Handbook of Chromatography Volume II (1990): Carbohydrates [1 ed.] 9781138506633, 9780203712733, 9781351363389, 9781351363372, 9781351363396, 9781138559417

Table of contents :

Section 1: CHROMATOGRAPHIC DATA 1. Gas Chromatography Tables 2. Supercritical Fluid Chromatography Tables 3. Liquid Chromatography Tables 4. Planar Chromatography Tables 5. Electrophoretic Data Section 2: DETECTION TECHNIQUES 1. Detection and Identification of Carbohydrates in Gas Chromatography: Use of Mass Spectrometry 2. Detection of Carbohydrates in Supercritical Fluid Chromatography: Use of Mass Spectrometry 3. Detection Methods for Liquid Chromatography 4. Detection Reagents for Planar Chromatography Section 3: SAMPLE PREPARATION AND DERIVATIZATION 1. Degradation of Polysaccharides and Glycoconjugates 2. Derivatization for Gas Chromatography 3. Derivatization for HPLC Section 4: LITERATURE REFERENCES 1. Bibliography 2. Reviews and Selected Journal References

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CRC Series in Chromatography Editors-in-Chief

Gunter Zweig, Ph.D. and Joseph Sherma, Ph.D. General Data and Principles

Lipids

Gunter Zweig, Ph.D. and Joseph Sherma, Ph.D.

Helmut K. Mangold, Dr. rer. nat.

Hydrocarbons Walter L. Zielinski, Jr., Ph.D.

Carbohydrates Shirley C. Churms, Ph.D.

Inorganics M. Qureshi, Ph.D.

Drugs Ram Gupta, Ph.D.

Phenols and Organic Acids Toshihiko Hanai, Ph.D.

Terpenoids Carmine J. Coscia, Ph.D.

Amino Acids and Amines S. Blackburn, Ph.D.

Steroids Polymers

Joseph C. Touchstone, Ph.D.

Charles G. Smith, Norman E. Skelly, Ph.D., Carl D. Chow, and Richard A. Solomon

Pesticides and Related Organic Chemicals Plant Pigments

Joseph Sherma, Ph.D. and Joanne Follweiler, Ph.D.

Hans-Peter Köst, Ph.D.

Nucleic Acids and Related Compounds Ante M. Krstulovic, Ph.D.

CRC Handbook of Chromatography Carbohydrates Volume II Editor

Shirley C. Churms, Ph.D. Research Associate Carbohydrate Research Group Department of Chemistry University of Cape Town Republic of South Africa

Editor-in-Chief

Joseph Sherma, Ph.D. Charles A. Dana Professor and Head Department of Chemistry Lafayette College Easton, Pennsylvania

First published 1991 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1991 by Taylor & Francis CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access w w w .copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organiza-tion that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. A Library of Congress record exists under LC control number: 80029541 Publisher’s Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-138-50663-3 (hbk) ISBN 13: 978-0-203-71273-3 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

CRC HANDBOOK OF CHROMATOGRAPHY SERIES PREFACE The CRC Handbook of Chromatography series began with two volumes that were published in 1972. These volumes, written by Dr. Gunter Zweig and myself, covered all compound types and chromatographic methods. The first volume on carbohydrates by Shirley C. Churms, published in 1982, was the third volume in the series, and the first in the current format in which each book is devoted to a particular class of compounds or chromatographic method. The first volume by Dr. Churms is among the best selling and most highly acclaimed in the entire series, which now numbers 25 volumes overall with the publication of this second book on carbohydrates. Dr. Churms is an internationally recognized expert on the chromatography of carbohydrates and I am gratified that she was willing and able to update her original coverage of the field at this time. Future volumes in the Handbook of Chromatography series are now being planned or written. These include additional volumes on pesticides, polymers, lipids, steroids, and hydrocarbons, and coverage for the first time of toxins, vitamins, and chiral separations. I would appreciate hearing from readers who have suggestions for topics or authors for subsequent volumes in the series. I would also be interested in receiving comments or corrections on the present volume or any others that have already been published.

Joseph Sherma, Ph.D. Easton, PA August 1990

PREFACE Since the compilation of CRC Handbook of Chromatography: Carbohydrates, Volume I, which was completed early in 1979, there have been many developments in the field, particularly in liquid chromatography, owing to the rapid proliferation of HPLC methods during the past decade. Gas chromatography, too, has been revolutionized by the increasing use of capillary columns and the advent of chiral phases and some novel methods of derivatization, which have greatly improved resolution of related sugars, including enantiomeric pairs. Derivatization has become important in HPLC, also, owing to the greatly enhanced sensitivity of detection possible by use of chromophoric and, especially, fluorescent deriv­ atives; this applies equally to PC and TLC. In addition to pre- and postcolumn derivatization, other methods of detection have been developed for HPLC: in particular, coupling of HPLC and MS is now a reality and electrochemical methods have become important, especially in the relatively new technique of ion chromatography, the application of which to analysis of mono- and oligosaccharides has produced some remarkable results. Another new technique which shows great promise in the analysis of oligosaccharides and glycoconjugates is super­ critical fluid chromatography. This new handbook, which has been compiled almost entirely from literature published during the period 1979 to 1989, has been structured to take cognizance of these developments. Since HPLC has been the main area of growth during the period under review, the section on liquid chromatography data has been greatly expanded, as have those on detection and derivatization methods in liquid chromatography. The vital importance of mass spectrometry in GC analysis is reflected in the inclusion of relevant mass spectral data for various deriv­ atives used, including some of the newer ones. These new methods of derivatization, and improvements in the older methods, are given in detail in Section III. Although application of supercritical fluid chromatography to carbohydrates is not yet widespread, its potential is great enough to justify the inclusion of a new subsection covering the data available at present, and the recent coupling of SFC to MS is discussed in Section II. To allow some more space for all this new material and a comprehensive list of important literature references relating to the new methods, it has been decided to omit the separate section on chromatographic materials that was included in Volume I. Developments in this area are so rapid at present that the currency of any such directory will inevitably be of short duration, and therefore to attempt to update the list published in Volume I seemed futile. Instead, relevant details of the packings or plates used are given in the footnotes to tables showing data obtained with these chromatographic materials, and the names of man­ ufacturers, from whom further details can be obtained, are also appended in such footnotes.

Shirley C. Churms, Ph.D. October 1989

THE EDITOR-IN-CHIEF Joseph Sherma, Ph.D., received a B.S. in chemistry from Upsala College, East Orange, NJ in 1955 and a Ph.D. in analytical chemistry from Rutgers University, New Brunswick, NJ, in 1958 carrying on his thesis research in ion exchange chromatography under the direction of the late William Rieman III. Dr. Sherma joined the faculty of Lafayette College, Easton, PA in September, 1958, and is presently Charles A. Dana Professor and Head of the Chemistry Department. At Lafayette, he teaches three courses in analytical chemistry. Dr. Sherma, independently and with others, has written or edited about 350 research papers, chapters, books, and reviews involving chromatography and other analytical meth­ odology. In addition to being Editor-in-Chief of the CRC Handbook of Chromatography series, he co-edits the series Analytical Methods for Pesticides and Plant Growth Regulators, previously published by Academic Press and now by CRC Press. He is co-editor for residues and trace elements of the Journal of the Association of Official Analytical Chemists and a member of the editorial board of the Journal of Planar Chromatography. He is consultant on analytical methodology for many companies and federal agencies. Dr. Sherma has received three awards for superior teaching and scholarship at Lafayette College and the E. Emmet Reid Award for excellence in teaching, presented at the Middle Atlantic Regional meeting of the ACS. He is a member of the ACS, AIC, Phi Lambda Upsilon, Sigma Xi, and AOAC. Dr. Sherma’s current research interests are in quantitative TLC, mainly applied to clinical analysis, pesticide residues, lipids, and food additives.

