APA = 6-aminopenicillanic acid. TABLE 45.1 ION-EXCHANGE CHROMATOGRAPHY O F D-PENICILLAMINE AND RELATED COMPOUNDS (PURDIE el al.) Compound
Time (min)
LCysteine Glutathione (ox.) DL- + meso-Lanthionine D-Penicillamine LCysteine-gluta thione disulphide LCystine LCy steine-D-penicillamiiie disulphide D-Penicillamine disulphide DL- f alloCystathionine L-lsoleucine LCys teine trisulphide LCysteine-D-penicillamine trisulphide LCysteine-L-homocysteine disulphide L-Homocysteine-D-penicillamine disulphide D-Penicillamine trisulphide LCys teine-L-homocys teine trisulphide L-Homocysteine D-Penicillamine tetrasulphde Cysteamine-glutathione disulphide L-Homocysteine trisulphide LCysteinecys teamine disulphide
24 9 275 296, 319* 331 3 94 473 5 04 545 548,554* 566 595 616 618 640 655 675 723 735 137 7 96 976
*Twin peaks.
983
PEMCILLINS AND CEPHALOSPORINS
v)
-0
EE3 3
30-
B
i ;
20-
W -1
a
5
'\
I
v) v)
z
c
'
lo-
a r
0
1
10
'.
1
20
30
40
50
30
40
50
, 60
I
I
70
80
H a c
4T
A
I
0
10
6
20
ELUATE.
6(
ml
Fig. 45.2. Separation of penicillins, 6-APA and a degradation mixture of 6-APA by gel chromatography (Dennen). Column: 39 X 1.0 cm. Sorbent: Sephadex G-10. Detection: measurement of p-lactam content was performed by reacting intact p-lactam with neutral hydroxylamine t o give the hydroxamate, followed by colour development with iron(lI1) chloride. The absorbance a t 304 nm represented polymeric material. Above: 14.9 pmoles of penicillin and 11.SO pmolcs of 6-APA applied to column; broken line = phenoxymethylpenicillin or benzylpenicillin; solid line = 6-APA; recovery of total p-lactam = 88%. Below: the starting material was 6-APA, which was degraded and then applied on to the column; solid line = intact p-lactam of 6-APA and polymeric material; broken line = 304-nm absorbing material. Peaks A, B and C represent polymeric material, penicillin or 6-APA dimer, and 6-APA, respectively.
Smith and Marshall separated high-molecular-weight materials, formed in aqueous solutions of 6-APA, benzylpenicillin, ampicillin and hetacillin, by repeated chromatography through Sephadex C-25 fine with water and 0.5% sodium chloride solution as eluents. Of the degradation products of penicillin, D-penicillamine is of practical importance. D-penicdlaniine, its derivatives and sulphur-containingamino acids were separated, using a Technicon Model NC-1 amino acid analyzer with a column of Chromobeads A cationexchange resin (150 X 0.6 cm), by Purdie et al. The column was maintained at 60°C throughout the runs as this temperature was satisfactory for the compounds being studied. An Autograd gradient elution device was used with 75 ml of buffer solution in each chamber as follows: 1 4 , pH 2.875; 5 , p H 3.80; 6, pH 3.80 + pH 5.0 (1:2); 7-9, pH 5.0. A buffer flow-rate of 35 ml/h was used. The results obtained are presented in Table 45.1 . References p . I005
984
ANTIBIOTICS
Cephalosporins and 7-ACA Cephalosporin C has been used directly in medicine and also for the preparation of 7-ACA, the intermediate of “semi-synthetic” cephalosporins. Woodward e f al. achieved the total synthesis of cephalosporin C, using CC on silica gel for the separation of the last of 18 intermediates in the series of reactions leading to cephalosporin C. Nagarajan er al. reported the isolation of penicillin N and of three new P-lactam antibiotics of the cephalosporin C type from two species of Srrepfomyces. The antibiotics present in the broth filtrate were concentrated by carbon and anion-exchange CC. The final purification was achieved by CC on cellulose and silica gel to yield the purified antibiotics. The structures of the three new cephalosporins and that of cephalosporin C are as follows: NH2
0
COOH
COOH 0 Cephalosporin C
R=H
II
X = OCCH3
0 A 1 6 8 8 6A
R-H
II
X = OCNH2
0 AI 6886 B
R-OCH,
!I
X = OCNH2
0 A16884A
II
R=OCH3 X = OCCH3
Upon acid degradation, antibiotic A16886B and A1 6884A yielded, in addition to a-aminoadipic acid, approximately three times more glycine than did the antibiotic A16886A or cephalosporin C. Brannon e f al. determined the origin of glycine obtained upon acid hydrolysis of A1 6886B-14C-6and A1 6886B-’4C-8. The results of these, chromatographic analyses are presented in Fig. 45.3. Thus the higher yield of radioactive glycine obtained from the 6-14C labelling compared with that from the 8-14C labelling indicates that only the presence of the methoxy group at C7 must be responsible for the significant increase of glycine and, therefore, for a mechanism of hydrolysis different from that with cephalosporin C. 7-ACA is prepared commercially by reaction of cephalosporin C with nitrosyl chloride in formic acid as solvent. Morin er al. (1969a) studied the reaction conditions in order to obtain higher yields of 7-ACA. Using CC, they found that the reaction mixture also contained other biologically active substances, which were purified and characterized. Desacetylcephaloglycine is known to be a metabolic product present in the blood and urine after treatment with cephaloglycin. In an acidic medium, cephaloglycin (I) is also converted into desacetylcephaloglycin (11, having antibiotic activity) and desacetylcephalo-
985
CARBOHYDRATE ANTIBIOTICS
n
150i
1
I\
&AAA
n
G LY CINE
I\
cpm 150.
100-
50 -
FRACTIONS
Fig. 45.3. Ninhydrin response and radioactivity profiles of column elution fractions of the acid hydrolyzates of p-lactarn antibiotics [6-"C] A168868 and [8-"C] A16886B (Brannon et el.).Column: 52 X 0.9 cni. Ion exchanger: Durrum DC-1A resin. Buffers: ( 1 ) 2%thiodiglycol, 0.2 N Na', pH 2.2 for application; (2) 0.5% thiodiglycol, 0.2 N Na', pH 3.23. Operating conditions: Beckman-Spinco Model 120 C amino acid analyzer equipped with a BioCal Model BC501 automatic sample injector was used; flowrate, 70 ml/h; temperature, 56.5"C. Detection: A ninhydrin flow of 35 ml/h was used when a ninhydrin response curve was desired; for radiogctive scintillation counting, 1-min fractions were collected directly from the column. a-AAA = a-aminoadipic acid; thick line = radioactivity; thin line = ninhydrin response curve.
sporin lactone (111, considerably less active). Kukolja separated the reaction mixture using a cellulose column, collecting the fractions with an automatic fraction collector and following the progress of the separation by TLC (detection with W light and a ninhydrin spray). Fractions containing I and trace amounts of 111 were followed by fractions containing the required 11.
CARBOHYDRATE ANTIBIOTICS This section includes antibiotics known as arninoglycosidic or aminocyclitol antibiotics.
Streptomycins Pharmaceutical preparations of streptomycin may contain salts of alkali metals as impurities. The behaviour of mixtures consisting of streptomycin sulphate and alkali References p. I005
986
ANTIBIOTICS
metal salts in gel filtration on Sephadex G-10 was investigated by Storl. Elution with deionized water resulted in a virtually salt-free product and the yield was about 95%. Streptomycin is eluted in two fractions with deionized water. The normal molecular sieve effect was obtained using buffer solutions (0.2 M ammonium acetate solution, pH 6.5) for elution. Streptomycin is inactivated (by adenylation or phosphorylation) by resistant bacteria, and experiments of Umezawa etal. ( 1 9 6 8 ~ )illustrated the use of IEC in similar studies. A sample of streptomycin sulphate was inactivated by a cell-free system obtained from Escherichia coli carrying R factor. The reaction mixture was then passed through a column of Amberlite IRC-50 (Na’) and the inactivated streptomycin was eluted with 0.5 N hydrochloric acid. The fraction that gave a positive Sakaguchi reaction and showed no antibacterial activity was collected and passed through a column of active carbon and the inactivated streptomycin was eluted with 0.2 N hydrochloric acid-methanol (1 :1). The fraction that gave a positive Sakaguchi reaction and negative biological activity was neutralized with Dowex 44 (OH-), and, after concentration under vacuum, the inactivated streptomycin was precipitated by the addition of 14 volumes of acetone and dried in vacuo. Part of the inactivated streptomycin in the reaction mixture passed through the column of IRC-50 resin, but it was adsorbed by another column of the same resin and purified by the same procedure as above. In total, 250 mg of inactivated streptomycin were obtained from the original sample (500 mg). It was characterized by PC and other analyses as adenylylstreptomycin. De Fabrizio developed a single and sensitive method for the determination of dihydrostreptomycin in complex pharmaceutical preparations (Sulpec and sulphaguanidine cum dihydrostreptomycin). The method entails the recovery of dihydrostreptomycin by cation-exchange CC with subsequent fluorimetric determination.
Neomycins The term neomycin usually refers to the amino sugar antibiotic complex, composed of two stereoisomers, neomycin B and neomycin C, and their degradation product, neamine (neomycin A). Commercial preparations contain neomycin B as the major constituent. “Extra” neomycins appear in varying amounts (always less than 1%) as constituents of many commercial neomycin samples. Three “extra” neomycins, neomycins D, E and F, were identified by Hessler e f al. as paromamine, paromomycin I and paromomycin 11, respectively. A commercial sample of neomycin sulphate was neutralized and subjected to IEC on Amberlite CG-50 (NH;) resin. Gradient elution with ammonia solution yielded two fractions, the first of which contained neamine, neomycin D and neomycin F, and the second neamine and neomycins D, F, B and E, in order of elution. The two fractions were then further separated by ion-exclusion chromatography on Dowex 1-X2 (OH-). Elution with water gave the individual components, which were identified as mentioned above. Majumdar and Majumdar (1970) isolated and characterized three phosphoamidoneomycins, which are converted into neomycin by Sfrepfomycesfradiae in the later stages of the fermentation. The compounds were isolated and separated using Amberlite IRC-50 (NG)and were characterized as neomycin B pyrophosphate, neomycin C
987
CARBOHYDRATE ANTIBIOTICS
01
15
I
I
I
I
25
35
45
55
1 -
65
75
85
E L U A T E , ml
Fig. 45.4. Separation of phosphorylated neomycin intermediates (Majumdar and Majumdar, 1970). Column: 6 5 X 1.4 cm. Ion exchanger: Amberlite IRC-50 “ti:), 200-400 mesh, equilibrated with 1500 ml of 18 mMamrnonia solution. Buffer: 90 mM ammonia solution. Opcrating conditions: elution volumes are apparent from the figure; 1.6-ml fractions were collected; flow-rate, 0.15 ml/min. Detection: the ammonium molybdate reagent of Hams and Isherwood was used and absorbances a t 660 mi were measured. A = Neomycin C dipyrophosphate complex; B = neomycin C pyrophosphate; C = neomycin B pyrophosphate.
pyrophosphate and neomycin C dipyrophosphate complex. Fig. 45.4 shows a typical pattern of their separation. A method for the quantitative analysis of the neomycin components in fermentation broths was devised by Majumdar and Majumdar (1967), and involves a combination of IEC with PC as described below. Reagents R-1 to R-5 were as follows: R-I: commercial carbon tetrachloride is washed with concentrated sodium hydroxide solution, layer-separated, treated with fused calcium chloride and distilled. Methanolacetic anhydride-carbon tetrachloride reagent (3:2:95) is prepared just before use. R-2: chlorine-carbon tetrachloride reagent. Chlorine is prepared from 100 ml of concentrated hydrochloric acid and 50 g of potassium permanganate, then passed through water and concentrated sulphuric acid and absorbed in 1 1 of carbon tetrachloride. About 5 g of barium carbonate and 5 g of fused calcium chloride are added and stored in a glassstoppered amber-coloured bottle. R-3: starch-iodine-pyridine reagent. Starch (1 g) and 0.25 g of potassium iodide are dissolved by heating in 3.5 ml of water and then 0.5 ml of this solution is added quickly to 50 ml of pyridine. Freshly prepared reagent is used. R-4: starch-iodirie-barium carbonate reagent. Starch (1 g) and 0.25 g of potassium iodide are dissolved by heating in 100 ml of water, then 2 g of barium carbonate are added to the cold solution and thoroughly shaken. After the solid particles have settled, the upper turbid portion is used for spraying the chromatograms. This reagent can be used for 5 to 6 days. R-5:starch-iodine-hydrochloric acid reagent. This reagent is prepared freshly by boiling 1 g of starch, 0.25 g of potassium iodide and 1 ml of 5 N hydrochloric acid in 100 ml of water. References p . 1005
988
ANTIBIOTICS
The resin is prepared as follows. Amberlite IRC-50, 200-400 mesh, is converted into the NH4 form by shaking with 1 N ammonia solution. The resin is suspended in water and allowed to settle and the upper turbid portion is decanted; this process is repeated until all finer particles are removed. The resin is then suspended in an equal volume of water, and 2 ml of the suspension are transferred to a funnel with its stem (I.D. 8 mm; column volume 1 ml) plugged with glass-wool and then washed with water until the washings are neutral. Such a column can be used several times. After each use, it is washed with 1 N hydrochloric acid and then converted into the ammonium form. N-Acetylneomycins are prepared in the following manner. The fermentation broth or the solution of the neomycin sample (containing about 4 mg of neomycins) is passed through an Amberlite IRC-50 (NH;) column, which is washed with 5 ml of water and eluted with 5 ml of 1 N ammonia solution. This eluate is spotted at points 20 mm apart along the baseline (7.5 cm from the end) of Whatman No. 4 paper (43 X 16 cm) in a volume sufficient to contain 4-12 pg of neomycin. Standard solutions of neomycin B and C and neamine-free bases are applied in the same concentration range. For N-acetylation, the papers are rolled and soaked in R-1 for at least 12 h, then air-dried for 1 h in order to remove acetic anhydride. The papers are developed for 24-36 h at 28°C with n-butanol-water-piperidine (84: 16:2) by the descending technique. The chromatograms are air-dried and placed for 1-2 h in a chamber with a water-saturated atmosphere at 37°C for humification and complete removal of piperidine from the papers by the same subsequent step. The papers are next rolled inside the chamber and quickly transferred to a cylinder and the filtered R-2 is poured in. The apparatus is kept in the dark for 20 min at room temperature, and the chromatograms are air-dried for 0.5 h at 4-6OC in the dark in order to remove excess of chlorine. The papers are divided into a number of parallel strips each 20 mm wide. One of a pair of strips is sprayed with R-3 and bluish pink spots are obtained by transferring it to a water-saturated atmosphere. For rapid work, detection with R 4 and marking of the wet spots with a ball-point pen is preferred. Rectangular areas corresponding to N-acetylneomycin B and C and N-acetylneamine are removed from the untreated paper strip with the help of the guide chromatogram and then cut in small pieces. Rectangles are cut from spot-free areas as a blank. The spots are then extracted for 30 min with 4 ml of water and 1 ml of R-5 in clean test-tubes and the absorbances of the coloured solutions are measured at 570 nm. The amount of neomycins is determined by reference to standard curves; all three neomycins can be determined by reference to a single standard curve. Theoretically, 2.56 p g of neomycin B or C are equivalent to 2.01 pg of neamine. The positions of the neomycins on the chromatograms relative t o N-acetylneamine are about 0.69-0.70 (N-acetylneomycin B), 0.35 (N-acetylneomycin C) and 1.O (N-acetylneamine). As little as 1 pg of neomycin base can be determined with an accuracy of + 0.7% and a 60-pg sample can be analyzed if the amount of the minor component present is very low. PBnasse eC al. prepared several mono-N-alcoyl derivatives of neomycin B and paromomycin. Sulphates of the derivatives were successfully purified by IEC on Amberlite IRC-50 (H') with 0.066-1 .ON ammonia solution as the buffer.
CARBOHYDRATE ANTIBIOTICS
989
Kanamycins Kanamycins A, B and C contain 3-amino-3-deoxy-D-glucose (3AG) in their molecules. 3AG exhibits antibacterial activity and was also isolated from Bucillus uminoglucosidicus by Umezawa e f ul. (1967a, b). The isolation procedure included LEC. Total syntheses of kanamycins were reported by Umezawa et al. (1968b, d, 1969a, b, c). Synthetic hepta-O-acetyltetra-N(2,4-dinitrophenyl)derivatives of the three kanamycins were purified on column of silica gel, deacetylated and the dinitrophenyl groups were removed by TEC using Dowex 1-X2 (OH-).
Gentamicins Wagman et al. described the preparative separation of gentamicins C, , C,, and C2 by use of the following cellulose and Chromosorb W chromatographic column procedures. I17 the cellulose column chromatography, Whatman No. 1 cellulose powder is packed in small segments in a column (I.D. 2.4 cm) to a height of 30 cm. The upper phase of the solvent system, consisting of chloroform-methanol-17% ammonia (2: 1: l ) , is run through the column until a yellow band of impurities emerges, and the column is allowed to drain. A 200-mg amount of gentamicin sulphate is mixed with 2 g of cellulose powder, packed on top of the cellulose in the column and wetted with a small amount of the upper phase. The lower phase is allowed to run through the column at the rate of 2 ml/min; 16-ml fractions are collected every 8 min. Aliquots of each fraction are spotted on filter-paper and tested with ninhydrin reagent in order to determine the presence or absence of antibiotic. Like fractions, converted into their free bases on the column, are pooled and evaporated to dryness. In the Chromosorb W column chromatography, 60-100 mesh Chromosorb W (JohnsManville, Denver, Colo., U.S.A.) is slurried with the upper phase of chloroform-methanol17% ammonia (2: 1 :1) and filtered using suction on a buchner funnel until excess of solvent is removed. The Chromosorb is packed into a column (I.D. 3 cm) to a height of 50 cm in 5 c m segments (cu. 150 g as dry Chromosorb). An alternative method is to pour the slurry into the column and to remove excess of solvent by suction from the bottom of the column. One litre of lower phase is run through the column in order to wash the Chromosorb. A 3-g portion of gentamicin base is dissolved in 10 ml of methanol, adsorbed on to the smallest possible amount of Chromosorb and dried under vacuum using a film evaporator. This mixture is packed on top of the column and wetted with a small amount of the lower phase. The column is eluted with the lower phase at the rate of 1 ml/min, collecting five fractions per hour. The fractions are tested as described for cellulose column chromatography. The component peaks are located, pooled, decolourized on IRA-401s (OH-) resin and evaporated to dryness. The results of the separation using a cellulose column are presented in Table 45.2. PC of the bases and bioautography against Staphylococcus aureus showed that the C I and Cz components were free from impurities and that C,, contained approximately 5% of Cz . Re-chromatography of the C1, fraction using the described column resulted in the isolation of this component free of C2. With Chromosorb W, the separation was as shown References p . 1005
990
ANTIBIOTICS
TABLE 4 5 . 2 CHROMATOGRAPHIC SEPARATION OF THE GENTAMICIN COMPLEX USING A CELLULOSE COLUMN (WAGMAN et a l . ) Starting material: 200 mg of the gentamicin complex sulphate ( 1 29 mg base equivalent). Total yield = 121.5 mg (94%). Component
Fraction Nos.
Weight (mg)
c,
12-19 23-33 38-49
58.2 50.8 12.5
c2
c,a
TABLE 45.3 CHROMATOGRAPHIC SEPARATION OF THE GENTAMICIN COMPLEX USING A CHROMOSORB W COLUMN (WAGMAN et al.) Starting material: 3 g of gentamicin complex base. Component
Fraction Nos.
Weight of purified fractions (g)
Antibiotic activity* (Pg/mg)
C,
35 -60 95-140 165-231
1.01 0.74 0.3 1
833 1050 1050
c*
C,,
*Average of four assays against complex standard.
in Table 45.3. PC of the bases and bioautography indicated that all components were free from microbiologically active impurities. IEC was used for the preparative separation of the entire gentamicin complex by Maehr and Schaffner. The strongly basic resin Dowex 1-X2, 100-200 mesh, was used without further purification or sizing. The resin was converted into the hydroxide form with 10 resin-bed volumes of 8%carbonate-free sodium hydroxide solution. All washings and elutions were made with distilled, carbon dioxide-free water. Typically, 35 g of gentamicin complex were dissolved in 30 ml of water and washed into the resin bed, the column flow-rate not exceeding 0.7 cm/min; manual fraction collection, according to the continuously recorded conductivity of the effluent (using an Industrial Instruments Model RC-16B conductivity bridge, operated at a frequency of 1000 Hz, and a Leeds & Northrup Sidomax H instrument recording the 0-10 mV range). Of the 10 fractions collected, the first appeared as a sharp peak in the conductivity diagram, exhibiting a high ash content with only traces of biological activity. All of the following fractions displayed high activity against Staphylococcus aureus. Fraction aliquots were analyzed by TLC on nonactivated layers of Brinkmann (Westbury, N.Y., U.S.A.) silica gel G, 0.75 mm thickness, developed with the solvent system chloroform-methanol28%ammonia (2: 1 : 1). In parallel experiments, the spots were located by bioautography and with ninhydrin, the correlation affording qualitative agreement between ninhydrin
99 1
CARBOHYDRATE ANTIBIOTICS
TABLE 45.4 SUMMARY OF CC AND TLC CHARACTERISTICS OF GENTAMlCIN ANTIBIOTICS (ADAPTED FROM MAEHR AND SCHAFFNER) Figures are TLC RF X 100 values. ~
~~
Column effluent volume (ml) B**
A*
19502670 42 54 58
26703155 18 24
31553485 9 17 26 64
34854175 13 39
41754770 22
47705110
51105550
-
26 45
55509340 18
934010120 59
*A = Mixture of the three major antibiotics, gentamicins C,, C, and D. **B contained gentamicin A as the major constituent.
colour and zones of inhibition. Sixteen different active components of the entire gentamicin complex were found (Table 45.4).
Lincomycin group
IEC was used by Thomas et al. for the purification of tritiated lincomycin, and it seems that this procedure is applicable to the purification of all lincomycins. Clindamycin is 7-chloro-7-deoxylincomycin prepared by chemical modification of the sugar moiety of lincomycin. Argoudelis et al. (1969) described the microbial transformation of clindamycin into Ndemethylclindamycin. A mixture of the two substances was separated by CC on silica gel (Merck, Catalogue No. 7734). A column was prepared from 600 g of the sorbent in chloroform-methanol (6:1), and 6 g of the crude mixture were chromatographed using the same eluent. The fractions obtained were analyzed by TLC; clindamycin was eluted first, followed by N-demethylclindamycin. During studies on the possibility of Streptomyces lincolnensis methylating N-demethylclindamycin to give clindamycin, it was found that N-demethylclindamycin had been transformed into a new bioactive material, compound A. When CM-Sephadex was used, Ndemethylclindamycin and lincomycin (the latter being produced by S. lincolnensis) were adsorbed, while compound A passed through the column. It was found to be N-demethyl-N-hydroxymethylclindamycin (Argoudelis et al. , 1972). Recently, urinary excretion products of clindamycin in rats and dogs were isolated and purified by means of LCC, TLC and countercurrent distribution (Sun). Celesticetins are antibiotics produced by S. caelestis. Four members of this group, desalicetin and celesticetins B, C and D, were separated by counter-current distribution and/or silica gel CC (Argoudelis and Brodasky). References p.1005
992
ANTIBIOTICS
Other aminoglycosides Aminoglycosidic antibiotics were separated by high-speed LLC on a Carbowax 750 column with nhexane-isopropanol as the mobile phase. The hydroxy groups of the antibiotics interact with the Carbowax to retain the sample. The antibiotics are sufficiently soluble in isopropanol that a small amount in the mobile phase will elute the compounds satisfactorily. Retention times are governed hy the concentration of isopropanol in the mobile phase (Schmit). Fukagawa et at. (1968a, b) devised procedures, including IEC, for the isolation and purification of kasugamycin and [“C] kasugamycin from fermentation broths. Various Dowex ion exchangers were used for the purification of semi-synthetic derivatives of kasugamycin by Cron et al. On the basis of PC and bioautography, nebramycin was differentiated from the related antibiotics neomycin, paromomycin, kanamycin and gentamicin. Thompson and Presti separated the nebramycin complex as follows. A preparation of nebramycin complex was dissolved in deionized water and, after adjusting the pH of the solution to 4.5 with sulphuric acid, partial decolourization was achieved bv treatment for 1 h with Darco G-60 carbon. The carbon was removed by filtration and the filtrate was charged on 7 1 of Amberlite IRC-50 (NH;) resin in a 105 X 9.2 cm column. After the column had been thoroughly washed with deionized water, the resin was eluted with 0.1 N ammonia solution at a flow-rate of 20 ml/min. The eluate was collected in portions of 900 ml. Following the elution of components 1-5, the concentration of the eluent was increased to 0.3 N in order to elute component 6 more efficiently. The eluates were pooled on the basis of bioautographic purity and potency and the pooled eluates were concentrated and dried in uacuo to yield the amorphous free bases of the respective nebramycin components. Four components of the complex nebramycins 2 , 4 , 5 and 6 were characterized satisfactorily. In studies of the structure of nebramycin factor 6 (now called tobramycin), Koch and Rhoades isolated the amino sugar nebramine by CC on Bio-Rad 1-X4 (OH-) with water as the eluent. Lividomycins have been investigated by Mori et al. Two of them, containing 2-amino2,3-dideoxy-D-glucose, were named lividomycins A and B. One further compound, a new member of the paromomycin group, was provisionally designated as antibiotic No. 2230-C, while the fourth compound, No. 2230-D, was identified as paromomycin I. Two methods were used for their separation. . (a) Crude lividomycins, ca. 25 mg, are dissolved in 5 ml of water and the solution is adsorbed on a 4 0 X 1 cm CM-Sephadex C-25 (NH;) column. After the column has been thoroughly washed with deionized water, active portions are obtained by gradient elution between 200 ml of 0.1 2 N a n d 25 ml of 0.35 N ammonia solution at a flow-rate of 25 ml/h at 27°C. All fractions, each containing 3 ml, are assayed by the paper disc method against Bacillus subtilis ATCC 6633. The active fractions are lyophilized and weighed. Four peaks of the active fractions are designated as No. 2230-C, lividomycin A, No. 2230-D and lividomycin B, respectively. ( b ) A solution of about 6 g of crude powder in 600 ml of deionized water is adsorbed on an Amberlite CG-50 Type I (NH;) column (30 X 3 cm). After the column has been thoroughly washed with deionized water and 0.08 N ammonia solution, the compounds
993
CARBOHYDRATE ANTIBIOTICS
No. 2230-C, lividomycin A, No. 2230-D and lividomycin B are eluted stepwise from the column with 0.1 N , 0.12 N , 0.1 5 N a n d 0.17 N ammonia solution, respectively. The eluates are pooled on the basis of biological activity as in (a) and concentrated under reduced pressure. Further purification of the antibiotics is carried out by CC on a column of Dowex 1-X2 (OH-), 2 0 0 4 0 0 mesh, using deionized water for development. After detection, each active fraction is collected, concentrated at below 40°C and finally lyophilized. The free bases are obtained as amorphous powders. A new antibiotic complex, butirosin, was isolated from a strain of Bacillus circuluns by Dion et al. The complex was separated into butirosins A and B by means of Dowex 1-XI or 1-X2 ( O H ) resin. The 3-1 amount of resin (Cl-, 50-100 mesh) in a 50 X 2 cni column was converted into the hydroxide form with 16 1 of 2 N sodium hydroxide solution, washed with ca. 9.5 1 of water, treated with 17.5 1 of 5% boric acid, and washed with several hold-ups of water. Butirosin (6.01 g) in 15 ml of water was added to the prepared resin column. After percolation of the sample, the column was washed with 6.42 1 of water at a flow-rate of ca. 420 ml/h. The column was then developed with 4 1 of 1% boric acid, 4 1 of 2% boric acid, and finally with several hold-ups of 5% boric acid. Portions of the eluate fractions were lyophilized, and the residues were analyzed as the N-acetyl derivatives for butirosin A and B content by PC. Most of the butirosin A (ca. 4.5 g), free from B, was found in fractions in the initial effluent volume of 1.5-4.95 1. Most of the butirosin B (0.6 g) was found in the effluent volume of 2.64-4 1 after application of 5% boric acid to the column. The butirosin A and B, present in the effluent volumes from the Dowex 1 columns, were purified by means of Amberlite IRC-50 (NH;), XE-243 (free base form) and sometimes Dowex 1-XI 6 resin columns. TABLE 45.5 APPLICATIONS OF LIQUID COLUMN CHROMATOGRAPHY IN SEPARATIONS OF AMINOGLYCOSIDIC ANTIBIOTICS Compounds
Sorbent or ion exchanger
Eluent
Reference
Benzene-diethyl etheracetone (1 : I :1) Isopropanol-2 N ammonia (5: 1 ) Not given n-Propanol-pyridineacetic acid-water (10: 15:3: 10)
Kunze et al.
Liquid-solidchromatography
Moneomycin D
Oxalic acidSilica gel Silica gel
Ribostamycin Yazumycin
Silica gel Cellulose
a-lipom yoin
Ion-exchange chroma tography Bio-Rad AG 1-X2 Hygromycin B M o neom y ci ns Dowex 1 (CI-) Nojir im y cin SF-701 Y azumycin References p . 100.5
Dowex 1-X2 (OH-) Amberlite IRC-50 (Na') Amberlite IRC-50(Hi)
Water 0.6% KCl in methanolwater ( 4 : l ) Water 0.5 N HCl 0.3 N HCl
Schacht and Huber Ito et al. Akasaki and Abe
Neuss et al. Schacht and Huber Inouye et al. Tsuruoka et al. Akasaki and Abe
994
ANTIBIOTICS
Khokhlov and Reshetov developed an effective general procedure for the separation of streptothricin mixtures based on IEC on CM- cellulose with a sodium chloride gradient. By means of this method, they demonstrated that all of the available preparations of antibiotics of this type were mixtures of six streptothricins differing in the number of LQ-lysine residues. Taniyama et al. (1971a, b) modified the method of IEC on CMcellulose by replacing sodium chloride with volatile pyridine-acetic acid buffer, and also developed gel chroma tographic techniques on Sephadex LH-20. Using these procedures they separated individual racemomycins A, C, B and D, and demonstrated their identity as streptothricins F, E, D and C, respectively. They also showed by the same methods the identity of the antibiotics racemomycins A and C as yazumycins A and C (Taniyama et . above techniques, in combination with TLC on cellulose, were used by al., 1 9 7 1 ~ )The Khokhlov and Shutova in order to confirm the chemical structure of streptothricins. Applications of CC in the isolation, purification and separation of some other carbohydrate antibiotics are given in Table 45.5.
Biotransformations of aminoglycosidic antibiotics Strains of Escherichia coli that are resistant to various aminoglycosidic antibiotics are known to carry R factors with genetic information to produce enzymes that transform these antibiotics. Three kinds of transformations are known: adenylation, acetylation and phosphorylation. In studies on transformation by E. coli carrying R factors, IEC is widely used in order to isolate and to purify the transformed compounds from reaction mixtures (Benveniste and Davies; Umezawa et al., 1968a, c). In addition to IEC, cellulose phosphate paper binding assays were developed and used to monitor the phosphorylation (Ozanne et al.), adenylation (Benveniste et al.) and acetylation (Benveniste and Davies). These assays can be combined with IEC analysis.
MACROCYCLIC ANTIBIOTICS Macrolides Banaszek et al. described the separation of erythromycins on a preparative scale as follows. Silica gel, < 0.08 mm (Merck), 1 part by weight, is treated with 0.5 part of formamide in 1.5 parts of acetone. Acetone is removed in a rotatory evaporator, and the gel is suspended in benzene and introduced into a column. The substance to be separated, preferably in benzene solution, is placed on the column in the proportion of 1 :100 with reference to the gel. The eluents are (I) n-hexdne or benzene-chloroform or methylene chloride-ethanol (30&40:50-60: 5); and (11) n-hexane-chloroform or methylene chloride-ethyl acetate-ethanol (35:30:30:5). The chloroform used must be free from phosgene and hydrogen chloride. For detection, Bacillus subtilis ATCC 6633 can be used for testing the biological activity using a paper disc soaked with aliquots of the fractions. When the column is packed with similarly impregnated aluminium oxide, the eluent used is n-hexane-chloroform or methylene chloride-ethanol (70:20:5). The small amounts of
995
MACROCYCLIC ANTIBIOTICS
0
19
49
95
ELUATE, ml
215
Fig. 45.5. Separation of pristinamycins IA,.lB, ]Iq, Ilg (Preud’homme et 01.). Column: 15 X 1 .O cm. Adsorbent: Celite 545 (Johns-ManviUe) mixed w t h the bottom layer of the eluent. Eluent: Cyclohexane-dioxane-distilled water (3:4:3), upper layer. Operating conditions: elution volumes are apparent from the figure; the flowrate of 1 ml/min was controlled by nitrogen pressure. Detection: Spectrophotometric at 260 nm. A , = Pristinamycin IA; A, = pristinamycin IB; B, = pristinamycin IIA; B, = pristinamycin IIB. To separate a 5-mg sample, 5 g of Celite were wetted with 2.3 ml of the stationary phase. Elution was carried out with a total volume of 250 ml of eluent.
formamide in the fractions from the column are removed by washing with water. Pristinamycin was fractionated into five components belonging to two different chemical groups. The three components of group I are amphoteric cyclopeptides and the two components of group I1 are neutral macrolides (Preud’homme et al.). Except for pristinamycin I,, the other pristinamycins were separated by means of CC on Celite (Fig. 45.5). Megalomicin complex was partially purified on a column of Sephadex LH-20 with aqueous ethanol as the eluent. TLC on silica gel C with the solvent system chloroformmethanol (3:2) indicated that the complex is composed of four major components, A, B, C, and C 2 . Separation of C, and C2 from the complex was accomplished by CC on silica gel developed with the above solvent system (TLC). Components A and B, eluted together, were resolved by CC on Florisil. Elution was carried out with ethyl acetate followed by increasing amounts of acetone in ethyl acetate. Megalomicin was eluted first, followed by component A (Weinstein et aZ.). Sixteen-membered macrolides, 18-dihydroleucomycinA3, 9-dehydro-18-dihydroleucomycin A3 and tetrahydrotylosin were separated on columns of silica gel using benzeneacetone (5:l; 6-3:l for tetrahydrotylosin) as the eluents (Omura ef ul., 1972). The same adsorbent and eluent (1O:l) were used for acetyl-leucomycins (Omura et al., 1968). TLC and CC showed that the antifungal antibiotic fiiipin contains a minimum of eight filipin-like pentaenes. Three of the components, which seemed to constitute 96% of the complex, were crystallized after partition CC on siliceous earth and adsorption CC on silica gel (Bergy and Eble). References p . I005
996
ANTIBIOTICS
[“C] Brefeldin A, which is identical with cyanein (Betina eta/.), was purified on a column of silica gel with chloroform-acetic acid esters (1 :1) as the eluents (Handschin et a[.). The fungal macrolides phomin and 5-dehydrophomin were separated on columns of alumina (activity 11) with methylene chloride-methanol (998:2) as the eluent, or on a silica gel column with the above solvents (98:2 and 98: 1) as the eluents (Rothweiler and Tamm).
Other macrocyclic antibiotics Rlfamycin SV was isolated from a mutant of Streptomyces mediterranei strain and was purified by CC on silica gel by Lancini and Hengeller. Rifampicin undergoes deacetylation on alkaline treatment, affording the corresponding deacetyl derivative without substantial loss of antibacterial activity. Under milder alkaline conditions, rifampicin yields two additional products in which the acetyl group migrates to two different positions in the molecule. The reaction products, after extracting the rifamycins from the reaction mixture with chloroform, and the remaining rifampicin were recovered by CC on a pH 6.0 (McIlvaine) buffered silica gel by stepwise elution with chloroform containing 1 4 % of mzthanol (Maggi et al.). LLC was used for the separation of some impurities in a 3-formylrifampin sample. The optimum partition ratio range was obtained conveniently by adjusting the carrier polarity. It was found that the optimum carrier for this separation with a Zipax polyamide column was n-hexane-ethanol(3 : 1). This combination permits the low-level detection of the desired impurities (a quinone, rifampin and X32) and at the same time provides a good measurement of the very polar, relatively high-molecular-weight major constituent, 3-formylrifampin (Kirkland). Kinoshita and Umezawa reported the total synthesis of dehexyldeisovaleryloxyantimycin A,, which established a possible general synthetic pathway to the members of antimycin A. The final product was purified by CC on silica gel with n-hexane-ethyl acetate (4:3) as the eluent.