THE EDITOR Shirley C. Churms (bom Macintosh) graduated at the University of Cape Town, South Africa, and then undertook research, at the same University, on cation-exchange processes in aqueous monoethanolamine, for which the degree of Ph.D. was awarded in 1962. She spent her postdoctoral year at the Imperial College of Science and Technology in London, England, as the holder of the Ohio State Fellowship, awarded by the International Federation of University Women; during this period she carried out an extensive survey of the properties of inorganic ion-exchangers and commenced an investigation of cation- and anion-exchange on amphoteric hydrated alumina. In 1964 she returned to the University of Cape Town and during the next 4 years she continued her research on inorganic ion-exchangers, supervised a group of research students working on various aspects of ion-exchange, and lectured in the Department of Chemistry at the University. In 1967 the work of the ion-exchange group was recognized by the South African Chemical Institute in the award of the African Explo­ sives and Chemical Industry medal for one of its publications. A change in research interests occurred in 1968, when Dr. Churms was appointed Research Associate in the newly formed Carbohydrate Chemistry Research Unit, sponsored by the South African Council for Scientific and Industrial Research, which functioned in the Department of Chemistry (from 1974 until 1988 in the separate Department of Organic Chemistry), under the direction of Professor Alistair M. Stephen. The Unit, which became the Carbohydrate Research Group in 1985, used chromatographic methods, particularly gas chromatography, very extensively in analyses of the products of degradative studies of polysaccharides, and Dr. Churms developed a special interest in the application of gelpermeation (now steric-exclusion) chromatography in these structural studies. Publication of a major review on this topic in Advances in Carbohydrate Chemistry and Biochemistry in 1970 resulted in an invitation from Dr. Erich Heftmann to contribute a chapter on chromatography of carbohydrates to the Third Edition of the well-known book on chro­ matography of which he is the Editor. She has subsequently remained a co-author in the Fourth Edition and, recently, the Fifth Edition of this seminal work. She was Volume Editor, and a major contributor, for Volume I of the present Handbook, published by CRC Press in 1982. To date, Dr. Churms has been the author of seven major reviews and, in addition to the chapters for Heftmann’s Chromatography already mentioned, co-author (with Professor Stephen) of a further three chapters to be published in books dealing with various aspects of polysaccharide chemistry. She has been author or co-author of over 50 published papers and numerous presentations at national and international conferences, and is currently a member of the Editorial Board for the Symposium Volumes of the Journal of Chromatog­ raphy.

DEDICATION This volume is dedicated to Professor Alistair M. Stephen, in deep gratitude for many happy and productive years under his inspiring leadership, and to colleagues, past and present, in the Carbohydrate Research Group at the University of Cape Town, whose cooperation and camaraderie have helped to ease the high pressure of carbohydrate chromatography.

Shirley C. Churms October 1989

ACKNOWLEDGMENTS The financial support of the University of Cape Town and the Foundation for Research Development of the South African Council for Scientific and Industrial Research is gratefully acknowledged. I thank the University of Cape Town for a grant of study leave during the writing of this book and Professor Alistair M. Stephen for his patience and encouragement, as well as for many helpful discussions of aspects of chromatography. I am indebted also to Mrs. Evelyn Rossmeisl, of the Inter-Library Loans Department in the University of Cape Town Library, for her invaluable assistance in obtaining some of the literature reviewed in this book, and to Dr. Bill Edwards, of the Dionex Corporation, Sunnyvale, California, for kindly sending me some literature on ion chromatography from sources not otherwise ac­ cessible to me. Special thanks are due to Mrs. Patsy Alexander and Mrs. Jean Goode for their excellent processing of the manuscript. The cooperation of the editorial staff of CRC Press is also gratefully acknowledged.

LIST OF ABBREVIATIONS Ac Ara Asn atm Cl d Da DEAE DP El Et f FAB fmol FI Fru Fuc Gal GalA GalN GalNAc GalNAc-ol GC GLC Glc GlcA GlcN GlcNAc GlcNAc-ol Glc-ol Gly h HPLC I.D. k' Kav Kd kPa LC M Mn Mw Man mCi Me meq min mM mmol

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

acetyl arabinose asparagine atmosphere chemical ionization days daltons diethylamino ethyl degree of polymerization electron impact ethoxyl furanose fast atom bombardment femtomole flame ionization detector fructose fucose galactose galacturonic acid 2-amino-2-deoxygalactose (galactosamine) 2-acetamido-2-deoxygalactose (N-acetylgalactosamine) 2-acetamido-2-deoxygalactitol (N-acetylgalactosaminitol) gas chromatography gas liquid chromatography glucose glucuronic acid 2-amino-2-deoxyglucose (glucosamine) 2-acetamido-2-deoxyglucose (N-acetylglucosamine) 2-acetamido-2-deoxyglucitol (N-acetylglucosaminitol) glucitol glycine hour(s) high-performance liquid chromatography inner diameter capacity factor = (tr “ tG) / tO SEC partition coefficient = (Ve — VO) / (Vt - VQ) alternative SEC partition coefficient = (Ve — V0) / Vi kilopascal liquid chromatography molecular weight (molar mass); in Table EL 1 = mobility number-average molecular weight weight-average molecular weight mannose millicurie methyl milliequivalent minute(s) millimolar millimole

MPa ms MS m/z n.a. NeuAc ng nm nmol p PC pg pmol ppb ppm r RF Rha RI s SCOT SEC Ser SFC SS Thr TLC TMS tO tr Tyr U UV v Ve Vi VQ Vt w WCOT Xyl

= megapascal (S.I. unit of pressure) = millisecond = mass spectrometry = mass/charge ratio = not available = N-acetylneuraminic acid (sialic acid) = nanogram = nanometer = nanomole = pyranose = paper chromatography = picogram = picomole = parts per billion = parts per million = relative retention time = migration distance relative to solvent front = migration distance relative to glucose = rhamnose = refractive index detector = second = support-coated open tubular = steric-exclusion chromatography = serine = supercritical fluid chromatography = stainless steel = theonine = thin-layer chromatography = trimethylsilyl = retention time of solvent front = retention time = tyrosine = units (of enzyme) = ultraviolet absorbance = volume = elution volume = internal solvent volume in SEC = void volume = total volume in SEC = weight = wall-coated open tubular = xylose

Other abbreviations are defined in the test.

TABLE OF CONTENTS Volume II SECTION I — CHROMATOGRAPHIC DATA 1.1 Gas Chromatography Tables........................................................................................... 3 1.11 Supercritical Fluid Chromatography Tables............................................................... 67 1.111 Liquid Chromatography Tables................................................................................... 71 I.IV Planar Chromatography Tables...................................................................................249 I. V Electrophoretic Data................................................................................................... 267 SECTION II — DETECTION TECHNIQUES 11.1

Detection and Identification of Carbohydrates in Gas Chromatography: Use of Mass Spectrometry.......................................................................................... 277 11.11 Detection of Carbohydrates in Supercritical Fluid Chromatography: Use of Mass Spectrometry.......................................................................................... 285 11.111 Detection Methods for Liquid Chromatography......................................................287 II. IV Detection Reagents for Planar Chromatography...................................................... 315 SECTION III — SAMPLE PREPARATION AND DERIVATIZATION 111.1 Degradation of Polysaccharides and Glycoconjugates............................................323 111.11 Derivatization for Gas Chromatography................................................................... 339 111.111 Derivatization for HPLC............................................................................................ 349 SECTION IV — LITERATURE REFERENCES IV. I Bibliography.................................................................................................................365 IV.II Reviews and Selected Journal References................................................................369 Index

381

Section I Chromatographic Data

Carbohydrates

3

Section I.I.