TETRACYCLINES AND RELATED ANTIBIOTICS Tetracyclines Griffiths studied the adsorption characteristics of tetracycline (TC), 4-epi-anhydrotetracycline (EATC) and anhydrotetracycline (ATC) on Sephadex gel under various conditions of pM and salt concentration. The adsorption of the compounds was strong in acidic solvents but diminished as the pH was increased. In the alkaline region of pH 8.59.5, the ATC and EATC epimer were eluted at different rates from the column. An increase in the salt concentration resulted in stronger adsorption of the compounds to the gel, but did not influence their separation efficiency. In Fig. 45.6 are shown the elution diagrams of mixtures of TC, ATC and EATC at various pH values. At pH 2.5, ATC and EATC displayed equal and maximal retardation on
997
TETRACYCLINES AND RELATED ANTIBIOTICS
0.6
0.2 11.0 1.0
0.6 0.2 11.0 1.o
0.6 0.2
0
10
20
30
0
10
20
30
40
T U B E NUMBER
Fig. 45.6. Gel chromatography of mixtures of tetracycline, anhydrotetracycline and 4-epi-anhydrotetracycline (Griffiths). Column: 25 X 1.8 cm. Sorbent: Sephadex G-25 F (particle size 2 0 - 8 0 pm). Buffers: ( I ) dilute hydrochloric acid, pH 2.5; ( 2 ) 0.04 Mphosphate buffer, pH 7.7; ( 3 ) 0 . 0 5 Tris buffer, pH 8 . 5 , 9.0 and 9 . 5 . Collection of samples was carried out with an LKB Radi Rac fraction collector and 4 . 4 d aliquots were collected. Detection: the absorbance at 2 7 3 nm was measured. The columns were equilibrited with the above buffers, and the column with the Tris buffer was re-equilibrated with several solvents containing Tris buffer at various pH values. T = Tetracycline; A = anhydrotetracycline; E = 4-epi-anhydrotetracycline.
the column but did not show evidence of separation from each other. At pH 9.0 and 9.5, TC and the two derivatives produced constant elution patterns and optimal separation between ATC and EATC. Fig. 45.7 depicts the elution diagram for the TC and the derivatives eluted separately on Sephadex columns equilibrated with phosphate buffer of pH 7.7 and Tris buffer of pH 9.0. It can be seen that the elution positions of the separate compounds agree closely with the admixture elution in Fig. 45.6. The sensitivity of the separation of the components is evident, since trace amounts of ATC impurities could be detected in the TC and EATC products. With a low salt molarity and a constant pH of 9.0, ATC and EATC showed weak adsorption to the column. As the salt concentration was increased, the ATC and EATC exhibited stronger adsorption, as evidenced by their delayed elution. Evidence of broadening of the TC peak in Tris-sodium chloride solvent was observed (Fig. 4 5 . 8 ~ ) . The findings on the separation of the ATC and EATC epimer on Sephadex are of both theoretical and practical importance because they reveal a refined chromatographic mechanism operating under optimal solvent conditions. The results are of immediate practical interest because the toxic nature of EATC requires its analytical determination in pharmaceutical preparations for human use. Recently, an automated LCC method for References p . 100.5
ANTIBIOTICS
998
6 z
U m
0.6
a
0.2
u
11.0 7
zm
0
10
20
30
40
TUBE NUMBER
Fig. 45.7. Gel chromatography of tetracycline, anhydrotetracycline and 4-epi-anhydrotetracycline eluted separately on Sephadex at pH 7 . 7 and 9.0 (Griffiths). Column, sorbent, buffers, operating conditions and detection as in Fig. 45.6. __ ,Tetracycline; ---.-., anhydrotetracycline; - - - - -, 4-epi-anhydrotetracycline. Fig. 45.8. Gel chromatography of tetracycline; anhydrotetracycline and 4-?pi-anhydrotetracyclinein solvents of different salt concentrations (Griffiths). Column, sorbent, operating conditions and detection as in Fig. 45.6. T = Tetracycline; A = anhydrotetracycline; E = 4-epi-anhydrotetracycline. a, 0.025 M T r i s buffer, pH 9.0; b, 0.050M Tris buffer in 0.025 M sodium chloride solution, pH 9.0; c, O.OSOM Tris buffer in 0.2 M sodium chloride solution, pH 9.0.
determining the tetracycline antibiotics was reported by Ascione et al. The method was applicable to crystalline tetracyclines and their various pharmaceutical dosage forms.
Anthracyclines Several Streptomyces strains produce yellowish red pigments of the anthracyclinone group and their glycosides, anthracycline antibiotics. Silica gel (Merck), modified with acids or alkali salts, is mostly used for the CC of these antibiotics and their aglycones. The modifications of the sorbent according to Brockmann et al. (1965) are as follows: oxalic acid-silica gel, 1 kg of silica gel ( Li' > Mg" > Ca2' (water as eluent), while from Sephadex G-10, Mgz+is eluted first followed by a mixture of Li', Na' and Ca2'. For this reason, in addition to size differences among the ions, which appear to be the main determining factor, side-effects such as adsorption (Egan, Lindqvist), ion exclusion (Neddermeyer and Rogers, Pecsok and Saunders), restricted diffusion (Ackers) and ion exchange (Ortner and Pacher) must be also taken into account. Another phenomenon is the dependence of the elution volumes on the nature of the background electrolytes employed (Ueno et al., 1970a). For the elution of both anions and cations, water or aqueous solutions of different
I089
SIMPLE INORGANIC COMPOUNDS
salts are used. Yoza and Ohashi found that elution with salt solutions does not lead to complete separation owing to the formation of tails. These tails can be depressed to a minimum by using an acidic eluent, which can change the order of elution of the ions. The eluent also affects the symmetry of the elution curves (Yoza and Ohashi), which is ascribed both to the multicomponent character of metal ions in solution and to the polyfunctional character of the gel. As a typical example of application of gel chromatography, the following method for the separation of alkaline earths on Sephadex C-15 (Ueno et al., 1970a) is described. For preparation of the column, Sephadex G- 15 (particle size 40- 120 pm) suspended in the eluent is allowed to swell for 2 days and undesirable fine particles are removed by decantation. A deaerated (under reduced pressure) suspension of the swollen gel is then poured into a column (60 X 1.5 or 90 X 1.5 cm) partially filled with the eluent and, after the gel bed has reached a height of about 5 cm, the outlet at the bottom is opened so as to allow the eluent to flow at a rate of approximately 30 ml/h. The addition of the gel is continued until the gel bed reaches the desired height. The bed volume is adjusted to 100 or 150 ml. In order to protect the surface of the gel from disturbance, a disc of filter-paper is placed on the top of the bed. After packing, 500 ml of the eluent are passed through the column in order to settle the gel bed. The separation procedure is as follows. A 1-ml volume of a 0.01 Msample solution (chlorides) is placed on the column bed just as the layer of the eluent (0.1 M potassium chloride-0.01 M hydrochloric acid) is soaking into the bed. As soon as the solution has entered the bed, about 4 ml of the eluent are added and elution is started at a constant flow-rate of 25-35 ml/h. The effluent is collected in fractions of 1 ml with a drop count fraction collector (see Fig. 51.1). The amounts of the samples in the effluents are determined by EDTA titration.
FRACTION NUMBER
Fig. 5 1.1. Gel permeation chromatographic separation of magnesium, calcium and barium (Ueno et al., 1970a).
References p. 1111
1090
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
Ion-exchange chromatography In order to achieve efficient separations, the following general requirements must be met: (1) the concentration of the sample applied on the column (this amount depends on the column capacity) should be low ( Th(1V) > U(V1) As(lI1) > Sn(1V) > Sb(III)** As(1II) > Te(1V) > Cd(I1) > Bi(lll)*** Al(II1) > Fe(II1) > As(II1) > Mo(V1) Cr(II1) > Fe(I1I) Fe(1lI) > Re(VII)** Ge(1V) > Sn(IV)**
Amberlite CG-4B, I4 x 1 cm
0.1 - 11.4 M HC1
Kuroda et al.
Ga(II1)
Amberlyst XAD-2 0 + IPE, 6 X 1.2 cm Amberlyst A-26, 6 x lcm Amberlyst A-26, 14 x 1 cm Amberlyst XAD-2 + TOPO, 11 X 1.4 cm Dowex SOW-X8, 6 X 1.2 cm Amberlyst XAD-2 + TOPO, 10 x 1.3 cm
Pb(l1) > Zn(I1) Ni(I1) > Co(l1)
> Cu(I1) > Zn(I1) > Cd(I1)
Ti(1V)
References p . I I I 1
Strelow el al. (1969) 3 M HNO,
Fritz and Latwesen
'
(Continued on p . 1096)
1096
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
TABLE 51.1 (continued)
Ion exchanger (sorbent )
Cations*
La(II1)
> Ti(IV) > Th(IV) > Zr(IV)
Eluent
References
Amberlyst XAD-2 + TOPO, 13 X 1.2 cm Dowex 50W-X8, 7 X 1.2 cm
Ni(I1) > Mg(I1) > Ca(l1) Pd(I1) > Pt(IV) > Au
Daiflon M-300, + TBP, 30 X 0.92 cm
Cd(I1) > Zn(I1) > Fe(II1) > Cu(I1) > Co(I1) > Mn(1I) > Tl(II1) > In(II1) > Ga(II1) > Al(II1) > Yb(II1) V(V) > Fe(II1) > U(V1) > Ti(IV) > Ca(I1) > Ba(I1)
Bio-Rad AG SOWX8, 17 x 2.1 cm
Be(I1) > Fe(II1) > Cr(II1) > Co(I1I) 8 Be(I1) > Cu(I1) > Ru(II1) > Co(II1) Be(I1) > Cu(I1) > AI(II1) > Cr(1II) > Ru(II1) > Co(II1) § § 3 w
Kieselguhr, SO x 0.27 cm; stationary phase, H,O-EtOHTMP (0.343: 0.641 :0.016, w/w)
$9
§§i't
5§5
HCl, HNO,
' H,O-EtOH-TMP (0.0007:0.022: 0.977, w/w)
Akaza el al. (1970)
Strelow et al. (1971)
Huber er al.
*Cations listed in order of elution. **Cations eluted with 1 M NaOH. ***Cations eluted with 3 M H,SO,. §For details, see original paper. §§High-speed chromatography (cations separated as acetylacetonates), ligand present in mobile phase. @§Flow-rate, 1.7 ml/min. ?Flow-rate, 1.9 ml/min. ttFlow-rate, 1.1 ml/min.
Anions
Inorganic phosphoms compounds The application of chromatography to the inorganic chemistry of phosphorus includes in principle either the separation of compounds that have phosphorus atoms in different oxidation states or the separation of phosphates that contain a different number of phosphorus atoms in a molecule. Lower 0x0 anions of phosphorus are advantageously separated on Dowex 1-X8 (Benz and Paiaxo; Pollard et al., 1962, 1963). For the complete separation, gradient elution is necessary (Koguchi et al. ; Pollard et al., 1962, 1963). Due to the nature of the cornpounds to be separated, changes in pH alter the order of elution. For example, at pH 6 the
1097
SIMPLE INORGANIC COMPOUNDS
order of elution was found to be HPOj- > HPO:-, whereas at pH 11 the HP0:- anion was eluted before HP0:- (Pollard er al., 1962). The adsorbability of phosphorus 0x0 anions.also depends on the concentration of the eluting agent used. This dependence (logarithm of concentration), which has a linear character, allows the calculation of the position of the elution peak in gradient elution (Koguchi er al.). For the separation of lower 0x0 anions of phosphorus, Pollard et al. (1967) used gradient elution chromatography on cellulose. The results obtained by TLC can be used in column chromatography, but the sample must be applied as a solid (adsorbed on cellulose) and, in addition, the water content in the solvent must be lower than that used in TLC. In order to obtain a narrow band, the minimum volume of sample must be applied. Condensed phosphates that differ in molecular weight can also be separated using Dowex 1-X4(Ohashi et al.), but gel chromatography, in which the molecular-sieve effect appears to be the main factor, is more convenient. Ueno et al. (1970b) studied the separation of linear polyphosphates in detail and found: (1) elution with water results in low distribution coefficients with ortho, di- and triphosphates; (2) electrostatic repulsions between 0x0 anions and the gel matrix can be reduced by elution with potassium bromide solution; (3) the distribution coefficients of 0x0 anions increase with increasing concentration
I F R A C T I O N NUMBER
Fig. 51.6. Elution curves for linear phosphates (P, -P,* = number of phosphorus atoms) (Ueno ef al., 1970b). Column: 150 ml (volume of bed). Sorbent: Sephadex G-25. Eluent: 0.1 M KCI, pH 7.0. Phosphorus concentration in sample solution = 3 . lo-’ 5 .lO-’M for individual phosphates and 0.01 - 0.02 M for the polyphosphate fractions. One fraction = 1.08 ml. ~
References p . I I I I
1098
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
TABLE 51.2 SEPARATION O F LOWER PHOSPHORUS O X 0 ANIONS (POLLARD ef al., 1963) Column: 5 0 X 1.5 cm. Ion-exchanger: Dowex 1-X8 (Cl-), 100-200 mesh. Elution: potassium chloride buffer solution, pH 6.8. Potassium chloride concentration: (a) in mixing vessel, 0.05 M (750 ml); (b) in reservoir, 0.2 M.Flow-rate: 6 0 ml/h. Temperature: 18°C. -~ ~
~~~~
~~~
Compounds separated*
H2PO;
150 260 330 590 680 720 890 990 1180
w0:-
HP0:H,P,O:HP20:H P 0:HP,0,3 p20;pq-p3-p4
Retention volume (rnl)
**
* Listed in order of elution. ** Na,P,O,. TABLE 51.3 SEPARATION O F DIFFERENT ANIONS Anion*
I-
I-
> Br- > C1-
> Br- > C1'
(Mo,O,,)~-*
**
Sorbent (ion exchanger)
Eluent
References
Hydrous zirconium oxide 12 cm x 0.075 cm'
0.15-1 M KNO3 flow-rate 110 cm/h
Tustanowski (1967a)
Alumina 29.5 cm x 0.075 cm2
0.2 M KNO,, flow-rate 20-70 ml/h
Tustanowski (1967b)
Cellulose-20* *
Water
Brown and Chitumbo
Kel-F + tri-n-butylphosphate
HCl-H,O
Akaza ef al. (1969)
Sephadex (3-10 and LH-20, Bio-Gel P-2
HZSO,
Streuli and Rogers
DE AE-cellulose
0.02-0.1 M NH,CNS 55
Ishida and Kuroda
t
*Anions listed in order of elution. **For preparation, see original paper. ***Different types of molybdates, the composition of which depends on pH. §Eluted with 0.1 MNaOH-0.1 M NaCI. 6 OpH 3 for ReO;, pH 5 for MOO:-.
COORDINATION AND ORGANOMETALLIC COMPOUNDS
1099
of the eluting agent; (4) the higher distribution coefficients of both K+ and Cl- compared with those of 0x0 anions cause the phosphates to be accompanied by background electrolyte; (5) only at phosphorus concentrations lower than 0.01 Mare the elution curves symmetrical (see Fig. 5 1.6); (6) the pH does not affect the distribution coefficients; (7) the elution volume increases with increasing gel porosity. As expected, polyphosphates can be also separated using a Bio-Gel P-2 column, from which they are eluted in order of decreasing molecular weight (Neddermeyer). As an example of this type of separation, the separation of 0x0 anions on Dowex 1-X8 (Pollard et al., 1963) is described. A 1-ml volume of a mixture of anions containing 2 mg of phosphorus per anion per millilitre is applied on the column (50 X 1.5 cm). As soon as no liquid remains above the bed of Dowex 1-X8 (Cl-) resin (100-200 mesh), 2 ml of a 0.2 M buffered solution of potassium chloride (the pH of which is adjusted to 6.8 by the addition of 25 ml of a 2 M solution of ammonium acetate to 1 litre of potassium chloride solution) are added. Anions are further eluted by gradient elution (see Table 5 1.2) according to Grande and Beukenkamp. Fractions of 10 ml are collected and their phosphorus contents determined. Examples of the use of chromatography for the separation of some other anions are shown in Table 51.3. COORDINATION AND ORCANOMETALLIC COMPOUNDS
General survey The choice of the best chromatographic method is affected by the character of the compounds to be separated. While the use of ion-exchangers is very effective for charged complexes, the separation of non-ionic metal complexes is difficult and different sorbents are employed. However, some of these materials behave simultaneously as weak ion exchangers. A typical example is silica gel, the silanol sites (= Si-OH) of which act as a strong hydrogen-bond donor (Basila). For this reason, this material will strongly adsorb species that are hydrogen-bond acceptors and therefore, in such instances, the use of solvents capable of forming strong hydrogen bonds is necessary (Jursik). According to Burwell et al., competition for the coordination sphere between the silanol or siloxane sites and ligands occurs during the chromatography of metal complexes. From this it follows that any effect that increases the association between the complex and the Si-OH centre will increase the tendency of the silanol (siloxane) group to penetrate into the coordination sphere of the central atom. This means on the one hand that solvents with low dielectric constants will increase the adsorption of solutes and on the other hand such solvents (or those with a low flow-rate) will make the adsorption irreversible (Bradley and Pantony). The adsorption of metal complexes on silica gel is also affected by its activity. In general, greater mobility is observed on hydrated than on dehydrated silica gel (Hathaway and Lewis, Jursik). References p . I 1 I I
1100
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
Apart from silica gel, alumina is most frequently used as a sorbent. As far as its adsorption mechanism is concerned, it is similar to that of silica gel, because the surface of alumina also contains hydroxyl groups. However, stronger adsorption of metal complexes can be expected on alumina owing to the different coordination numbers of aluminium and silicon. Geometrical isomers Geometrical isomerism of coordination compounds is the consequence of the coordination of different kinds of donor atoms, which leads to a different orientation of molecules in an electric or magnetic field. Such metal complexes are characterized by a permanent dipole moment, the value of which indicates the polarity of the metal complex. In addition, the different orientation of alkyl groups of a symmetrical bidentate ligand coordinated to a metal ion also gives rise to geometrical isomerism. The results show that it is only the polarity of the metal complex or the character of the ligands that influences the chromatographic behaviour of geometrical isomers, and that the different dipole moments of geometrical isomers result in trans-isomers having a higher mobility than cis-isomers (King and Walters). The observed lower mobility of cisisomers is due to their ability to bind two sorbent sites (e.g., silanol centres of silica gel or functional groups of an ion exchanger), while trans-isomers bind only one sorbent site (Druding and Hagel). The different chromatographic behaviour of geometrical isomers is not only related t o their different dipole moments. Legg, for example, described the separation of [Co(EDDA) (pn)]' isomers, (EDDA = ethylenediamine-N,N'-diacetate; pn = propylenediamine) for which the geometrical isomerism arises only as a result of the different orientation of the methyl groups in propylenediarnine. The chromatography of isomers is accompanied by a series of negative effects, such BS substitution of labile bonded ligands for the hydroxyl groups of silica gel. Isomerization promoted by the sorbent must also be taken into account (Kauffman et aZ.). For the efficient separation of isomeric pairs, which depends on the interaction of solvent, sorbent and solute, the following ideal conditions can be derived: (1) isomers must have maximally different dipole moments; (2) isomers must be soluble in both polar and non-polar solvents; (3) an ideal solvent is one which does not promote isomerization; for example, the following equilibrium: cis-[Co(en)&12]+ + truns-[Co(en)zC12]+ (en = ethylenediamine) is strongly influenced by solvents (Tobe and Watts, 1962, 1964), and this observed influence of polarity has been attributed to the dipolar character of the cis-isomer (Fitzgerald and Watts; Pearson e l ul.; Tobe and Watts, 1962, 1964); (4) an ideal sorbent binds the cis-isomer sufficiently firmly, but there must still be the possibility of eluting it with a polar solvent; (5) the sorbent must be isomerically inert; in this sense, it must be noted that silica gel is a sorbent that sometimes catalyzes isomerization (Kauffman et a[.); (6) faster flow-rates (>2 ml/min) give diffuse bands (Girgis and Fay).
COORDINATION AND ORGANOMETALLIC COMPOUNDS
1101
As far as the practical use of column chromatography for the separation of geometrical isomers is concerned, two examples are given below. (1) Separation of isomers of [Co(EDDA)(en)] (Legg and Cooke, 1965). To a column containing 9 0 ml of Dowex 50W-X8 (Na’), 50- 100 mesh, 3 mmoles of this complex dissolved in 100 ml of water are added. The column is then allowed t o swell with water and the adsorbed complex is eluted with 0.5 M sodium perchlorate solution at the rate of 0.1-0.5 ml/min. The trans-isomer is eluted first. (2) Separation of isomers of tris(benzoylacetonato)chromium(III) (Girgis and Fay). A column (84 X 2.8 cm) equipped with an electric vibrator is packed with a slurry of Florisil(180 g) in n-hexane and allowed to drain under continuous vibration. Then 0.5 g of the complex dissolved in the minimum volume of benzene-n-hexane (1 : 1) is added and elution is carried out with benzene-diethyl ether (19: 1) at a flow-rate of 2 ml/min. After eluting the first band from the column (trans-isomer), the flow-rate is increased to 8 ml/min and the cis-isomer is obtained. For other examples of the separation of geometrical isomers, see Table 5 1.4. +
Optical isomers and diastereoisomers Neglecting the origin of the metal complex chirality, these compounds may contain one or more centres of chirality, which affects the choice of both solvent and sorbent. Considering the separation of optical antipodes, according to the classical method (fractional crystallization of diastereoisomers), another centre of chirality is introduced into the racemate molecule so that enantiomers are transformed into diastereoisomers that differ from each other in their physico-chemical properties. In chromatography, this can be achieved either by using a chiral sorbent or a chiral solvent. The first instance, which is the more frequently used, is based on the fact that both enantiomers, e.g. (+)-M and (-)-M (M = metal atom), are bonded on the chiral sorbent, which can be expressed by (+)-Ads, with the formation of both (+)-Ads-(-)-M and (+)-Ads-(+)-M. However, diastereoisomers formed in such a manner are not equally stable, so that one enantiomer may be adsorbed more strongly than the other. As typical examples of the separation of enantiomeric metal complexes on chiral sorbents (cellulose, starch, etc.), four methods of resolution are described below. (1) Resolution of [Co(EDDA)(B)] (B = N-methyl, N-ethyl, N,N’-dimethyl or N,N’-diethylenediamine) (Legg and Douglas). For the preparation of the column, 20 g of Cellex CM-cellulose (Bio-Rad Labs., Richmond, Calif., U.S.A.) are stirred with 400 ml of 0.01 Msodium perchlorate solution, allowed to settle for 30 min and then the process is repeated. A further 100 ml of 0.1 Msodium perchlorate solution are added and this suspension is quickly stirred and immediately and continuously poured into the column (50-70 X 2.5 cm). The cellulose is allowed to settle for about 30 min and a plug of glasswool is inserted in order to protect the cellulose surface. The column is washed with 100 ml of 0.01 Msodium perchlorate solution and then with 100 ml of water at a flowrate of 2-4 ml/min. Then 0.2-0.3 g of the complex dissolved in 100 ml of water are loaded on to the column at a rate of 1-2 ml/min. The 3-4-cm layer of adsorbed +
References p . 1111
1102
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
complex is eluted with 0.01M sodium perchlorate solution and the eluate is collected in 15-ml fractions. (2) Resolution of triaquotribenzo(b,j; j)( 1,5,9)triazacycloduodecinenickel(II) nitrate on cellulose (Taylor and Busch). A suspension of microcrystalline cellulose (Avicel, technical grade) in water is poured into a column (60X 2.2,65 X 2.2 or 100 X 2.0cm) and water is allowed to filter through the bed at a rate of approximately 0.2 myinin. The column is washed with water for 24-26 h at a flow-rate of 10 ml/min. A saturated aqueous solution of the complex containing a few drops of 0.0 I M nitric acid is then loaded on to the column. The adsorbed complex is eluted with water as one broad band at a rate of 0.5 ml/min and collected into several fractions. The best separation was achieved with a column size of 65 X 2.2 cm. (3) Resolution of the same complex as in (2) on starch (Taylor and Busch). Before use, the starch is stirred with 0.01M nitric acid and decanted or filtered. A starch suspension in 0.01M nitric acid is added t o the column and washed with 0.01M nitric acid for 48 h. In order to achieve a flowrate of 0.2 ml/min (suitable flow), air pressure is applied. A 10-ml volume of a saturated 0.01M nitric acid solution of the complex is then placed on the column and eluted with 0.01 M nitric acid as one broad band, which is separated into fractions. (4) Resolution of cobalt(II1) or chromium(II1) tris-(0-diketonates) on D-(+)-mannit01 or D-(+)-sorbit01 (MarkoviC and Schweitzer). A thin slurry of D-(+)-mannit01 (or D-(+)sorbitol) (sieved to 80 mesh) in a benzene-ligroine (1:6)mixture is poured into a column so as to form a 124 X 5 cm bed. After removing the excess of solvent by flow through the column, 0.102-0.120 g of sample dissolved in 15 ml of benzene is applied t o the column. The adsorbed complex is then eluted with benzene-ligroine (1:6)solvent and the eluate is separated into several fractions. Chromatography on the chiral sorbents usually does not lead to the total resolution of enantiomers (Girgis and Fay; Jursik et al., 1973;MarkoviC and Schweitzer), with the exception of the cobalt(II1) trinuclear complex (Brubaker et al.). Also, comparison of the sorbents shows that the separation on cellulose is not as efficient as that on starch (Taylor and Busch). The most effective resolution on starch, which also depends on the amount of sorbent used, can be attained with a water-soluble racemate (Krebs and Diewald). Furthermore, the presence of a larger number of polar groups in the metal complex is necessary in order to achieve a multi-site adsorption (Krebs and Rasche). Resolution of enantiomers, as mentioned above, can also be achieved in principle by using a chiral solvent, in which racemic metal complexes anti-racemize (Bosnich). As the mechanism of this anti-racemization includes a stereospecific substitution reaction (Smith and Haines) with the solvent being coordinated as a bidentate ligand, it is possible to assume preferential adsorption of one of the enantiomers on the achiral sorbent. However, the application of this method, which seems to be promising, has not yet been reported. In addition to the described chiral sorbents, alumina or silica gel can also be employed, but before using it, it must be covered with a layer of optically active material, e.g. (+)tartaric acid (Piper). In other instances, Amberlite IRA-400 with chiral ionogenic groups was used (Gillard and Mitchel). By modifying the latter method, the racemate is transformed into diastereoisomers, which are then separated chromatographically (Dhar et al., 1958,1959).
COORDINATION AND ORGANOMETALLIC COMPOUNDS
1103
As can be seen from the two examples given below, for the resolution of diastereoisomeric metal complexes, where differences in non-bonding interactions in individual stereoisomers are utilized, alumina, silica gel and ion exchangers are most frequently used. (1) Resolution of tris-[(+)-3-acetylcamphorato]cobalt(III) on alumina (Springer et a l ) . Approximately 500 g of acid-washed alumina suspended in benzene are poured into a column (bed size 40 X 4 cm) and, as soon as the benzene has soaked into the alumina, 7 g of the complex dissolved in 25 ml of benzene are added to the column. When all of the complex has been adsorbed, the column is filled with benzene so as to form a column of 1 15 cm above the bottom of the alumina (this benzene layer maintains a flow-rate of approximately 10 ml/min). Elution with benzene separates the complex into two bands (geometrical isomers). Each band is collected in two fractions, each of which contains two isomers and, by repeated chromatography under the same conditions (on a smaller scale), pure optical isomers are obtained. (2) Resolution of [C~(gly)~(pn)]and [Co(gly)(pn)z] (gly = glycine; pn = propylenediamine) on Dowex 50W-X8 (Kojima and Shibata, 1970). The reaction mixture, containing both complexes dissolved in water, is added to the column of Dowex 50W-X8 (Na’), 14.5 X 8 cm, and the adsorbed complexes form a compact band at the top of the column. The column is allowed to swell with water. The adsorbed bands are eluted with a 0.5 M solution of sodium chloride at a rate of about 0.6 ml/min and, after elution for about 24 days, five bands develop, four of which are collected separately. The band remaining at the top of the column is then eluted with a 2 M solution of sodium chloride for about 36 days, so that the fifth band is separated into two more bands, which are eluted separately. The resolution of both enantiomers and diastereoisomers naturally depends on their minimum rate of isomerization and racernization.
’
’+
Relationship between chromatographic behaviour and configuration of optical isomers The resolution of enantiomeric metal complexes on chiral sorbents resembles the method of the “less soluble diastereoisomers”. It seems that here also there exists a relationship between the adsorptivity of individual enantiomers (diastereoisomers) and their configurations, so that, for example, the less adsorbed enantiomers (diastereoisomers) will have the same configuration. However, this would be valid only for sorbents or metal complexes with the same adsorption sites. In addition, the dependence of the order of elution of isomers on the kind of eluent (Krebs and Diewald) and kind of sorbent (Markovik and Schweitzer) must be also taken into account. The order of elution of isomers is also affected by the ligand configuration, as was demonstrated by Schoenberg et al. As an example, Piper observed that on alumina modified by (+)-tartaric acid, (+)isomers of Co(II1) and Cr(1II) acetylacetonates are less adsorbed. Similar results were obtained when these complexes were resolved on a D-lactose column (Fay et al.). Several racemic complexes, e.g. [&(en), ] 3 + , [Co(pn), ] ’+, [Cr(en), ] 3 + and [Rh(en), ] 3 + , were separated into enantiomers on Amberlite IRA400in the (+)-tartarate form. The first isomers eluted were: (-)-[Co(en),] 3 + , (-)-[Co(pn),] 3 + , (-)-[Cr(en),] and (+)[Rh(en),] Because of the similarity of the adsorption sites in these complexes, it can
’+.
References p . 1111
’+
CL
TABLE 51.4 SEPARATION OF GEOMETRICAL ISOMERS ?Lpe of compound
M%
Compounds separated*
Co(bzacac), ] [ Cr(bzacac), ] [ Co(tfaca3, ]
[ Cr(tfacac),] [ Co (tmb 1,1 lCr(tmb), I I Co(MHDTH),] [Co(dien),] 3+ [Co(dien),]
+ '
+ 0
P
Sorbent (ion-exchanger)
Eluent
Isomer eluted first
References
Florid Florid Florisil Florisil Florisil FIorisil Alumina Cellulose phosphate
Benzene-diethyl ether (19:1) Benzene-diethyl ether (19:l) Benzene-n-hexane (1: 1) Benzene-n-hexane (1:1) Benzene-n-hexane (3: 1) Benzene-n-hexane (3: 1) Benzene-n-hexane (2:3) n-Butanol-HCl-H,O (200: 15 :1 5) 0.3 M sodium tartrate
trans trans trans trans trans trans trans trans
Girgis and Fay Girgis and Fay Girgis and Fay Girgis and Fay Girgis and Fay Girgis and Fay Gordon and Holm Keene et al.
s-cis* *
Keene and Searle
SE-Sephadex C-25
c (
2 0
P
n P
5
-0 0
0 0
P
U
I
Dowex 1-x10 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 1-X2 Dowex 50W-X8 Bio-Rad AG 50W-X2 Dowex 1-X8
0.5 M NaClO, 0.5 M KBr 0.5 M NaCIO, 0.5 M NaClO, 0.5 M NaClO, 0.5 M NaClO, 0.25 M NH,Br 0.5 M NaCl 0.1M NaCl
Alumina Alumina
Matsuoka et aL (1967)
z
z
Matsuoka etaL (1967) Legg and Cooke (1965) Legg and Cooke (1965) Legg and Cooke (1966) Legg and Cooke (1966) Freeman and Liu Kojima and Shibata (1970) Yamada e t ai. Tiethof and Cooke Ford and Sutton Bridges and Chang
0
0.3 M HNO,
trans trans trans trans trans trans trans trans trans trans trans trans
Ethanol-water (85:15) Water
trans trans
qillard e t al. Celap e t al.
0
0.5-0.075 M NaCl0, 2.2 and 2.5 M NaCl
0 2: P U
0 ~
$ 0
54 P
r c n n
5
0
C
2:
F1
Alumina Silica gel 'p
Alumina
4
4 4 4
Silica gel Alumina Silica gel
Benzene Acetone Benzene Acetone Benzene Acetone Acetone-benzene (0.5: 100) Acetone Acetoneebenzene (7: 100) Acetone Acetone-benzene (17: 100) Acetone
trans cis trans cis trans cis trans cis trans cis trans cis
Kauffman et al.
0 ;d
n
*z
0
* bzacac = benzoylacetonate; tfacac = trifluoroacetylacetonate; tmb = 1,l ,l-trifluoro-4-p-methoxyphenyl-2,4-butanedionate; MHDTH = 5-methylhexane2,Cdithionate; dien = diethylenetriamine; ox = oxalate; gly = glycinate; IDA = iminodiacetate; MIDA = methyliminodiacetate; dap = L- or DL-a, p-diaminopropionate; asp = aspartate; N,N'-DMEEN = N,N'-dimethylethylenediamine;py = pyridine; val = valinate; ala = alaninate; n-Bu = n-butyl; Et = ethyl.
** Symmetrical-cis.
54 9
r
t 0
s
0
TABLE 51.5 RELATIONSHIP BETWEEN THE STRUCTURE OF COBALT COMPLEXES AND THE ORDER OF ELUTION OF ISOMERS USING SORBENTS Compounds separated*
Sorbent
Eluent
Sign of rotation of less adsorbed isomer
cis- I Co (gly), 1 trans-[Co(gly), ] trans- [ Co(sarc), 1 trans [ Co(a-isobu t ), ]
Starch
10%KCI Water Water Water
-+ + -
References
Douglas and Yamada Douglas and Yamada Jursik etal. (1972) Jursik ef al. (1973)
CI
(Continued on p . I 106)
TABLE 51.5 (continued) Compounds separated*
Sorbent
Eluent
trans [ Co( L - V ~ )] ,
Alumina
Ethanol-acetone (1:2) Ethanol-water ( 9 5 5 ) Isopropanol-water (4: 1) Isopropanol-water (4: 1) Isopropanol-water (3:2) Isopropanol-water (3:2) Methanol
Cellulose
n-Butanol**-lO M HCI(97:2) n-Butanol**-10 M HCI (97:2) n-Butanol**-lO M H C l ( 9 7 ~ 2 )
trans[ Co(L-leu), ]
trans- 1Co(L-norleu) ,] trans-[Co { ( 2~ , 3~ ) - i l eu }J trans- [Co(L-leu), (gly)] trans-[ Co( L-leu)(gly), ] trans-[ Co(L-SluOMe), ]
Sign of rotation of less adsorbed isomer
References
Shibata eta.! (1966) Gillard and Payne Jursik and Hijek Jursik and Hijek Jursik e t al. (1970) Jursik e t al. (1970) Gillard and Payne -
Dwyer et al. Dwyer et al. Dwyer et al.
* Sarc = sarcoshate; a-isobut = a-aminoisobutyrate; leu = leucinate; norleu = norleucinate; ileu = isoleucinate. ** Saturated with water.
TABLE 51.6 RELATIONSHIP BETWEEN THE STRUCTURE OF COBALT COMPLEXES AND THE ORDER OF ELUTION OF ISOMERS ON ION EXCHANGERS Compounds separated*
Ion exchanger
Elution
[Co3N6S6 1 [Co(EDDP)(en)] c~s-[CO(IDA),][ Co(EDTA)] -
Cellex CM Dowex 50W-X8 Cellex AE Cellex AE
0.1 M NaCl 0.35 M NaClO, 0.01 M NaCl 0.01 M NaCl
+
Sign of rotation of less adsorbed isomer
References
Brubaker et at. Schoenberg et al. Legg and Douglas L e g and Douglas
Cellex AE Dowex I-XI0 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 Dowex 50W-X8 DOWCX1-X2 CM-cellulose
Legg and Douglas Matsuoka et al. (1970) Kojima and Shibata (1970) Kojima and Shibata (1970) Kojima and Shibata (1971) Kojima and Shibata (1971) Yamada et al. Broomhead and Grumley
0.0 1 M NaCl 0.07 M KCI 0.5 M NaCl 2 M NaCl 0.1 -0.4 M NaCIO, 0.1-0.4 M NaCIO, 0.1 M NaCl 0.4 M CH,COOH
* I Co,N, S,,1 = hexakis(2-aminoethanethiolo)tricobalt(III) bromide; EDDP = ethylenediaminediaminopropionate;IDA = iminodiacetate; TRDTA = trimethylenediaminetetraacetate;ox = oxalate; ser = serinate; aspH = monoanion of aspartic acid; asp = aspartate; phen = 1,lo-phenanthroline. ** M = Ir(IlI), Rh(III), Cr(Ill), Co(l1l); X = CI or Br.