GAS CHROMATOGRAPHY TABLES As in Volume I, these tables are grouped according to the type of volatile derivative used in gas chromatography (GC), with the derivatives arranged in alphabetical order for easy reference. In the present volume the emphasis is mainly on capillary GC, although in many cases retention data for chromatography on equivalent packed columns are included for comparison. The new tables do not necessarily supersede those in Volume I; many should be regarded rather as supplementary to the earlier compilations, as indicated in footnotes to the present tables. A few of the derivatives included in Volume I have not been used extensively in recent years and are, therefore, omitted here, while several new derivatives are included for the first time in this volume. Of special interest are those permitting resolution of enantiomers, which has also been achieved by the use of recently developed chiral phases. Tables devoted to this important aspect will be found at the end of Section I.I.

4

CRC Handbook of Chromatography

TABLE GC 1 Peracetylated Alditols, Aminodeoxyalditols, and Inositols: GLC on Packed Columns Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 N2; 40

PI T2 He; 20

P2 T3 N2; 20

P3 195 N2; 45

P4 195 N2; 40

158 0.4 Packed Glass FI 1

100 0.2 Packed Glass FI 2

183 0.18 Packed Glass FI 3

200 0.3 Packed Glass 4

250 0.4 Packed SS FI 5

Parent compound

r*

r*

rb

i*

r*

Rhamnose Fucose Ribose Arabinose Xylose Mannose Galactose Glucose 2-Amino-2-deoxyglucose 2-Amino-2-deoxymannose 2-Amino-2-deoxygalactose 2-Acetamido-2-deoxyglucitol 2-Acetamido-2-deoxygalactitol 2-Acetamido-2-deoxymannitol myo-Inositol cA/ro-Inositol neo-Inositol mwco-lnositol 5cy//o-lnositol

_

_

_

—

—

—

—

—

0.46 0.59 0.77 0.83 0.91 0.57d

0.52 0.64 0.79 0.84 0.91

—

—

—

—

—

—

0.73d

—

—

0.18 0.20 0.27 0.30 0.39 0.78 0.93 1.00 0.46c 0.48c 0.54c

—

0.32

—

—

—

—

—

—

—

—

—

—

—

—

1.00

1.00

— — — —

1.44f 1.55f 1.55f

fi

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

— — —

— 0.69 0.79 0.87 — — —

1.00 0.72 0.81 1.17 1.53

a t, relative to myo-inositol hexaacetate (39.2 min,1 8.3 min,2 25 min5). b t, relative to peracetylated D-gluco heptitol (10.5 min). c t, relative to glucitol hexaacetate (53.94 min). d As 2,5-anhydrohexitol acetates (2,5-anhydromannitol and -talitol from 2-amino-2-deoxyglucose and -galactose) produced by deamination. e A-methylated (HCHO/NaBH3CN) between reduction and acetylation. f For simultaneous analysis of acetamidodeoxyhexoses as O-methyloxime acetates see Table GC 7, Reference 3. Packing

Temperature

PI = 3% SP-2340 on Supelcoport® (100— 120 mesh). P2 = 3% diethylene glycol adipate, stabilized, on Chromosorb® W HP (100— 120 mesh). P3 = 2% EGSS-X on Chromosorb® W AW DMCS (60—80 mesh). P4 = 0.5% QF-1 + 0.5% LAC-2R-446 on Chromosorb® W HP (80— 100 mesh). T1 = 150 —> 220°C at 2°C/min. T2 = 190 —* 260°C at 5°C/min. T3 = 210 240°C at 2°C/min.

Carbohydrates REFERENCES 1. Anastassiades, T., Puzic, R., and Puzic, O., Modification of the simultaneous determination of alditol acetates of neutral and amino sugars by gas-liquid chromatography, J. Chrom atogr., 225, 309, 1981. 2. Lehrfeld, J., Gas chromatographic analysis of mixtures containing aldonic acids, alditols, and glucose, Anal. C hem ., 56, 1803, 1984. 3. Mawhinney, T., Simultaneous determination of 7V-acetylglucosamine, ^-acetylgalactosa­ mine, A-acetylglucosaminitol and N-acetylgalactosaminitol by gas-liquid chromatography, J. C hrom atogr., 351, 91, 1986. 4. Kiho, T., Ukai, S., and Hara, C., Simultaneous determination of the alditol acetate deriv­ atives of amino and neutral sugars by gas-liquid chromatography, J. Chrom atogr., 369, 415, 1986. 5. Irving, G. C. J., Gas chromatography of inositols as their hexakis-O-acetyl derivatives, J. C hrom atogr., 205, 460, 1981.

5

6

TABLE GC 2 Peracetylated Alditols and Aminodeoxyalditols: GLC on Capillary Columns

Material

PI T1 He; 5

P2 205 He; la

P3 T2 h 2; 1*

P3 T3 H2; n.a.

P4 T4 He; 1.5-

P4 T5 H2; 1.3-

P5 T6 H2; 9

P5 T6 H2; 9.5

P6 T7 He; 10

P7 275 He; 0.6-

P8 T8 He; 0.6-

P9 T9 N2; 2.5«

35 0.3

25 0.25

12 0.2

24 0.3

25 0.25

6 0.2

28.5 0.5

28.5 0.5

24 0.75

30 0.25

30 0.25

25 0.25

Coated

WCOT

WCOT

Coated

Coated

SCOT

SCOT

Coated

Coated

Fused silica FI 3

Fused silica FI 4

Glass

Glass

Glass

Glass

Glass

FI 5

FI 7

FI 8

FI 9

FI 10

Bonded phase Fused silica FI 11

WCOT

Glass

Bonded phase Fused silica FI 6

FI 12

Detector Reference

FI 1

Fused silica FI 2

Parent compound

rb

rb

rb

r*

r*

r*

r*

I*

r*

r*

rb

r*

Glycerol Erythritol

— 0.44 0.52

— — 0.25

— — 0.23

— — —

0.12 0.27 0.37

_

0.08 0.26 0.37

_

— — —

— — —

_

0.28 0.39

0.29f —

— — —

0.58 0.59 — 0.61 0.62 — — 0.65 0.75

0.30 0.32 0.33 0.36 0.40 — — 0.44 —

0.31 0.34 0.36 0.38 0.42 — — 0.48 —

— — —

0.38 0.39 0.48 0.49 0.52 0.59 0.61 0.63 0.64

0.52 0.54 — 0.63 0.65 — — 0.72 0.74

0.39 0.41

0.41 0.43

— —

0.55 0.57

0.40f 0.44f

— —

—

—

—

—

—

—

0.50 0.53 — — 0.63 0.66

0.53 0.56 — — 0.66 0.68

0.63 0.65

0.46' 0.47*

—

—

— — —

— 0.53f —

— — — — — —

— — 0.92

0.58 0.74 0.82 0.86

0.62 0.78 0.84 0.89

0.65 0.70 0.80 0.75

— 0.78 —

— 0.72 — —

— 0.75 — —

— — — —

— 0.77f — —

2-Deoxy-D-erythro-

pentose Rhamnose Fucose 6-Deoxyglucose Ribose Arabinose 1,4-Anhydromannitolg 1,5-Anhydromannitolg Xylose 2-Deoxy-D-arabino-

hexose 2-Deoxy-D-/y;c0-hexose Allose 4-0-Methylglucoseh Altrose

—

—

0.63c —

— 0.68c — — — — —

0.39 0.51

—

—

0.35f — — 0.48f — — — — —

Glass

— — —

—

CRC Handbook of Chromatography

Phase Temperature (°C) Gas; flow rate (ml/min) Column Length, m Diameter (I.D.), mm Form

Mannose Talose Galactose Glucose Idose myo-Inositol 2,5 -Anhydromannose1 2,5 -Anhydrotalose' 2-Amino-2-deoxyglucose 2-Amino-2-deoxygalactose 2-Amino-2-deoxymannose 2-Amino-2-deoxyallose 2-Amino-2-deoxytalose 2-Amino-2-deoxyaltrose 2-Amino-2-deoxyidose 2-Amino-2-deoxyglucose 2-Acetamido-2-deoxyglucose 2-Acetamido-2-deoxygalactose 2-Acetamido-2-deoxymannose