0
;4
E!
2 >
3 0 %J
n > z 0
5-I >
t:
k TABLE 5 I .7 RELATION BETWEEN THE STRUCTURE OF FERROCENES A N D THE ORDER OF ELUTION OF ISOMERS Compounds separated*
Sorbent
Eluent
References
2-Acetyl-l,l'-dimethylferrocene 3-Acetyl- 1 ,I1-dimcthylferrocenc l~Acetyl-2-ethylferrocenc 1-Ace tyl- 1'-e tliylfcrrocene 3-Ace tyl-3-ethylferroccne 1,l'-Dimethylferrocene-2-carbosylicacid* * (RR)-(r-[ 2-Trimcthylsilylferroccnyl] cthyldimethylarsine
Alumina
Benzene
Reinehart
Alumina
Benzene
Rosenblum and Woodward
Alumina Alumina***
Benzene Ligroineisopropanol (20: I )
Westman and Reinehart Marquarding et al.
(RS)-a-[2-Trimc thylsilylferrocenyl j ethyldime thylarsine
* Listed in order of elution. ** Separated as p-brombenzyl csters. *** Modified with a 2% aqueous solution of ammonia.
8
1108
INORGANIC, COORDINATION AND ORGANOMETALLlC COMPOUNDS
be concluded that these isomers will have the same configuration (Gillard and Mitchel). With the exception of tris-(@diketonato)metal complexes, adsorptivity can be correlated with the different alkyl group chelate ring conformation. This is apparent for Co(lI1) complexes of C-substituted diamines and amino acids. In all instances (except for both Co(gly)3 and Co(y-gluOMe), (where gluOMe is the methyl ester of glutamic acid) the (-)-isomer is less strongly adsorbed (see Table 5 1S).The sign of the Cotton effect indicates isomers with axial (pseudo-axial) arrangements of alkyl groups. The exception of Co(gly), is due to the absence of alkyl groups. This fact, however, does not reflect the influence of the chiral sorbent because the (-)-isomer of Co(a-isobut), (isobut = amino isobutyric acid) is the first to be eluted from a starch column (Jursik et al., 1973). On the other hand, the unusual order of elution of Co(y-gluOMe)3 (see Table 51 5 )may be due to the presence of a polar side-chain in which, for example, hydrogen bonding may occur. The effect of the latter on the order of elution of isomers, the importance of which was observed by Matsuoka et al. (1970), must not be overlooked. The above examples indicate that an equatorial disposition of alkyl groups ((+)-isomers) facilitates the adsorption of the corresponding isomer on both chiral and achiral sorbents (Jursik and Hrijek). However, the resolution of optical isomers using ion exchangers shows the opposite behaviour (see Table 51.6). Schoenberg et al. and Kojima and Shibata (1970) ascribed this effect to the possible steric interactions of ligand alkyl groups with the resin.
Ferrocenes Most of the chromatographic applications concern the separation of reaction mixtures (see, for example, Dormond and Decombe, Hughes et al., Nesmeyanov et al., Shiga e t al. ). In several investigations, chromatography was used for the purification of the reaction products (Combs et al., King et al., Reich-Rohrwig and Schlogl). Another very useful application includes the stereochemistry of ferrocenes. Falk e t al. (1969), for example, separated isomeric ferrocenophan carboxylic acid diphenylamides on a silica gel column (30 X 4 cm). Elution with benzene gave the a-isomer, while the 0-isomer was eluted with benzene-ethanol (1 0: 1). Other examples include the separation of cis- and trans-isomers of bis(a-ketotetramethy1ene)ferrocene on alumina (the cis-isomer is eluted first) (Falk and Schlogl, 1965), isopropylferrocene carboxylic acid amides (Schlogl and Fried) and isomers of acetylmethylferrocene (Benkeser e t al., Hill and Richards, Nagai eta/.). On the other hand, attempts to separate a mixture of 1-ferrocenyl2-propyl acetate and 2-ferrocenyl-1-propyl acetate using an alumina column proved unsuccessful (Nugent et al.) . Table 51.7 lists a few applications showing the relationship between the structure and chromatographic behaviour of ferrocene derivatives. From Table 5 1.7, it follows that a-isomers are less strongly adsorbed than 0-isomers. This fact can be utilized for the study of the structure of ferrocene derivatives. Both chiral ferrocenes and ferrocenes with the chirality centre outside the ferrocene part can be partially resolved, as shown in the resolution of a-acetylmethylferrocene on acetylcellulose by Falk and Schlogl(l966). A 300-mg amount of racemic complex
1109
COORDINATION AND ORGANOMETALLIC COMPOUNDS
dissolved in benzene is added to the column (400 X 3 cm) containing 700 g of partially acetylated cellulose. The adsorbed complex is then eluted with benzene and fractions of effluent are collected (the (+)-isomer is eluted first). Similarly, N,N‘-diferrocenylcarbodiiinide possessing axial chirality can be resolved (Schlogl and Mechtler).
Me tallocenes The application of chromatography to other metallocenes is similar to its application to ferrocenes. An example is the separation of the a- and 0-isomers of methylcymanthreonylpropionic acid on alumina (activity 11-111). When elution is carried out with a benzene-light petroleum mixture, the a-isomer is eluted first (Gowal and Schli5gl, 1968a). On alumina, the a- and 0-isomers of arninoacetylcymanthrene were also separated (Egger and Niluforov). As far as resolution of racemic complexes of “metallocene asymmetry” is concerned, the following procedure is a typical example, involving the resolution of (1-tetralone)chromium tricarbonyl on acetylcellulose (Falk et al., 1966). To the glass column (400 X 3 cm), 700 g of partially acetylated cellulose suspended in benzene are added. The cellulose bed thus formed is then washed with benzene and 250 mg of racemate is applied. TABLE 51.8 APPLICATIONS OF COLUMN CHROMATOGRAPHY IN METALLOCENE CHEMISTRY Compounds separated*
C,H ,ReC, H, CH,COC, H ,ReC, H CH, COC,H, ReC, H,
* Listed in order of elution. References p . 1111
Sorben t
Elution
References
.$lumina (5% H,O)
n-Hexane
Fischer e t al. (1969)
Alumina (4% H,O) 1500 x 2 cm
Benzene Benzenediethyl ether ( 1 : l )
Fischer and Wehner
Alumina (4% H,O) 1500 X 2 crn
Benzene
Wehner et al.
Alumina (6% H,O) 1500 x 2 cm
Benzene
Wehner et al.
Alumina (activity 11)
n-Hexane
Fischer and Schneider
Sephadex G10 40 X 2 ern
Water
Fischer and Schneider
1110
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
TABLE 51.9 APPLICATIONS O F COLUMN CHROMATOGRAPHY IN THE CHEMISTRY OF METAL CARBONYLS AND ORGANOMETALLIC COMPOUNDS Compounds separated*
Sorbent
Eluent
References
trans-[(CO),ML,] ** cis-[(CO),ML,] **
Alumina 70 X 3 cm
Light petroleum (b.p. 30-60°C)
Grim and Wheatland
trans-[ (CO),-Cr-(1-acetoxytetralin)]
Alumina (deactivated)
Diethyl etherlight petroleum (b.p. 40-60°C) (1:12) (1:l)
Jackson and Mitchell
Alumina (deactivated) 80 x 2.5 crn
n-Pentane
Haszeldine ef al.
Acid alumina (activity 111) 6 0 x 2.5 cm
n-Hexane
syn-[CH,S-Fe-(CO), ] [CH,S-Fe,-(CO),, 1
Coleman e t al. (1967)
2,7-DOX-Fe-(CO), 2,7-DOX-[ Fe-(CO), 1
Alumina (activity 111)
Light petroleum
Fischer e t al. (1967)
(CO),-0s2-I, (CO),-Os,-l,
Florisil
n-Hexane Dichloromethanen-hexane (1:l)
Green e t al.
MT-Cr-(CO) , MXL-Cr-(CO), TO-Cr-(CO),
Carbowax 400 on Porasil C
2,2,4Trimethylpentane
Veening e t al.
trans- [ PtCl (Et, P), 1
Alumina 35 x 2 cm
Diethyl etherlight petroleum (b.p. 40-60°C) (20:80)
cis-[ (CO),-Cr-(1-acetoxy teiin)] C,HF, C ,HF, C,HF, CJF,
(2H)Mn(CC,, (4H)Mn(CO) UH)Mn(CO), O(4 H)Mn(CO)
,
,
anti- [ CH, S-Fe-(CO), ]
,
trans-[Pt(n-C,H,)CI(Et,P), ] trans-[ PtH(Et ,P),Cl] cis-IPt(n-C3H,),(Et,P),1
Chatter al.
* Listed in order of elution. DOX = dimethyloxepin; MT = mesitylene; MXL = m-xylene; TO = toluene. ** M = W or Mo; L = alkyl, arylalkyl or diarylphosphine.
The adsorbed complex eluted witqbenzene forms a band 1 m in length. The effluent is collected in fractions and these are further purified by TLC. Other applications, with the exception of purification processes (see Fischer et al., 1969; Gowal and Schlogl, 1968b; Herberich and Michelbring), concerning the separation of the reaction mixtures are listed in Table 5 1.8. Applications of column chromatography in the chemistry of organometallic compounds and metal carbonyls are summarized in Table 5 1.9.
REFERENCES
1111
REFERENCES Abe, M., Bull. Chem. SOC.Jap., 4 2 (1969) 3701. Abe, M. and Ito, T., Bull. Chem. SOC.Jap., 4 0 (1967) 1013. Ackers, G. K., Biochemistry, 3 (1964) 723. Akaza, I., Bull. Chem. SOC.Jap., 39 (1966a) 980. Akaza, I., Bull. Chem. SOC.Jap., 39 (1966b) 585. Akaza, I., Kiba, T. and Kiba, T., Bull. Chem. SOC.Jap., 43 (1970) 2063. Akaza, I., Kiba, T. and Taba, M., Bull. Chem. SOC.Jap., 42 (1969) 1291. Araki, S., Suzuki, S., Hobo, T., Yoshida, T., Yoshizaki, K. and Yamada, M., Bunseki Kagaku (Jap. Anal.), 17 (1968) 847. Basila, M. R.,J. Chern Phys., 35 (1961) 1151. Benkeser, R. A., Nagai, Y. and Hooz, J.,Bull. Chem. SOC.Jap., 36 (1963)482. Benz, C. and Paiaxo, L. M., Chim. Anal. (Paris), 50 (1968) 247. Birney, D. G., Blake, W. E. and Meldrum, P. R., Talanta, 15 (1968) 557. Bosnich, B., J. Amer. Chem. SOC.,89 (1967) 6143. Bradley, M. P. T. and Pantony, D. A., Talanta, 16 (1969) 473. Bridges, K. L. and Chang, J. C., Inorg. Chem., 6 (1967) 619. Broomhead, J. A. and Grumley, W., Inorg. Chem., 10 (1971) 2002. Brown, W. and Chitumbo, K.,J. Chromatogr., 63 (1971) 478. Brubaker, Jr., G. R., Legg, J. I. and Douglas, B. E., J. Amer. Chem. Soc., 88 (1966) 3446. Burwell, Jr., R. L., Pearson, R. G., Haller, C . L., Tjok, P. B. and Chock, S. P., Inorg. Chem., 4 (1965) 1123. k l a p , M. B., Niketid, S. R., Janjid, T. J. and Nikolid, V. N., Inorg. Chem, 6 (1967) 2063. Chatt, J., Coffey, R. S., Cough, A. and Thompson, D. T., J. Chem. Soc., A , (1968) 190. Coetzee, C. J. and Rohwer, E. F. C. H., Anal. Chim. Acta, 44 (1969) 293. Coleman, J. M., Wojcicki, A., Pollick, P. J. and Dahl, L. F., Inorg. Chem.. 6 (1967) 1236. Coleman, J. S., Asprey, L. B. and Chisholr.1, R. C., J. Inorg. Nucl. Chem., 31 (1969) 1167. Combs, C. S., Ashmore, C. I., Bridges, A. F., Swanson, C. R. and Stephens, W. O., J. Org. Chem., 34 (1969) 1511. Cziboly, C., Zsinka, L. and Szirtes, L., Magy. Kem. Lapja, 24 (1969) 470; Anal. Abstr., 19 (1970) 3700. De Bruyne, R. C., J. Inorg. Nucl. Chem., 32 (1970) 348. Dhar, S. K., Doron, V. and Kirschner, S., J. Amer. Chem. SOC.,80 (1958) 753. Dhar, S. K., Doron, V. and Kirschner, S., J. Amer. Chem. SOC.,81 (1959) 6372. Dormon, A. and Decombe, J.,Bull. SOC.Chim. Fr., (1968) 3673. Douglas, B. E. and Yamada, S., Inorg. Chem., 4 (1965) 1561. Druding, L. F. and Hagel, R. B., Anal. Chem., 38 (1966) 478. Druding, L. F. and Kauffman, G. B., Coord. Chem. Rev., 3 (1968) 408. Dwyer, F. P., MacDermott, T. E. and Sargeson, A. M., J. Amer. Chem. SOC.,85 (1963) 2913. Dybczyfiski, R., J. Chromatogr., 71 (1972) 507. Egan, B. Z., J. Chromatogr., 34 (1968) 382. Egger, H. and Nikiforov, A.,Monatsh. Chem., 99 (1968) 2296. Falk, H., Hofr, 0. and Schlogl, K., Monatsh. Chem., 100 (1969) 624. Falk, H. and Schlogl, K., Monatsh. C h e m , 95 (1965) 266. Falk, H. and Schlogl, K., Tetrahedron, 2 2 (1966) 3047. Falk, H., Schlogl, K. and Steyrer, W., Monatsh. Chem., 97 (1966) 1029. Faris, J. P., J. Chromatogr., 26 (1967) 232. Fay, R. C., Girgis, A. Y. and Klabunde, U., J. Amer. Chem. SOC.,92 (1970) 7056. Fidelis, 1. and Siekierski, S., J. Chromatogr., 17 (1965) 542. Fischer, E. O., Kreiter, C. G., Ruhle, H. and Schwarzhaus, K. E., Chem. Ber., 100 (1967) 1905. Fischer, E. O., Louis, E., Bathelt, W. and Muller, J., Chem. Ber., 102 (1969) 2547. Fischer, E. 0. and Schneider, R. J. J., Chem. Ber., 103 (1970) 3684.
1112
INORGANIC. COORDINATION AND ORGANOMETALLIC COMPOUNDS
I,'ischer, Fa.0. and Wclincr, H. W., Chem. Ber., 101 (196X) 454. titzpcrald, W. K. and Watts. D.W.,L Amer. Chem. SOC.,89 (1967) 821. Ford. D. C. and Sutton, C., Inorg. Chem., 8 (1969) 1544. Frache, R. and Dadonc. A . , Chronzatographia. 5 (1972) 581. I*recman,W. A . and Liu, C. I:.. Inorg. Chem., 7 (1968) 764. I.ritz. J . S.. Frazee, K . T. and Latwesen, G., Talanta. 17 (1970) 857. I..ri(z, 1. S. and Latwesen, G., Talanta, 17 (1970) 81. Ckrunchio, V . and Strazza, C;. G., Cliromarogr. Rev., 8 (1966) 260. Gillard. K. D. and Mitchcl. P . R.,Struct. Bonding (Berlin). 7 (1970) 49. Gillard. R. 0. and Payne, N . C..J. Chem. Soc., A . (1967) 1197. Gillard, R. D.,Payne. N . C. a n d Phillips, D. C., J. Chcm. Soc., A . (196X) 973. (;irgis, A . Y. and I:ny, It. C.. J. Amer. Chem. Sac.. 92 (1970) 7061. Gordon, 11, J . C. and Holm. K. H..J. Amer. Chern. Soc.. 92 (1970) 5219. (hw31. t i . and SchlBgl. K.. Moizotsh. Chem., 99 (1968a) 267. C h v a l , tl. and Sctil-opl. K., Monatsh. Chern., 99 (196811) 972. Grandc. J . E. and Bcukenkamp, J., Anal. Chem.. 28 (1956) 1497. Green. M.. Bruce. M. I., Cookc. M. and Wcstlakc, D.J.. J. Chcm. Soc.. A , (1969) 98'1. Grim, S.0. and Whcattand, I). A.,Inorg. Chcm., 8 (1969) 1716. Ilaszcldine. R: N . . Fields. R., Green. M., Harrison, T.. Jones, A . and Levcr, A. B. P., .I. Chern. Soc., A . (1970) 49. Hathaway, B. J. and Lewis, C. I;., J. Cliem. Soc., A . (1969) 1176. tlcrhcrich, G . 6. and Michelhring, K., Chem. Ber., 103 (1970) 3615'. Hill. 1.1. A:and Richards, J. H . . L Amer. Chem. Soc.. 83 (1961) 4216. Huber. J . P. K.. Kraak, J. C. and Vcening, H., Anal. Chem., 44 (1972) 1554. Hughcs. K. 1 . . Vuurcn, P. J . V . . Fletterick, K. J. and Heinvald, J . , Chem. Cornmun., (1970) 883. Ishida, K., Kiriyama, T. and Kuroda, R., Anal. Chirn Acta, 41 (1968) 537. Ishida, K. and Kuroda, R.,Anal. Chem., 39 (1967) 212. Jackson, W. R. and Jennings, W. B . , J Chem. Soc., B , (1969) 1221. lursik, I:., Separ. Sci.. 3 (1968) 235. Jursik. F. and Hijek, B.. Collect. Czech. Chern. Cornmun., 38 (1973) 907. Jnrsi'k, I:., Pctr8, F. a n d Ikijck, B., Proc. 3rd Co1r.f.on Coordination Chemistry. Drhrwen, 1970, Vol. 2, A k a d h i a i Kind&, Budapest, p . 251. Jursik, F., S$korovi, D. and Hijck, B., unpublished results, 1972. Jursik. I;.. Wollnianovk D. and Hijck, 8.. Collect. Czech. Chem. Cornrnun.. 38 (1973) 3627. Kaufhiian. G . B., Pinncll, K . P. and Takahashi, L. F., Inorg. Chern.. I (1962) 544. Kccne, I:. K. and Searle, C . H., Inorg. Chem., 11 (1972) 148. Kccnc. F. R.. Searle, C . H., Yoshikawa. Y., Imai, A. and Yamasaki, K.. Chem. Comnzitn.. (1970) 784. Kcrtcs, S. and Ledcrcr, M.,Anal. Chim. Acta, 15 (1956) 543. King, E. L. and Waltcrs. R. R.. J. Amer. Chem. Soc., 74 (1952) 4471. King. K. B., Houk. L. W. and Pannell, K. H:. Inorg. Chem., 8 (1969) 1042. Koguchi. K.. Waki, H. and Ohashi, S., J. Chromatogr., 25 (1966) 398. Kojirna. Y.and Shibata. M.,fnorg. Chem.. 9 (1970) 238. Kojima. Y. and Shibata, M.-Inorg. Chem.. 10 (1971) 2382. Korkisch. J . . Separ. Sci., 1 (1966) 159. Korkisch. J. and Klakl, ti., Talanta, 15 (1968) 339. Korkiscli. J. and Klakl. H., Tafanfa. 16 (1969) 377. Krcbs, H. and Diewald. J., Z. Anorg. Allg. Chem.. 287 (1956) 98. Krebs. H. and Raschc, K., Naturwissenschajten, 4 1 (1954) 63. Kuhn, G. and Hoyer. E., Z. Anal. Chem., 228 (1967) 166. Kuroda, R., Ishida, K. and Kiriyama, T., Anal. Chem., 40 (1968) 1502. Ledercr, M. and Ossicini, L . . J . Chromatogr., 13 (1964) 188. Lcgp, J. I., Chem. Commun.. (1967) 675. Lcgg, J. 1. and Cooke. D. W., h o r g Chem., 4 (1965) 1576. Legg, J. I. and Cooke. D.W.. Inorg. Chem., 5 (1966) 594
REFE;RENCES
1113
Lcgg. J . 1. and Douglas. B. IT.. Inorg. Chmi., 7 (1968) 1452. Lindqvist. 8 . . Acra Chern. Scarid.. I 6 ( 1 962) 1794. Linhard. M., Weigcl. M. and I:lygarc, H . . Z . Anorg. Allg. Chun., 263 ( 1 9 5 0 ) 233. Marcus, Y.and Lyal, L.,.1. Inorg. Nucl. Cbeni., 32 (197U) 2040. Marcus, Y . and Kertcs, A . S., lo17 l?xcbnnge andSolvent Extraction ofMetal Complexes, Wilcy. New York. 1969. Markovii. V. and Scliwcitzer. G. K.,J. Inorg. Nr1c.l. Chem.. 3 3 (1971) 3197. Maryuarding, D., Klucazck, H.. Gokcl. C.,Hoffmann, P. and Ugi, I . , J. Amcr. Clicni. Soc., 9 2 ( I 070) 5389. Martin. A . J . P. and Syngc. R. L. M., Bioheni. J . , 35 ( 1 9 4 1 ) 1358. I r t . D. L . and B o s m r t . W.. .I.Chromafogr..32 ( 1 9 6 8 ) 195. Matsuoka. N . . Hidaka. 1. and Shimura. Y.. Birll. Chem. Soc. Jap.. 40 (1967) 186X. Matwoka, N.. Hidaka, J . and Shimura, Y.. Inorg. Cbem.. 9 (1970) 719. Mulnir. F..Horwith. A. and Khalkin, V. A.,J. Chrornarogr., 26 (1967) 225. Muzarelli, R. A. A.. Aduan. Cbromatogr., 5 (1968) 127. Nagai. Y . , Hooz. I . and Bcnkescr, R. A., Bull. Cliern. Sor. J a p , 37 (1964) 53. Mcddcrmcycr, P. 4..Diss. Ahstr., B , 29 (196X) 2782. Ncddcrmeycr. P. A. and Rogers. t.. 8.. Anal. Chem.. 41 ( I 969) 94. Nesmcyanov, A . N.. Polovyanyuk. 1. V., Lokshin, 8. V., Makarova, L. G. and Chapovskij, V. A.. Z h . Ohsbch. Khim., 37 (1967) 2015. Ncumann, 9. I;.. Paxson. J. R. and Cuinmishey. C. J., J. Inorg Nucl. Chern., 30 (1968) 2243. Nickless, G . , Lob. Pract., 16 (1967) 1238. Nicklens, G..Advan. Cliromatogr.. 7 (1968) 121. ’ Nugcnt, M . J., Kummer. R. and Richards. J. H..J. Arner. Chem. Soc.. 91 (1969) 6141. Ohashi. S.. Tsuji, N., Ucno. Y.,Takeshita, B. and Muto, M.,J. Cbrornatogr.. 50 ( 1 970) 349. O’Laughlin, J. W. and Jcnsen, D. F., J. Cbromatogr., 32 (1968) 567. Orlandini, K. A.. Inorg. Nucl. Cbern. Lett.. 5 (1969) 325. Ortner, H. M. and Pachcr, 0..J. Chromatogr., 71 (1972) 55. Ossicini, L. and Lederer, M., J. Cbromatogr., 17 (1965) 387. Pearson. R. G., Henry. P. M . and Basolo, F., J. Arner. Cbem. Soc., 79 (1957) 5379, 5382. I’ecsok. R. L. and Saundcrs, D., Separ. Sci., 3 (1968) 325. Piper, T. S., J. Amer. Cbem. Soc., 8 3 (1961) 3908. Pollard, F. H., Nickless, G . and Murray, J. D., J. Clirornarogr., 27 (1967) 271. Pollard, I:. H., Nickless, G . and Rothwell, M. T., J. Cliromatogr., 10 (1963) 212. Pollard, F. I%, Rogers, D. E., Rothwcil, M. T. and Nickless, G., J. Cbromotogr., 9 (1962) 227. Reich-Rohrwig, P. and Schlogl. K., Monntsb. Cbem., 99 (1968) 1752. Reinehart. A. B.. J. Amer. Chem. Soc., 79 (1957) 5749. Roscnblum. M. and Woodward, R. B., J. Amer. Cliern. Soc., 80 ( I 958) 5443. Saundcrs, D. l,.,Diss.Absrr.. B , 29 (1969) 2783. Schlogl, K. and Fried, M.,Monats/i. Giem., 95 (1964) 558. Schliigl. K. and Mechtler, H.. Angew. Oiem., 78 ( I 966) 606. Schmitt, D. H. and Fritz, I . S . , Talanta, 1 5 (1968) 515. Schoenberg, L. N., Cooke, D. W. and Liu, C . F., Inorg Cbem., 7 (1968) 2386. Schwab, G. M. and Jockers, K., Angew. C b e m , 5 0 (1937) 546. Shcrma, J . , J . ChromatoEr., 26 (1967) 327. Shibata. M . . Nishikawa. J . and Nishida, Y., Bull. Cliem. Soc. Jap., 39 (1966) 2310. Shibata, M., Tanabe. I., Okuda, K. and Kadota, K . , Bull. Cbern. SOC.Jap., 4 1 (1968) 2627. Shiga. M., Tsunashima, M.. Konu, H., Motoyama, I. and Hata. K.,BuZl. Cbem. Soc. Jap., 4 3 (1970) 841. Smith, A. A. and Haines, R. A., J. Amer. Cbem. SOC.,91 ( 1 969) 6280. Springer, Jr., C. S., Feibush, B. and Sievers, R. E.,Inorg. Cbern.. 10 (1971) 1242. Strelow, F. W. E., Coetzee, J. H. J. and Van Zyl, C. R.. Anal. Cbem., 40 (1968) 197. Strelow, F. W. I:., Van Zyl, C. R. and Bothma, C. J . C., Anal. Cbim. Acra, 4 5 (1969) X I . Strclaw. F. W. E., Victor, A. H., Van Zyl. C. R. and Eloff. C., Anal. Chem., 4 3 (1971) 870.
1114
INORGANIC, COORDINATION AND ORGANOMETALLIC COMPOUNDS
Strculi, C. A. and Rogers, L. B., Anal. Chem., 40 (1968) 653. Taylor, L. F. and Busch, D. H., J. Amer. Chem. Soc., 89 (1967) 5372. Tiethof, J. A. and Cooke, D. W., Inorg. Chem., 11 (1972) 315. Tobe, M. L. and Watts, D. W., J. Chem. Soc., London, (1962) 4614. Tobe, M. L. and Watts, D. W., J. Chem. Soc., London, (1964) 2891. Tsitovich, J. K., Izv. Vyssh. Ucheb. Zaved., Khim. Khim. Tekhnol., 11 (1968) 758;C.A.. 69 (1968) 99730~. Tustanowski, S . , J. Chromatogr., 31 (1967a) 268. Tustanowski, S., J. Chromatogr., 31 (1967b) 270. Ueno, Y., Yoza, N. and Ohashi, S., J. Chromatogr., 52 (1970a) 321. Ueno, Y., Yoza, N. and Ohashi, S., J. Chromatogr., 52 (1970b)469. Vanderdeelen, J., Anal. Chim. Acta, 49 (1970) 360. Veening, H., Greenwood, J. M., Shanks, W. H. and Willeford, B. R., Chem. Commun., (1969) 1305. Wehner, H. W., Fischer, E. 0. and Miiller, J., Chem. Ber., 103 (1970) 2258. Westman, B. C. and Reinehart, A. B.,Acta Chem Scand, 16 (1961) 1199. Winget, J. 0. and Lindstrom, R. S., Separ. Sci.,4 (1969) 209. Wbdkiewicz, L. and Dybczyikki, R., J. Chromatogr., 32 (1968) 394. Yamada, S., Hidaka, J. and Douglas, B. E., Inorg. Chem., 10 (1971) 2187. Yoza, N. and Ohashi, S., J. Chromatogr., 41 (1969) 429.
Chapter 52
Isotopes and radioactive compounds J. DRSATA and I. M. HAIS
CONTENTS Isotopic effects in liquid column chromatography ................................... Separation of radioactive substances .............................................. References .................................................................
1115
1125 1125
ISOTOPIC EFFECTS IN LIQUID COLUMN CHROMATOGRAPHY There have been relatively few reports in recent years describing even the partial separation of chemically identical substances that differ only in atomic or molecular weight. Thus Knyazev el al. applied the “stationary zone” method of Spedding et al. to the separation of the isotopes of iron-54 to iron-58 on a column of the cation-exchange resin Dowex 50-X4 (H?).When 15% citric acid buffered with ammonia to pH 3.5 was used, the head fraction was enriched with iron-54. With 0.75 N hydrofluoric acid and 0.75 N ammonium fluoride solution, the head fraction was enriched with iron-58. The isotopes in question have been differentiated by mass spectroscopy. The effect of the isotopic composition of the solutes on their ion-exchange behaviour has been noted for 3H-labelled compounds (Gottschling and Freese) and I4C-labelled compounds (Piez and Eagle 1955, 1956). This observation was confirmed by Gaitonde and Nixey, who reported the enrichment of 14C-labelledamino acids in the tail fraction on
EP
1.0
Glu
m W
u z
4
m
GlY
0.5
s 0
4
Ala
52 127
38
54
145
r
l
60
70
l
l
80
90
204
I
l
l
l
l
I
I
1
100
110
120
130
140
150
180
170
FRACTION NUMBER ( 1
ml
1
Fig. 52.1. Resolution of I4C- and ‘2C-labelledamino acids on Chromobeads. A cation-exchange resin by elution with lithium citrate buffer, pH 3.0 (Gaitonde and Nixey). The numbers of the elution profile indicate the specific radioactivity of the amino acid in the fraction as a percentage of the mean specific radioactivity of that amino acid. The mean specific radioactivities (dpm/pmole) of each amino acid determined by pooling all fractions representing that amino acid were as follows: aspartate, 24,790; threonine, 13,820; serine, 7830; glutamate, 14,820; glycine, 13,260; and alanine, 26,210.
References p . I 1 25
1115
TABLE 52. I SEPARATION OF RADIOACTIVE SUBSTANCES NAA = neutron activation analysis Separated substances or nuclides
purpose
Column
Mobile p h a x
References
\ o l e \ on tIc.rcL1l~lllC I
z
U
SEPARATION OF RADIOACTIVE SUBSTANCES
1125
cation-exchange resins (Fig. 52.1) and in the head fractions on anion-exchange resins. If this phenomenon is not borne in mind and specific radioactivities are estimated from individual fractions, gross errors may ensue. It is therefore necessary to pool all of the fractions under the peak for the estimation of specific radioactivity. It is possible that the gain in resolving power (increase in the number of theoretical plates per column) that has been achieved in liquid column chromatography recently and is still increasing, will, within a short time, be reflected by more success in separations based on minute differences in distribution coefficients between substances that differ in isotope composition.
SEPARATION OF RADIOACTIVE SUBSTANCES In this book, most of the separations of radioactively labelled compounds have been covered in the various chapters in the special part, according to the chemical nature of the compounds (mostly organic). Applications to organic compounds and biochemicals are illustrated by several examples at the end of Table 52.1. Most of the other examples included in Table 52.1 belong to the realm of radiochemistry. The products of nuclear fission have been separated by means of LCC. Neutron activation analysis (NAA) has often been supplemented by chromatographic separation, either before activation (Morgan e l d.)in order to remove components (often those which occur in bulk) that might interfere in the interpretation of the NAA results or, more frequently, after activation. In some instances, chromatography has been used to separate unstable products that have then been subjected to further chromatographic analysis. NAA is involved in a substantial part of Table 52.1. In experiments with 3H- or I4C-labelledsubstances, the position of 3 H , 0 or 14C0, (HCO;), respectively, on the elution curves should be established in order to avoid misinterpretation of the chromatograms.
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1126
ISOTOPES AND RADIOACTIVE COMPOUNDS
Hellung-Larsen, P. and Frederiksen, S., Anal. Biochem., 40 (1971) 227. Huber, J. F. K. and Van Urk-Schoen, A. M., Anal. Chim. Acta, 58 (1972) 395. Joon, K., den Boef, R. and de Wit, R., J. Radioanal. Chem., 8 (1971) 101. Joshi, B. D., Patel, B. M. and Page, A. G., Anal. Chim. Acta, 57 (1971) 379. Keenan, R. W., Zishka, M. K. and Nishimura, J. S., Anal. Biochem., 47 (1972) 601. Knyazev, D. A., Dobizha, E. V. and Klinskii, G. D., Isotopenpraxis, 6 (1970) 130. Krtil,J., Bezd&k, M. and Mencl, J., Radioanal. C h e m , 1 (1968) 369. Kulesza, A., Nukleonika, 14 (1969) 216. KyrS, M. and Kadlecovi, D.,J. Radioanal. Chem., 1 (1968) 103; C A . , 68 (1968) 111002a. Lee, H. M., Anal. Chim Acta, 41 (1968) 431. Liessens, J., Dams, R. and Hoste, J.. Anal. Chim. Acta, 45 (1969) 213. Loomeijer, F. J. and Kroon, A. M., Anal. Biochem., 49 (1972) 455. Lowe, J. T., US.A t . Energy Comm.,1969, Rep. No. DP-1194, 29 pp.; C.A., 72 (1970) 27327k. Machiroux, R and Mousty, F., Anal. C h i n Acta, 42 (1968) 371. Massart, D. L. and Hoste, I., Anal. Chim. Acta, 42 (1968) 15. Moller, P., 1.Inorg. Nucl. Chem., 32 (1970) 2413. Morgan, D. J., Black, A. and Mitchell, G. R., Analyst (London), 94 (1969) 740. Natsume, H., Umezawa, H., Suzuki, T., Ichikawa, F., Sato, T., Baba, S. and Amano, H., J. Radioanal. Ozem., 7 (1971) 189. Neirinckx, R., Adams, F. and Hoste, J., Anal. Chim. Acta, 43 (1968) 369. Neirinckx, R., Adarns, F. and Hoste, J., Anal. Chim. Acta, 46 (1969a) 165. Neirinckx, R., Adams, F. and Hoste, J., Anal. Chim. Acta, 48 (1969b) 1. Neirinckx, R. D., Anal. Chim. Acta, 58 (1972) 237. Norton, E. F. and Stoenner, R. W., Anal. Chim. Acta, 55 (1971) 1. Piez, K. A. and Eagle, H., Science, 122 (1955) 968. Piez, K. A. and Eagle, H., J. Amer. Chem. Soc., 78 (1956) 5284. Pinajian, J. J . and Raman, S . , J. Inorg. Nucl. Chem., 30 (1968) 3151. Porcellati, G. and Di Jeso, F., J. Label. Compounds, 3 (1967) 206. Sebesta, F . , J . Radioanal. Chem., 7 (1971) 41. Souliotis, A. G., Analyst (London), 94 (1969) 359. Spedding, F. H., Powell, J. E. and Svec, H. J.,J. Amer. Chem. SOC.,77 (1955) 6125. SpkviEkovi, V. and Kiivinek, M . , Radiochem. Radioanal. Lett., 3 (1970) 63. Strand, L. J., Swanson, A. L., Manning, J., Branch, S. and Marver, H. S., Anal. Biochem., 47 (1972) 457. Talvitie, N. A., Anal. Chem., 43 (1971) 1827. Testa, C., Anal. Chim. Acta, 50 (1970) 447. Wakat, A. and Griffin, C., Radiochem. Radioanal. Lett., 2 (1969) 351. Wong, K. M., Anal. Chim.Acta, 56 (1971) 355. Wood, A. W., McCrea, M. E. and Seegmiller, J. E., Anal. Biochem., 48 (1972) 581. Zappacosta, S. and Rossi, G., Immunochemistry, 4 (1967) 122. Zhukovskii, Yu. G., Katykhin, G. S., Martynov, A. L. and Nikitin, M. K., Radiokhimiya, 10 (1968) 252. Zirna, S. and Giacintov, P.,J. Radioanal. Chem., 7 (1971) 19. Ziv, D. M., Shestakov, B. I. and Shestakova, I. A., Radiokhimiya, 10 (1968) 738.
Subject index*9** A ABMC, see rn-Aminobenzyloxymethylcellulose Absorbance 148 Absorption maxima of metalloproteins 803 Acetic acid-aniline-orthophosphoric acid method 482,483 Acidflex 477,539,541 Activity coefficient 46, 50, 51, 235, 236 Activity of sorbents, determination 290, 291 Adsorbent(s) 174-182 Adsorbent activity 177, 178 Adsorbents, physical characteristics 174-176 Adsorbents with non-porous support 175 Adsorption 176-179 Adsorption centres 177,178 Adsorption chromatography 6, 37 Adsorption distribution constant 23, 253 Adsorption energy 93,94, 252 Adsorption isotherm 93,94, 176, 177 Adsorption isotherm for affinity chromatography 93,94 Adsorption kinetics, one-site 37 Adsorption layer, interfacial 22 Adsorption potential 177 AE-Cellulose, see Aminoethylcellulose Aerogels 189 Aerosil788 Affinant 89,92-94,215-227 Affinant binding 94-96,369,370 Affinant capacity 95,96 Affinant coupling 220-223 Affinity chromatography 89-97, 369-376, 799,800 Affinity chromatography, elution 372-375 Affinity chromatography, practice 369-376 Affinity chromatography, preservation of sorbents 375, 376 Affinity chromatography, principles 89-91 Affinity chromatography, sorbents 21 5-227 Affinity chromatography, sorption conditions 370-372 Affinity sequences 76 Agar 192,194 Agarose 194- 196,219-223 Agarose-acrylamide gels 196 Agarose-bound antigens 223 Agarose-bound enzymes 222
Agarose-bound haptens 223 Agarose-organomercury derivatives 799 Agarose, stability 222 Agarose with bound antibodies 799 Agarose with bound concanavalin A 799 Agarose with bound soya bean trypsin inhibitor 89,90 Agarose, with covdently bound pyridoxamine phosphate 81 8 Aggregation of the phases 5 Alamine336 932 Albumin, methylated 827 Albumin-Kieselguhr, methylated 862 Alcoa F-20 632 Alginic acid (sorbent) 643- 645 Alumina 180,181,184 Alumina activity determination 290, 291 Alumina, impregnated with silver nitrate 624, 625 Alumina, impregnated with sulphoxide 634 Alumina, porous 202 Alumina-Byflo Supercel596 Amberlite 326,327,330-333,336-338,340, 341,344-347, 354 Amberlite LA 601 kmberlyst 336, 337 Aminex 340-343 Amino acid analysis, computerization 687, 688 Amino acid analyzers 675-688 Amino acid analyzers, Beckman-Spinco 682,683 Amino acid analyzers, Bender-Holbein 685 Amino acid analyzers, Carlo Elba 685 Amino acid analyzers, Durrum 686,687 Amino acid analyzer, Jeol Model JLC-6AH 684 Amino acid analyzers, LKB 684 Amino acid analyzers, Mikrotechna Model AAA 881 685,686 Amino acid analyzers, non-automated 676 Amino acid analyzers, Perkin-Elmer 685 Amino acid analyzers, Phoenix 685 Amino acid analyzers, Technicon type 680-683 Amino acid standards 696,697 pAminobenzylcellulose 21 8, 350 rn-Aminobenzyloxymethylcellulose 2 18 Aminoethylcellulose 21 8, 350 Ammonium molybdophosphate 204,352
*Compiled by H. BeEvlibvl. **For some general terms which occur throughout the book, reference is given only to the page where the particular term is explained or t o the chapter or paragraph dealing with the term in question. Sorbents are referred to without further specification (particle sue, degree of cross-linking, etc.). All specifications available to us are summarized in tables in the part edtitled Practice of liquid chroma-. tography. 1127
1128 Amphoteric ion-exchangers 70 Anaerobic column system 808 Anaerobic laboratory 809 Anakrom AB diatomaceous earth 601 Analcite 203 Analytical property of system 146,148 Analyzers, non-automated 676 Anion exchangers 70 Anthrone-sulphuric acid method 476,477 Anthrone-sulphuric acid method, automated 526 Anti-DNP antibody as column packing 769 Antimicrobial agents for mobile phase 266, 267 Antinitrotyrosine-Sepharose 768 Apatite 203 Apophyllite 203 Apparatus for gel chromatography 304-311 Apparatus for liquid chromatography, operation 283-300 Apparatus for liquid chromatography, preparation 283-285 Apparatus for liquid chromatography, schematic diagram 284 Application of sample 111,112, 139-143, 145, 295-297,306,307,360-363 Aquapak 198,199,473,525 Argentation chromatography 85, 595,596,604, 626 ARW-7 Detergent 538 Autograd 499 Automated chromatography, DNP-amino acids 720-726 Automated chromatography, Dns-amino acids 727-731 Automated chromatography, enzymes 81 1 Automated chromatography, nucleic acids conponents 836-839 Automated chromatography, peptides 746 Automated chromatography, polynucleotides 880 Automated detection methods 475 Automated separation 637,638 Automatic evaluation of chromatograms 399 Automatic programmer 676 Automatic sample introduction devices 121, 142,143 Automation of determination 116 Axial diffusion 60, 61
SUBJECT INDEX
BAC, see Bromoacetylcellulose Basic processes in chromatography 11-23 BD-Cellulose, see Benzoyl-diethylaminoethylcellulose Bead form of ion exchangers 75 Beckman carbon analyser 437 Beckman resin 342,343,491 Bed, chromatographic 5 , 6 Bed, cylindrical, equation 14 Beidellite 203 Benzoyl-diethylaminoethylcellulose350, 870, 87 5 Benzoyl-naphtho yl-diethylaminoet hylcellulose 350,875 Bessel function 30 Binary single-phase liquid system 21 Bioaffiiity chromatography, see Affinity chromatography Bio-Beads 198,199,421 Bioclar G 966 Bio Deminrolit 338 BioGel 190, 342 Bio-Gel A 195, 222 Bio-Gel CM 225 Bio-Gel P 196,225 Bio-Gel with bound 3’-(4-aminophenylphosphoryl)deoxythymidine-5’-phosphate 95 Bio-Glass 201 Bio-Rad 326,327,332,333,336-338,340344,346-348,352 Bio-Rex 326-331,334,335,338, 344-347 Biuret reaction 801 BND-Cellulose,see Benzoyl-naphthoyl-diethylaminoethylcellulose Boundary conditions of elution chromatography 29 Break-through curve 80 Brij-35 499,693 Bromoacetylcellulose 218 Brushite 342, 343 Brush-type sorbents 187 Bubble flow meter 119 Bubble-trap 108 Buffering of ion exchangers 355 Buffers for amino acid analysis 692-695 Buffers, preservation 695 Buffers, volatile 265, 266
SUBJECT INDEX
1129
C
Charcoal-Celite mixture 483,485,486,495,
Calcium phosphate 342,529,788,817 Calibration curve 64 Calibration of GPC systems 58,63-65 Calibration technique, absolute 395 Calibration, universal, for GPC 1059 Cancrinite 203 Capacitance detector 148 Capacity break-through 80 Capacity factor 236,247 Capacity of ionexchangers 74,80 Capacity of the bound affinant 96 Capacity ratio 8, 32, 124 Capillary columns 126,144,726 Carbon molecular sieves 181 Carbon-Nuchar 467 Carboraffin 467 Carboxymethoxypropyl Sephadex 600 Carboxymethylcellulose 207,348,349
Chelating ion-exchangers 70,211 Chelex 340,815 Chemical potential 48,234,235 Chemically bound stationary phases 186,187 Choice of the system 239 Chromatogram, analytical utilization 377-401 Chromatographic data handling 401 Chromatographic systems, classification 5,6 Chromatography, basic quantities 8-10 Chromatography, definition 4 Chromatography, fundamental concepts 3-10 Chromatography, history 3,4 Chromatography, invention 3 Chromatography, mechanism 4 Chromatography, non-linear 6 Chromatography, principle 4,5 Chromatography, techniques 7, 8 Chromatronix 431,432 Chromex 691 Chromic acid and carbazole assay 481 Chromobeads resin 497,498,691 Chromosorb 184,425,426 Chymotrypsin bound to Spheron 216 Citrate buffers for amino acid analysis 693,694 Classical LC 102, 123-127 Cithrate nickel 7-picoline thiocyanate 658 Clathrates 428,658 CMCellulose, see Carboxymethylcellulose CM-Sephadex 35 1 Coefficient of the mass transfer resistance in the stationary and in the mobile phase 125 Co-ions 71, 72 Colorimetry, semiautomated 674 Colorimetry equipment 672,673 Colour yields of amino acids 706 Column accessories 106-1 10 Column adaptors 108 Column, all-glass 669 Column, anaerobic 808 Columns, capillary 126, 144,726 Columns, heavy loaded 185 Column, material 143 Column, method of filling 110, 11 1, 144 Columns, segmented 113,710 Column and flat-bed chromatography, comparison 382,383 Column and flat-bed techniques, combination
496,634
Carboxymethyl-NeoCel348,349 Cation exchangers 70 Celite, siliconetreated 438 Celite, silylated 585 Celite-calcium phosphate 537 Celite-charcoal mixture 483,485,486,495, 496,634 Celite-Microcel mixture 840,843,846 Cell, forms 150 Cell volume 148,149 Cellex 348-350 Cellex AE 218 Cellex PAB 218 Cellulose coated with calcium phosphate gel
789 Cellulose derivatives, see under the names of the respective derivatives Cellulose for affinity chromatography 217,218 Cellulose gels 194 Cellulose ion-exchangers 207,348-351,367 Cellulose phosphate 348,764-766 Cellulose phosphonic acid 348 Cellulose with covalently bound antigen 91 Cellulose with covalently bound DNA 871 Cellulose with ether-bound resorcinol residues
91 Centrifugal chromatography 597 Centrifugal force in LCC 162-164, 167 Chabazite 203 Charcoal 181,182,184,467 Charcoal, elution from 254 Charcoal, graphitized 181
384-386 Column dimensions 104,105,143,144 Column dimensions, nomogram 104 Column efficiency 140,144,249-252 Column efficiency in GPC 61-63
1130 Column equilibration 770 Columns for classical LC 103-1 07 Columns for gel chromatography 307, 308 Columns for high-efficiency LC 143-145 Columns for IEC 356-360 Columns for IEC, dimensions 357 Column form 105,144 Column length, calculation 83 Column packing 110,111,144,291-295, 310,311,358-360,673,674,688,727 Column preparation 110, 111, 144,291-295 Column producers 106, 107, 110 Column regeneration 729 -731 Comparison of LC and TLC 596 Complex-forming mobile phase 267, 268 Compressibility coefficient 53 Computer program 400,401,687,688 Con A-Sepharose 222,523,799 Concavalin A immobilised on Sepharose 222,
523,799 Concentration excess of the solute component in the interfacial region 22 Concentration profile of solute 16 Conductivity detector 148,158,309 Connecting tubes 107 Continuous gel chromatography 31 1 Continuous gradient elution 670 Continuous LC 121,122 Controlled-pore glass 201 Convective transport 5 Copper-Bio-Rex 892 Corasil 184 Counter-current chromatography 1 62-167 Counter-ions 71,72,77 Cross-linking 339 Cross-linking of polydextran ion exchangers
784 Cross-linking of the matrix 70,73,74 Custom Research Resin 691 Cyanogen bromide activation 219, 220 Cycling of ion-exchanger 72, 354, 355 Cylindrical bed equation 14 Cysteine-sulphuric acid method 479,480,526 Cystine-containing peptides, detection 748,749
D Darco 467,484,486 Darco-charcoal-Celite mixture 577 Darcy law 15 De Acidite 334-337,346 Dead volume 9,126,144 DEAE-Cellulose, see Diethyhminoethylcellulose DEAE-Sephadex 351
SUBJECT INDEX
Deaeration of ion exchangers 356 Decalso 961,963 Decantation of ion exchangers 354, 355 Deflection refractometer 153 Degassing 128,129,289 Degree of branching, determination by GPC
316,317 Degree of cross-linking 73 Demixing effects 257,258 Demonstration experiment in LC 123 Denaturation of nucleic acids 860 Desalting 750,751 Desalting of nucleosides 833 Desalting of proteins 776 Detection methods, automated 475 Detection minimum of sample 148 Detection of proteins 800-805 Detector(s) 126,145-162 Detector, capacitance 148 Detector, cell volume 148,149 Detector, concentration sensitive 389,390 Detector, conductivity 148, 158,309 Detector, destructive 155, 391 Detector, disc 148 Detectors, evaluation 162 Detector, fluorescence 891 Detector, fluorimetric 148, 151 Detector, heat of sorption 148, 159, 160 Detector, IR-absorption 148,151 Detector, mass-sensitive 389,390 Detector, micro-adsorption 159,160 Detector, minimum detection of a sample 148 Detector, moving-wire 309 Detector, noise 147 Detector, non-destructive 154,389, 390 Detector, permittivity 157, 158 Detector, photometric 151 Detector, polarographic 148, 160, 161 Detector, properties 148 Detector, range of linearity 147,148 Detector, refractometric 148, 151-154 Detector, selective 384 Detector, selectivity 148 Detector, solute transport 154-156 Detector, spectrophotometer 151 Detector, temperature dependence 148 Detector, W-absorption 116,148-151, 308 Detector, wire with alkali FID 148,155 Detector, wire with FID 148,154-156 Detectorsfor classical LC 116 Detectors for GPC 308,309 Detectors for radioactive substances 161,404-
408 Detectors for radioactive substances, a-radiation
407,408
SUBJECT INDEX
Detectors for radioactive substances, y-radiation
404 Detectors for radioactive substances, GeigerMuller 404,405 Detectors for radioactive substances, liquid scintillation 406,407 Detectors for radioactive substances, semiconductor 407 Detectors for radioactive substances, solid-phase scintillation 405,406 Detector response 146-148 Detector response and solute concentration
387-391 Detector role in quantitation 388-391 Detector sensitivity 146,147 Detector sensitivity, absolute 146 Detergents 781 Detergent gradient 798 Dextran gels 193 Diagram temperature-composition of a binary system 18 Diaphragm pulse damper 139 Diatomaceous earth 184 Dielectrical constant 148,154,157 Diethylaminoethoxypropyl Sephadex 600 Diethylaminoethylcellulose 349,3 50 Differential flow meter 120 Differential form of the chromatogram 8,9 Differential refractometer 148, 151-154, 309 Diffusion 5 Diffusion, axial 60,61 Diffusion, general rules 15, 16 Diffusion, lateral 5 Diffusion, longitudinal 5, 35, 36,60,61,83 Diffusion, longitudinal, in the mobile phase 36 Diffusion, longitudinal, in the stationary phase
37 Diffusion, restricted 60 Diffusional transport 15, 16 Diffusion coefficient 17,60,125 Diffusion coefficient, eddy 36 Diffusion coefficient, effective 34 Diffusion-controlled kinetics 37 Diffusion in liquids 17 Diffusion within the phases 15-17 5-Dimethylaminonaph thalene-1-sulphonyl chloride 804 2,4-Dinitrophenylsulphenylchloride reagent
768 Dipolar ion-exchangers 70 Dipole-dipole interaction, Keesom forces 46 Disc detector 148 Dispersion forces (London) 46 Displacement chromatography 7, 8,81, 563
1131 Displacer 7 Dissociation constant 77 Distribution between phases 234 Distribution between two phases, mechanism 6 Distribution constant 5-9, 32,45,49,82,234,
254 Distribution constant, anomalies 780 Distribution constant, dependence on temperature and pressure 52,55 Distribution constant, thermodynamic 49 Distribution constant in liquid-solid system 54 Distribution constant in proteins 775,776 Distribution equilibrium 5 Distribution isotherm 6 DNA as adsorbent 868,871,873 Donnan equation 77,78,83 Dowex 326-329,334-337,340,341,344-347 Drift of the baseline 149 Drop counters 119 Droplet counter-current chromatography 163 Dry column extraction 657 Dry column technique 110,112, 113,598 Duolite 328-331,336,337,344-347,932 Durrum ion-exchangers 342, 343,691 Dye-extraction assay procedure 890,891
E ECTEOLACellulose 349,350 Effective equilibrium 83 Effective hydrodynamic volume 59,65 Effective plate number 42 Effective theoretical plates 124 Effluent 80 Effluent analysis 115-120 Einstein’s equation 16,34, 36-38 Electrolytical current 148 Electrolytic conductivity 148, 158 Electrophoresis 6 Electrostatic bonds, role in protein separation
782 Eluents for gel chromatography 303, 304 Eluotropic series 242, 254 Elution, dry column technique 112,113 Elution centrifuge 167 Elution curve, calculation 705 Elution chromatography 7,8,29, 81, 82 Elution flow-rate 11 5 Elution for amino acid analysis 674,697-702 Elution for amino acid analysis, single column system 700-702 Elution for amino acid analysis, two-column system 697-700
1132 Elution gradient 113-115 Elution techniques 112-115 Elution volume, anomalies 780 Elution volume in recycling 778 EMA, see Ethylene-maleic anhydride copolymf:I Enthalpy 47 Entropy 47 Enzacryl224, 225 Enzite-agarose 222 Enzite-CMC-hydrazide 218 Enzite-EMA 219 Epimers separation 554 Equi-eluotropic series 260 Equilibration of column 770 Equilibration of solute 31 Equilibration of solute between phases 17-23 Equilibrium constant 93,94 Equilibrium in binary two-phase liquid systems 17,18 Equilibrium in liquid-solid system 20-23 Equilibrium in ternary two-phase liquid system 19,20 Equipment for classical LC 103-110 Estradiol-Sepharose 222 Ethylene-maleic anhydride copolymer 219 Evaluation of fractions in IEC 366 Exclusion chromatography 59 External solution 79 Extinction coefficients of proteins 803 Extra-column zone broadening 42, 43
F Factise 958 Fick’s laws 16 Film diffusion 79, 82, 83 Flame ionization detector 483 Flat-bed techniques, combination with LCC 384-386 Florisil 182 Florisil, silvered 596 Florisin 889 Flow, linear 364 Flow, non-uniformity of 36 Flow, volumetric 364, 365 Flow measurement 118-120 Flow-meter, bubble 119 Flow-meter, differential 120 Flow of mobile phase 11-15, 31, 35 Flow-rate 5,115, 125 Flow-rate measurement 298,299,364-366 Flow-rate programming 129,245, 246 Fluorescein isothiocyanate 804
SUBJECT INDEX Fluorescence 148 Fluorescence labelling 804 Fluorimetric detector 116, 148, 151, 891 Folin-Ciocalteau reagent 800 Folin-Lowry method 748 Fractionation of ion-exchangers 353, 354 Fractionation of peptides 751 Fraction collectors 116-118,671 Franconite 889 Fresnel refractometer 153 Frontal analysis 81 Frontal chromatography 7 , 8 Frontal injection 257 Frontal zone 257,258 Fugacity 5 1 Functional groups of ion exchangers 208-210
G Galactomannate (sorbent) 192 Gas chromatography, as detector for LC 161, 162 Gas connection 109 Gas-flow counters 404 Gauss theorem 26 GE-Cellulose, see Guanidoethylcellulose Geiger-Muller detectors 404,405,411 Gel filtration 59 Gel packing 187-215,301-303 Gel packing, general aspects 187, 188 Gel packing, types 189-204 Gel permeation chromatographic data 312-317 Gel permeation chromatography 57-67 Gel permeation chromatography, calibration of column systems 63-65 Gel permeation chromatography, column efficiency 61-63 Gel permeation chromatography, combination with various techniques 312 Gel permeation chromatography, continuous 311 Gel permeation chromatography, physical basis of the separation process 59-65 Gel permeation chromatography, practice 301-323 Gel permeation chromatography, principle 51,58 Gel permeation chromatography, side-effects in peptide separation 752-755 Gel permeation chromatography, theory 59-65 Gel permeation chromatography, universal calibration 64 Gels 187-215
SUBJECT INDEX Gels, aerogels 189, 201, 202 Gels, calcium phosphate 529 Gels, dextran 192-196 Gels, glycol methacrylate 197 Gels, heterogenous 189-192 Gels, homogenous 189,190 Gels, hydrophilic 189,192,193,198 Gels, hydroxyalkyl methacrylate 226 Gels, organophilic 189, 198-201 Gels, polyacrylamide 196, 197 Gels, polysaccharide 192-196 Gels, polystyrene 198, 199 Gels, rigid 189, 201, 202 Gels, semi-heterogenous 189-191 Gels, semi-rigid 189 Gels, separation ranges 320 Gels, soft 189 Gels, special 198 Gels, vinyl acetate 198-200 Gels, xerogels 189 Gibbs adsorption isotherm 21,22 Gibbs free energy 45,46,48, 50, 51 Gibbs phase rule 18, 19 Glass 227 Glass, porous 201, 202 Glass with controlled pore size 781, 782 Glauconite 203 Glycol methacrylate gels 197 Glycosylex A 223,799 Gradients, classification 270, 271 Gradient, concave 130 Gradient, continuous 130,131,271 Gradient, convex 130 Gradient, discontinuous (stepwise) 130, 131, 27 1 Gradient, disproportional 272,273,276 Gradient, exponentionall30,271-275 Gradient, extended 271 Gradient, linear 130 Gradient, multicomponent 272, 27 3 Gradient, pH 276, 277 Gradient, proportional 130, 272, 273, 275, 276 Gradient, stepwise 130, 131, 271 Gradient apparatus 114, 129,720 Gradient calculation 270-277 Gradient chromatography 129 Gradient elution 113-115,246-248, 363, 364 Gradient elution, theory 277 Gradient formation 271 -273 Gradients for peptide separations 767 Gradient technique 148 Grading device 287-289 Grain size 75, 353 Graphite 202 Gravity 12 Group contributions to the distribution constant 380-382 Guanidine in eluent 743,751,764,780 Guanidoethylcellulose 349
1133
H Haeviside unit step function 30 Half-exchange time 79, 80 Hamilton resin 691 Hamilton’s hydraulic method 353, 354 Harmotome 203 Heating-baths 672 Heat of sorption detector 148, 159, 160 Height equivalent to a theoretical plate 10, 33, 82,83,125,126,144,149,310 Helix counter-current chromatography 162-165 Henry’s law 50 HETP,see Height equivalent to a theoretical plate Heulandite 203 High-efficiency LC 102, 123-127 High-efficiency LC, techniques 123-1 67 High-speed LC 40 Histone-Kieselguhr 861 History of chromatography 3 , 4 Homoionic ion-exchanger 71 Hostalen 618 Hostalen, purification of 599 Hydrodynamic separation mechanism 62 Hydrodynamics of the mobile phase 11 Hydrodynamic volume, effective 59, 65 Hydrodynamic volume parameter 1065 Hydrogen bonding 47 Hydrophobic binding 534 Hydrophobic contact 531 Hydrophobization of Kieselguhr 598 Hydrophobization of Sephadex 599 Hydroxyalkoxypropyl Sephadex 599 Hydroxyalkyl methacrylate gels 226 Hydroxyalkyl methacrylate gel with bound chymotrypsin 91 Hydroxyapatite 203, 343, 788, 789, 813, 814, 817 Hydroxyapatite, preparation of 862 Hydroxyapatite chromatography, mechanism 865 Hydroxypropyl Sephadex 598,599 Hyflo Supercel, siliconized 576 Hyperchromic effect 860
I Ideal chromatographic column, behaviour 30 Ideal linear chromatography, concept of 31-33 Identification 37 7-386 Identification by means of retention data 378380 U t e s 202 Imac 345-347 Indicator-Bio Deminrolit 338
SUBJECT INDEX
1134 Induction (Debye) forces 47 Influent 80 Injection port 139-144 Instrumentation for LC 101-168 Integral time of the chromatogram 8 , 9 Integrators 399,400 Interaction s o h te- phases 45 -48 Interactions solute-solvent 6 Internal standard technique 396 Intramedic PE 240 539 Intrinsic viscosity 65 Introduction of sample 111, 112, 295-297, 306,307,360-363 Ion exchange 69-87 Ion exchange, affinity 75,76 Ion exchange, amphoteric ions 76 Ion exchange, equilibrium 75-80 Ion exchange, kinetics 77-80 Ion exchange, non-aqueous solution 85, 86 Ion exchange, reactions 75 Ion exchange, selectivity 76 Ion-exchange chromatography, column operation 80-83 Ion-exchange chromatography, elution 363,364 Ion-exchange chromatography, fundamentals 69-87 Ion-exchange chromatography, mobile phase 261-269 Ion-exchange chromatography, practice 325 368 Ion-exchange chromatography, principles 69-72 Ion-exchange chromatography, terminology 69-72 Ion-exchange crystals 204, 352 Ion-exchange particle 79 Ion-exchange potential 76 Ion exchanger(s) 69-75,202-215 Ion exchanger, amphoteric 70, 71 Ion exchanger, anion 70,73 Ion exchangers, buffering 355 Ion exchanger, capacity 74 Ion exchanger, cation 70,73 Ion exchanger, cellulose 348-351, 367 Ion exchanger, characterization 73-75 Ion exchanger, chelating 70,71,211 Ion exchangers, choice 325-353 Ion exchanger, classification 70,73 Ion exchangers, columns 356-360 Ion exchangers, cycling 354,355 Ion exchangers, deaeration 356 Ion exchangers, decantation 354,355 Ion exchanger, dipolar 70,71 Ion exchangers, filling of columns 358-360
Ion exchanger, functional groups 208-210 Ion exchangers, grain size 35 3 Ion exchanger, homoionic 7 1 Ion exchangers, inorganic 202-204 Ion exchangers, liquid 215 Ion exchangers, macroreticular 213,214 Ion exchangers, methods for fractionation 353, 354 Ion exchanger, monofunctional 71 Ion exchangers, organic 205-215 Ion exchangers, packing 688-692 Ion exchanger, particle form 75,212-215 Ion exchanger, particle size 75, 82 Ion exchangers, pellicular 213,214 Ion exchanger, polydextran derivatives 207, 208, 351,367 Ion exchanger, polyfunctional71 Ion exchanger, porosity 73, 74 Ion exchanger, porous form 212-215 Ion exchangers, purification procedure 689 Ion exchangers, redox 21 1 Ion exchangers, regeneration 366-368 Ion exchanger, selective 70,71 Ion exchangers, solid 21 2 Ion exchangers, special 210-212, 340, 350 Ion exchanger, spherical beads 212, 21 3 Ion exchangers, storage 366-368 Ion exchanger, swelling 73 Ion exchanger, titration curves 74 Ion-exchange resins 207 Ion-exchange resins, interchangeable 344- 347 Ion exclusion 83, 84 Ionization current 148 Ion retardation 84 Ion-sieve process 83, 84 IR-Absorption detector 148, 151 IR-Spectrometer 309 Isoelectric fractionation of proteins 783 Isoelectric point of proteins 783
K Kaolin 889 Kaolinite 203 Kieselguhr, acid-washed 7 16 Kieselguhr, hydrophobization of 598 Kieselguhr, methylated albumin-coated 863 Kieselguhr, poly-L-lysinecoated 862, 863, 867 Kieselguhr, siliconized 598 Kieselguhr with bound hexamine cobalt (11) salt 868 Kavits retention index 382 Kazeny-Carman equation 15,126 KU-2 cation-exchanger 438, 548
SUBJECT INDEX
L Langmuir adsorption isotherm 22 Lewatit 328-331,334-337,345-347 LFS pellicular anion-exchange resin 663 Ligand-exchange chomatography 85 linear chromatography 6 Linear chromatography, ideal, concept of 31-33 Linear flow 364 Liquid chromatograph 127-167 Liquid chromatograph, scheme, 127, 128 Liquid chromatography, classical 102-1 23 Liquid chromatography, continuous 121,122 Liquid chromatography, coupling with other analytical methods 384-386 Liquid chromatography, high-efficiency 123167 Liquid chromatography, high-speed 40 Liquid chromatography, instrumentation 101168 Liquid chromatography, preparative 120-122 Liquid chromatography, quantitation 386-401 Liquid chromatography, techniques 101-414 Liquid chromatography-mass spectrometry 162 Liquid ion exchangers 215 Lithium buffers for amino acid analysis 693, 694 Loading of sample 360,361 Locular counter-current chromatogaphy 164167 Longitudinal diffusion 5, 35, 36, 60, 61, 83 Longitudinal diffusion coefficient 125 Low-activity sample 409, 410 Lowry method for protein detection 800
M Macroreticular resins 74, 85, 213 Magnesium oxide 182 Magnesium silicate(s) 182 Magnesium silicate, hydrated 897 Magnesium trisilicate 182 Magnesol 182, 897 Manual evaluation of chromatograms 398 Mass transfer in mobile phase 38 Mass transfer resistance coefficient 250 Mass transport, convective solute 11 Matrix of ion-exchanger 69,73 Merckogel 190, 199, 200 Merckogel SI 202 Mesh number 286 Mesh screens 75
1135 Methacrylate gels 226 Methyl Sephadex 599 Micro-adsorption detector 159, 160 Migration of the zone 4-7,32 Migration velocity 32 Miscibility of phases 245 Mixed bed resins 211 Mixers 670 Mixers, multi-chamber 671 Mixing coil 5 38 Mobile and stationary phases equilibrium 234-238 Mobile phase(s) 5 , 7 , 8, 233-280 Mobile phase, antimicrobial agents 266, 267 Mobile phase, antioxidants 267 Mobile phase, complex forming 267, 268 Mobile phase, cross-section 9 Mobile phase, detergents 267 Mobile phase; hydrodynamics 11 Mobile phase, ionic strength 261-263 Mobile phase, pH 26 1-263 Mobile phase, programming 245-248 Mobile phase, properties 248-252 Mobile phase, requirements 248, 249 Mobile phase, reservoirs 127-129 Mobile phase, selectivity 258-261 Mobile phase, temperature 269 Mobile phase, viscosity 25 1 Mobile phase, volume 9 Mobile phase flow 35 Mobile phases for ion-exchange chromatography 26 1-269 Mohile phase strength 252-259 Moderator 177, 178 Molar extinction coefficient 802 Molecular convection 25 Molecular diffusion 25 Molecular sieve chromatography 3 17-321 Molecular sieves, application in amino acid analysis 692 Molecular size, separations according t o 578 Molecular weight, polydispersity 316 Molecular weight, retention volume dependence 58 Molecular-weight determination 312, 313 317-321, 5 2 6 , 5 2 7 , 5 9 0 , 7 7 7 , I 7 8 , 8 6 2 , 8 7 3 Molecular-weight distribution 62 Molecular-weight distribution, calculation 313-316 Molecular-weight effect of phases 237, 238 Molecular-weight heterogeneity of nucleic acids 86 1 Montmorillonite 203 Moving-wire detector 309