0.89 — 1.02 1.00 1.08 — — — —

0.92 — 1.01 1.00 1.08 — — 1.74

0.98c — 1.02e — — 1.00c — — —

0.74 — 0.79 0.86 0.99 1.00 — — —

0.83 — 0.87 0.92 — 1.00 — — —

0.78 0.79 0.83 0.90 1.03 1.00 — — —

0.79 _ 0.84 0.90 — 1.00 0.63 0.76 —

—

—

1.92

—

—

—

—

—

—

1.96

—

—

—

— — —

— — —

— — —

—

—

— — —

— —

— —

— —

— —

—

—

—

—

—

—

—

0.87f 0.90f — 1.00f _ _ —

0.90 0.93 — 1.00 _ _ —

0.88f _ 0.97f 1.00f — — _ _ —

—

—

—

—

1.22

—

—

—

—

—

1.36

— — —

— — —

— — —

— — —

— — —

— — —

0.83 1.03 1.05

— —

— —

— —

— —

— —

— —

— —

1.13 1.31

1.45c

—

—

—

—

—

—

—

—

—

1.54e

—

—

—

—

—

—

—

—

—

1.56c

—

—

—

—

—

—

—

—

—

—

0.77f _

Split ratios 1:100,2*310 1:20,5 1:4,6 1:50,11 1:5.12 tr relative to glucitol hexaacetate (65 min,1 25.8 min,2 15.2 min,3 11.3 min11). tr relative to myo-inositol hexaacetate (18.6 min,4 36.8 min,5 21.5 min,6 18.1 min,7 16.7 min,8 7.8 min,9 7.0 min10). t, relative to acetylated 2-amino-2-deoxyglucitol (71.5 min.). For simultaneous analysis of neutral and amino sugars as O-methyloxime acetates see Table GC 7, Reference 7. For simultaneous analysis of alduronic acids as 7V-alkylaldonamide acetates see Table GC 6, References 3 and 4. Produced on reductive cleavage of polysaccharides. Produced on carboxyl reduction of 4-0-methylglucuronic acid. Produced on nitrous acid deamination of 2-amino-2-deoxyglucose and -galactose.

0.86 _

_

— — — — — _ _ 1.00

Carbohydrates

a b c d e f g h '

0.95 — 0.98 1.00 — — — — —

7

Temperature

PI =9: 1 mixture of N-propionyl-L-valine-/eri-butylamide polysiloxane + Witconal® LA23 (surfactant). P2 = FFAP. P3 = Carbowax 20M. P4 = OV-275. P5 = Silar IOC. P6 = SP-2330, 1.0-pm film. P7 = SP-2380 (stabilized phase, partially cross-linked), 0.20-p.m film. P8 = DB-1701 (J and W Scientific, Folsom, California). P9 = Poly A-103. T1 = 80 —> 200°C at 4°C/min; held at 200°C. T2 = 185 -► 200°C at l°C/min; held at 200°C for 2 min; 200 220°C at20°C/min. T3 = 80 —> 180°C at 20°C/min; held at 180°C for 1 min; 180 —> 210°C at 2°C/min;held at 210°C for 1 min; 210 —> 230°C at 10°C/min; held at 230°C. T4 = 165 —» 215°C at 20°C/min; held at 215°C. T5 = 170°C for 4 min; 170 230°C at 4°C/min. T6 = 190°C for 4 min; 190 230°C at 4°C/min; held at 230°C for 8 min. T7 = 200°C for 2 min; 200 -> 235°C at 3°C/min. T8 = 220 -> 270°C at l°C/min. T9 = 175°C for 50 min; 175 210°C at 0.5°C/min. REFERENCES

1. 2. 3. 4. 5.

6.

Green, C., Doctor, V. M., Holzer, C., and Oro, J., Separation of neutral and amino sugars by capillary gas chromatography, J. C hrom atogr., 207, 268, 1981. Oshima, R., Yoshikawa, A., and Kumanotani, J., High-resolution gas chromatographic separation of alditol acetates on fused-silica wall-coated open-tubular columns, J. C hrom atogr ., 213, 142, 1981. Oshima, R., Kumanotani, J., and Watanabe, C., Fused-silica capillary gas chromatographic separation of alditol acetates of neutral and amino sugars, J. C hrom atogr. , 250, 90, 1982. Neeser, J.-R., G.l.c. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines and sialic acids, Carbohydr. R es., 138, 189, 1985. Klok, J., Cox, H. C., de Leeuw, J. W., and Schenck, P. A., Analysis of synthetic mixtures of partially methylated alditol acetates by capillary gas chromatography, gas chromatography-electron impact mass spectrometry and gas chromatography-chemical ionization mass spectrometry, J. Chrom atogr ., 253, 55, 1982. Blakeney, A. B., Harris, P. J., Henry, R. J., Stone, B. A., and Norris, T., Gas chromatography of alditol acetates on a high-polarity bonded-phase vitreoussilica column, J. C hrom atogr ., 249, 180, 1982.

CRC Handbook of Chromatography

Phase

8

TABLE GC 2 (continued) Peracetylated Alditols and Aminodeoxyalditols: GLC on Capillary Columns

7.

Blakeney, A. B., Harris, P. J., Henry, R. J., and Stone, B. A., A simple and rapid preparation of alditol acetates for monosaccharide analysis, Carbohydr. R es., 113, 291, 1983.

8.

Henry, R. J., Blakeney, A. B., Harris, P. J., and Stone, B. A., Detection of neutral and amino sugars from glycoproteins and polysaccharides as their alditol acetates, J. Chrom atogr ., 256, 419, 1983. 9. Lehrfeld, J., Simultaneous gas-liquid chromatographic determination of aldoses and alduronic acids, J. C hrom atogr. , 408, 245, 1987. 10. Sidisky, L. M., Stormer, P. L., Nolan, L., Keeler, M. J., and Bartram, R. J., High temperature partially cross-linked cyanosilicone capillary column for general purpose gas chromatography, J. Chromatogr. S ci ., 26, 320, 1988. 11. Walters, J. S. and Hedges, J. I., Simultaneous determination of uronic acids and aldoses in plankton, plant tissues, and sediment by capillary gas chromatography of /V-hexylaldonamide and alditol acetates, Anal. C hem ., 60, 988, 1988. 12. Kontrohr, T. and Kocsis, B., Separation of eight epimeric 2-aminoaldohexoses as aminohexitol acetates by gas chromatography and analysis of bacterial heteroglycans containing rare 2-aminoaldohexoses and 2-aminouronic acids, J. C hrom atogr., 291, 119, 1984.

TABLE GC 3 Partially Methylated Alditol Acetates: GLC on Capillary Columns Phase Temperature Gas; flow rate (ml/ min) Column Length, m Diameter (I.D.), mm Form Material

P3 T3 He; 0.6

P4 T4 n.a.a

P5 T5 H2; n.a.

P6 T6 H2; n.a.

P7 T7 H2; n.a.

P8 T8 He; 0.8

P9 P10 T9 T10 He; n.a.a He; n.a.

P ll T il He; n.a.

P12 T12 He; n.a.