,
1136
N Nalcite 345-347 Naphthoyl-diethylaminoethylcellulose 870 Natrolith 203 Negative sorption effect 568,569 Nernst fdm 79 Ninhydrin detection of peptides 745, 746 Ninhydrin method, fluorescent 748 Ninhydrin reagent, preparation 695,696 Nitrocellulose 861, 862, 866 Nitro-y-globulin-Sepharose 768 Noise 147-149 Nomogram for column dimension 104 Nomogram for flow calculation 104 Non-electrostatic binding of proteins to ion exchangers 531 Non-equilibrium in the interparticle mobile phase 38 Non-equilibrium in the intraparticle mobile phase 38 Non-equilibrium in the sorbent 37 Non-linear chromatography 6 Norit P 467 Normalization technique 398 Nucleoside analyser 842 Number of theoretical plates 10, 35, 124, 125 Nylon 717, 725,126,903
0 Obstructive factor 37, 39 Open system 779 Optical density in proteins 803 Optically active resins 212 Optical rotation 475 Orcinol-sulphuric acid method 476 Organomercurial adsorbents 769 Organophilic gels 198-201 Ostion 691 Overlayering of sample loading 361, 362 Oxalic acid impregnation 461 p,p'-Oxydipropionitrile 433,434
P PAB-Cellulose, see p- Aminobenzylcellulose Packed bed, porosity 13, 14 Packed column, flow of mobile phase 11-15 Packed column, hydrodynamic properties 1 3 Packing of column 110,111,144,291-295, 310,311,358-360,673,674,688,727
SUBJECT INDEX
Packing of column, dry 110,111 Packing of column, wet 111 Particle diameter 125, 174 Particle diffusion 79, 80,82, 83 Particle form of ion-exchangers 75 Particle size 82, 285-290 Particle size of ion-exchangers 75 Partition chromatography 6, 37 Partition chromatography on ion-exchangers 83,84 Partition coefficient 31, 58 Peak areas 7 Peak broadening 126 Peak capacity 42 Peak maxima 7 Peak spreading 6 1 PEICellulose, see Polyethyleneimine-cellulose Pellicular ion-exchange resins 21 3 Periodate oxidation method 480,481 Perlon 903,904 Permaphase ETH 431,432,661 Permaphase ODS 425 Permeability constant 126 Permittivity detector 157, 158 Permutit 338,345-347 pH, role in separation of proteins 783, 785 pH gradients 276,277 Phase diagram of a binary two-phase liquid system 18 Phase equilibria 124 Phenol reagent, see Foline-Ciocalteau reagent Phenol-sulphuric acid method 477,478 Phosphocellulose, see Cellulose phosphate Phosphonic acid cellulose 348 Photometric detectors 151 Picric acid (sorbent) 418 Picric acid with alumina as sorbent 418 pK,' values 835 Planar chromatography 6, 8 , 9 Plaskon CTFE 601,605 Plate height contributions 39 Plate model, representation of 34 Polarity of a liquid phase 239 Polarographic defector 148, 160, 161 Polarography 658 Polyacrylamide gels 196, 197, 223-226 Polyacrylamide gels, binding of proteins 224-226 Polyacrylamide gels, stability 225 Polyamide 663,727 Polycar 903,912 Polydextran, ion-exchange 207,208, 351, 367 Polyethylene glycol lauryl ether, see Brij-35 Polyethyleneimine-cellulose 349, 350
SUBJECT INDEX
Polyethylene powder 618 Polylysine-Kieselguhr 862, 863, 867 Polysaccharide gels 193-196 Polystyrene gels 198, 199 Polyvinyl acetate gels 198-200 Poly-N-vinylpyrrolidone (sorbent) 834, 840, 903,909,911 Polyvinylpyrrolidone Polycar 9 11 Poragel 190, 198, 199 Poragel PN 601, 619 Porapak 423 Porasil202 Pore diameter 76, 174, 175 Pore volume 174, 175 Porosity, internal 15 Porosity of packed bed 13, 14 Porous alumina 202 Porous glass 60, 201, 202 Porous ion exchanger 212, 213 Porous layer bead 175 Porous silica 202 Porter-Silber reagent 603 Potassium hexacyanoferrate(II1) method 479 PQ-28 resin 663 Pre-columns 186,283 Preparative chromatography 120-122,708710,787 Preparative column 120 Preparative column, packing 31 1 Preparative segment columns 120 Pressure drop 125, 126 Pressure effect on retention volume 238 Pressure pulses 137, 138 Procion Brilliant Red M,B 483 Programme for recycling 779 Programme of amino acid analysis 678,679 Programmer, automatic 676 Programming of the composition of mobile phase 245-248 Programming of the solvent flow 245, 246 Programming of the temperature 247, 248 FTFE capillary columns 597 Pulse-damping device 137-139, 538 Pumps 109,110,131,133-139,306, 307,670 Pumps, diaphragm 135-137 Pumps, gradient forming 671 Pumps, mechanical 136 Pumps, membrane 133 Pumps, peristaltic 133 Pumps, piston 133-137 Pumps, pneumatic 136 Pumps, pulsating 133 Pumps, pulse-free 133 Pyridinium acetate as mobile phase 765 Pyridoxamine phosphate bound to agarose 818
1137
Q QAE-Sephadex 35 1 Quantitative analysis 386-401 Quantitative analysis, absolute calibration technique 395 Quantitative analysis, automatic evaluation 399-401 Quantitative analysis, internal standard technique 396 Quantitative analysis, manual evaluation 398 Quantitative analysis, normalization technique 398 Quantitative analysis, standard addition technique 396, 397
R Radiation detector 161 *Radiation detectors 407,408 7-Radiation detectors 404 Radioactivity, continuous measurement 116 Radiochromatographic techniques 403-41 4 Radiochromatography, detection modes 408413 Radiochromatography, effluent monitoring 4 10-4 12 Radiometry of collected fractions 412, 413 Raoult’s law 46, 50 Raoult’s law, deviations 17 Rate of diffusion of ions 79 Reactor 677 Recycling technique 31 1,778-781 Redox resins 21 1 Reference state 50 Refractive index 148, 153, 154 Refractometric detector(s) 148, 151-154 Refractometric detectors, deflection 153 Refractometric detectors, Fresnel 153 Refractometric detectors, thermostatting 152 Refractometry 116 Regeneration of ion-exchanger 72 Relationship between structure and chromatographic behaviour 380-383 Relative retention data 379, 380 Resex 345-347 Resins 205-207 Resins, chelating 340-343 Resins, ion retardation 340, 341 Resins, macroreticular 213 Resins, mixed bed 21 1 Resins, optically active 2 12
1138 Resins for biochemical analysis 340-343 Resolution 10,40-43, 124, 236, 250 Resolution factor 63,65 Response factors and specificity of detection 391-394 Restricted diffusion 60 Retardation factor 9, 32, 34 Retardion 340 Retention 8,45-55 Retention, relative 124, 236, 237 Retention data, relative 379, 380 Retention data in column and flat-bed systems 382,383 Retention equation 32 Retention index 382 Retention theory 235 Retention time 8, 10, 32,124, 378, 379 Retention time, dead 32 Retention volume 9, 33,58,235, 236 Retention volume, dead 9 Reversed-flow technique 61,65 Reynolds number 12,13 RF values 10 Rigid gels 201,202 Root-mean-square radius of gyration 59 Rubber, chlorinated 719 Rubber, granulated 198 Rysorb 184
S Saccharose, powdered (adsorbent) 627 Sag (Ago-Gel) 195 Sagarose 473 Salting-out chromatography 458,459,544,658 Sample application 111, 112, 139-143, 145, 295-297,306,307,360-363 Sample introduction devices, automatic 142, 143 Sample preparation 295-297 Sample preparation for amino acid analysis 704,705 Scintillation detectors, liquid 406,407,411 Scintillation detectors, solid-phase 405,406,411 SECellulose, see Sulphoethylcellulose Sedimentation of silica gel 289 Segment column 113,710 Selective ion-exchangers 70 Selectivity of detector 148, 384 Selectivity of ion-exchange process 76 Selectivity of the liquid-liquid systems 239245 Selectivity of the mobile phase 258-261
SUBJECT INDEX
Semiconductor detectors 407,412,413 Separation efficiency 5, 10, 124, 125, 140,690 Separation ranges of different gels 320 Sephadex 57,60, 190, 193, 199, 200, 208 Sephadex derivatives, see under the names of the respective derivatives Sephadex, hydrophobization of 599 Sepharose 190, 195,219-223,798 Sepharose, cyanogen bromide-activated 91 Sepharose-E-aminocaproyl-PTA 822 Sepharose derivatives, see under the names of the respective derivatives Sepharose with bound 3'-(4-aminophenylphosphoryl) deoxythymidine-5'-phosphate 95 Septum 141,142 SE-Sephadex 351 Sieving af sorbents 285,286 Silica gel 179, 180, 184 Silica gel, activation 180 Silica gel, adsorption centres 179, 180 Silica gel, deactivation with trimethylchlorosilane 438 Silica gel, impregnated with oxalic acid 461 Silica gel, preparation 180 Silica gel, regeneration 290 Silica gel, sedimentation 289 Silica gel, silver nitrate-impregnated 595, 596, 624,628,658 Silica gel, surface 179, 180 Silica gel-Celite mixture 596, 717 Silica gel columns, buffered 716 Silica gel particles classification 289 Silica gel SIL-X 6 15 Silica, porous 202 Silver nitrate-impregnated sorbents 595, 596 SIL-X Silica gel 6 15 Single column system for amino acid analysis 679 Size range of dry copolymer beads 75 Snake-cage resin 84, 2 11 Sodium deoxycholate 781 Sodium dodecyl sulphate 781 Solenoid-operated double-way valve 670 Solubility chromatography 789 Solubility coefficients 239-245 Solubilization chromatography 45 8 Solubilization of proteins 774 Solute concentration 29 Solute diffusion, longitudinal 31 Solute equilibrium concentration 20 Solute interaction 45-48 Solute mass balance 25-31
SUBJECT INDEX
Solute mass balance in an idealized column 28 Solute mass fluxes in the interparticle space of the column 26 Solute standard state 54 Solute transport detectors 154-156 Solvaflex 539 Solvent characteristics 240, 241 Solvent manipulation 132 Solvent programming 129-132, 245, 246 Solvent purification 757 Solvent systems for LCC 183-186 Sonntag’s equation 12, 14 Sorbent(s) 170-231 Sorbents, classification 170-173 Sorbents for affinity chromatography 215-227 Sorbents for gel chromatography 187-215 Sorbents for liquid-liquid chromatography 182-187 Sorbents for liquid-solid chromatography 174-1 82 Sorbent sorting 285-290 Sorption, random 170 Sorption equilibrium 4,5, 35,48 Sorption equilibrium thermodynamics 48-55 Sorption isotherm 6 Specific conductance 474 Specific flow 104 Specific surface area 174, 176, 177, 184, 216 Specificity of detection 391 -394 Spherix 69 1 Spheron 190-192,197,200,216,226 Spherosil202 Spreading factor 61,62 Spreading function 63 SP-Sephadex 351 Staionit FN 965 Standard addition technique 396 Standard state 50 Starch 192, 194, 627 Starting condition procedure 363 Stationary liquid film 6 Stationary phase 7, 8, 182-187 Stationary phase cross-section 9 Stationary phase programming 129, 132 Stationary phase volume 8 Stepwise elution 363 Stereoisomers separation 551 Steric exclusion 60 Stochastic theory of chromatography 60 Stokes’ radius 777 Structure and chromatographic behaviour 380-383 Styragel 198, 199 Sulphoethylcellulose 348
1139
Supports for LLC 182-1 87 Surface 174- 178 Surface, chemical character 176-179 Surface, chemical modification 178 Surface affinity 177 Surface area 174, 175, 184 Surface-etched beads 184 Surface free energy 20 Surface porosity, controlled 183, 185 Surface tension of the liquid 20 Swelling of ion-exchangers 73 Syphon flow meter 119 Syringes, high-pressure 141 System, binary liquid mixture-solid adsorbent 21-23 System, binary single-phase liquid 21 System, binary two-phase liquid-solid 21
T Tailor-made gel 302, 303 Talc 889 Taurodeoxycholate 781 TEAECellulose, see Triethylaminoethylcellulose Technicon AutoAnalyzer, for enzyme analysis 811 Technicon ion-exchange resin 471,474,487, 488 Technicon-type analyzer 680 -682 Technicon Varigrad gradient mixer 67 1 Techniques of liquid chromatography 101-414 Teflon 6 645 Temperature effect in LCC 299, 775 Temperature effect on retention volume 237, 238 Temperature of mobile phase 269 Temperature programming 129,132, 247, 248 Tension, interfacial 21 Ternary system 19 Theoretical plate, concept of 33-35, 82 Theoretical plate height 250, 251 Theoretical plates number 10, 35, 124, 125 Thermal chromatography on a hydroxyapatite column 879 Thermal contribution of the relative retention 242 Thermal movement, translational 20 Thermodetection LC 436 Thermostats (for column) 145 Thiourea with diatomaceous earth 428 Time-delay coil 538 Time of analysis 125 Titration curves of ion-exchangers 74
1140
SUBJECT INDEX
W
Transport, convective 16 Transport, diffusional 15 Tri-n-butyl phosphate column 918 Triethylaminoethylcellulose 349 Trinitrophenyliminodinitrophenyl group resin 71 Triton X-100 781 Tung's equation 62 Two-column system for amino acid analysis 676 -6 80 Tygon 47 8
Water regain values 74 Wavelengths of maximum absorption, proteins 803 W A X anion exchanger 892 Wet packing 111 Wire, with alkali FID 148,155 Wire, with FID 148,154-156 Wofatit 345-347 W.R., see Water regain values
U
X
Ultragrad 114, 115 Ultramarine, lasurite 203 Underlayering of sample loading 362,363 Universal calibration for GPC 1059 Universal calibration parameter 65 Urea effect 531,743,751, 849 UV-Detector 116, 148-151,308 UV-Fluorescence of proteins 803, 804
Xerogels 189 X value of ion-exchanger 206
V Vaives 109 Van Deemter equation'61 Varigrad 114,115,639,681 Varigrad gradient mixer, programming 680 Varigrad gradient mixer, Technicon 67 1 Velocity of flow 5 Velocity of zone migration 32 Velocity profiles 5 Viscosity coefficient, dynamic 17 Viscosity of the mobile phase 25 1 Void volume 58 Volumetric flow 364,365 Vydac 431,432
Z Zeo-Karb 328-331, 345 Zeolites 203,204 Zerolit 346 Zimmermann reagent 603 Zipax 184,214,566,601 Zirconium molybdate 352 Zirconium oxide, hydrated 204,352 Zirconium phosphate 204, 352 Zirconium tungstate 352 Zone 4 , 8 Zone broadening, extra-column 42,43 Zone movement 4-7 Zone spreading 4,5, 33 Zone spreading, dynamics of 35-40
List of compounds chromatographed*
A Abietal632 Abietal, dehydro- 632 Abietic acid, dehydro- 631 Ablastmycin 1004 Absinth 627 7-ACA, see Cephalosporanic acid, 7-aminoAcenaphthene 419,424 Acetaldehyde 457-459 Acetaldehyde, 2,4-dinitrophenylhydrazone457 Acetamide 660 Acetamide, N, N-dimethyl- 660 Acetamide, N-methyl- 660 Acetanilide 663 Acetic acid 546, 547,564, 567,569-571 Acetic acid, chloro- 564,567, 571 Acetic acid, dichloro- 564 Acetic acid, 2-(2,4-dichlorophenoxy)-564 Acetic acid, 2,4-dinitrophenylhydrazide 548 Acetic acid, 2-(2-hydroxyphenyl)- 553 Acetic acid, 2-(3-hydroxyphenyl)- 553 Acetic acid, 2-(4-hydroxyphenyl)- 553 Acetic acid, phenyl-, see Phenylacetic acid Acetic acid, trichloro- 564,571 Acetic acid, trimethyl-, 2,4-dinitrophenylhydrazide 548 Acetohexamide 936 Acetoin 435,459 Acetone 457,459,460 Acetone, dihydroxy-, phosphate 5 16 Acetophenone 448,458 Acetophenone, 4-amino- 643,644 Acetylcholinesterase 822 N-Acetyl-p-glucosaminidase 812 Aconitic acid 552, 554, 561,567 Acridine 921-923 Acrylamide 660 Acrylamide, N-tert.-butyl- 660 Acrylic acid 57 1 ACTH, see Hormone, adrenocorticotropic Actinium 1121 Actinomycins 1000 Adamantanone 461 Adenine 834, 835,837,841 Adenosine 833-835, 837,841,847, 852
Adenosine, deoxy- 518,833,841, 845-847 Adenosine, deoxy-, N6-methyl- 833 Adenosine, N6,N6-dimethyl-833 Adenosine, N6-(cis4-hydroxy-3-methylbut-2eny1)- 833 Adenosine, N6-(A24sopentenyl)- 8 33, 84 3 Adenosine, N6-(A2-isopentenyl)-2-methylthio833 Adenosine, 1-methyl- 833 Adenosine, 2-methyl- 833 Adenosine, 2'-O-methyl- 833 Adenosine, N6-methyl- 833 Adenosine, N-( 9-(p-D-ribofuranosyl-9H-purin6-yl) carbamoyl] -L-threonine- [ N-(nebularin6-ylcarbamoyl)]-L-threonine 833 Adenosine, 2'(3')-O-ribosyl- 833 Adenosine monophosphate 943 Adenosine 2'-phosphate 835 Adenosine 3'-phosphate 835 Adenosine 5'-phosphate 834, 835, 841, 841, 851,852 Adenosine polyphosphates 839 Adenosine 5'-pyrophosphate 835, 838, 841, 852 Adenosine triphosphate 835,841,852,942 Adenylic acid 849, 878 Adian-Sene 629 Adipic acid 564, 567 Adipic acid, 2-amino- 699, 706,985 Adipic acid, dimethyl ester 460 ADP, see Adenosine 5'-pyrophosphate Adrenaline 642, 646,650-654 Adrenaline, N-methyl- 646 Aesculin 899 Aflatoxin(s) 9 12-915 Aflatoxin B, 912,914 Aflatoxin B, 912,914 Aflatoxin G, 912, 914 Aflatoxin G, 912,914 Aflatoxin M I 914 Aflatoxins from cottonseed 913 Agmatine 640,646 Air pollutants 427 Aklavinone 999 Aklavinone, 7-deoxy- 999 a-Alaskene 627 PAlaskene 627 Albene 625 Albumin from bovine serum 780, 781, 800
*Compiled by H. BeEvifovi. 1141
1142 Albumin from human serum 800,803 Albumin from human serum, structural studies 768 Alcohols 431-439 Alcohols, aliphatic 431-439 Alcohols, amino derivatives 649-650 Alcohols, aromatic 431 -439 Alcohols, diterpenic, from Dacrydium bidwillii 633 Alcohols, esters with pyruvic acid 2,4-dinitrophenylhydrazone 437 Alcohols, polyhydric 501, 503 Alcohols, terpenic 633 Aldehydes, 455-464,631,632 Aldehydes, aliphatic 456-458 Aldehydes, aliphatic, 2,4-dinitrophenylhydrazones 456,457 Aldehydes, cyclic 456-458 Alditols 467,481,487,501-504 Alditols, amino derivatives 498 Aldobionic acids 481,514 Aldobiuronic acids 513, 564 Aldolase 87 1 Aldolase from chicken breast muscle 824 Aldonic acids 481,514,551,554, 555,559,560 Aldosterone 615,617 Aldrin 1010,1012,1013,1016,1017 Aldrin, 6,7-dihydro- 1014 Alkaloids 887-894 Allonic acid 557,558 AUosamine 497 Allose 488 see Mycinose AUose, 2,3-di-O-methyl-6-deoxy-, AUuronic acid 512,513 Ally1 alcohol 433,435 AUylamine 638,640 Allylestrenol610 Altritol, 1,4-anhydro- 509 Altritol, 1,5-anhydro- 509 Altritol, 3,6-anhydro- 509 Altronic acid 557-559 Altrose 488 crAltrose, 3-acetamido-2,4di-O-acetyl-3,6dideoxy-, methyloside 469 crAltrose, 4,6-0-benzylidene-2-O-tosyl-, methyloside 469 Aluminium 657,1094-1096 Amaranth 1035 Amblyonin 632 Amentoflavone 903 Amides 651-664 Amines 637-655 Amines, aliphatic, mono-, di- and polyamines 637-643
LIST OF COMPOUNDS CHROMATOGRAPHED Amines, aromatic 643-645 Amines, biogenic 650 -654 Amines, mixtures of aromatic amines and aliphatic polyamines 645 Amines, primary 639 Amino acid(s) 651,665-711 Amino acids, in analysis of nucleic acid components 837 Amino acids, separation of amino sugars 49950 1 Amino acid derivatives 713-739 Amino acid derivatives, bound with 0-hydroxyaquotriethylenetetraminecobalt(II1) 738 Amino acid derivatives, 4-dimethylamino-3,5dinitrophenyl-hydantoins 737 Amino acid derivatives, S-dimethylaminonaphthalene1 -sulphonyl- 726 -73 1 Amino acid derivatives, 2,4-dinitrophenyl- 165, 714-726 Amino acid derivatives, 3,5-dinitrophenylthiohydantoins 737 Amino acid derivatives, hydantoins 731-734 Amino acid derivatives, methoxycarbonyl compounds 738 Amino acid derivatives, phenylthiohydantoins 734-736 Amino acid derivatives, pipsyl compounds 737 Amino alcohols 649 Amino sugars 496-501 Amino sugars, N-acetyl- 467 Amino sugars, antibiotics 482 Aminotransferase, tyrosine 818 Aminotransferase, L-tyrosine-2-oxoglutarate 818 Ammonia 638,642,646,698,700,701,703, 706 Ammonium compounds 649,650 AMP, see Adenosine 5’-phosphate Amphetamines 892 Amphomycin 1003 Ampicillin 983 Amurensin 908 n-Amy1 alcohol 436 terf.-Amy1 alcohol 436 Amylamine 638,640, 642 Amylopectin 495 cY-Amyrin, palmitate 631 PAmyrin, acetate 63 1 PAmyrin, palmitate 631 Analgesics 663, 892 Anaphylatoxin 797 Androgens 604,605 Androsta-l,4-dien-3-one, 17phydroxy- 608 Androstane, 17-mercapto- 604
LJST OF COMPOUNDS CHROMATOGRAPHED Androstane-3a, 17pdiol604 Androstenediol613 Androstenedione 603,613,616 Androst-4-ene-3,17-dione604,608 Androst-4-ene-3,17-dione, [7’-H] 605 Androst-4-ene-3,l T-dione, 1I@-hydroxy-608 Androst-4-ene-3,ll ,l7-trione 608 Androst-4-en-3-one, 17whydroxy; see Epitestosterone Androst-4-en-3-one, 17p-hydroxy-, see Testosterone Anhydrase, carbonic 871 Anhydrosaccharinic acid 515 Aniline 568,643,644 Aniline, 4-aminodimethyl- 644 Aniline, 2-chloro- 643, 644 Aniline, 2-nitro- 643, 644 Aniline, 3-nitro- 643-645 Aniline, 4-nitrO- 643, 644 o-Anisidine 643,644 Anisole 423,451 Anisole, alkyl derivatives 447 Anserine 706,722 Antheraxanthin 1047 &-Antheraxanthin 1047 Anthocyanidins 9 10 Anthocyanin pigments 909,910 Anthocyans 909-912 Anthracene 418,424,425 Anthracene, 9,lO-dihydro- 424 Anthracene, methyl- 422 Anthracene, octahydro- 424 Anthracene, 1,2,3,4-tetrahydro- 424 Anthncyclines 998,999 Anthracyclinone 998 Anthranilic acid 549,552,553,568,643,644, 647,649 Anthranilic acid, 3-hydroxy- 553, 649 Anthranilic acid, 3-methoxy- 649 Anthranilic acid glucuronide 647 Anthraquinone 455,462,1034 Anthraquinone, 2-tert-butyl- 462 Anthraquinone, 1,4-dimethyl- 462 Anthraquinone, 2-ethyl- 462 Anthraquinone, 2-methyl- 462 Anthraquinone1,S-disulphonicacid 928 Anthraquinonel,6-disulphonicacid 928 Anthraquinone-l,7-disulphonicacid 928 Anthraquinone-1 ,8-disulphonic acid 928 Anthraquinones in natural materials 461 Anthraquinonesulphonic acid 927,928 Antibiotics 979- 1007 Antibiotics, amino acid analogues 1000-1003
1143 Antibiotics, aminoglycosidic 994 Antibiotics, carbohydrate 985 -994 Antibiotics, p-lactam 980, 981 Antibiotics, macrocyclic 994-996 Antibiotics, nucleoside 999 Antimony 1094, 1095,1117 Antimycin A 996 Antimycin A, desacetyl- 1004 Antimycin A ,,dehexyldeisovaleryloxy- 996 Antiplasmin 796 6-APA, see Penicillanic acid, 6-aminoApigenin 908 Apigenin, 6,8-di-C-glucopyranosyl derivatives 903 Apigenin, oxidation products 906 Apigenin derivatives 904 Apigenin-7glucoside 899, 904 Apigenin-7-glucuronide 908 Apigenin-7-prutinoside 903 Apiin 899,908 Apolipoproteins 781 Apyrimidinic acid, dephosphorylated 880 Aquamycin 1004 Arabinan 525 Arabinitol470,502-504 Arabinobiose 485 Arabinogalactan 526 Arabinonic acid 514, 559 Arabinose 485-488,491,492,495,502,503,512 Arabinose, 3-O-a-arabinopyranosyl- 485 Arabinose, 3-0-p-glucopyranosyl- 485 Arabinose, methyl ether 510 WArabinose, 2,3,5-tri-O-benzoyl-, bromide 508 PArabinose, 3,4-O-isopropylidene-, benzyloside 508 PArabinose, 1,3,5-tri-O-benzoyl- 508 PArabinose, 2-0-(2,3,5-tri-O-benzoyl-a arabinofuranosy1)-, benzyloside 508 PArabinose, 2-0-(2,3,5-tri-O-benzoyl-c+ arabinofuranosyl)-3,4-O-isopropylidene 508 Arabinuronic acid 557,558 3-a-Arabofuranosido-7-a-rhamnofuranoside 901 Arabonic acid 563 Arachidonic acid 576 Arbutin 444,899 Arginine 638,640,642,646 Aristeromycin 1004 Aromadendrin 902,908 o-Arsanilic acid 643, 644 pArsanilic acid 643,644 Arsenic 1095,1117
1144 Arsine, (RR)-or[ 2-trimethylsilylferrocenyl] ethyldimethyl- 1107 Arsine, (RS)-(~-[2-tRmethylsilylferrocenyl] ethyldimethyl- 1107 Artemazulene 627 AS, see Sulphates, alkylAscochlorin 1004 Ascorbic acid 492, 962, 975, 976 Ascorbic acid, dehydro- 975,976 Ascorbic acid, dehydro-, osazones 975 Aspartic acid 567 Asphaltenes 1065 Asphalts 418,420,1065 Aspon 1022 ATPase 798 Atrazine 1027 Atrovenetin 1004 Aurone 899 Avicularin 908 Avidin 376 Aza-heterocyclics 92 1-9 23 6-Azapseudouridine 843 Aza-steroids 620 6-Azauridine 843 Azinphos-ethyl 1023 Azinphos-methyl 1012, 1023 Azobenzene 290 Azobenzene, 4-amino- 290 Azobenzene, o,o'-dihydroxy- 1035 Azobenzene, 4-methoxy- 290 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, allylamide 660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-amyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-butylamide 660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-butyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene, n-decyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, di(n-butyllamide 659,660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, diethylamide 659,660 Azobenzoic acid, N, Ndimethyl-p-aminobenzene, dimethylamide 659,660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene, di(n-propyllamide 659,660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, ethylamide 660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, ethyl ester 437 Azobenzoic acid, N,Ndimethyl-p-aminobenzene-, n-hexylamide 660
LIST OF COMPOUNDS CHROMATOGRAPHED Azobenzoic acid, N,Ndimethyl-p-aminobenzene-, n-hexyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, isobutyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, isopropyl ester 437 Azobenzoic acid, N,N-dimethyl-p-aminobenzene-, methylamide 660 Azobenzoic acid, N,Ndimethyl-p-aminobenzene-, methyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-nonyl ester 437 Azobenzoic acid, N,N-dimethyl-p-aminobenzene-, n-octyl ester 437 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-propylamide 660 Azobenzoic acid, N, N-dimethyl-p-aminobenzene-, n-propyl ester 437 Azo compounds 657-664 Azomethine dyes 1035 Azulenes 627 Azulenium salts 627
B Bacilysin 1003 Bacitracin 780, 871 Bacteriochlorophyll 1042, 1044 Bacteriophage(s1 1081, 1082 Bacteriophage M 12 1081 Bacteriophage M 13 1081 Bacteriophage QP 1081, 1082 Bacteriophage T4 1081 , ~ 1082 Bacteriophage L ~ X , 1081, Baicalein 908 Baicalin 908 Bakkoside 908 Balsams 624 Barium 1089,1094-1096,1121 Bence-Jones protein, K-type, structural studies 762,763 Bencdones protein, S-aminoethylated, structural studies 759 Benzaldehyde 457 Benzaldehyde, 2-hydroxy- 444 1,2-Benzanthracene, 9,lO-dimethyl- 419 Benz(cu)anthracene-7,12-dione 419 Benzanthracene, peroxo derivatives 453 Benzene 421,424-426,443 Benzene, n-butyl- 427 Benzene, set-butyl- 427 Benzene, tert.-butyl- 427 Benzene, 1,2-diethyl- 427
LIST OF COMPOUNDS CHROMATOGRAPHED Benzene; 1,3-diethyl- 427 Benzene, 1,4-diethyl- 427 Benzene, diisopropyl- 422 Benzene, dioctyl- 422 Benzene, ethyl- 422,423,426 Benzene, n-hexyl- 427 Benzene, isopropyl-, see Cumene Benzene, 4-isopropyl-1-methyl- 427 Benzene, nitro- 448 Benzene, n-nonyl- 427 Benzene, n-octyl- 427 Benzene, n-pentyl- 427 Benzene, n-propyl- 426 Benzene, tridecyl- 422 Benzene, 1,3,5-triethyl- 427 Benzene, 1,2,4-trimethyl- 427 Benzene, 1,3,5-trimethyl- 427 Benzene-l,3-disulphonic acid, 4,5-dihydroxy932 Benzenesulphonic acid 930,935 Benzenesulphonic acid, 2-amino-, see Orthanilic acid Benzenesulphonic acid, 4-amino-, see Sulphanilic acid Benzenesulphonic acid, 4-chloro-, sodium salt 935 Benzenesulphonic acid, 2,5-dimethyl-, sodium salt 435 Benzidine 644 Benzofluorene 41 8 Benzoic acid 443,546,550, 552,553,565, 568 Benzoic acid, 2-amino-, see Anthranilic acid Benzoic acid, 3-amino- 549, 643-645 Benzoic acid, 4-amino- 549,552, 553, 571, 643-645 Benzoic acid, chloro-, separation of 0-,rn- and p isomers 549 Benzoic acid, 3,4-dichloro- 1028 Benzoic acid, 2,3-&hydroxy- 568, 569 Benzoic acid, 2,4-dihydroxy- 568, 569 Benzoic acid, 2,5-dihydroxy- 569 Benzoic acid, 2,6-dihydroxy- 569 Benzoic acid, 3,4-dihydroxy- 569, 570 Benzoic acid, 3,5-dihydroxy- 569 Benzoic acid, 2-hydroxy-, see Salicylic acid Benzoic acid, 3-hydroxy- 443, 552, 553, 569 Benzoic acid, 4-hydroxy- 443, 5 5 2 , 553, 568, 569 Benzoic acid, 4-hydroxymethyl- 549 Benzoic acid, nitro-, separation of 0 - , m-and p isomers 549,658 Benzene, nitroethyl-, separation of 0,rn- and p isomers 658
1145 Benzoic acid, 2,3,4-trihydroxy- 569 Benzoic acid, 3,4,5-trihydroxy- 569 Benzoperylene 418 Benzophenone, dichloro- 460 Benzophenone derivatives 896 7,8-Benzoquinoline 922 Benzo[ clquinoline 923 Benzo[jJquinoline 923 Benzo[h]quinoline 921,923 Benzoquinone 46 1 Benzoylglucuronide, 3,4-dichloro- 1028 Benzoyl peroxide 451 Benz(a)pyrene 418,425 Benz(e)pyrene 425 Benzyl alcohol 60,431-434,436, 510, 568 Benzyl alcohol, a,ordimethyl- 434 Benzyl alcohol, a,a’-dimethyl- 432 Benzyl alcohol, a-methyl- 432,434 Benzyl alcohol, a-phenyl- 434 Benzylamine 646 Benzylamine, 3,4-dimethoxy- 646 Benzylamine, 3-ethoxy-4-hydroxy- 646 Benzylamine, 4-hydroxy- 646 Benzylamine, 4-hydroxy-3-methox y- 646 Benzylamine, 4-methoxy- 646 Benzyl chloromethyl sulphide 934 Benzylglucuronide, 3,4-diChlOro- 1028 Beromycin B 999 Beromycin C 999 Beryllium 1095,1096,1121 Betaine 649 Betaine-aldehyde 649 Beyerene 628 BHC 1013 Biacetyl, see Diacetyl [ 2.1.1 ] Bicyclohexane, l-vinyl-5,5-dimethyl- 625 Bidrin 1022 Bile acids 538,589,597,598,617,618 Bile acids, glycine-conjugated 589 Bile acids, unconjugated 583 Biliproteins 1041, 1047, 1048 Biochanin A 904 Biogenic amines 650-654 Biopterin 972, 973 Biopterin, dihydro- 973 Biotin 970, 971 Biphenyl, see Diphenyl PBisabolene 627 Bismuth 1094,1095,1122 Biuret 660,663 Blancoic acid 901 Bleomycin 1001 Bleomycin A, 1003
1146 Bleomycin Cu-Bt complex 1001,1002 Blood cells 1082,1083 Blue VRS 1035 Bone marrow cells 1084,1085 Boranes 947,949 Boranes, ligand derivatives 949,950 Boranes, substituted 947 Boron compounds 945-951 Brassicosid 903 Brassidin 903 Brefeldin A 996 Bromides, alkyl658 Bromine 1098, 1119 Bromophos 1012,1013,1022,1023 Brucine 891 Buffadienolides618 Bufotenin 646 Butane 426 Butane, 2,2-dimethyl- 426 Butane, 2,3-dimethyl- 426 Butane, 2-methyl- 426 Butane, 1-nitro- 658 1,3-Butanediol435 1,4-Butanediol435 2,3-Butanediol438 2,3-Butanediol, meso- 438 l-Butanol433,435,437 1-Butanol, 2-amino- 638 1-Butanol, 2-nitro- 658 2-Butanol435,437 1-Butene, 2,3-dimethyl- 426 1-Butene, 2-methyl- 426 2-Butene-1-01, see Crotyl alcohol 3-Butenoic acid, 2-amino4methoxy- 1000 Butirosin A 993 Butirosin B 993 tert.-Butyl alcohol 433,435,436 Butylamine 638,640,642 2,3-Butylene glycol 435 Butyraldehyde 459,460 Butyraldehyde, 2,4-dinitrophenylhydazone 457 Butyric acid 546,548, 567,569-571 Butyric acid, 2-amino- 699, 706 Butyric acid, 4-amino- 700, 706 Butyric acid, 2,3-dihydroxy- 562 Butyric acid, erythro-2,3-dihydroxy- 557 Butyric acid, threo-2,3-dihydroxy- 5 14 Butyric acid, 2,4-dihydroxy- 514, 555, 556, 558,562 Butyric acid, 3,4dihydroxy- 514, 555 Butyric acid, 2,4-dihydr oxy-3,3-dimethyl- 557 Butyric acid, 2,4-dinitrophenylhydrazide548 Butyric acid, 2-hydroxy- 557,558
LIST OF COMPOUNDS CHROMATOGRAPHED Butyric acid, 3-hydroxy- 557, 558 Butyric acid, 4-hydroxy- 557 Butyric acid, 2-hydroxy-2-methyl- 551, 558 Butyric acid, 2-methyl- 558 Butyric acid, 2-methyl-, 2,4-dinitrophenylhydrazide 548
C Cadaverine 640,641,646 Y-Cadinene 626,627 Wadinene 627 Cadmium 1095,1096,1118,1122 Caeruloplasmin 803 Caffeic acid 570 Caffeine 892 Caffeine alkaloids 892 Calamendiol633 Calamenenes 627 Calciferols 957-960 Calcium 1088,1089,1094-1096 Campesterol606 Camphene 625 Canavanine 642 Cannabichromene 898 Capric acid 569,570 Caproic acid, 2,4-dinitrophenylhydrazide 548 Caprolactam, oligomers of 1069 Caprylic acid 548 Captan 1013 Carbazide, diphenyl- 663 Carbazole 419,922 Carbohydrate antibiotics 985-994 Carbohydrates 438,465-522,551 Carbonic anhydrase, see Anhydrase, carbonic Carbonyl compounds, complexes with ehydroxysulphonic acids 455,457 Carbophenothion 1023 Carboranes 948,950 Carboranes, ligand derivatives 949,950 Carboranes, substituted 948 Carboxylase, apopyruvate 827 Carboxylase, pyruvate 827, 828 Carboxylic acids, dinitrophenylhydrazides 548 Carboxylic acids, higher 575-580 Carboxylic acids, hydroxy derivatives, automated analysis 556 Carboxylic acids, lower 543-573 Carboxypeptidase 9 1 Carboxypeptidase B, structural studies 768 Carcinogenic substances 417 Cardanol444 Cardenolides 618
LIST OF COMPOUNDS CHROMATOGRAPHED Cardiac glycosides 618 A3-Carene625 Carmoisine 1035 Camosine 638,700,706,722 Carotenek) 589 aCarotene 1042,1047 @Carotene 961, 1042, 1043,1047 Carotenoids 582, 1040-1047 Caryophyllene 626 Caryophyllene oxide 626 Casbene 628 Castor oil 585 Catalase 87 1 Catechin(s) 896, 899 Catechin, degradation products 906 Catechin-7-arabinoside 900 Catechol444,445 Catecholamines 650-654 Catena polyphosphates 1097 Celesticetin B 991 Celesticetin C 991 Celesticetin D 991 Cellobionic acid 514 Cellobiose 489-491 Cellobiulose 490 Cellobiuronic acid 5 13, 5 15 Cellodextrin 494 Cellohexaose 484,488,489,494 Cellopentaose 489 Cellotetraose 489,491 Cellotriose 489,491 Cells 1075-1085 Cells from the spleen 1083, 1084 Cellulose derivatives 1065 Cellulose esters 1065 Cellulose nitrate 1065 Cephalin 588 Cephalocillanic acid, 7-amino- 981 Cephalocillins 981 Cephaloglycin 984 Cephaloglycin, desac.ety1- 984 Cephalosporanic acid, ?’-amino- 980,984,985 Cephalosporin(s) 980-985 Cephalosporin, desacetyl-, lactone 984 Cephalosporin C 984 Cephalosporin P, 980 Ceph-3-em-4-oic acid, 3-methyl-7-(2-phenoxyacetamido)-, methyl ester 981 Ceramide, aminoethyl phosphate 583, 589 Ceramide, dihexosides 583 Ceramide, polyhexosides 583, 589 Cerebroside sulphate 583, 589 Cerebrosides 583, 584, 588, 589 Ceruloplasmin 779
1147
Cesium 1090, 1095, 1121 Chalcone 897,899 Chalcone, 2-methyl-2’,4,4‘,6’-tetrabenzoy1-3methoxy- 905 Chamazulene 627 Chamazulene, 3,6-dihydro- 627 Chamazulene, 5,6-dihydro- 627 Chitaric acid 557 Chitonic acid 557, 558 Chlorbenside 1012 Chlordane 1012, 1013 Chlorfenson 1012 Chlorfenvinphos 1012, 1022, 1023 Chlorine 1098, 1120 Chlorobactene 1042 Chlorogenic acid 564, 569, 570, 899 Chloromandinone acetate 6 10 Chlorophillidea 1045 Chlorophillide b 1045 Chlorophyll(s) 589,1041-1045 Chlorophyll u 1042-1045 Chlorophyll a’ 1042 Chlorophyll b 1042-1045 Chlorophyll b’ 1042 Chlorophyll c 1042,1044 Chlorophyll c 1044 Chlorophyll c 2 1044 Chlorophyll d 1042, 1044 Chlorophyll-protein complexes 1045, 1046 Chloroprene 1070 Chlorpropamide 936 Chloroprophan 1026 Chlorothiophosphate, 0,O’dimethyl- 1016 Chlorthion 1022 Cholesta-5,7-diene, 30-hydroxy- 606 Cholestane-la, 2cu-diol606 Cholestane-lp, 2pdiol606 Cholestan-3p-01,4-14C606 Cholest-5-ene, 3p-acetoxy- 606 Cholest-7-ene, 3p-acetoxy- 606 Cholest-8( 14)-ene, 30-acetoxy- 606 Cholest-S-en-23-one, 3phydroxy- 607 Cholesterol 422, 582, 584,606,607, 959 Cholesterol, 7-dehydro- 958, 959 Cholesterol, 1 7 q 20adihydroxy- 607 Cholesterol acetate 584 Cholesterol esters 582 Cholesterol octadecenoate 584 Cholesterol sulphate 606, 607 Choline 649 Choline, lysophosphatidyl- 582, 583 Choline, phssphatidyl- 582-584,589 Chondroitin sulphate A, see Chondroitin-4sulphate
,
1148 Chondroitin sulphate B, see Dermatan sulphate Chondroitin sulphate C, see Chondroitin-6sulphate Chondroitin-4-sulphate 529 -5 37 Chondroitin-6-sulphate 529, 530, 532-537 Chondroitin sulphuric acid 531 Chroman, cis-3,4-dihydroxy-6-methoxy-2,2dimethyl- 898 Chroman, rrans-3,4-dihydroxyd-methoxy-2,2dimethyl- 898 Chroman, 7-methoxy- 898 Chromano-(3,4-d)-isooxazole,7-methoxy- 898 Chromanone, 5,7-dihydroxy-2,2-dimethyl897 Chroman-rl-one, 3,3-dimethylisothio- 898 Chroman-4-one, 6-hydroxy-2,2-dimethyl- 898 Chroman-4-one, 7-methoxy- 898 Chroman-4-one, 3-methylisothio- 898 Chromene, cinnamoyl-, C-methylated 904 Chromene, dihydro-(4’-methylpent-3’-enyl)-5hydroxyd-carbethoxy-7-penty l- 898 Chromene, 2,2-dimethyl-5-hydroxy-6-acetyl898 Chromium 1095,1096,1116,1117,1119 Chromium, cis-(1-acetoxytetra1in)tricarbonyl1110 Chromium, frans-(1-acetoxytetra1in)tricarbonyl1110 Chromium, tricarbonyl(mesity1ene) 1110 Chromium, tricarbanyl(1-tetralone) 1109 Chromium, tricarbonyl(to1uene) 1110 Chromium, tricarbonyl(rn-xylene) 1110 Chromium(III), dibromo-bis-( 1,lO-phenanthroline)-, cation 1107 Chromium(III), dichloro-bis-(1,lo-phenanthroline)-, cation 1107 Chromium(III), cis-tris-(benzoylacetonato)- 1104 Chromium(III), trans-tris-(benzoylacetonato) 1104 Chromium(III), tris-(pdiketonat0)- 1 102 Chromium(III), tris-(trifluoroacety1acetonato)1104 Chromium(III), tris-(l,l ,l-trifluoro-4-p-methoxyphenyl-2,4-butanedionato)-1104 Chromone(s) 897,898 Chromone, 2-methyl-5,7-dihydroxy- 898 Chromone, 2-methyl-6,8-di-C-prenyl-5,7dihydroxy- 898 Chromone, 2-methyl-7-prenyloxy-5-hydr0xy898 Chromone, 2-methylthio- 908 Chrysene 418,419,421,425 Chrysosplenetin 908 Chrysosplenin 908 Chymotrypsin 89-91,370,372
LIST OF COMPOUNDS CHROMATOGRAPHED Chymotrypsinogen 871 Chymotrypsinogen A 780 Cigarette smoke 421,458 Cinerubin A 999 Cinnamic acid 568 Cinnamic acid, 4-hydroxy- 553 Cinnamic acid, 4-hydroxy-3-methoxy- 553 Cinnamyl alcohol 431-433 Cirsimarin 908 Cirsimaritin 908 Citraconic acid 561,567 Witraurin 1047 Citric acid 549,551,552,554,562,564,567, 571 Citrulline 699,706 Clindamycin 991 Clindamycin, Ndemethyl- 991 Clindamycin, N-demethyl-N-hydroxymethyl99 1 Clupeine, structural studies 763 CMP, see Cytidine S‘-phosphate Coal tar 421 Cobalamide 975 Cobalamin(s) 974,975 Cobalamin, cyano-, see Vitamin B I I Cobalamin, hydroxy- 974,975 Cobalamin, methyl- 974,975 Cobalt 1094-1096,1116-1119,1121 Cobalt(III), aspartato-bis-(1-propylenediamine), cation 1107 Cobalt(III), bis-(oc,pdiaminopropionato)-, cation 1104 Cobalt(III), bis-(diethy1enetriamine)-,cation 1104 Cobalt(lII), bis-(ethylenediaminel-propylenediamine-, cation 1106 Cobalt(III), trans-bis-glycinato-leucinato-1106 Cobalt(III), bis-(g1ycinato)propylenediamine-, cation (1103, 1104,1107 Cobalt(III), bis-(hydrogenaspartato) (l-propylenediamine), cation 1107 Cobalt(lII), diamine(ethy1enediamine-N,N’diacetatol-, cation 1104 Cobalt(III), dibromo-bis-(1 ,lO-phenanthroline)-, cation 1107 Cobalt(III), cis-dichloro-bis-(ethylenediamineb, cation 1100 Cobalt(III), trans-dichloro-bis-(ethylenediaminel-, cation 1100 Cobalt(III), dichloro-bis-( 1,lO-phenanthroline)-, cation 1107 Cobalt(III), diethylenetriamine(iminodiacetato)-, cation 1104
LIST OF COMPOUNDS CHROMATOGRAPHED Cobalt(III), diethylenetriamine(methyliminodiacetat0)-, cation 1104 Cobalt(IIl), ethylenediamine-bis-(glycinatob, anion 1104 Cobalt(III), ethylenediamine-bis-(propylenediamine)-, cation 1106 Cobalt(III), ethylenediamine-N, N‘-diacetato(N,N’-diethylethylenediaminek, cation 1 101 Cobalt(l11), ethylenediamine-N,N’-diacetato(ethylenediamine), cation 1101 Cobalt(III), ethylenediamine-N,N’-diacetato(N-ethylethylenediamine), cation 1101 Cobalt(III), ethylenediamine-N,N’-diacetato(N-methylethy1enediamine)-,cation 1101 Cobalt(IIl), ethylenediamine-N,N’-diacetato(propy1enediamine)-, cation 1100 Cobalt(III), ethylenediamine(N, ”-dimethylethy1enediamine)-bis-nitro-,cation 1104 Cobalt(III), ethylenediamine(ethy1enediamine diaminopropionato)-, cation 1106 Cobalt(III), trans-glycinato-bis-leucinato 1106 Cobalt(III), glycinato-bis-(propy1enediamine)-, cation 1103, 1107 Cobalt(III), tetraamineazido-, cation 1087 Cobalt(III), tri-, hexakis(2-aminoethanethiolo)-, bromide 1106 Cobalt(III), tris-[ (+)-3-acetylcamphorato]- 1103 Cobalt(III), tris-(p-alaninato)- 1104 Cobalt(III), trans-tris-(aaminoisobutyrato)1105 Cobalt(III), cis-tris-(benzoy1acetonato)-1104 Cobalt(II1). trans-tris-(benzoy1acetonato)- 1104 Cobalt(II1). tris-(6-diketonat0)- 1102 Cobalt(III), tris-(ethylenediamine), cation 1103 Cobalt( HI),cis-tris-glycinato- 1105 Cobalt(III), trans-tris-glycinato- 1105 Cobalt(III), trans- tris-(2L, 3L-isoleucinato) 1106 Cobalt(III), rmns-tris-leucinato- 1 106 Cobalt(III), trans-tris-(7-methoxyg1utamato)1106,1108 Cobalt(III), tris-(5-methylhexane-2,4-dithionato)1104 Cobalt(III), trans-tris-norleucinato- 1106 Cobalt(III), tris-(propy1enediamine)-,cation 1103,1106 Cobalt(III), frans-tris-sarcosinato- 1105 Cobalt(IlI), tris-(tnfluoroacety1acetonato)- 1104 Cobalt(III), tris-(l,l ,l-trifluoro-4-p-methoxyphenyl-2,4-butanedionato)-1104 Cobalt(III), tris-valinato- 1104 Cobalt(III), trans-tris-valinato- 1106 Cobaltate(III), bis-asparatato-, anion 1104, 1107 Cobaltate(III), bis-(g1ycinato)oxalato-, anion 1104
1149
Cobaltate(III), cis-bis-(iminodiacetat0)-,anion 1106 Cobaltate(III), ethylenediaminetetraacetato-, anion 1106 Cobaltate(III), oxalato-bis-serinato-, anion 1107 Cobaltate(III), trimethylenediaminetetraacetato-, anion 1107 Cobamamide 974 Cocoa butter 585 Coenzyme A 971 Coenzyme A, a-carboxy- 971 Coenzyme A, a-methyl- 971 Coenzyme A, p-methyl- 971 Coenzyme A, stearyl- 971 Columbium 1120 Communal 632 crCopaene 626 Copolymer(s) 1063, 1064 Copolymer ethylene-1-butene 1063 Copolymer isoprene-styrene 1063 Copolymer styrene-divinylbenzene 1067 Copolymer 4-vinyldiphenyl-isoprene1063 Copper 1094-1096,1116-1118 Coprostanol606 Coronene 418,422 Corrinoids 962, 973-975 Corticosteroids 603,614-617 Corticosterone 615-617 Corticosterone, 1 1-dehydro- 615 Corticosterone, deoxy- 615 Cortisol 616, 617 Cottisol, 11-deoxy- 615,617 Cortisone 615-617 Cortisone, 21-acetate 615 Cortisone, 60-hydroxy- 61 5 Cosmosiin 908 Coumaphos 1023 Creatine 642 Creatinine 700, 706 rn-Cresol443 o-Cresol443-445, 1070 pCresol448, 553, 1070 Crotonic acid 571 Crotyl alcohol 433 Crufomate 1023 Crustecdysone 619 Cryptoxanthin 1042,1047 Cumarone 905 Cumene 423,427 Curare alkaloids 892 c-Curcumene 627 pCurcumene 627 y-Curcumene 627 ar-Curcumene 626, 627
1150 Cyanein, see Brefeldin A Cyasterone 619 Cycloartanone, 24-methylene, 632 Cycloartenol palmitate 631 Cycloartenone 632 Cyclobalanone 632 Cyclobutanone derivatives 460 a-Cyclodextrin 484 pCyclodextrin 484 Cyclohexane 422,423,426 Cyclohexane, ethyl- 426 Cyclohexane, methyl- 426 Cyclohexanol436 Cyclohexanone 459 Cyclohexene 426 1-Cyclohexene 3,4,5-trihydroxy-l-carboxylic acid 552 Cyclopentane 426 Cyclopentane, 1,3-dicyclopentyl-2-dodecyl419 Cyclopentane, methyl- 426 Cy clopent anone 4 5 9 Cyclopentene 426 Cyclopropylamine 638 Cycloserine 1000 Cymathrene, or-aminoacetyl- 1109 Cymathrene, p-aminoacetyl- 1109 Cystathionine 699, 706,982 Cysteamine-glutathione disulphide 982 Cysteine, S-carboxymethyl- 706 Cysteine-cysteamhe disulphide 982 Cysteine-glutathione disulphide 982 Cysteine-homocysteine disulphide 982 Cysteine-homocysteine trisulphide 982 Cysteine-penicillaminne disulphide 9 82 Cysteine-penicillaminetrisulphide 982 Cysteine trisulphide 982 Cytidine 833,835, 841,847 Cytidine, NQacetyl- 833 Cytidine, deoxy- 833,841,845-847 Cytidine, deoxy-, 5-hydroxymethyl- 833 Cytidme, deoxy-, 5-methyl- 833 Cytidine, N4,02-dimethyl-833 Cytidine, 2’-O-methyl- 833 Cytidine, 3-methyl- 833 Cytidine, 5-methyl- 833, 835 Cytidine, 2-thio- 833 Cytidine 2‘-phosphate 835 Cytidine 3’-phosphate 835 Cytidine 5’-phosphate 834, 835, 841, 847, 851, . 852 Cytidine 5’-pyrophosphate 835, 852 Cytidine 5’-triphosphate 835, 852 Cytidylic acid 848
LIST OF COMPOUNDS CHROMATOGRAPHED
Cytidylic acid, 5-hydroxymethyl- 848 Cytochrome(s) 798, 803, 1047 Cytochrome b , 794 Cytochrome c 781,794,871,966 Cytochrome c,,, 793 Cytochrome c from horse, structural studies 768 Cytosine 834, 835, 837, 841 Cytosine, 5-methyl- 835, 851
D 2,4-D 1013, 1017 Daidzein 90 1, 904 Daidzein glucoside 901 DDD 1010,1016,1017 DDE 1010,1012,1013,1026 DDT 1010,1012,1013,1016,1017,1026 Decacyclene 422 Decalin 422 Decane 422,423 Decanoic acid 571 Decanol433,436 1-Decarboxylase L-glutamate from E. coli 823 Decene-1 423 Dehydrogenase, triphosphorydine nucleo tide isocitrate, from B. stearothermophilus 815 Delphmidm 909,910 Delphinidin-3,5-diglucoside91 1 Delphinidin-3-glucoside910 Delphinidin-3-rutinoside 9 10 Demeton 1018 Demeton-0-methyllO22, 1023 Demeton-S 1022,1023 Demeton-S-methyl 1022, 1023 Demeton-S-methyl sulphone 1022 Demeton-S-methyl sulphoxide 1022 Dendrolasin, dehydro- 630 Deoxyribonucleic acids, see DNA Deoxyribonucleosides 837 Dermatan sulphate 529,530, 533, 534, 536 Dextrans 80,495, 525, 526 Diacetone alcohol 459 Diacetyl435,459 Diamines 637-643 Di-n-amyl ether 451 Diazinon 1012,1013,1018,1022,1023 Diazoketones, bis- 460 Diazosulphonate 934 Dibenzo[a,c]phenazine 92 1,923 Dibrom 1023 Di-n-butyl ether 451 Dichlorofenthion 1023
LIST OF COMPOUNDS CHROMATOGRAPHED Dichlorvos 1022,1023 Dicumyl peroxide 423 Dicyanodiamide 663 Di-n-dodecylether 423 Dieldrin 1010, 1012-1015,1017 Diethanolamine 650 Diethylamine 638, 642 Diethylene glycol 438 Diethylene glycol, monomethyl ether 433 Diethyl ether 423 Diethyl ketone 459 N,N-Diferrocenylcarbodiimide 1109 Digitoxose 487 Diglyceride, diglycosyl- 583 Diglyceride, monoglycosyl- 583 1,2-Diglycerides 609,610 1,3-Diglycerides 609,610 Diglycidyl ether 453 Diglycollic acid 567 Dihydrogendiphosphite 1093 Dihydrogendiphosphate 1098 Dihydrogenhypophosphate 1098 Diisoamyl ether 451 Diisopropyl ether 451 Dimefox 1022, 1023 Dimethoate 1013, 1020, 1022, 1023 Dimethylamine 638,642 Dioctadecyl ether 423,584 Dioctyl ether 423 Diol esters 584 Dioxathion 1012 Diphenoquinone, 3,3’-dihydroxy- 447 Diphenyl421,423,425 Diphenyl, phydroxy- 444 Diphenyl, polychlorinated 1013, 1026 Diphenylamine, 2-nitro- 657 Diphenyl-4-carboxylic acid 928 Diphenyl4,4‘dicarboxylicacid 928 Diphenyl4,4’-disulphonicacid 928, 930 Diphenyl ether 451 Diphenyl sulphone, 4-amino-4’-acetamido933 Diphenyl sulphone, 4,4’-diacetamido- 933 Diphenyl sulphone, 4,4’diamino- 933 Diphenyl-4-sulphonic acid 928, 930 Diphenyl-4-sulphonic acid, 4’-hydroxy 930 Diphosphate 1097,1098 Diphosphopyridine nucleotide 852 Dipropylene glycol 433 Di-n-propyl ether 451 [ 1,2-6:3,4-b' ] Dipyran-4(3H),10(9H)-dione, 5-hydroxy-2,2,8,8-tetramethylbenzo897
1151
[ 1,2-b: 3,4-b’]Dipyran-5-01, 3,4,9,10-tetrahydro6-isobutyl-2,2,8,8-tetramethylbenzo-898 [ 1,2-b :3,4-b’]Dipyran-5-01, 3,4,9,10-tetrahydro2,2,8,8-tetramethylbenzo- 898 Disulfoton 1012, 1020, 1022, 1023 Diterpenes 629 Diuron 661, 1029 DNA 862,864,866,868-873 DNA, anticodon strand 873 DNA, circular 859 DNA, cross-linked 865 DNA, denatured 866, 867, 872 DNA, double-stranded 865 DNA, heat-denatured 865 DNA, high-molecular-weight 870 DNA, mitochondrial862 DNA, native 867 DNA, single-stranded 859 DNA, structural studies 836, 848 DNA, supercoiled 859 DNA, transformation, from B. subtilis 867 DNA, transforming, from H. influenzae 864, 869 DNA from B. subrilis 861, 868, 870 DNA from B. subtilis, denatured 869 DNA from B. subtilis, native 870 DNA from calf thymus 864, 866, 880, 881 DNA from E. coli, denatured 868 DNA from H.influenzae, alkalidenatured 865 DNA from h phage 862 DNA from phage T2 864 DNA from v X , , ~phage 862 DNA from polyoma virus 864 DNA from S. cerevisiue mitochondria 864 DNA from S. cerevisiae nuclei 864 DNA from T-even phages 848 DNA-RNA complex 866 DNA-RNA hybrid 859 DNA satellite 865 DNase 873 Dodecane 421-423 Dodecanedisulphonic acid 928 Dodecanesulphonic acid 928 Dodecanoic acid 571 Dodecanol436 Dolineone 915,916 Dolineone, 12a-hydroxy- 915,916 DOPA, see Phenylalanine, 3,4-dihydroxyDOPAC, see Phenylacetic acid, 3,4dihydroxyDopamine 638,642,646,650-653 Dursbane 1013 Dyes 1033-1037 Dysprosium 1092, 1095, 1117
1152
E Ecdysone 6 19 Eicosane 422 Elemane 631 PElemene 627 Endosulfan A 1012,1013 Endosulfan B 1012 Endrin 1012, 1013,1016 Enduracidin 1003 Enniatin B 1003 Enpressuflavone 903 Enzyme(s)807-830 Enzyme, fast cathode-migrating 825 Enzyme, slow migrating 825 Ephedrine alkaloids 892 Epiandrosterone, dehydro- 61 3 [ 2-'4C]Epicatechin, 5,7,3',4'-tetramethyl- 905 16-Epiestriol 612 17-Epiestriol 612 16,17-Epiestriol612 Epilaccishelloic acid 630 Epinephrine, see Adrenaline Epinine 646 16-Epiphyllocladan-15-one631 Epishyobunone 631 Epitestosterone 604 Epoxide resins 1070 Epoxides, terpenic 629, 630 9,lO-Epoxystearic acid, methyl ester 579 Epoxystearic acids 576 Equilenin 61 2 Erbium 1095 Ergocalciferol, 25-hydroxy- 95 9 Ergosterol 958 Ergot alkaloids 892, 893 Erosnin 915,916 Erosone 915,916 Erythritol438.470, 502-504 Erythrocuprein 803 Erythromycin 994 Erythronic acid 514, 560 Erythrose 488, 502 Essential oils 623-635 Estradiol610-613 17a-Estradiol612 170-Estradiol, see Estradiol Estradiol, ethynyl- 610 Estradiol, l7a-glucosiduronic acid 612 Estradiol, 17p-glucosiduronic acid 602 Estradiol, monopropionate 609, 610 Estradiol3-benzoate 610 Estradiol cyclopentylpropionate 609, 610 Estradiol dipropionate 609, 610
LIST OF COMPOUNDS CHROMATOGRAPHED
Estra-l,3,5(10),6,8-pentaen-17-one,3-hydroxy-, see Equilenin Estra-l,3,5(1O)-triene-3,17pdiol, see Estradiol Estra-l,3,5(10)-triene-l6,17dione, 3-hydroxy612 Estra-l,3,5 (1O)-trien-l6-one, 3,17phydroxy612 Estra-l,3,5(10)-trien-17-one, 3-hydroxy-, see Estrone Estra-l,3,5( lO)-triene-3,16a,l7a-triol, see 17-Epiestriol Estra-l,3,5(1O)-triene-3,16a,l7p-triol,see Estriol Estra-1,3,5(lO)-triene-3,16~,17~~-triol, see 16,17Epiestriol Estra-l,3,5(10)-triene-3,16p,l7~triol, see 16-Epiestriol Estrenolone, vinyl- 610 Estriol610-612 Estrogens 594,601,605-612 Estrone 610-613 Ethane, diphenyl- 423 Ethane, 1,1,2-triphenyl- 419 Ethanediol435 Ethanediol, dioctadecanoate 584 Ethanediol, dioctadecyl- 584 Ethanediol, octadecyl-, acetate 584 Ethanediol, octadecyl-, octadecanoate 584 Ethanol 433,435-437 Ethanol, 2-phenyl- 432,434 Ethanolamine 638,642,646,650,700,706 Ethanolamine, lysophosphatidyl- 583, 589 Ethanolamine, N-methyl- 638 Ethanolamine, phosphatidyl- 582-584, 589 Ethanolamine, phosphoryl- 11.23 Ethers 451-453,629,630 Ethidium bromide 1123 Ethion 1013,1022,1023 Ethoate-methyl 102 3 Ethyl acetate 460 Ethylamine 638,640,642,646 Ethylamine, 2,2'-dithiobis- 646 Ethylamine, 2-methoxy- 638 Ethylamine, N-methyl- 638 Ethyl-n-butyl ether 451 Ethylene glycol 436,438,439,470, 502 Ethylene glycol, oligomers 437,452 Ethylenediamine, N-acetyl- 638 Ethylestrenol610 Ethyl glycosides 496 Ethynodiol diacetate 610 Eugenin 897 Euparin 908 Europium 1095, 1117,1122 allo-Evodionol 898 aflo-Evodionol, dihydro- 898
LIST OF COMPOUNDS CHROMATOGRAPHED
F PFarnesene 627 Farnesylacetic acid, geranyl esters, separation of isomers 630 Fatty acids 582, 583, 587, 589 Fatty acids, unsaturated 585 Fenchlorphos 1022, 1023 Fenitrothion 1012, 1020, 1022, 1023 Fenthion 1019,1022 Fenthion 0-analogue 1019 Fenthion 0-analogue sulphone 1019 Fenthion sulphone 1019 Fenthion sulphoxide 1019 Fenuron 661,1029 Ferna-7,9(1l)-diene 629 Fern-7-ene 629 Fern-8-ene 629 Fern-g(ll)-ene 629 Ferredoxin 1047 Ferrocene(s) 1108, 1109 Ferrocene, 2-acetyl-l,l’-dimethyl- 1107 Ferrocene, 3-acetyl-l,l’-dimethyl- 1107 Ferrocene, I-acetyl-1’-ethyl- I107 Ferrocene, 1-acetyl-2-ethyl- 1 107 Ferrocene, 3-acetyl-3-ethyl- 1107 Ferrocene, acetylmethyl- 1108 Ferrocene, cis-bis-(or-ketotetramethy1ene)- 1 108 Ferrocene, trans-bis-(or-ketotetramethylene1108 Ferrocene-2-carboxylic acid, 1,l ‘-dimethyl1107 Ferrocenophan carboxylic acid diphenylamides 1108 Ferrocene carboxylic acid amides, isopropyl1108 1-Ferrocenyl-2-propyl acetate 1108 2-Ferrocenyl-1-propyl acetate 1108 Ferulic acid 570 Fibrinogen 792 Filic-3-ene 629 Filipin 995 Fisetin 908 Flavanone(s) 897, 899 Flavanone, 3-methyl-3‘-methoxy-4’,5,7-trihydroxy- 905 Flavan-(4or-ylthio) acetic acid 905 Flavan-(4or-ylthio) acetic acid, 3-hydroxy-, methyl ester 905 Flavan-(4a-ylthio) acetic acid, 7-methoxy- 905 Flavine-adenine dinucleotide 965, 966 Flavine nucleotides 838 Flavins 965,966 Flavone 899,908
1153
Flavone, 3’,5-dihydroxy-4’,7-dimethoxy902 Flavone, 4,5-dihydroxy-3’,6,7,8-tetramethoxy902 Flavone, 4,5-dihydroxy-6,7,8-trimethoxy902 Flavone, C-p-glucopyranosyl-(6)-O-mono-pglucoside-(7)-5,7,4’-trihydroxy-900 Flavone, 6-C-p-glucopyranosyl-5,7,4’-trihydroxy900 Flavone, 3,3’,4‘,5,6,7,8-heptamethoxy902 Flavone, 3‘,4’,5,6,7,8-hexamethoxy902 Flavone, 5-hydroxy-6,7,4’-trimethoxy901 Flavone, 3’,4’,5,6,7-~entamethoxy902 Flavone, 3’,4‘,5,7,8-~entamethoxy902 Flavone, 4’,5,6,7,8-pentamethoxy902 Flavone, 4’,5,7,8-tetramethoxy- 902 Flavone, 5,5‘,7-trihydroxy-3’,4’,6,8-tetramethoxy- 900 Flavone, 3-veratrylidene-7-methoxy-, hydrogenated products 906 Flavon-3-[ p-galactopyranoside tetraacetate], 3,5,3’-trihydroxy-7,4’-dibenzyloxy-905 Flavonoid(s) 896-898 Flavonol 899 Flavonol, dihydro- 899 Flavon-3-p-[ 6-O-a-rhamnosyl-glucoside, 3,5,3’trihydroxy-7,4’-dimethoxy-906 Flavoproteins 798 Flavoyladorinin 902 Fluoranthene 422,424,425 Fluoranthene, 1,2,3,4-tetrahydro- 424 Fluorene 419 Fluoren-9-one 458 Fluorine 1098 Folic acid 553,962,972,973 Formaldehyde 457-459,470,502 Formaldehyde, 2,4-dinitrophenylhydrazone 457 Formic acid 546, 547, 549,554,559, 567 Formycin A 999 Formycin B 999 Formononetin 901,904 Frideline 632 Fructosamine 497 Fructose 487,488,490492,495, 943 p-Fructose, 2,3:4,5-di-O-isopropylidene495 Fructose-l,6-diphosphatasefrom rabbit liver 810 Fructose 1,6-diphosphate 515-518 Fructose 1-phosphate 517 Fructose 6-phosphate 515-517 Fruit juices 551 FSH, see Hormone, follicle stimulating Fucitol 504 Fucosamine 497
1154
Fucose 487,495,512 a-Fucosidase 8 12 Fucoxanthin 1042 Fukugetin 908 Fumaric acid 549,562,564,566,567,571 Furadane 1027 Furan derivatives 915 Furan, dibenzo- 418 Furano[ 3',2':2,3]pterocarpan 905 Furano [ 3',2 ' :2,3] pterocarpan, 8,9-dimethoxy-, hydrogenated 905 a-apio-Furanose, 1,2 :3,5di-O-isopropylidene510 Furanoterpenes 630 Furans, terpenic 629,630 Furazolidone 916 Furfural457,458,491 Furfural, 5-hydroxymethyl- 458,491 Furfuryl alcohol polymers 1070 Furoic acid, 2-tetrahydro- 557 Furospongin-2, dihydro- 630 Furospongin-2, tetrahydro- 630
LIST OF COMPOUNDS CHROMATOGRAPHED
a-Galactose, 1,2:3,4-di-O-isopropylidene-6-O(methy1thio)methyl- 510 PGalactose, 2,3-di-O-methyl-, methyloside 5 10 a-Galactose, ethyloside 505 @-Galactose,ethyloside 505 Galactose, 6-O-(p-glucopyranosyluronicacid)513 Galactose, methyl ether 510 a-Galactose, [methyl 3,4-O-isopropylidene-2-0(2,3,4,6-tetra-O-acetyl-p-galactopyranosid)] uronate, methyloside 508 a-Galactose, methyloside 505 PGalactose, methyloside 505 bromide a-Galactose. 2,3,4,6-tetra-O-acetyl-, 905 PGalactose, 2,3,6-tri-O-methyl-, methyloside 5 10 a-Galactosidase 812 pGalactosidase 812 Galacturonic acid 512,513,515,559, 560 Gallic acid 570 Gallium 1093,1095,1096,1116 Gangliosides 588 Genistein 904, 908
Genkwanin-6-C-&glucopyranosyl-X"-O-mono-
G Gadolinium 1095,1117 Gaillardipinnatin 632 Galactaric acid 561 Galactitol470,495,502-504 Galactonic acid 514, 559, 560 Galactonic acid, 2-deoxy- 558 Galactonic acid, 6-deoxy- 557-559 Galactonic acid, 2,3,5-tri-O-methyl- 514 Galactosamine 496,497,499, 500, 501, 638, 700,706 Galactosamine, N-acetyl-, 1-phosphate 51 6 Galactosaminitol490 Galactosaminoglycans 533 Galactose 485-488,491,492,495, 502, 503, 512 @Galactose,2-O-acetyl-l,6-anhydro-3-0(3,4,6-tri-0-benzoyl-2-deoxy-2-dichloroacetamido-p-glucopyranosy1)- 508 *Galactose, 6-O-acetyl-l,2 :3,4-di-O-isopropylidene 5 10 Galactose, 2-amino-2-deoxy-498 Galactose, 3-amino-3,6-dideoxy- 497 PGalactose, 3,6-anhydro-2-0-methyl-, methyloside 510 Galactose, 2-deoxy- 487 a-Galactose, 3-deoxy-3-fluoro-l,2:5,6-di-Oisopropylidene- 5 10
glucoside 903 GentamicinW 989-992 Gentamicin C, 990 Gentamicin C,, 990 Gentamicin C, 990 Gentiobiose 484,491 Gentiotetraose 484 Gentiotriose 484 Germacrene A 627 Germanium 1095 Gerniarin 901 Gestagens 594,605,613,614 Gibberellins 634 y-Globulin, human 871 Glucagon 780 Gl~cito1470,502-504 Glucitol, 1,S-anhydro- 509 a-Glucoisosaccharinic acid 514 PGlucoisosaccharinic acid 514 a-Glucometasaccharinic acid 5 14 PGlucometasaccharinic acid 514 Gluconic acid 495,514,515,557,563 Gluconic acid, 2,5-anhydro-, see Chitaric acid Gluconic acid, 2-0x0- 515,555, 563 Gluconic acid, 5-0x0- 515,555,563 Gluconic acid, 2,3,4,6-tetra-O-methyl- 514 Gluconic acid 6-phosphate 5 1 7 a-Glucosaccharinic acid 5 14 Glucosamine 494,496-498,500, 501,638, 642,699,700,703,706