25 0.25

25 0.22

30 0.25

30 0.25

30 0.28

22 0.25

20 0.25

85 0.25

15 0.24

30 0.25

20 0.30

50 0.33

50 0.33

Coated

Bonded phase Fused silica MS 2

Coated

Coated

SCOT

WCOT

WCOT

WCOT

WCOT

WCOT

WCOT

WCOT

Glass

Glass

Glass

Glass

Glass

Glass

MS 3

FI 4

FI 5

FI 5

FI

Fused silica MS 7

Glass

MS 3

Fused silica MS 6

FI 8

Fused silica FI 9

Bonded phase Fused silica FI 9

r*

I*

r0

rc

r°

Pd

Pe

Pe

1.23 — 1.07 1.14

1.42 — 1.24 1.39

1.89 — 1.40 1.55

1.48 — 1.26 1.45

1.55 — 1.14 1.30

Glass FI 1

r*

Parent sugarValditol r* Rhamnose/rhamnitol 1-O-methyl 2-O-methyl 3-O-methyl

P2 T3 He; 0.6

1.52 0.94 1.30 1.50

1.25 —

1.14 1.22

5

_

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

Carbohydrates

Detector Reference

PI PI T1 T2 He; 1.5a He; 0.8

9

10

TABLE GC 3 (continued) Partially Methylated Alditol Acetates: GLC on Capillary Columns r*

r°

r*

r*

r*

r*

r*

r°

rc

Pd

Pe

P'

4-0-methyl 5-0-methyl 1,2-di-0-methyl 1,3-di-0-methyl 1,4-di-0-methyl 1,5-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 3,4-di-0-methyl 2,3,4-tri-0-methy 1 Fucose/fucitol 1-0-methyl 2-0-methyl 3-0-methyl 4-0-methyl 5-0-methyl 1,2-di-0-methyl l,3-di-0-methyl 1,4-di-0-methyl l,5-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 3,4-di-0-methyl 2,3,4-tri-0-methyl Ribose/ribitol 1-0-methyl 2-0-methyl 3-0-methyl 4-0-methyl 5-0-methyl 1,2-di-0-methyl l,3-di-0-methyl 1,4-di-0-methyl 1,5-di-0-methyl

1.18 — — — — — 0.96 0.96 0.94 0.67 1.30 — 1.17 1.26 1.25 — — — — — 1.04 1.01 1.06 0.78 — — — — — — — —

__ — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — —

__ — — — — — — — — 0.54 — — — — — — — — — — — — — 0.69 —

— — — — — — — — — — — — — — — — — — — — — — — 0.82 —

__ — — — — — — — — — 1.23 — — — — — — — — — — — — 0.74 —

__ — — — — — — — — — 1.11 — — — — — — — — — — — — 0.87

1.12 — — — — — 0.91 0.93 0.91 0.69 1.25 — 1.08 1.15 1.15 —

1.32 — — — — — 0.98 — 0.94 0.58 1.50 — 1.30 1.47 1.45 —

1.47 — — — — — 0.94 1.00 0.89 0.54 2.08 — 1.46 — — —

1.36 — — — — — 1.00 0.97 0.93 0.69 1.58 — 1.32 1.52 — —

—

—

—

—

— — — 1.11 1.07 1.15 0.79

—

—

—

—

—

—

—

—

—

—

— — — 1.10 1.05 1.15 0.73 — —

— — — 1.03 1.08 — 0.65

—

— — — 0.93 0.96 0.97 0.75 —

1.24 — — — — — 0.78 0.83 — 0.39 1.53 — 1.15 1.32 — — — — — — 0.83 0.90 0.90 0.50

— — — — — —

— — — — — —

—

—

—

—

—

—

— — — —

— — — —

— — — —

— — — —

— — — —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.41 1.14 0.70 0.76 0.86 0.70 1.02 0.99 0.98 0.60 1.59 0.98 1.38 1.59 1.56 1.29 0.71 0.77 0.88 0.78 1.17 1.08 1.21 0.76 2.00 1.32 1.63 1.59 1.63 1.32 0.91 0.85 1.01 0.85

—

—

— — — —

— — — — — —

— — — — —

— — — — —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

CRC Handbook of Chromatography

Parent sugarb/alditol r*

— — — — — — — — — — — — — — — — 1.49 — 1.33 1.37 1.36 1.17 — — —

— 1.09 1.10 1.01 1.11 0.94 — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

—

—

—

— — —

—

—

— — — — — — — — — — — — — — — — — — 2.23 — — — —

— — —

1.32 — 1.09 —

0.94 — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — __ — — — — — — — — — — — — — 1.26 — 1.09 1.12 1.10 0.97 — — — — 0.88 0.90 0.81 0.90 0.80 — — — — — — —

__ __ __ — — — — — — — — — — — — — 1.84 — 1.57 1.62 1.60 1.75 — — — — 1.17 1.18 1.04 1.20 0.94 — — — — — — —

__ __ __ — — — — — — — — — — — — — 2.62 — 1.82 1.94 — — — — — — 1.12 1.20 0.98 1.17 0.94 — — — — — — —

__ __ __ — — — — — — — — — — — — — 2.16 — 1.69 1.79 — 1.34 — — — — 1.19 1.21 1.07 1.44 0.98 — — — — — — —

__ __ __ — — — — — — — — — — — — — 1.62 — 1.16 1.26 — 0.91 — — — — 0.74 0.77 0.57 — 0.57 — — — — — — —

11

1.08 1.04 1.01 1.08 0.85 0.91 0.50 0.54 0.55 0.56 0.46 0.55 0.55 0.56 0.54 0.50 2.09 1.36 1.75 1.82 1.80 1.40 0.97 0.94 1.08 0.86 1.26 1.27 1.10 1.30 0.98 1.02 0.59 0.64 0.59 0.64 0.49 0.58

Carbohydrates

2,3-di-O-methyl 2,4-di-O-methyl 2,5-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 4,5-di-0-methylb 1,2,3-tri-O-methyl 1,2,4-tri-O-methyl 1,2,5-tri-O-methyl 1,3,4-tri-O-methyl 1,3,5-tri-O-methyl 1,4,5-tri-0-methylb 2,3,4-tri-O-methyl 2,3,5-tri-O-methyl 2,4,5-tri-0-methylb 3,4,5-tri-0-methylb Arabinose/arabinitol 1-O-methyl 2-O-methyl 3-O-methyl 4-O-methyl 5-O-methyl 1,2-di-O-methyl 1,3-di-O-methyl 1,4-di-O-methyl 1,5-di-O-methyl 2,3-di-O-methyl 2,4-di-O-methyl 2,5-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 4,5-di-0-methylb 1,2,3-tri-O-methyl 1,2,4-tri-O-methyl 1,2,5-tri-O-methyl 1,3,4-tri-O-methyl 1,3,5-tri-O-methyl 1,4,5-tri-0-methylb

12

TABLE GC 3 (continued) Partially Methylated Alditol Acetates: GLC on Capillary Columns r*

r*

r°

r*

r*

r*

r*

r*

2,3,4-tri-0-methyl 2,3,5-tri-0-methyl 2,4,5-tri-0-methylb 3,4,5-tri-0-methylb Xylose/xylitol 1-0-methyl 2-0-methyl 3-0-methyl 4-0-methyl 5-0-methyl 1,2-di-0-methyl 1,3-di-0-methyl 1,4-di-0-methyl 1,5-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,5-di-0-methyl 3,4-di-0-methyl 3,5-di-0-methyl 4,5-di-0-methylb 1,2,3-tri-0-methyl l,2,4-tri-0-methyl 1,2,5-tri-0-methyl 1,3,4-tri-0-methyl l,3,5-tri-0-methyl 1,4,5-tri-0-methylb 2,3,4-tri-0-methyl 2,3,5-tri-0-methyl 2,4,5-tri-0-methylb 3,4,5-tri-0-methylb Mannose/mannitol 1-0-methyl 2-0-methyl 3-0-methyl

0.81 0.70 — — 1.65 — 1.44 1.43 1.44 — — — — — 1.18 1.12 — 1.18 — — — — — — — — 0.86 — — — 1.87 — 1.73 1.84

— — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— 0.59 — — — — 2.70 2.65 — — — — — — 1.48 —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 1.66 —

— — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 1.37 — 1.30 —

0.68 0.60 — — 1.31 — 1.11 1.12 1.11 — — — — — 0.89 0.87 — 0.89 — — — — — — — — 0.66 — — — 1.91 — 1.73 1.83