LIST OF COMPOUNDS CHROMATOGRAPHED Glucosamine, N-acetyl-, 1-phosphate 516 Glucosaminitol499 Glucosaminouronic acid 497 Glucose 472,484-486,488-495, 502, 503, 506,509,512,516 Glucose, 2-acetamido-2-deoxy- 494, 495 Glucose, 2-acetamido-2-deoxy-3,4,6-tri-Omethyl-, butyloside 508 @Glucose, 2-0-acetyl4,6-0-benzylidene, methyloside 51 1 Wlucose, 3-O-acety1-4,6-0-benzylidene5 11 Glucose, 2-amino-2-deoxy-, see Glucosamine 498 Glucose, 2-amino-2-deoxy-3,4-di-O-methyl498 Glucose, 2-amino-2-deoxy-3,6-di-O-rnethyl498 Glucose, 2-amino-2-deoxy-4,6-di-O-methyl498 Glucose, 2-amino-2-deoxy-3-0-methyl498 Glucose, 2-amino-2-deoxy-4-0-methylGlucose, 2-amino-2-deoxy-6-O-methyl498 Glucose, 2-amino-2-deoxy-3,4,6-tri-O-methyl49 8 Glucose, 3-amino-3,6-dideoxy- 496, 497 K l u c o s e , 1,6-anhydro- 484,488 @Glucose, 1,6-anhydro-, benzyl ether 510 @Glucose, 4,6-0-benzylidene-, methyloside 507,511 Glucose, 6-chloro-6-deoxy-, methyloside, separation of a- and p-anomers 505 Glucose, 2-deoxy- 472,487 Glucose, 6-deoxy- 488 wGlucose, 6-deoxy-, methyloside 505 @Glucose, 6-deoxy-, methyloside 505 Glucose, 6-deoxy-, methyloside, separation of a-and p-anomers 505 @Glucose, 2,3-di-O-acetyl-l,6anhydro5 11 @Glucose, 2,4-di-O-acetyl-l,6-anhydro5 11 @Glucose, 3,4-di-O-acetyl-l,6-anhydro51 1 @Glucose, 2,3-di-O-acety1-4,6-O-benzylidene511 Glucose, 3,6-diamino-3,6-dideoxy505 methyl&lucose, 3,6-diamino-3,6-dideoxy-, oside 505 pGlucose, 3,6-diamino-3,6-dideoxy-, methyloside 505 495 &lucose, 1,2:5,6-di-O-isopropylidene@Glucose, 2,3-di-O-tosyl- 507 *Glucose, 1,2-0-ethylene- 509 Wlucose, 1,2-0-ethylene- 509 Glucose, 2-0-(2-hydroxyethyl)- 509 &lucose, 1,2-O-isopropylidene- 495 a-Glucose, methyloside 495, 505 @Glucose, methyloside 495, 505 Glucose, methyloside, separation of a- and panomers 505 Glucose, 2,3,4,6-tetra-O-benzyl- 506
1155
@Glucose, 2-0-tosyl- 507 Wlucose, 3-0-tosyl- 507 Glucose 1,6-diphosphate 5 17, 5 18 Glucose 1-phosphate 515-518 Glucose 6-phosphate 515-517 Glucose 6-phosphate dehydrogenase 810 Glucose 6-phosphate ketol-isomerase 825 oc-Glucosidase 812 PGlucosidase 812 Glucuronicacid495, 512, 513, 515, 559, 560 Glucuronic acid, 4-0-methyl- 5 12, 51 3, 5 15 Wlucuronidase 812 Glutamic acid 567, 1124 Glutamine 1124 Glutaric acid 549,554, 561, 567 Glutaric acid, 2-0x0- 554, 567 Glutathione 722, 982 Glyceraldehyde 502, 5 15 Glyceraldehyde 3-phosphate 516 Glyceric acid 495,514,567 Glyceric acid 1,3-diphosphate 5 17 Glyceric acid 2,3-diphosphate 516 Glyceric acid 3-phosphate 516 Glycerides 589,609 Glycerol 438,439,470, 502-504 Glycerol, diacyl- 582,587 Glycerol, 1,2-dioctadecyl-, octadecenoate 584 Glycerol, 1,2-dipalrnitoyl- 587 Glycerol, 1,3-dipalmitoyl- 587 Glycerol, dipalmitoyllinoleyl- 5 86 Glycerol, dipalmitoyloleyl- 586 Glycerol, diphosphatidyl- 583, 589 Glycerol, monoacyl- 582,587 Glycerol, monoepoxytrioleyl- 5 87 Glycerol, l-octadecyl-, dioctadecenoate 584 Glycerol, palmitoyldioleyl- 586 Glycerol, palmitoyloleyl- 586 Glycerol, palmitoyloleyllinoleyl- 5 86 Glycerol, phosphatidyl- 583, 589 Glycerol, l-stearoyl- 587 Glycerol, 2-stearoyl- 587 Glycerol, stearoyldioleyl- 586 Glycerols, triacyl- 582, 585,587 Glycerols, triacyl-, mercury adducts 585 Glycerol, trioctadecyl- 584 Glycerol, trioleyl- 586, 587 Glycerol, tripalmitoyl- 586 Glycerol, tristearoyl- 586 Glycerol esters 584-588 Glycerol lipids 584 Glycerol trioctadecenoate 5 84 Glycerophosphoethanolamine 699,706 Glycine 571 Glycine, dimethyl- 649
1156 Glycine, vanilloyl- 548 Glycolaldehyde 502 Glycolic acid 514,559, 562, 567 Glycolipids 583,584,588 Glycopeptides 53 8-541 Glycopeptides, structural analysis, automated 538 Glycoproteins 538-541,799 Glycoproteins, structural analysis, automated 538 Glycosaminoglycans 5 29 -542 Glycosaminoglycans, anionic 5 29 Glycosaminoglycans, cetylpyridinium complexes 529,530 Glycosaminoglycans, molecular-weight estimation 537 Glycosides 504-506 Glycosides, complex 506 Glycosides, phenolic 506 Glycosyl diglycerides 589 Glyoxal457 Glyoxylic acid 515, 559,567 Glyoxylic acid, 3,4-dihydroxyphenyl- 548 Glyox ylic acid, 4-hydroxy-3-methoxy-phenyl548 GMP, see Guanosine S’-phosphate Gold 1096,1117 Griseofulvin, dehydro: 1004 Guaiacol444,445 Guaiazulene 627 Guaioxide 629,633 Guaioxide, 4-hydroxy- 629 Guaioxide, 6p-hydroxy- 633 Guaioxide, 7orhydroxy- 633 Guanidine(sl657-664 Guanidine, nitro- 663 Guanidine, pentafluoro- 662 Guanine 834, 835,837,841 Guanosine 833-835,837,841,847 Guanosine, deoxy- 833,845-847 Guanosine, 2‘-deoxy- 841 Guanosine, N’, NZ-dimethyl- 833 Guanosine, 1-methyl- 833 Guanosine, 2‘-O-methyl- 833 Guanosine, 7-methyl- 833 Guanosine, N*-methyl- 833 Guanosine 2’-phosphate 835 Guanosine 3’-phosphate 835 Guanosine S’-phosphate 834, 835, 841, 847, 851,852 Guanosine 5’-pyrophosphate 835, 852 Guanosine 5’-triphosphate 835, 852 Gulonic acid 514 Gulonic acid, 3,5,6-tri-O-methyl- 514 Gulosamine 497 Gulose 488
LIST OF COMPOUNDS CHROMATOGRAPHED Guluronic acid 512, 513, 515 Gums 525,526 Guthion-methyl 1013
H Haemocyanin 793, 803 Haemoglobin 785,794,803 Haemoproteins 803 Haemovanadin 803 Hafnium 1116, 1117 Haloxon 1023 Hashish components 898 Hemicelluloses 462 Heparin 529-531,534,535 Heparin sulphate 535,537 Heparin sulphuric acid 531 Heptachlor 1010, 1012, 1013, 1016 Heptachlor epoxide 1010,1012,1013 Heptaldehyde 460 Heptane 421-423,426 Heptanoic acid 571 l-Heptanol433,435,436 3-Heptanol433 1-Heptene 423,426 Heptonic acid, glycero-gulo- 514 Heptonic acid, glycero-manno- 514 aHeptose, xylo-, 4,6dideoxy-, methyloside 506 Hepturonic acid, gluco-, 6-deoxy- 557 orHept-6-ynose, gluco-, 3-0-benzyl-6,7-dideoxy1,2-O-isopropylidene- 51 1 PHept-6-ynose, ido-,3-0-benzyl-6,7-dideoxy1,2-O-isopropylidene- 5 1 1 Hesperetin 908 Hesperidin 908 Hetacillin 983 Hexachlorobenzene l u l j Hexadecane 421 Hexadecanedisulphonic acid 928 Hexadecanesulphonic acid 928 1,s-Hexadiene 426 Hexamethylenediamine 640 Hexan-2,s-dione 459 Hexane 422,423,426 Hexane, 2,4-dimethyl- 426 Hexane, 2-methyl- 426 Hexanoic acid 571 l-Hexanol435,436 Hexan-2-one, 6,6-diarylbicyclo [ 3.1.01 460 Hexatriacontane 421,422 Hexene-1 426 5-Hexenoic acid, 2-amino-rl-methyl- 1000 Hexonic acid(s) 555
LIST OF COMPOUNDS CHROMATOGRAPHED
Hexonic acid, arabino-, 2-deoxy- 557 Hexonic acid, lyxo-, 2-deoxy- 557 Hexonic acid, lyxo-, 3-deoxy- 557 Hexonic acid, ribo-, 2,6-dideoxy- 557 Hexonic acid, xylo-, 3-deoxy- 557 Hexosamine 482,497,537 Hexosaminuronic acids 497 Hexose, lyxo-, 2-deoxy- 487 PHexose, ribo-, 1,6-anhydro-4-deoxy- 509 Hexose, ribo-, 4-deoxy- 509 Hexose, ribo-, 2,6-dideoxy- 487 Hexose, xylo-, 3-deoxy-l,2:5,6-di-O-isopropylidene- 5 10 Hexulose 487 a-Hex-5-ulose, xylo-, 3-O-benzyl-l,2-O-isopropylidene- 468 Hexulosonic acids 51 3 5-Hexulosonic acid, arabino- 513 Hexulosonic acid, 3-deoxy- 515 5-Hexulosonic acid, lyxo- 5 13 5-Hexulosonic acid, ribo- 5 13 4-Hexulosonic acid, xylo- 5 13 Hexuronic acids 513,555 Hexylamine 638 Hinokiflavone 903 Hippuric acid 553 Hippuric acid, 2-amino- 647, 649 Hippuric acid, 4-amino- 643,644 Hippuric acid, 3,4-dichloro- 1028 Hippuric acid, 2-hydroxy-, see Salicyluric acid Histamine 638,640,646,650, 651 Histamine, N-acetyl- 646 Histamine, 1-methyl- 646 Histidine 638, 646, 651 Histidine, 1-methyl- 638,646, 700, 706 Histidine, 3-methyl- 638, 700, 706, 722 Histones 795, 868 Holmium 1095, 1117 Holothurinogenin, 17-deoxy-22,25-oxido- 632 Holothurinogenin, 17-deoxy-22,25-oxido-, acetate 633 Holothurinogenin, 22,25-oxido- 6 32 Holothurinogenin, 22,25-oxido-, acetate 633 Homocitrulline 732 Homocysteine 982 Homocysteine-penicillamine disulphide 982 Homocysteine trisulphide 982 Homocytidine 843 Homoflavoyladorinin 902 Homogentisic acid 553 Homoorientin 901, 908 Homouridine 843 Homovanillic acid, see Phenylacetic acid, 4-hydrox y-3-methoxy-
1157
Homovitexin 901 Hopane, 17,2l-epoxy- 630 Hop-17(2 1)-ene, 30-acetoxy- 63 1 Hop-22(29)-ene 629 Hormone, adrenocorticotropic 789, 790 Hormone, follicle stimulating 790, 791 Hormone, light-adapting 791 Hormone, plipotropic 791 Hormone, y-lipotropic 790 Hormone, luteinizing 776, 787,792 Hormone, melanocyte stimulating, a 790 Hormone, melanocyte stimulating, p 790 Hormones, peptidic 752-754 Hormone, red pigment concentrating 791 Hormone, thyroid stimulating 791 Humulene 626 Humulene dioxide 626 Humulene epoxide 626 Humulenol-I1 626 Hyaluronic acid 529-536 Hydrazo compounds, aromatic 657-664 Hydrindene 424 Hydrocarbons 417-429,582, 584, 589 Hydrocarbons, fluorinated 428 Hydrocarbons, halogen derivatives 41 7-429 Hydrocarbons, paraffinic 41 7-429 Hydrocarbons, polycyclic 41 7 -429 Hydrocarbons, terpenic 624-626, 628, 629 Hydrocarbons, tricyclic 626 Hydrocarbons, triterpenic from P. vulgare 630 Hydrochloric acid 546,571 Hydrogendiphosphite 1098 Hydrogenhypophosphate 1098 Hydrogenphosphate 1097, 1098 Hydrogenphosphite 1097, 1098 Hydrolases 818-823 Hydroquinone 443-445 Hydroxylamine 638 Hydroxylysine 700, 701, 706 allo-Hydroxylysine 700, 701 Hydroxyproline 699,701, 706 Hygromycin B 993 Hyperin 908 Hypoxanthine 835,837,841,851
I Idonic acid 557, 563 Idose, 3-amino-3,6-dideoxy- 497 Iduronic acid 5 15 Iminobispropylamine 640
Immunoglobulin,780,782,787,803,1123 Incensol629
1158 Incensoloxide 629 Incensoloxide, benzoate 629 Indane 419,422 Indene 424 Indigo carmine 1035 Indium 1093,1095,1096,1118 Indole 647,922 Indole, 3-phydroxyethyl- 1003 Indole, 3-methyl- 647 Indole-3-acetic acid 647, 648 Indole-3-acetic acid, 5-hydroxy- 647,648,651 Indole-3-acetic acid, 5-hydroxy-, methyl ester 920 Indole-3-acetic acid, methyl ester 920 Indole-3-acetonitrile 648 Indole-3-aceturic acid 648 Indole-3-carboxaldehyde 648 Indoxyl sulphate 647 Influenza virus 871 Inosine 833, 835, 837, 841 Inosine, deoxy- 518, 841 Inosine, 1-methyl- 833 Inosine 5'-phosphate 835, 851, 852 Inositol, hexaphosphate 51 7 Inositol, phosphatidyl- 583, 589 myo-Inositol, see Myoinositol Inositol polyphosphates 942, 943 Insect hormones, steroidal 619, 620 Insecticides, natural '1027 Insulin 780,87 1 Insulin, A-chain 780 Insulin, B-chain 780 Insulin, structural studies 770 Iodine 1098,1119,1122 Iodobenzene-psulphonyl chloride 7 37 Ion01433 Iridium(III), dibromo-bis-(1,lo-phenanthroline)-, cation 1107 Iridium(III), dichloro-bis-(1,lO-phenanthro1ine)-, cation 1107 Iron 1094-1096,1116-1119,1121 Iron, di-, hexacarbonyl(2,7-dimethyloxepin) 1110 Iron(II), hexacyano-, anion 1098 Iron(III), hexacyano-, anion 1098 Iron, tricarbonyl(2,7-dimethyloxepin)1110 Isano oil 585 Isoamylamine 638,640,642,646 Isobavachin 898 Isobutanol435 Isobutylamine 638,640 Isobutyric acid 567 Isobutyric acid, 3-amino- 699, 700, 706
LIST OF COMPOUNDS CHROMATOGRAPHED Isobutyric acid, 2,4-dinitrophenylhydrazide 548 Isobutyric acid, 2-hydroxy- 557 Isobutyric acid, 2-hydroxymethyl- 557 Isocalamendiol633 20-Isocholesterol, 17a, 20p-dihydroxy- 607 Isocitric acid 567 Isocryptomerin 903 Isodrin 1012 Isoelliptol isoflavone 905 Isoflavone 899 Isoflavone, 7-p-glucosyl-5,7-dihydroxy-4'methoxy 901 Isoflavone, 5-malonyl-7-pglucosyl-5,7-dihydroxy4'-methoxy- 900 Isoincensol benzoate 629 Isoincensoloxide 629 Isoleucine, N-methyl- 1000 do-Isoleucine 1000 do-Isoleucine, N-methyl- 1000 Isomaltose 493 Isomaltotriose 493 Isomerase(s) 825, 826 Isomerase, phosphcglucose 826 Isomuramic acid 497 Isooctane 423 Isopapuanic acid 901 Isophthalic acid 566 Isopimaradiene 628 Isopimaradienol633 Isopimaral 632 Isopimaric acid 631 Isopropanol433,435 aIsopropylidenecyclotriveratrylenesa(8) and P(8) 453 PIsopropylidenecyclotriveratrylenesa-(8) and p(8) 453 Isoquinoline 922 Isorhamnetin-3-mono-p-glucoside 903 PIsorhodomycinone 999 I soscutallar ein 9 02 Isoshyobunone 631 Isosinensetin 902 Isovaleric acid, 2,4-dinitropher:ylhydrazide548 Isovaleric acid, 2-hydroxy- 557, 558 dsovetivene 627 Isoxanthopterin 973 Itaconic acid 561,567
J Juglanin 908
LIST OF COMPOUNDS CHROMATOGRAPHED
K Kaempheritrin 308 Kaempherol908 Kaempferol-3-rhamnoside 904 Kaempferol-3-6-sophoroside 906 Kaempferol-3-p-sophoroside,7,4’-dibenzyl- 905 Kalafungjn 1004 Kamala seed oil 585 Kanamycin 992 Kanamycin A 989 Kanamycin B 989 Kanarnycin C 989 Kasugamycin 992 Kaurene 628 Kaurenetetraol, see Lasiodonin Kaurenetriol, see Lasiokaurin Kaurenolide, 7,18-dihydroxy- 632 Kaurenolide, 7-hydroxy- 632 Kaurenolide, 7,16,18-trihydroxy- 6 32 Kelthane 1013 Keratan sulphate 529, 531-533, 536 Ketones 455-464 Ketones, aliphatic 456-458 Ketones, aliphatic, 2,4-dinitrophenylhydrazones 456 Ketones, cyclic 456-458 Ketones, terpenic 631,632 Kojic acid 492 Kymenoxin 900 Kynuramine 646 Kynurenic acid 647-649 Kynurenine 647-649 Kynurenine, N-acetyl- 647,649 Kynurenine, N-acetyl-3-hydroxy- 649 Kynurenine, 3-hydroxy- 648,649
L Laccishelloic acid 630 Lactic acid 481,514,516, 552, 559, 560, 562, 563,567,571 Lactic acid, 3-(4-hydroxyphenyl)- 553 Lactic acid, lactyl- 552 Lactic aldehyde 457 Lactobionic acid 514 Lactones, diterpenic, from G. fujikuroi 632 Lactones, terpenic 632, 633 Lactones, triterpenic 632 Lactose 491,492 Laevulinic acid 492,515 Laevulinic acid, 6-amino- 1124 Laminaribiose 484, 495
1159 Laminaripentaose 495 Laminaritriose 484,495 Lanatosides 618 Lanostan-l 16-01, 36-acetoxy- 629 Lanthanum 1092,1095,1096,1117,1121 Lanthionine 982 LAS, see Sulphonates, alkylbenzeneLasiodonin 633 Lasiokaurin 633 Laspartomycin 1003 Lauric acid 549, 553 Lauric acid, cholesteryl ester 422 Lead 1095,1122 Lecithin 588 Lehman enzyme 861 Leucine 871 Leucoanthocyanidins 896,911 Leucomycin A,, 9-dehydro-18-dihydro- 995 Leucomycin A,, 18-dihydro- 995 Levulinic acid 559, 560 LH, see Hormone, luteinizing Ligases 826-829 Lignin 455,461-463 Ligninsulphonic acid 455,463 Ligninsulphonic acid, calcium and lithium salts 463 Ligularane, 8,8a-epoxyfurano- 630 Liguloxide 629 Limonene 625 Linarin 908 Lincomycin 991 Lincomycin, 7-chloro-7-deoxy-, see Clindamycin Lincomycin group of antibiotics 991, 992 Lindane 1010,1012,1016, 1017 Lindebein 1003 Linoleic acid 586 Linoleic acid, hydroperoxide 577 Linoleic acid, methyl ester 577 Linoleic acid, methyl ester, hydroperoxide 579 Linolenic acid 569 Linuron 1029 Lipase 821 Lipase from R.arrhizus 819, 820 Lipids 581-592 Lipids, acidic 589 Lipids, neutral 581-583, 585-588, 781 Lipids, polar 588-590 Lipids from serum 583 a-Lipomycin 993 Lipopolysaccharides 5 88 Lipopolysaccharide C 496 Lipoproteins 589 0-Lipoproteins 788 Lipoprotein complexes 781
1160 Liquiritin 908 Lithium 1088, 1094 Lividomycin A 992, 993 Lividomycin B 992, 993 Longifolene 626,627 Lmocerin 908 Lumazine 973 Lumazine, 6,7-dimethyl-8-ribityl- 966 Lumazine, 6-methyl-7-hydroxy-8-ribityl- 966 Lupenol palmitate 631 Lutecium 1095,1117, 1122 Lutein 1042, 1047 Luteolin 908 Luteolin-7-glucoside 904,906,908 Luteolin-7-P-neohesperidoside904, 906 Lyase(s) 823-825 Lyase, fructose-l,6-diphosphateD-glyceraldehyde3-phosphate 824 Lymphocytes 1083 Lynestrenol610 Lysine 638 Lysolecithin 588 Lysozyme 754, 786 Lysozyme, nitrosyl-, structural studies 769 Lysozyme, structural studies 754,766 Lysozyme from rat liver 812 Lyxonic acid 514 Lyxose 487,488,490,492,495 PLyxose, 1,5-anhydr@2,3-O-isopropylidene509 Lyxose, 2,3-O-isopropylidene-, methyloside 509 Lyxuronic acid 557
M Macroglobulins 782 Macrolides 994-996 Macromomycin 1003 Magnesium 1088,1089,1094-1096 Malathion 1012, 1013, 1018, 1022, 1023 Malathion 0-analogue 1022 Maleic acid 552,560-562, 564, 566, 567, 571 Malic acid 549, 551,552,561,562,564, 571 Malonic acid 552,562,564,567 Maltenes 1065 Maltobionic acid 514 Maltodextrins 484 Maltohexaitol495 Maltohexaose 495 Malt01557 Maltose 486,488,491,493,495 BMaltose, 1,2,6,2',3',4',6'-hepta-O-acety!-5 11
LIST OF COMPOUNDS CHROMATOGRAPHED
p-Maltose, octa-0-acetyl- 51 1 Maltotetraose 486 Maltotriose 486,493 Maltoundecaose 493 Malvidin 910 Malvidin-3,5-diglucoside91 1 Mandelic acid, 3,4-dihydroxy- 548,653 Mandelic acid, 4-hydroxy- 553 Mandelic acid, 4-hydroxy-3-methoxy- 548, 553, 653 Manganese 1094,1096, 1116, 1118 Manganese, pentacarbonyl-o-(2H-hexafluorocyclopent-3-eny1)- 1110 Manganese, pentacarbonyl-o-(4H-hexafluorocyclopenteny1)- 1110 Manganese, pentacarbonyl-o-(SH-hexafluorocyclopenty1)- 1110 Manganese, pentacarbonyl-u-(4H-tetrafluoropent-1-en-3-onyl) 1110 Mannan 525 Mannitol470, 502-504 Mannitol, 1,4-anhydro- 509 Mannobiose 488 Mannonic acid 5 14 Mannonic acid, 2,5-anhydro-, see Chitonic acid Mannonic acid, 6-deoxy- 5 14 Mannosamine 497 Mannose 470,487,488,490-492,495,502, 503,512 Mannose, 2-acetamido-2-deoxy- 495 Mannose, 3-amino-3,6-dideoxy- 497 orMannose, 3-acetamido-3,6-dideoxy-, methyloside 507 orMannose, 2-0-acetyl-3,4,6-tri-O-benzyl-, methyloside 468 Mannose, 4-0-p-glucopyranosyl- 49 1 orMannose, methyloside 495, 505 pMannose, methyloside 505 orMannose, 3,4,6-tri-O-benzyl-, methyloside 468 Mannose 1,6-diphosphate 517 orMannosidase 812 Mannuronic acid 512,513, 515 Manool633 Manoyl oxide 632 Mecarbam 1022,1023 Megalomicin A 995 Megalomicin B 995 Megalomicin C, 995 Megalomicin C, 995 Meisenheimer complex 747 Melibionic acid 5 17 Melinacidin 1003 Mercaptans 933 Mercury 1094,1122
LIST OF COMPOUNDS CHROMATOGRAPHED
Mesaconic acid 567 Mescaline 646 Mesotartaric acid 564 Mestranol610 Metallocarboranes 949,950 Metallocenes 1109, 1110 Metalloproteins 793, 803 Metanephrine 646 Metanephrine, N-methyl- 646 Methacrylic acid, methyl ester 423 Methane, bis(difluoroamino)difluoro- 662 Methane, dichloro- 427 Methane, diphenyl- 423 Methane, nitro- 658 Methane, tetrachloro- 427 Methane, trichloro- 427 Methane, tris(difluoroamino)fluoro- 662 Methanilic acid 643,644 Methanol 433,435,437 Methanol, (cis-2,2-dimethyl-3-hydroxy-6methylene-cyc1ohexane)- 629 Methionine sulphone 698, 706 Methionine sulphoxide 698,699, 701, 706 Methioxy ketoxime 934 Methoxychlor 1012, 1013,1017 Methylamine 638, 640, 642 Methyl amyl ketone 458 Methyl butyl ketone 458 N-Methylcarbamate, 3,4-dichlorobenzyl- 1028 N-Methylcarbamate, 3,5-dichlorobenzyl- 1025 Methyl decenyl ether 453 Methyl ethyl ketone 457,459,460 Methyl heptyl ketone 458 Methyl hexyl ketone 458 Methyl isobutyl ketone 458,460 Methyl isopropyl ketone 459 Methyl metacrylate, oligomeric 1068 Methyl nonyl ketone 458 Methyl octyl ketone 458 Methyl pentenyl ether 453 Methylprednisolone 21-phosphate 617 Methyl propyl ketone 459 Mevinphos 1022,1023 Mobam 1025 Molybdate(s) anion(s) 1098 Molybdenum 1095,1116,1117,1119 Molybdenum, cis-alkylarylphosphine(tetracarbony1)- 1110 Molybdenum, frans-alkylarylphosphine(tetracarbony1)- 1110 Molybdenum, (rpcyclopentadieny1)cycloheptatrienyl- 1109 Molybdenum, bis-(q-cyclopentadieny1)dihydrido 1109
1161
Molybdenum, cis-dialkylphosphine(tetracarbony1)- 11 10 Molybdenum, trans-dialkylphosphine(tetracarbony1)- 11 10 Molybdenum, cis-diarylphosphine(tetracarbony1)1110 Molybdenum, trans-diarylphosphine(tetracarbony1)- 1110 Moneomycin D 993 Monoamines 637-643 Monoglycerides 609,610 Monosaccharides 474,483-496 Monuron 661,1029 Morin 908 Morphine alkaloids 893 Morphothion 1022,1023 MSH, see Hormone, melanocyte stimulating Mucopolysaccharides, see Glycosaminoglycans Muramic acid 497 Mycinose 488 Mycotoxins 912-915 Myoglobin 780, 793 Myoinositol503,518,943 Myoinositol, glycerol phosphoryl943 Myoinositol diphosphate 518, 943 Myoinositol hexaphosphate 942 Myoinositol monophosphate 518,943 Myoinositol pentaphosphate 51 8,943 Myoinositol tetraphosphate 518, 943 Myoinositol triphosphate 518,943 Myoinositol tripyrophosphate 943 Myrcene 625 Myricetin 908 My ricet in-3-rhamno side 9 04 Myristic acid 571 Myxoma virus 87 1 Myxoxanthin 1042 Myxoxanthophyll 1042
N NADP, see Nicotinamide-adenine dinucleotide phosphate Naphthalene 419,421,423-425,427 Naphthalene, dimethyl- 422 Naphthalene, 2,6-dioctyl- 41 9 Naphthalene, 2-hydroxy- 448 Naphthalene, 5-hydroxy-l,2,3,4,10,1 O-hexa-
chloro-6,7-epoxy-l,4,4~~,5,6,7,8,8ol-octahydro1,4-end0-5,8-exodimethano1014 Naphthalene, 1-hydroxy-6,7,8-trimethoxy447 Naphthalene, trimethyl- 422 Naphthalene-1 ,S-disulphonic acid 928
1162 Naphthalenedisulphonic acid, 2-methyl- 928 Naphthalene-1-sulphonic acid 932 Naphthalene-2-sulphonic acid 927,928,932, 935 Naphthalene-1-sulphonic acid, 6,7-dihydroxy932 Naphthalene-2-sulphonic acid, 6,7-dihydroxy932 Naphthalene-1-sulphonic acid, 2-m et h yl- 9 2 8 PNaphthol, see Naphthalene, 2-hydroxy2-Naphthol-3,6-disulphonicacid 9 32 2-Naphthol-6-sulphonic acid 932 2-Naphthol-8-sulphonic acid 932 Naphthol yellow S 1035 1-Naphthylamide 644 2-Naphthylamide 644 1-Naphthylamine 643 2-Naphthylamine 64 3 Narcissin 908 Naringenin 902,903 Naringenin, 4’-y,ydimethylaUyl- 905 Naringenin-7-0-[cellobioside heptaacetate] 905 Naringenin-7-prutinoside 903 Naringenin-7-&[rutinoside hexaacetate] 905 Naringin 905 Naringrnin 908 Neamine, see Neomycin A Nebramycin 992 Neburon 661 Nelumboside 908 Neoabietal632 Neodymium 1095,1120 Neofucoxanthin a 1042 Neofucoxanthin b 1042 Neohopa-l1,13(18)-diene 629 Neohop-12-ene 629 Neohop-l3(18)-ene 629 Neolinarin 908 Neomycin(s> 986-988,992 Neomycin A 986 Neomycin A, N-acetyl- 988 Neomycin B 986,988 Neomycin B, N-acetyl- 988 Neomycin B pyrophosphate 987 Neomycin C 986,988 Neomycin C, N-acetyl- 988 Neomycin C dipyrophosphate 987 Neomycin C pyrophosphate 987 Neomycin D 986 Neomycin E 986 Neomycin F 986 Neomycin sulphate 986 Neopterin 973 Neotelomycins 1003
LIST OF COMPOUNDS CHROMATOGRAPHED Neotenone 915,916 Neotenone, dehydro- 915,916 Neoxanthin 1042,1043,1047 Neptunium 1123 Neuraminic acid, N-acetyl- 495 Neurophysin 753 Neurotoxin 797 Niacin 962 Niacinamide 962 Nickel 1094-1096 Nickel(II), triaquatribenzo(b,f,j) (1,5,9) triazacycloduodecine-, nitrate 1102 Nicotinamide 838,967 Nicotinamide-adenine dinucleotide phosphate 967 Nicotinic acid 967, 968 Nicotinic acid, esters 967 Nifuroxime 916 Niobium 1120 Nitrates, alkyl658 Nitric acid 567 Nitrilotriacetic acid 566 Nitrite, sodium 658 Nitrocellulose 657 Nitro compounds 657-664 Nitroglycerine 657 Nitrosamines 657-664 Nobiletin 902 Nobiletin, 5-0-desmethyl- 902 Nojirimycin 993 Nonane 422,423,426 Nonanic acid 571 Nonanol436 Noradrenaline 642,646,650-654 Noradrenaline, isopropyl- 653,654 Noradrenaline, 0-methyl- 653 Noradrenaline, 0-methylisopropyl- 654 19-Norandrost-4-ene-3,l’I-dione 608 19-Norandrost-4-en-3-one,19phydroxy-, see 19-Nortestosterone Norepinephrine, see Noradrenaline Norethindrone 610 Norethinodrel610 Normetanephrine 646 19-Nortestololact one 604 19-Nortestosterone 608,610 19-Nortestosterone, 17pdecanoate 610 19-Nortestosterone, 17pphenylpropionate 6 10 19-Nortestosterone, 17ppropionate 610 Nuciferal 631 Nuclease 9 1 Nuclease, Neurospora 86 1 Nuclease, staphylococcal 95 Nucleic acids 859-886
LIST OF COMPOUNDS CHROMATOGRAPHED Nucleic acid, desalting components 834 Nucleic acids, methods of hydrolysis 832 Nucleic acids, sequence analysis 880-882 Nucleic acid components, automated analysis 836 Nucleic acids from E. coli infected with MS2 phage 870 Nucleosides 831-857, 871 Nucleoside antibiotics 999 Nucleoside triphosphates 1123 Nucleotides 831-857 Nylon 6 1069 Nylon 66 1063, 1069
0 Octacosane 422 Octadecanal584 Octadecane 422 Octadecanoic acid, methyl ester 584 9-Octadecanoic acid, 12-0xo-, methyl ester 579 Octadecanol acetate 584 Octadecanol octadecanoate 584 Octane 422,426 Octanoic acid 571 Octanol436 Octene-1 426 Octitol503 Octopamine 646 Octulose 1,8-diphosphate 517 Octulose 1,8-diphosphate, 5-deoxy- 51 7 Octulose 1&diphosphate, 2-keto-5-deoxy- 518 Octulose 8-phosphate 517 Oils 418 Oils, polymerized 579 Oiticica oil 585 Olean-12-en-27-oic acid, 3phydroxy- 634 Olefis 659 Oleic acid 586 Oleic acid, methyl ester 577 Oleic acid, thermal dimers 577 Oligonucleotides 847-851,862,878-882 Oligonucleotides from an Azoto bacrer nuclease 878 Oligophenylenes 1066 Oligosaccharides 469,473,474,483-496 Oligostyrenes 1068 Oligothymidylic acid 851 Ombuoside 906 Ononin 901 Opium alkaloids 893 Orange G 1035 Orcinol444,445 Organophosphorus pesticides 1012
1163
Orientin 901 Ornithine 638, 706 Orthanilic acid 643,644,930-932 Orthophosphate 942 Osajaxanthone 907 Ovalbumin 780, 781 0xalic acid 560, 562, 563, 567,571 Oxaloacetic acid 515, 567 Oxidase, milk xanthine 814, 815 Oxides, sesquiterpenic 629 Oxidoreductases 813-816 Oxindole alkaloids 893 0 x 0 compounds 455-464 0x0 compounds, 2,4-dinitrophenylhydrazones 455 0x0 compounds, oximes 455 Oxoformycin B 999 Oxydemeton-methyl1023 Oxytocin 752-754,771
P Pachyrrhizin 915,916 Pachyrrhizone 915,916 Pachyrrhizone, dehydro- 915 Pachyrrhizone, 12u-hydroxy- 915, 916 Paecilomycerol 1004 Palladium 1096 Palmitic acid 571,586 Palmitic acid, cholesteryl ester 422 Palm oil 586 Panepoxydione 898 Pantothenic acid 971 Pantothenic acid 4-phosphate 971 Pantotheno1971 Papaver alkaloids 893 Papuanic acid 901 Paraffins 595,624 Paraoxon 1013 Paraoxon-methyl 1013 Parasitic01 914 Parathion 1012,1013,1018-1020,1022,1023 Parathion 0-analogue 1022 Parathion-methyl 1013, 1016, 1019, 1020 Paromamine, see Neomycin D Paromomycin 988,992 Paromomycin I, see Neomycin E Paromomycin 11, see Neomycin F Patuletin-3-0-glucoside 900 Patuletin-3-rutinoside 900 Patulitrin 900 Pectic acid 512 Pectoliuarin 908
1164
LIST OF COMPOUNDS CHROMATOGRAPHED
Pedaliin 908 Pedalitin 908 Penicillamine 982,983 Penicillamine disulphide 982 Penicillamine tetrasulphide 982 Penicillamine trisulphide 982 Penicillanic acid, 6-amino- 980-982 Penicillanic acid, 6-amino-, polymers 983 Penicillin(s) 980 -9 85 Penicillin, benzyl- 981,983 Penicillin, phenoxymethyl-, sulphoxide methyl ester 981 Penicillins, semisynthetic 980 Penicillin G 981 Pentadecene, 1-phenyl- 419 Pentane 422,423,426 Pentane, 2,2-dimethyl- 426 Pentane, 2,3-dimethyl- 426 Pentane, 2,4-dimethyl- 426 Pentane, 3-ethyl- 426 Pentane, 3-methyl- 422,426 Pentane, 2,2,4-trimethyl- 422,426 1,3-Pentanediol, 2,2,4-trimethyl- 435 2,3-Pentanedione 459 2,CPentanedione 459 Pentanoic acids 548 Pentanol435 Pentanol, 2-hydroxymethyl-2-nitro- 658 Pentanol, 2,2,4-trimethyl- 433 Pentatriacontane 422 Pentene-1 426 Pentene-1, 2-methyl- 422,423,426 Pentene-l,3-methyl- 426 Pentene-l,4-methyl- 422,423 Pentene-1, 2,4,4-trimethyl- 426 Pentitol, glum-, 1-C-cyclohexyl-2,3:43-di-0isopropylidene- 51 1 Pentitol, munno-, 1-C-cyclohexyl-2,3:4,5-di-Oisopropylidene- 5 11 Pentonic acid, erythro-, 2-deoxy- 557 Pentonic acid, erythro-, 3-deoxy- 557 Pentoses 483 Pentose, erythro-, 2-deoxy- 487,495 Pentulose 485 Peonidin-3,s-diglucoside91 1 Peonin chloride 910 Pepsin 752 Pepsin, structural studies 753 Peptides 741-772 Peptides, cysteine-containing 769, 770 Peptides, 2,4-dinitrophenylsulphenylchloride derivatives 768 Peptides, nitrotyrosine-containing 768
Peptides, reaction products with 2,4,6-trinitrobenzenesulphonic acid 747 Peptide antibiotics 1000-1003 Peracetate, tetradecalylphenyl- 45 3 Perchlorate ammonium 657 Peroxides 451-453 Peroxide hydrogen 658 Perrhenate anion 1098 Perseitol 503 Perseitol-octitol503 Persicogenin 902 Perthane 1013 Pesticides 1009-1 03 1 Pesticides, carbamate 1024- 1029 Pesticides, chlorinated 1014-1018 Pesticides, phosphorus 1018-1 024 Petrolenes 418,420 Petroleum products 418 Petunidin 910 Petunidin-3,5-diglucoside9 11 Peucenin 898 Phellamurin 908 Phenanthrene 419,421,423-425 Phenanthrene, dihydro- 422,424 Phenanthrene, octahydro- 422,424 Phenazine 923 Phefiethyl alcohol 1003 Phenetole 423 Phenkapton 1023 Phenol 443-446,448,553,568,1070 Phenols 441 -449 Phenol, N-acetyl4amino- 446 Phenols, alkyl derivatives 444,447 Phenols, alkyl, ethylene oxide adducts 447 Phenol, 2-amino- 643,644 Phenol, 3-amino- 643, 644 Phenol, 4-amin0- 446,643,644 Phenol, 4-tert.-butyl- 448 Phenol, 2-chloro- 443-445 Phenol, 3-chloro-. 443 Phenol, 4-chloio- 443 Phenols, chloro derivatives 446 Phenol, 2,4-dichloro- 443 Phenol, 2,3-dihydroxy- 443 Phenol, 3,s-dihydroxy- 443 Phenol, 2,4-dimethyl- 443,447 Phenol, 2J-dimethyl- 434 Phenol, 2,6-dimethyl- 443,447 Phenol, 2,4-dinitro- 443,448 Phenol, 2,6-dinitro- 448 Phenols, halogen derivatives 447,448 Phenols, hydroxylated alkyl derivatives 447 Phenols, isopropyl derivatives 447 Phenol, 2-nitro- 443-448
LIST OF COMPOUNDS CHROMATOGRAPHED Phenol, 3-nitro- 443,446-448 Phenol, 4-nitro- 443,446-448 Phenol, pentachloro- 1015 Phenol, 2,4,6-trichloro- 443 Phenol, trinitro- 448 Phenolcarbolic acids 548 Phenol-formaldehyde resin 1062 Phenolglucinol444, 445 Phenolic acids 568,569 Phenol red 754 Phenol-4-sulphonic acid 930 Phenoxyacetic acid, 2,4-diChlOIO-, sodium salt 1027 Phenoxyacetic acid, 2,4,5-trichloro- 1026 Phenylacetic acid 546 Phenylacetic acid, 3,4-dihydroxy- 652, 653 Phenylacetic acid, 4-hydroxy-3-methoxy- 55 3, 653 Phenylalanine, 3,4-dihydroxy- 65 1-653 m-Phenylenediamine 644 o-Phenylenediamine 644 pphenylenediamine 644 p-Phenylenediamine, N-phenyl- 644 Phenylethylamine 638,640,646 Phenylethylamine, 3,4-dimethoxy- 646 Phenylethylamine, 3-hydroxy-4-methoxy- 646 Phenylethylamine, 4-methoxy- 646 Phenylpropionitrile oligomers 107 1 Phenylpropylthio acetate, 3-0-benzoyloxyphenyl-1-p-methoxy-, methyl 905 Pheophorbide u 1045 Pheophorbide b 1045 Pheophytin(s) 1043 Pheophytina 1042,1045 Pheophytinb 1045 Phleomycin 100 1 Phleomycin A 1001 Phleomycin B 1001 Phleomycin C 1001,1002 Phleomycin D, 1001 Phleomycin D, 1001 Phleomycin E 1001 Phleomycin F 1001,1002 Phleomycin G 1001 Phleomycin H 1001 Phleomycin I 1001 Phloretic acid 553 Phloridzin 899 Phloroglucinol445 Phomin 996 Phomin, 5-dehydro- 996 Phorate 1012, 1019,1022, 1023 Phorate 0-analogue 1022 Phorate sulphone 1022
1165
Phorate sulphoxide 1022 Phorbides 589 Phosalone 1023 Phosphamidon 1020,1022,1023 Phosphatase, acid 81 2 Phosphate 1097 Phosphate, tributyl 1023 Phosphate, triethyl 1023 Phosphate, trimethyl 1023 Phosphatidic acid 583, 589 Phosphitin 942 5’-Phosphodiesterase I 812 Phosphodiesterase I1 812 Phosphoethanolamine 699, 706 Phosphoglycerides 590 Phospholipids 581,582,588-590,781 Phospholipids, methylated 590 Phospholipids, molecular-weight determination 590 Phospholipids, oxidized 588 Phosphonic acid esters 1018 Phosphoric acid 567 Phosphorothiolic acid esters 1018 Phosphorothiolothionic acid esters 1018 Phosphorothionic acid esters 1018 Phosphorus compounds, inorganic 1096-1099 Phosphorus compounds, inorganic, P4-P3-P4-acid, pentasodium salt 1098 Phosphoserine 699, 706,722 Photodieldrin 1013 3-Photozerumbone 616 +-Photozerumbone, dihydro- 626 Phthalic acid 566, 568 Phthalide, 3,3-di-rerr.-butyldiperoxy45 3 Phycobilin-protein complex 1041, 1047, 1048 Phycocyanin(s) 1047, 1048 Phycocyanin, 4110- 1047 Phycoerythrins 1047, 1048 Phyllocladane, 15a,16-epoxy- 631 Phyllocladene 629 Phytin 589 Phytosphingosine 6 50 Picene 421 Picolinic acid 647 Picric acid 568, 747 Pigments of plastids 1039-1049 Pimaradiene 628 a-Pinene 625 PPinene 625 Pinocembrin-7-pneohesperidoside903 Pipendine 638,642 Piperine alkaloids 893 Pipsyl chloride 737 Plantaginin 908
1166 Plasmalogens 590 Plasmide from chromosomal DNA 867 Plasminogen 792 Plastics 1051-1073 Plastocyanin 803 Plastoquinone 455 Platinum 1096 Platinum(II), cis-bis-(n-propy1)-bis-(triethylphosphine) 1110 Platinurn(II1, trans-chlorohydrido-bis-(triethylphosphine) 1110 Platinum(II), dichloro-bis-(diethy1thio)-1105 Platinum(II), dichloro-bis-pyridine- 1105 Platinum(II), dichloro-bis-(n-tributy1phosphine)1105 Platinum(II), trans-dichloro-bis-(triethylph0sphine)hydrido- 1110 Platinum(II1, trans-n-propylchloro-bis-(triphenylphosphine) 1110 Pleraplysillin 630 Plutonium 1122, 1123 PMMA, see Poly(methy1 metacrylate) Polonium 1122 Polyadenilic acids, structural studies 849 Polyamides 1062 Polyamines 637-643 Polybutadiene 1056, 1057 Poly(1-butene) 1061 Polychloroprene 1056 Polycondensates 1062, 1063 Polydimethylsiloxane 1064 Polyesters 1062, 107 1 Polyether polyols 438 Polyethylene 1058- 106 1 Polyethylene glycol(s) 438,431 -439, 1062, 1067 Polyethylene glycol, monoalkyl phenyl ethers 45 3 Polyethylene glycol adipate 1071 Polyethylene glycol esters of fatty acids 587 Polyglycols 1062 Polyhydroxylic acids, erythro and threo-isomers 577 Polyisobutene 1056 Polyisobutylene 1057 Polyisoprene 1056, 1057 Polymerase, RNA 375 Poly(methy1 metacrylate) 1053 -1 055 Polymorphonuclear neutrophils 1082,1083 Polymyxin P 1003 Polynucleotides 878-882 Polynucleotides, doublestranded 864 Polynucleotides, random-coiled 864 Polynucleotides, triple-stranded 864
LIST OF COMPOUNDS CHROMATOGRAPHED
Polyolefins 1057-1061 POlyOlS 431-439, 471 Polyoxin(s) 999 Polyoxin A 999 Polyoxin B 999 Polyoxin E 999 Polyoxin F 999 Polyoxin C 999 Polyoxin H 999 Polyphenolic substances 896, 898 Polyphosphates, 838, 1097 Polypropylene 1061 Polypropylene glycol 1062 Polyribonucleotides 839 Polysaccharides 473,476,483, 523-528 Polysaccharides, branched-chain 523 Polysaccharides, protein complexes 529-542 Polysaccharides, structural studies 487 Polystyrene 1052-1057 Poly(styrene-co -butadiene) 1063 Polysulphone 1065 Polyuretane oligomers 1070 Polyvinyl chloride 1056 Poly(4-vinyldiphenyl) 1063,1064 Poly(4-vinyldiphenylpolyisoprene) 1064 Poly(2-vinylpyridine) 1065 Ponasterone 6 19 Ponceau 3R 1035 Ponceau 4R 1035 Ponceau 6R 659 Ponceau SX 1035 Poncirin 905 Porphyrin(s) 917 -9 19 Porphyrin A 918 Porphyrin B 9 18 Porphyrin C 9 18 Porphyrin D 9 18 Porphyrin E 918 Porphyrin F 9 18 Porphyrin C 9 18 Potassium 1088, 1090, 1094, 1095, 1121 Praseodymium 1092, 1095 Pregnane derivatives 598 Pregnanetriol6 13 Pregn-4-ene-3,2O-dione, 1lp,21-dihydroxy-, see Corticosterone F’regn4-ene-3,20-dione, 17a,2l-dihydroxy-, see Cortisol, 1l-deoxyPregn-4-ene-3,20-dione,2 1-hydrox y-, see Corticosterone, deoxyPregn-4-ene-3,20-dione, 11p,l70c,21-trihydroxy-, see Cortisol Pregn-4-ene-3,11,2O-trione,17a,2 1dhydroxy-, see Cortisone
LIST OF COMPOUNDS CHROMATOGRAPHED
Pregn-4-ene-3,11,20-trione, 21-hydroxy-, see Corticosterone, 1l-dehydroPregn-4-ene-3,11,20-trione, 6p,17a,21-trihydroxy-, see Cortisone, 60-hydroxyPregnenolone 605,613 Pregn-4-en-3-one, 2OP,21-dihydroxy- 6 15 Pregn-S-en-20-one, 3&17adihydroxy- 607 Pregn-4-en-3-one, 20p-hydroxy- 6 14 Pregn-5-en-20-one, 3P-hydroxy; see Pregnenolone Primeverose 485 Primeverulose 485 Pristinamycin I A 995 Pristinamycin IB 995 Pristinamycin Ic 995 Pristinamycin IIA 995 Pristinamycin IIB 995 Proanthocyanidins 910 Prochamazulenogens 627 Progesterone 603,609, 610,613,614, 617 Progesterone, l7or-hydroxy- 614 Progestins, see Gestagens Promethium 1118,1120 Propane, 1,2-diamino- 640 Propane, 1,3-diamino- 640,641 Propane, 2-nitro- 658 1,2-Propanedio1435 1,3-Propanediol, 2,2-dimethyl- 435 1,3-Propanediol, 2-hydroxy-2-methyl- 65 8 l-Propanol433,435-437 2-Propano1437 1-Propanol, 3-amino- 638 1-Propanol, 2-methyl- 437 2-Propanol, 2-methyl- 437 1-Propanol, 2-methyl-2-nitro- 658 1-Propanol, 3-phenyl- 434 2-Propanol, 2-phenyl- 432 Prophan 1026 Propionaldehyde 459 Propionaldehyde, 2,4-dinitrophenylhydrazone 457 Propionic acid 546,567,571 Propionic acid, 2,4-dinitrophenylhydrazide 548 Propionic acid, 2-hydroxy-, see Lactic acid Propionic acid, 3-hydroxy- 557 Propionic acid, or-methylcymanthreoyl- 1109 Propionic acid, p-methylcymanthreoyl- 1109 Propionic acid, 2-methyl-2,3-dihydroxy- 5 14 Propylamine 638,640,642 Propylamine, 2-hydroxy- 638 Propylamine, 3-methylmercapto- 638 Propylamine, 3-methylmercapto-, sulphoxide 638 Propylene glycol 436,438,439 Prostaglandins 588
1167 Protactinium 1116 Protamine 868 Proteins 60,583, 773-806 Proteins, aggregated 781 Proteins, globular type 777 Proteins, membrane 781 Proteinase, alkaline, from A. fluvus 821 Proteins from blood 792 Proteins from plasma membranes 799 Protein oligomers 781 Prothrombin 792 Protocatechuic acid 548 Protocatechuic aldehyde 548 Protochlorophyll a, 4-vinyl- 1043 Protochlorophyllide(s) 1042, 1043, 1045 Protoporphyrin dimethyl ester 917, 1045 Pseudomonic acid 1004 Pseudouridine 85 1 Pseudouridine, S-(p-D-ribofuranosyl) uracil 833 Pseudouridine monophosphate 85 1 Pseudouridylic acid 848, 852 Pteridine, 2-amino-4-hydroxy- 97 3 Pteridine, methyl tetrahydro- 972 Pterin, 6-hydroxymethyl- 973 Pterind-carboxylic acid 973 Purine 851 Purine, 6-dimethylamino- 835 Purine, 6-methylamino- 835 Purine alkaloids 893 Purine bases 834,839-842 Purine bases, analogues 839-842 Purine bases, methylated 839 Puromycin 999 Puromycin, 0-demethyl- 999 Putrescine 640, 641,646 Pyrazone 1015 Pyrene 418,419,422,424,425 Pyrene, dihydrc- 424 Pyrene, sym.-hexahydro- 424 Pyrethrins 1029, 1030 3(2H)-Pyridazinone, 4-amino-5-chloro-2-phenyl1015 3(2H)-Pyridazinone, 5-amino-4-chloro-2-phenyl1015 3(2H)-Pyridazinone, 4,5-dichloro-2-phenyI1015 Pyridine 920-922 Pyridine, 2-(methoxyiminomethyl)- 920 Pyridine, 2-methyl-4-amino-5-hydroxymethyl964 Pyridine-2-aldoxime 920 Pyridine-2-carboxamide 920 Pyridine-2-carb~xylicacid 920 Pyridinium methanesulphonate, 2-hydroxyiminomethyl-N-methyl- 920
1168
Pyridone, N-methyl- 920 Pyridoxal968-970 Pyridoxalamine5‘-phosphate970 Pyridoxal-5‘-phosphate 970 Pyridoxamine 646,968-970 Pyridoxine 962,969,970 Pyridoxine-5’-phosphate 970 Pyridoxol966,968 C-(2-Pyridyl)-N-methylaldonitrone920 Pyrimidine 851 Pyrimidine, 2-amino- 85 1 Pyrimidine, 2-methyl-4-amino-5-formyl964 Pyrimidine, 2-methyl-4-amino-S-formylaminc~ methyl- 963 Pyrimidine, 2-methyl-4-amino-5-methoxymethyl964 Pyrimidine, 2-methyl-4-aminomethyl- 964 Pyrimidine bases 834,839-842 Pyrimidine bases, analogues 839-842 Pyrimidine bases, methylated 839 Pyrimithate 1022,1023 Pyrocatechol443,446 Pyrochlorophyll Q 1042 Pyrochlorophyll b 1042 Pyrogallol444.445 4-Pyrone derivatives 896-909 Pyropheophytin Q 1042, 1043 Pyrophosphate 942 Pyrrole 922 Pyrrolidine 638, 642,646 2-Pyrrolidine, N-vinyl- 660 5-Pyrrolidone-2-carboxylicacid 567 Pyruvic acid 516, 554, 559, 567 Pyruvic acid enol phosphate 516 Pyruvic aldehyde 457
Q Quercetagitrin 900 Quercetin 908 Quercetin-3-glucoside,7,4‘-dibenzyl- 905 Quercetin-3-1hamnoside, see Quercitrin Quercitrin 899, 904,908 Quercitrin-4’-glucoside899 Quinaldic acid 647 Quinic acid 552, 557, 564,567 Quinoline 922 Quinolinic acid 564,647. Quinones 455,459-461,960,961 pQuinone 462 Quinone, hydroxy- 445
LIST OF COMPOUNDS CHROMATOGRAPHED
R Racemomycin A 994 Racemomycin B 994 Racemomycin C 994 Racemomycin D 994 Radioactive compounds 1115-1126 Raffinose 472,490,491,495 Rapeseed gum 588 Rare earths 1091 Raspberry pigments 912 Rauwolfia alkaloids 893 Reductase, 3-hydroxy-3-methylglutaryl coenzyme A, from Pseudomonas 813,814 Reichstein’s substance S, see Cortisol, 1l-deoxyResins 624 Resin from B. curteri 629 Resistomycin 1004 Resol type resin 1062 Resorcinol443-445,657 a-Resorcylic acid 553 Retinene 957 Retinoic acid, see Vitamin A, (acid) Retinol, see Vitamin A , (alcohol) Reynoutrin 908 Rhamnitol504 Rhamnose 485-488,491,492,494,495 Rhamnose, 2-O-(c~-gakctopyranosyluronicacid)513 Rhenium 1095, 1116 Rhenium, acetophenon(q-cyclopentadieny1)1109 Rhenium, benzene(acety1-q-cyclopentadieny1)1109 Rhenium, benzene(q-cyclopentadieny1)-1109 Rhodate(III), diaquatetrachloro-, anion 1104 Rhodium(III), dibromo-bis-(l,lO-phenanthroline)-, cation 1107 Rhodium(III), dichloro-bis-(l,l O-phenanthroline)-, cation 1107 Rhodium(III), tris-(ethy1enediamine)-,cation 1103 p-Rhodomycinone 999 Rhodopsin 956 Rhoifolin 899,908 Rhubarb pigments 912 Ribitol470,502,504 Riboflavine 962,965,966,971 Riboflavine S’-phosphate 965, 966 3-Ribohexulose 488 Ribonic acid 514 Ribonucleic acid, see RNA Ribonucleosides 837 Ribose 484,486-488,490-492,495
1169
LIST OF COMPOUNDS CHROMATOGRAPHED
Ribose, deoxy- 487 Ribose 1,s-diphosphate 517 Ribose, 3-0-a-glucopyranosyl- 485 &Ribose, 2,3-O-isopropylidene-, methyloside 509 @Ribose, 2,3-O-isopropylidene-, methyloside 509 Ribose, 2,3,4-tri-O-benzyl- 510 pRibose, 2,3,4-tri-O-benzyl-, benzyloside 5 10 Ribose 5-phosphate 515, 517 Ribose 5-phosphate, deoxy- 5 17, 5 18 Ribosomes 1076,1077 Ribostamycin 993 Ribothymidylic acid 852 Ribulose 5-phosphate 517 Riburonic acid 557 Ricinoleate 579 Rifampicin 996 Rifampin 996 Rifampin, 3-formyl- 996 Rifamycin SV 996 Risnagin, 3-methoxy- 906 RNA 862,866, 871-878,1124 RNA, denatured 872 RNA, Qp 881 RNA, ribosomal 877 RNA, 16s 874 RNA, structural studies 836,847,850 RNA, viral 859,874 RNA, viral, double-stranded 873 mRNA 859,873,878 rRNA 859, 868,870,872,877,878 rRNA, high-molecular-weight 874 rRNA, plant 864 rRNA, 5 s 878 rRNA, 16s 866 rRNA, 18s 877 rRNA, 23s 866, 874 rRNA, 28s 877 tRNA 859,862,866,868,870-872,874-877 tRNAAla from yeast 880,881 tRNAAla I from yeast 853 tRNA, aminoacyl 875-877 tRNAArg 877 tRNAA'g 'I1 from yeast 853 tRNAASP from yeast 853 tRNAGIU from E. coli 853 tRNA1le from T. utilis 853 tRNAMet 877 tRNAPhe from E. coli 853,881 tRNAPhe from E. coli, structural studies 854 tRNAPhe from wheat germ 853 tRNAPhe from yeast 853 tRNASer 877
tRNASe' from rat liver 853 tRNASer from yeast 880 tRNASer I from yeast 853 tRNASer from yeast 853 tRNA structural studies 844, 845 tRNA 'r from yeast 853 tRNATY' from T. utilis 853 tRNATYr from yeast 853 tRNAVal from yeast 853,880-882 tRNAVal I from T. utilis 853 tRNA from E. coli 864,874,877, 878 RNA from Ehrlich ascites tumour 864 RNA from phage 859 rRNA from A phage 870 RNA from plant virus 864 RNA from poliomyelitis virus 873 RNA from poliovirus 871,872 RNA polymerase 375 RNase 871 RNase, aminoethylated, structural studies 758 RNase, pancreatic, from chicken 810 RNase, pancreatic, structural studies 768 RNase H 861 RNase-S-peptide, structural studies 768 Robinin 908 Rock extract 428 Ronnel 10 13 Rotenone 915,916 Rubbers 1056,1057 Rubbers, natural 1057 Rubidium 1090,1095,1121 Rubrene 422 Rubromycin, derivatives of naphthoquinone 461 Ruthenium 1096 Ruthenium(III), tetraamine-bis-pyridine-, cation 1104 Rutin 899,908
S Sabinene 624 Saccharides, deoxy- 483-496 Saccharinic acids 5 14 Saccharose 472,490,491,495,496, 506, 509 Saccharose esters of fatty acids 587 Salicin 509 Salicylacetic acid 553 Salicyl alcohol 568 Salicylaldehyde 445,457 Salicylic acid 443-445, 552,553, 565, 568, 569 Salicylic acid, 4-amino- 643,644 Salicylic acid, 5-amino- 643
1170 Salicyluric acid 553 Saligenin 444,445 Salvigenin 901 Samarium 1095,1117 Sandaracopimaradiene 628 Sangivamycin 999 Saponaretin 900,901 Saponarin 900,908 Sarcosine 699,706,1000 Scandium 1117,1119 Scaposin 900 Schradan 1023 Sclarene 629 Scoparin 908 Scutellarin 908 Sebacic acid, dimethyl ester 460 Sedoheptulose 1,7-diphosphate 5 17 Sedoheptulose 7-phosphate 517 Selenonicotinamide 967 PSelinene 627 Sepiapterin 973 Serine, phosphatidyl- 583,589 Serinol638 Serotonin 638,640,646 Sesterterpenes 630 SF-701 antibiotic 993 Shelloic acid, dimethyl ester 630 Shikimic acid 557,564,567 Shyobunone 631 . Siccanochromenes 898 Siderophilin 803 Silicones 1064 Silicones, dimethyl- 1064 Silver 1116,1117,1122 Sinapic acid 570 Sinensetin 902 PSitosterol606 Skin lipids 588 Sodium 1088,1090,1094,1095,1116-1118, 1121 Somato-mammotropin 799 Sorbose 470,487,490,492 Southern been 871 Soyabean oil 578 Spermidine 640,641,646 Spermidine A, acetyl- 640 Spermidine B, acetyl- 640 Spermine 640,641 Spermine, acetyl- 640, 641 Sphingomyelin 582-584,588,589 Sphingosine 650 Sphingosine, dihydro- 650 Sphingosine esters 650 Spirostanes 618
LIST OF COMPOUNDS CHROMATOGRAPHED Squalene 421 Stachylose 495 Starch 525 Stearic acid 569-571 Stearic acid, cholesteryl ester 422 Stearic acid, mono- and diacetoxy derivatives 576 Stearic acid, mono-, di-, tri- and tetrahydroxy derivatives 576 Stellacyanin 793 Steroid acids 617 Steroidal glycosides 618,619 Steroids 593-622 Steroids, 17-ethynyl derivatives 614 Steroids, 17-hydroxy derivatives 603 Steroids, 16-keto derivatives 612 Steroids, 17-keto derivatives 602-605 Steroids, 17-keto derivatives, 2,4-dinitrophenylhydrazones 605 Sterol(s)589,602,604,609, 610 Sterol esters 589, 610 Stibium 1094, 1095, 1117 Stilbene derivatives 896 Streptomycin(s) 985,986 Streptomycin, dihydro- 986 Streptomycin sulphate 985 Streptothricin 994 Streptothricin C 994 Streptothricin D 994 Streptothricin E 994 Streptothricin F 994 Strobal632 Strontium 1094,1121 Strychnine 891 Strychnos alkaloids 893 Styrene 423, 1053 Styrene, a-methyl-, tetramer 1056 Styrene oligomers 31 1 Subcellular particles 1075-1085 Suberic acid 561 Succinic acid 549,564,567,571,1124 Sudan 1 1034 Sudan red 290 Sudan yellow 290 Sugars, peracetylated 468, 507 Sugar acetals 506,507 Sugar acids 507-515 Sugar derivatives 501 -519 Sugar esters 507 Sugar ethers 506, 507 Sugar phosphates 515-519 Sulfotep 1022,1023 Sulphadiazine 935 Sulphamerazine 935 Sulphamethazine 935
LIST OF COMPOUNDS CHROMATOGRAPHED
Sulphanilamide 643, 644 Sulphanilic acid 568,643,644,931, 932 Sulphaquanidine 986 Sulphatase, aryl- 812 Sulphatase, aryl-, from P. aeruginosa 819 Sulphatase, aryl-, from P.aeruginosa, isoenzymes 819, 820 Sulphates, alkyl- 928, 929 Sulphides 933, 934 Sulphohydrolase, arylsulphate 819 Sulpholipids 582, 583, 589 Sulphonamides 663 Sulphonates, alkylbenzene- 928-9 30 Sulphonazo I11 1035 Sulphones 933,934 Sulphonic acids 927-932 Sulphonomycin 1003 Sulphophthalein dyes 1036 Sulphoxides 932-934 Sulphuric acid 567, 928 Surfactants, non-ionic 438,453 Synephrine 646 Synthetase, methionyl-tRNA, from E. coli 826 Synthetase I, methionyl-tRNA 828 Synngic acid 553,569,570 Swertianol908 Swertijaponin 903 Swertisin 903
T Tachy ster 01-2, dihy dro- 9 59 Tagatose 487 Tagetin 908 Talboflavone, methylated 906 Talonic acid 514,558 Talonic acid, 2,5-anhydro- 557 Talosamine 497 Talose 488 Talose, 3-amino-3,6-dideoxy- 497 Taluronic acid 5 13 Tangeretin 902 Tangeritin 902 Tantalum 1116, 1117 Tartaric acid 551,552, 560, 562-564,567 Tartaric acid, dihydroxy- 561 Tartrazine 1035 Taurine 699,706,722 TCT, see Thyrocalcitonin TDE 1012,1013 Tea catechins 897 Technetium 11 16, 1119 Tellurium 1095
1171
Telodrin 1012 TEPP 1022,1023 Terbium 1095, 1117 Terephthalic acid 549, 550,565,566 Tzrephthalic acid, dimethyl ester 460 Temozide 901 Terpenes 62 3-6 3 5 pTerphenyl421 Testosterone 603-605,608,610, 614 Testosterone, methyl- 610 Testosterone cyclopentylpropionate 609, 610 Testosterone derivatives 604 Testosterone, 17p-propionate 609, 610 Tetracene 424 Tetracene, dihydro- 422,424 Tetracycline(s) 996-998 Tetracycline, anhydro- 996-998 Tetracycline, 4-epf-anhydro- 996 -998 Tetradecane 422 Tetradecanedisulphonic acid 928 Tetradecanesulphonic acid 928 Tetradifon 1012 Tetraethylene glycol 433 Tetralin 422,424,427 Tetramethylammonium 642 Tetrasiloxane, octamethyl- 1064 Tetronic acid, 3-deoxy-2-C-hydroxymethyl- 557 Tetrose 488 Tetrulose 485 Thallium 1093,1096 Theobromine 892 Theophylline 892 Thiamine 962-965,966 Thiamineacetic acid 963 Thiamine mononitrate 962 Thiamine phosphates 963,964 Thiazole-5-acetic acid, 4-methyl- 963 Thiometon 1022 Thionazin 1012, 1022, 1023 Thionazin 0-analogue 1022 Thionicotinamide 967 Thiopeptin A , 1001 Thiopeptin A, 1001 Thiopeptin A, 1001 Thiopeptin A, 1001 Thiopeptin B 1001 Thiosteroids 620 Thiourea 663 Thiouridine 834 Threitol 504 Threonic acid 514 Threose 488 a-Thujene 625 Thulium 1092,1095,1096,1118,1122,1123
1172
Thymidine 833,837,841,845-847,851 Thymidine 5'-phosphate 835,841 Thymidine 5'-pyrophosphate 841 Thiniidine 5'-triphosphate 841 Thymidylic acid 85 1 Thymine 834,835,837,841 Thymine, 1-p-D-ribofuranosyl-84 1 Thymol447 Thymol blue indicator 567 Thyrocalcitonin 790,802 Thyroglobulin, bovine 871 Tin 1095, 1117 Titanium 1094-1096,1117 Titanium dioxide 1118 Tobacco mosaic virus 60,782,871,1078,1079 Tobramycin 992 Tocopherol(s) 577,610,960,961 *Tocopherol 957,960,961 Tocopherol acetate 610 Tolazamide 936 Tolbutamide 936 Toluene 423,426 Toluene, 2-hydroxy-, see o-Cresol Toluene, 3-hydroxy-, see m-Cresol Toluene, 4-hydroxy-, see p-Cresol pToluenesulphenate 934 p-Toluenesulphonic acid 927,935 Toluic acid 549, 565 m-Toluidine 644 o-Toluidine 643, 644 pToluidine 643, 644 p Tolylsulphoxide 9 34 p-Tolylsulphoxide, methyl- 934 Torreyal631 Toxaphene 1012,1027 Toxin, A- 797 Toxin, B- 797 Toxin, E- 797 Toxins, see also Neurotoxin Toxins from A. flavus 91 2 Toxins from F. tricinctum 912 Toxins from scorpions, structural studies 754 Toxins from T.liguorum 912 Toyocamycin 999 Trachylobane 628 Transferase(s1 616-818 Transferase, carbamoyl-, from H. sulinurium 816 Transferase, omithine carbamoyl 817 Transferase I 818 Transferase I, aminoacyl-, from rat liver 817 Transferrin 780, 794 Trehalose 485,490 a, cr-Trehalose,6,6'-di-O-mesyl-485 a,aTrehalose, 6-0-mesyl- 485
LIST OF COMPOUNDS CHROMATOGRAPHED a,eTrehalose, per-0-benzyl- 506 a,p-Trehalose, per-0-benzyl- 506 p,pTrehalose, per-0-benzyl- 506 Tremuloidin 509 Triacetin 657 S-Triazine, 2-chloro-4-ethylamino- 1027 Tributyrin 569 Tricarballic acid 567 Tricetin-7-glucoside 904 Trichlorfon 1022,1023 Tridecane 422 Trifolin 908 Trifolirhizin 901 Triglycerides from oil 610 Trimesic acid 549 Trimethylamine 642 Trimethylamine, Naxide- 642 Triphenylcarbinol4 3 3 Triphosphopyridine nucleotide 85 2 Tripropylene glycol 433 Tristearin 569 Trithion 1013 Trithion-methyl1013 Tropine alkaloids 893 Tropolone 447,461 Truxene 422 Trypsin 89,90,373 Trypsin inhibitor 91, 780,793, 796 Trypsin inhibitor B 796 Tryptamine 646-648 Tryptamine, N,N-dimethyl- 646 Tryptamine, 5-hydroxy- 642,647, 648, 650, 65 1 Tryptamine, 5-methoxy- 646 Tryptamine, 5-methyl- 646 Tryptophan 647,648 Tryptophan, S-hydroxy- 647,648, 651 Tryptophan metabolites 645-649 Tryptophol, 5-hydroxy- 920 TSH, see Hormone, thyroid stimulating Tsushimycin 1003 Tuberactinomycin B, see Viornycin Tuberactinornycin N 1002 Tuberactinomycin 0 1002 Tungstate anion 1098 Tungsten 1116,1117 Tungsten, cis-alkylarylphosphine (tetracarbony1)1110 Tungsten, trans-alkylarylphosphine (tetracarbony1)- 1110 Tungsten, bis-(q-cyclopentadienyl)dihydrido1109 Tungsten, (q-cyclopent adienyl) cy clohept atrienyl- 1109
LIST OF COMPOUNDS CHROMATOGRAPHED
1173
Tungsten, cis-dialkylphosphine (tetracarbony1)1110 Tungsten, truns-dialkylphosphine (tetracarbony1)1110 Tungsten, cis-diarylphosphine (tetracarbony1)1110 Tungsten, trans-diarylphosphine (tetracarbony1)1110 Turanose 492 Tylosin, tetrahydro- 995 rn-Tyramine 646 o-Tyramine 646 pTyramine 638,640,642,646,652 pTyramine, 3-hydroxy-, see Dopamine p-Tyramine, 3-methoxy- 646 Tyrosinase 91 Tyrosine 652
Uridine 5’-diphospho-N-acetyl glucosamine 838 Uridinediphosphoaminosugar peptide 85 2 Uridinediphosphogalactose 852 Uridinediphosphoglucose 852 Uridinediphosphoglucuronic acid 85 2 Uridine 2’-phosphate 835, 851 Uridine 3’-phosphate 835 Uridine S’-phosphate 834, 835, 838, 841, 847, 851,852 Uridine 5’-pyrophosphate 835, 838, 852 Uridine S’-triphosphate 835, 852 Uridylic acid, 5,6-dihydro- 852 Uridylic acid, 5,6-dihydroxy- 848 Uridylic acid, 4-thio- 852 Uronic acids 468,481, 512, 513, 515, 551, 555, 559,564 Uroporphyrin I 919 Uroporphyrin 111 919 Urs-12-en-27-oicacid, 3phydroxy- 634
U Ubichromenol961 Ubiquinone 455 UDP, see Uridine 5’-pyrophosphate Umecyanin 793 UMP, see Uridine S’-phosphate Undecane 422 Undecanoic acid 571 Undecanol436 Uracil 834,835, 837,841 Uracil, 5-hydroxymethyl- 851 Uranium 1095,1096,1117,1120,1122,1123 Urea 648,657,660,663,664,699, 703, 706 Urea, terf.-butyl- 663 Urea, ethyl- 663 Urea, methyl- 663 Urea derivatives 661 -664 Uridine 833-835,837, 841,847 Uridine, 5-carboxymethyl- 833 Uridine, deoxy- 833, 841 Uridine, deoxy-, 5-(4’,5‘-dihydroxypentyl)-833 Uridine, deoxy-, 5-hydroxymethyl- 833 Uridine, deoxy-, 5-methyl-, see Thymidine Uridine, 5,6-dihydro- 833 Uridine, 5-hydroxy- 833 Uridine, 2’-O-methyl- 833 Uridine, 3-methyl- 833 Uridine, 5-methyl- 833 Uridine, 2‘-O-methylpseudo-[5-(2’-0-methylribosyl)uracil] 833 Uridine, 4-thio- 833 Uridine, 2-ethio-5-carboxymethyl-, methyl ester 833 Uridine, 2-thio-5-(N-rnethylaminomethyl)833
V Valeric acid 546, 548, 567, 571 Valeric acid, 2,s-dihydroxy- 557 Valeric acid, 3 ,S-dihydroxy-3-methyl- 55 7 Valeric acid, 2,4-dinitrophenylhydrazide548 Valeric acid, 2-hydroxy- 557, 558 Valeric acid, 4-hydroxy- 557 Valeric acid, 2-hydroxy-3-methyl- 557, 558 Valeric acid, threo-, 2,4,5-trihydroxy- 514 Valine, N-methyl- 1000 Vamidothion 1023 Vanadium 1095, 1096 Vanadium, dicarbonyl(q-cyclopentadieny1)-bis(tributylphosphine) 1109 Vanadium, dicarbonyl(q-cyclopentadienyl) diphosphine 1109 Vanadium, di-, pentacarbonyl-bis-(q-cyclopentadieny1)- 1109 Vanadium, disodium dicyano-dicarbonyl (17-cyclopentadieny1)- 1109 Vanadium, sodium cyano-tricarbonyl (q-cyclopentadieny1)- 1109 Vanadium, tetracarbonyl(qcyclopentadieny1) 1109 Vanadium, tricarbonyl(qcyclopentadieny1)phosphine 1109 Vanadium, tricarbonyl(q-cyclopentadieny1)tributylphosphine 1109 Vancomycin 1003,1004 Vanillic acid 548, 553,569,570 Vanillin 457,458,548 Vanilmandelic acid, see Mandelic acid, 4-hydroxy3-methoxy-
1174 Vasopressin 752, 754 Vasopressin-arginine 753 Vaucheriaxanthin ester 1042 Vegadex 1013 Veratrum alkaloids 893 Vinyl polymers 1053-1056 Violaxanthin 1042, 1047 Viomycin 1002 Viomycin, perhydro- 1003 Virus(es) 1077-1081 Virus, influenza 871 Virus, Mengo 1080 Virus, myxoma 871 Virus, stem mottle 1078, 1079 Virus, tobacco mosaic 60,781,871, 1078, 1079 Virus, turnip mosaic 1080, 1081 Virus X, potato 1078, 1079 Virus Y otato 1078 Virus Y $, potato 1079 Viscotoxins 796 Vi tamin(s) 953 -97 8 Vitamins, fat-soluble 955-962 Vitamins, pyridoxine group 968-970 Vitamins, water-soluble 962-978 Vitamin A 954,956 Vitamin A acetate 954 Vitamin A palmitate 954 Vitamin A, 955 Vitamin A,, methyl ester 957 Vitamin A, acetate 957 Vitamin A , (acid) 956,957 Vitamin A, (alcohol) 957 Vitamin A, palmitate 957 Vitamin A, 955 Vitamine B,, see Thiamine Vitamine B,, see Riboflavine Vitamin B, 966,968 Vitamin B,, 966,974,975 Vitamin C, see Ascorbic acid Vitamin D, 954,957-959 Vitamin D,, tritiated 959 Vitamin D, 954,958,959 Vitamin D,, [4-14C]-959 Vitamin D,, tsitiated 959 Vitamin E 954 Vitamin E acetate 954 Vitamin E succinate 954 Vitamin K 954,961 Vitamin K, 954,957,961 Vitamin K, 961,962 Vitamin K, 961 Vitexin 901 Volemito1503
LIST OF COMPOUNDS CHROMATOGRAPHED
W Waxes 584 Wogonin 908 Wood components 462 Wood extracts 623-635
X X32 antibiotic 996 Xanthine 835,837,841,851 Xanthone(s) 908,909 Xanthone, 2-C-allyl-3-allyloxy-1-hydroxy-907 Xanthone, 4-C-allyl-3-allyloxy-1-hydroxy- 907 907 Xanthone, 2-C-allyl-l,3-dihydroxyXanthone, 4-C-allyl-l,3-dihydroxy- 907 Xanthone, 3-allyloxy-1-hydroxy- 907 Xanthone, 2,4-di-C-allyl-1,3-dihydroxy907 Xanthone, 1,3-dihydroxy- 907 907 Xanthone, 1,5-dihydroxy-3,3-methoxyXanthone, 6-(3,3-dimethylallyl)-l ,S-dihydroxy907 Xanthone, 2,4-di-C-prenyl-l,3-dihydroxy-7methoxy- 906 Xanthone, 1-hydroxy-3,7-dimethoxy907 Xanthone, 3-hydroxy-l,2-dimethoxy907 907 Xanthone, 4-hydroxy-2,3-dimethoxyXanthone, 5-hydroxy-l,3-dimethoxy907 Xanthone, S-hydroxy-1,S-dimethoxy907 Xanthone, 2-hydroxy-1-methoxy- 907 Xanthone, 1-hydroxy-3-prenyloxy-7-methoxy906 Xanthone, 3-hydroxy-l,5,6-trimethoxy907 907 Xanthone, 8-hydroxy-l,2,6-trimethoxy907 Xanthone, 4-methoxy-2,3'-methylenedioxyXanthone, 2-C-prenyl-l,3-dihydroxy-7-methoxy906 Xanthophyll(s) 589,1042,1043 Xanthosine 835,841,851 Xanthurenic acid 647-649 Xanthurenic acid, 8-methyl ether 649 m-Xylene 426 o-Xylene 426 pXylene 423,426 m-Xylene-6-sulphonicacid, 5-amino- 930 Xylito1438,470,502-504 Xylobiose 488 Xylonic acid 514,556, 558 Xylosamine 497 Xylose 470, 485,487,488,490-492, 495, 502, 503,515 Xylose, 4-O-(a-galactopyranosyluronicacid)513
LIST OF COMPOUNDS CHROMATOGRAPHED
1175
Xylose, 2-0-(4-O-methyl-a-glucopyranosyluronic acid)- 5 13 Xylulose 1-phosphate, 5-deoxy- 5 18 Xylulose 5-phosphate 5 17 Xyluronic acid 557, 558
cu-Ylangene 626 Yohimbine alkaloids 893 Ytterbium 1096, 1117 Yttrium 1095
Y
Z
Yazumycin 993 Yazumycin A 994 Yazumycin C 994
Zeaxanthin 1042, 1047 Zerumbone 626 Zinc 1094-1096,1116-1118,1122 Zirconium 1096,1116,1120
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