0.78 0.65 0.66 0.61 2.55 1.70 1.98 1.96 1.98 1.70 1.13 1.10 1.25 1.05 1.39 1.25 1.25 1.39 1.10 1.13 0.65 0.65 0.68 0.71 0.59 0.68 0.80 0.71 0.65 0.65 2.98 2.21 2.64 2.97

—

— — — — — — —

1.48 — — — — — — — — 0.74 — — — — — —

—

—

— — — — — 2.07 — 1.91 —

0.75 0.63 — — 2.14 1.73 1.72 1.73 — — — — — 1.27 1.17 — 1.27 — — — — — — — — 0.78 — — — 2.40 — 2.23 2.38

Pd

Pe

Pe

0.67 0.56 — — 3.02 — 1.99 1.99 1.99 — — — — — 1.21 1.15 — 1.21 — — — — — — — — 0.69 — — — 6.63 — 4.79 5.49

0.81 0.73 — — 2.87 — 1.97 1.95 1.97 — — — — — 1.32 1.20 — 1.32 — — — — — — — — 0.84 — — — 4.20 — 3.28 4.12

0.36 0.23 — — 1.74 — 1.22 1.24 1.22 — — — — — 0.77 0.72 — 0.71 — — — — — — —

— 0.37 — — — 4.48 — 3.46 3.94

CRC Handbook of Chromatography

Parent sugarb/alditol r*

1.84 _ 1.56 — — — _ — 1.56 1.62 — 1.44 1.62 — 1.52 — 1.42 — — — — — — — — — — — 1.31 — 1.27 — 1.23 _ — 1.22 _ —

— _ _ — — — _ — 3.15 3.53 _ 2.56 3.45 — 2.92 — 2.47 — — — — — — — — — — — 1.85 — 1.75 — 1.68 _ — 1.61 _ —

— _ _ _ _ _ _ _ 2.48 2.81 _ 2.21 2.48 — 2.36 — 2.21 — — — _ — — — — — — — 1.61 — 1.55 — 1.63 _ — 1.44 _ _

— _ _ _ _ _ _ _ — 4.41 _ — 4.56 — — — — — — — _ — — — — — — — 2.16 — 2.01 — — _ — 1.81 _ _

— _ _ _ _ _ _ _

—

—

_

_

_ _ _

_

_

_

_

_

_

_ _ _ _ _ _

_ 1.12 _ — 1.10 _

_ _

_

— — — — 1.33 — 1.26

_

— 1.31 _

_

_ _ _ _ _ _ _ — — — — 1.14 — 1.11

_

_ 1.46

_ _ _ _ _ _ _

_ _ _ — — — — 1.53 — 1.34 _ 1.33 _

_ 1.19

_

— 1.26

1.43 1.60 — 1.50

_ 1.53

_ _ _

_

_

1.18 1.25 — 1.21

_

— —

1.56 1.63 _

1.53 1.80 — 1.66

_ _ _ — — — — 1.32 — 1.30 _ 1.24

_ _ 1.26

_ 1.43 1.59 — 1.51 _ 1.41 _ _ _

_

_

_ 1.82

_

1.60 — _ — —

_ _

_ 1.57

2.38 _ 1.96 —

_

_ _

_ _

_

1.83 _

_ 1.28 — 1.24

_

_

_ 1.99 2.05 _ 1.76 2.09 — 1.93 _ 1.75 _ _ _ _ _ _ _ — — — — 1.54 — 1.49 _ 1.39 — — 1.41 —

5.49 _ 3.53 — — — — _ 3.22 3.69 _ 2.60 3.28 3.69 2.91 _ 2.60 _

_ _ _ _ — — — — 1.81 — 1.76 _ 1.80 — — 1.62 — —

4.12 2.41 — — — — _ 2.47 2.70 1.99 2.77 2.70 2.35 1.96 _ — —

3.94 — 2.83 — — — — 2.67 2.97 — 2.16 — 2.97 2.43 — 2.26 — — —

_ _ — — — — 1.63 — 1.54 1.45 — — 1.44 — —

— — — — — 1.88 — 1.62 — 1.68 — — 1.59 — —

13

2.97 2.64 2.21 1.71 1.90 2.19 1.93 1.49 2.25 2.35 2.28 1.93 2.43 2.35 2.19 2.25 1.90 1.71 1.27 1.44 1.43 1.14 1.50 1.47 1.26 1.59 1.26 1.14 1.68 1.76 1.59 1.76 1.47 1.43 1.68 1.50 1.44 1.27

Carbohydrates

4-0-methyl 5-0-methyl 6-0-methyl 1,2-di-0-methyl 1,3-di-0-methyl 1,4-di-0-methyl 1,5-di-0-methyl 1,6-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,5-di-0-methyl 2,6-di-0-methyl 3,4-di-0-methyl 3,5-di-0-methyl 3,6-di-0-methyl 4,5-di-0-methylb 4,6-di-0-methyl 5,6-di-0-methyl l,2,3-tri-0-methyl l,2,4-tri-0-methyl l,2,5-tri-0-methyl 1,2,6-tri-0-methyl l,3,4-tri-0-methyl l,3,5-tri-0-methyl l,3,6-tri-0-methyl 1,4,5-tri-0-methylb 1,4,6-tri-0-methyl l,5,6-tri-0-methyl 2,3,4-tri-0-methyl 2,3,5-tri-0-methyl 2,3,6-tri-0-methyl 2,4,5-tri-0-methylb 2,4,6-tri-0-methyl 2,5,6-tri-0-methyl 3,4,5-tri-0-methylb 3,4,6-tri-0-methyl 3,5,6-tri-0-methyl 4,5,6-tri-0-methylb

14

TABLE GC 3 (continued) Partially Methylated Alditol Acetates: GLC on Capillary Columns r°

1,3,4,5-tetra-Omethyl*5 1,3,4,6-tetra-0methyl 2,3,4,6-tetra-0methyl 2,3,5,6-tetra-0methyl Galactose/galactitol 1-0-methyl 2-0-methyl 3-0-methyl 4-0-methyl 5-0-methyl 6-0-methyl 1,2-di-0-methyl l,3-di-0-methyl 1,4-di-0-methyl l,5-di-0-methyl 1,6-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,5-di-0-methyl 2,6-di-0-methyl 3,4-di-0-methyl 3,5-di-0-methyl 3,6-di-0-methyl 4,5-di-0-methylb 4,6-di-0-methyl 5,6-di-0-methyl 1,2,3-tri-0-methyl 1,2,4-tri-0-methyl 1,2,5-tri-0-methyl l,2,6-tri-0-methyl

0.99

_

0.80

r*

r*

r*

r*

r*

r*

r*

p"

Pe

Pe

_

_

_

_

_

_

_

_

_

_

_

—

—

—

—

—

—

—

—

—

—

—

—

0.99

0.95

0.98

1.00

0.98

0.99

1.00

1.01

—

—

—

—

—

3.19 2.34 2.80 3.18 3.18 2.80 2.34 1.82 2.04 2.23 2.04 1.61 2.52 2.54 2.48 2.04 2.68 2.54 2.23 2.52 2.04 1.82 1.48 1.54 1.60 1.20

1.93 — 1.78 1.90 1.90

— — —

—

—

—

2.13

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.66 1.68

3.70 3.97

2.70 3.16

—

—

—

—

—

—

1.48 1.72

2.62 4.27

2.32 3.02

3.19

—

—

—

—

—

—

—

—

—

—

—

—

3.01

2.50

—

—

—

—

1.62

1.55 —

5.28

—

1.00 —

1.38

1.01 —

—

1.00 —

0.98 —

1.00 —

2.50

7.42

4.72

4.78

—

—

—

—

—

—

1.78 1.87 1.87

2.33 2.48 2.48

5.18 6.18 6.18

3.69 4.64 4.64

3.65 4.17 4.17

—

—

—

—

—

—

2.12

4.10

2.65

2.92

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

2.17 2.19

3.62

1.28

1.61 1.68

—

2.97 3.13

—

—

—

—

—

—

—

—

—

—

1.45 1.67

1.85 2.25

2.74

2.14

2.20

—

—

—

—

—

—

—

3.04

2.43

2.41

—

—

—

1.63

1.52 —

1.88

1.64 —

—

1.21 —

1.97

0.98

1.63

—

1.51 —

1.98 —

2.84

2.67

2.37

—

—

2.14

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.49

1\45

1.59

1.20

1.50

1.85

CRC Handbook of Chromatography

Parent sugarb/alditol r*

1.69 1.56 1.28 1.73 1.28 1.20 1.98 1.93 1.73 1.93 1.56 1.60 1.98 1.69 1.54 1.48 1.14

— — — — — — 1.44 — 1.33 — 1.28 — — 1.32 — — —

— — — — — — 2.33 2.27 1.84 — 1.74 1.67 — 1.87 1.64 — —

— — — — — — 1.93 1.94 1.58 — 1.72 1.57 — 1.68 1.65 — —

— — — — — — — — — — 1.97 — — — — — —

— — — — — — 1.44 _ — — 1.28 — — 1.34 _ — —

0.92

—

—

—

—

1.14

1.06

1.11

1.11

1.10

1.02

1.05

3.48 2.51 2.86 3.03 3.26 2.91 2.54 1.90 2.01 2.38 2.10 1.76 2.45 2.33

1.97 — 1.78 1.83 1.90 — 1.66 — — — — — 1.64 1.60

— — — — — — — — — — — — 3.56 3.34

— — — — — — 1.59

— —

— —

— — — — — — 1.42 _ 1.26 _ 1.30 — — 1.30 _ — —

—

—

—

—

—

—

—

—

1.19

1.09

1.09

1.02

1.06

1.12

1.14

1.10

1.12

1.04

—

—

—

—

1.02

_

_

—

_

— — — — — — — — — — — — 2.60 2.63

— — — — — — 4.99 — — — — — 4.75 4.25

— _ — — — _ — — _ — — — — —

1.94 _ 1.76 1.82 1.85 _ 1.62 _ _ — — — 1.58 1.60

2.60

6.43 — 5.02 5.40 —

5.43 — 3.81 4.26 — — — _ — — — — 2.85 2.65

4.62 — 3.54 3.80 4.00 — 2.90

_

— — — — — — 1.18 _

— — 1.37 — — 1.40 _

_ — — 1.13 — — 1.13

_

2.18 _

_

1.38 _

— — — _

_ — — —

_ — —

_

_ — —

_ — — — — —

_ — — — — —

— — — — — — 1.78

—

1.48 — — 1.57 _ — —

1.91 — — _ — —

— — — — — — 2.00 — 1.69 _ 1.54 — — _ — — —

— — — — 2.38 — 1.84

1.59 _

_

2.37 2.44 2.44 _ — _

_

— _

— — — 2.15 —

_ — — — — 3.42 3.42

— — — — — — 2.13 _ 1.62 1.74 — — _ — — —

— — — — 2.72 2.80

Carbohydrates

l,3,4-tri-0-methyl l,3,5-tri-0-methyl l,3,6-tri-0-methyl 1,4,5-tri-0-methylb 1,4,6-tri-0-methyl l,5,6-tri-0-methyl 2,3,4-tri-0-methyl 2,3,5-tri-0-methyl 2,3,6-tri-0-methyl 2,4,5-tri-0-methylb 2,4,6-tri-0-methyl 2,5,6-tri-0-methyl 3,4,5-tri-0-methylb 3,4,6-tri-0-methyl 3,5,6-tri-0-methyl 4,5,6-tri-0-methy lb l,3,4,5-tetra-0methylb l,3,4,6-tetra-0methyl 2,3,4,6-tetra-0methyl 2,3,5,6-tetra-0methyl Glucose/glucitol 1-0-methyl 2-0-methyl 3-0-methyl 4-0-methyl 5-0-methyl 6-0-methyl 1,2-di-0-methyl 1,3-di-0-methyl 1,4-di-0-methyl 1,5-di-0-methyl 1,6-di-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl

( il

Parent sugarb/alditol 2.43 2.11 2.46 2.50 2.22 2.42 2.15 1.94 1.38 1.57 1.26 1.49 1.59 1.34 1.70 1.46 1.26 1.67 1.72 1.77 1.45 1.56 1.73 1.51 1.54 1.38 0.99 0.81 1.00

r*

r*

r* __ — — — — — — — — — — — — — — —

— — — — — — —

r*

r*

Pd

Pe

Pe

_

_

_

_

_

_

— — — 1.28 — — 1.29

— — — — — — — — — — — — — — — — — — — 1.11 — — 1.10

1.45 1.60 — 1.50 — 1.49 — — — — — — — — — — 1.33 — 1.28 1.24 — — 1.23

1.90 — — 1.97 — 1.93 — — — — — — — — — — — — 1.64 1.41 — — 1.44

2.71 — — 2.89 — — — — — — __ — — __ — — 1.93 — 1.89 1.68 — —

2.22 2.81 — 2.37 — 2.36 __ — — — __ — — __ — — 1.86 — 1.67 1.60 — —

—

2.24 — — 2.42 — 2.30 __ — — — — — — __ — — 1.67 — 1.76 1.43 — — 1.48

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.00

1.00

1.00

— 1.51 1.64 — 1.55 — 1.52 — — —

— 2.81 3.44 — 2.97 — 2.84 — —

— 2.27 2.52 — 2.38 — 2.29 — —

—

—

—

—

—

— — — — — — 1.34 — 1.36 1.25 — — 1.26

— — — — —

— — — — —

__ 3.39 — — 3.82 — — — — — — — — — — —

—

—

—

—

—

1.83 — 1.98 1.61 — — 1.62

1.61 — 1.61 1.53 — — 1.46

2.19 — 2.38 1.73 — — —

— — — 1.23 — — 1.25

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.00

1.00

1.00

1.00

1.00

1.00

1.00

__ — — — — — — — —

1.00

1.00

—

—

—

—

—

—

—

2.99

1.53

—

—

—

—

—

—

—

—

—

—

—

2.86

1.52

—

—

—

—

—

CRC Handbook of Chromatography

2,5-di-0-methyl 2,6-di-0-methyl 3,4-di-0-methyl 3,5-di-0-methyl 3,6-di-0-methyl 4,5-di-0-methylb 4,6-di-0-methyl 5,6-di-0-methyl 1,2,3-tri-0-methyl l,2,5-tri-0-methyl 1,2,6-tri-0-methyl 1,3,4-tri-0-methyl l,3,5-tri-0-methyl l,3,6-tri-0-methyl 1,4,5-tri-0-methylb 1,4,6-tri-0-methyl l,5,6-tri-0-methyl 2,3,4-tri-0-methyl 2,3,5-tri-0-methy 1 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 2,5,6-tri-0-methyl 3,4,5-tri-0-methylb 3,4,6-tri-0-methyl 3,5,6-tri-0-methyl 4,5,6-tri-0-methylb l,3,4,5-tetra-0methylb l,3,4,6-tetra-0methyl 2,3,4,6-tetra-0methyl 2-Acetamido-2deoxyglucose 3-0-methyl

r*

16

TABLE GC 3 (continued) Partially Methylated Alditol Acetates: GLC on Capillary Columns

6-0-methyl 3,4-di-0-methyl 3,6-di-0-methyl 4,6-di-0-methyl 3,4,6-tri-0-methyl l,3,5,6-tetra-0methyl 2-Acetamido-2deoxygalactose 3,4,6-tri-0-methyl

— —

1.50 1.47 1.42 1.47 1.34 1.19

— — — — — —

—

— —

— —

2.75 2.76 2.47 2.65 2.18 1.66

—

— — —

— — 2.00 — 1.78 1.45

— — — —

—

—

—

—

3.09

1.56

—

—

—

—

1.90

2.36

1.38

—

— —

— —

—

— — —

—

—

—

—

—

— —

— —

— —

—

—

—

—

— — —

— — — —

— — — — — —

— — — — — —

—

—

—

—

—

—

—

—

a Split ratios 1:20,1 1:60,4 1:50.7 b In Reference 1, derivatives were prepared by Haworth méthylation of alditols; some obviously have no counterpart among methylated sugars. c f relative to 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol (ca. 9 min,1 18 min4). d p = retention coefficient, based on retention time relative to two widely separated standards, l,4-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol (p = 1.00) and peracetylated 2-0-methyl-c7i/ro-inositol (quebrachitol; p = 4.30); p values calculated by computer program. e p values calculated from retention times relative to 1,4-di-0-acetyl-2,3,4,6-tetra-0-methylallitol (p = 0.80 on PI 1, 0.83 on P12) and peracetylated 2-0-methylchiro -inositol (p = 4.00 on PI, 3.00 on PI2). Phase

Temperature

17

P2 = OV-225. OV-275. P4 = Silar 10C. OV-17. P6 = Dexsil 410. Silar 9CP. P8 = SP-2100, 0.25-|xm film. OV-101. P10 == SP-1000, 0.22-p.m film. SP-2330, 0.20-(xm film. P12 == OV-1, 0.5-(xm film. CP-SÌ188, 0.22-|xm film. 165 —> 215°C at 2°C/min; held at 215°C. 150 250°C at 4°C/min; held at 250°C for 10 min. injection temperature 50°C; 50 —►182°C at 40°C/min; held at 182°C. 150 190°C at l°C/min. 100 -► 230°C at TC/min. 130 —> 240°C at 2°C/min. 100 -> 240°C at 3°C/min. 120 200°C at 2°C/min. 160 -* 210°C at 2°C/min; 210 240°C at5°C/min. injection temperature 60°C; operating temperatures 206°C. injection temperature 60°C; operating temperature 210°C. injection temperature 60°C; operating temperature 195°C.

Carbohydrates

PI = P3 = P5 = P7 = P9 = PII = T1 = T2 = T3 = T4 = T5 = T6 = T7 = T8 = T9 = T10 = T il = T12 =

TABLE GC 3 (continued) Partially Methylated Alditol Acetates: GLC on Capillary Columns

1. Klok, J., Cox, H. C., de Leeuw, J. W., and Schenck, P. A., Analysis of synthetic mixture of partially methylated alditol acetates by capillary gas chromatography, gas chromatography-electron impact mass spectrometry, and gas chromatography-chemical ionization mass spectrometry, J. Chrom atogr., 253, 55, 1982. 2. Bade, A., Harris, P. J., Hak, E. W., and Clarke, A. E., Capillary gas chromatography of partially methylated alditol acetates on a high-polarity bonded phase vitreous-silica column, J. C hrom atogr. , 315, 373, 1984. 3. Barreto-Bergter, E., Hogge, L., and Gorin, P. A. J., Gas-liquid chromatography of partially methylated alditol acetates on capillary columns of OV-17 and OV-225, Carbohydr. R es., 97, 147, 1981. 4. Shibuya, N., Gas-liquid chromatographic analysis of partially methylated alditol acetates on a glass capillary column, J. C hrom atogr., 208, 96, 1981. 5. Geyer, R., Geyer, H., Kiihnhardt, S., Mink, W., and Stirm, S., Capillary gas chromatography of methylhexitol acetates obtained upon methylation of Nglycosidically linked glycoprotein oligosaccharides, Anal. B iochem ., 121, 263, 1982. 6. Harris, P. J., Bacic, A., and Clarke, A. E., Capillary gas chromatography of partially methylated alditol acetates on a SP-2100 wall-coated open-tubular column, J. C hrom atogr., 350, 304, 1985. 7. Shea, E. M. and Carpita, N. C., Separation of partially methylated alditol acetates on SP-2330 and HP-1 vitreous silica capillary columns, J. C hrom atogr., 445, 424, 1988. 8. Lomax, J. A. and Conchie, J., Separation of methylated alditol acetates by glass capillary gas chromatography and their identification by computer, J. C hrom atogr., 236, 385, 1982. 9. Lomax, J. A., Gordon, A. H., and Chesson, A., A multiple-column approach to the methylation analysis of plant cell-walls, Carbohydr. R es., 138, 177, 1985.

CRC Handbook of Chromatography

REFERENCES

00

Carbohydrates

19

TABLE GC 4

Acetylated Aldononitriles from Neutral and Aminodeoxy Sugars Packing Temperature Gas; flow rate (ml/ min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 N2; 32

P2 T2 N2; 22

P3 T3 N2; 24

P4 T4 N2; 30

P5 T5 He; 0.5a

P6 T6 N2; 2a

P7 T7 N2; 2a

123 0.2

123 0.2

182 0.32

200 0.2

5000 0.02

2500 0.05

2700 0.03

Packed Glass FI 1

Packed Glass FI 1

Packed Ni

Packed Glass

Capillary Glass

FI 2

Fl

3

Capillary Fused silica FI 4

5

Capillary Glass FI 5

Parent sugar

rb

rb

rb

rb

rb

rb

rb

D-Erythrose 2,6-Dideoxy-D-r/òohexose 2-Deoxy-D-erythropentose L-Rhamnose L-Fucose D-Ribose D-Lyxose D-Arabinose D-Xylose 2-Deoxy-D-a r a b in o hexose 2-Deoxy-D-/yjcohexose D-Alióse D-Mannose D-Talose D-Glucose D-Galactosoe L-Idose

0.26 0.45

0.27 0.45

__ —

_

_

_

_

—

—

—

—

0.58

0.49

—

—

0.53

—

—

0.50 0.61 0.61 0.65 0.68 0.74 0.91

0.59 0.64 0.62 0.64 0.64 0.68 0.86

—

0.61 0.63 0.60

— — — —

— — — —

0.60e 0.68e

0.37 0.46 0.47 0.50 0.54 0.62

—

0.96

0.86

0.90 0.93 0.93 1.00 1.04 1.07 1.28

0.93 0.97 0.97 1.00 1.03 1.07 1.30

—

D -g ly c ero -D -g lu c o -

heptose 2,5-Anhydromannosed 2,5-Anhydrotalosed 2-Amino-2-deoxy-Dglucose 2-Amino-2-deoxy-Dmannose 2-Amino-2-deoxy-Dgalactose 2-Acetamido-2deoxy-D-glucose 2-Acetamido-2deoxy-D-galactose b

0.54c — —

—

FI

0.62 0.63

0.77 0.80

—

0.61 0.62 0.77

—

—

—

—

—

—

—

—

—

—

—

—

0.97e

0.88

0.93

0.96

0.98

—

—

—

—

—

1.00e 1.06e

1.00 1.05

1.00 1.05

1.00 1.06

1.00 1.06

— —

— —

— —

— —

— —

—

—

0.58

—

—

—

— —

—

—

0.75

—

—

—

— 1.20

—

—

—

—

—

—

—

—

1.30

—

—

—

—

—

—

1.34

—

—

1.63

1.22

—

—

—

—

—

—

1.34

—

—

—

—

—

Split ratios 1:35,4 1:20.5 t,. relative to peracetylated glucononitrile (20.9 min on P I,1 15.1 min on P2,1 6.7 min,2 25.7 min,3 14.2 min,4 10.4 min on P6,5 10.0 min on P75). c For simultaneous analysis of amino sugars as O-methyloxime acetates see Table GC 7, Reference 2. d From nitrous acid deamination of 2-amino-2-deoxyglucose and -galactose.

20

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE GC 4 (continued) Acetylated Aldononitriles from Neutral and Aminodeoxy Sugars Packing

Temperature

PI P2 P3 P4 P5 P6 P7 T1 T2 T3 T4 T5 T6

= 3 % neopentyl glycol succinate on Chromosorb® W (60 — 80 mesh). = 2% OV-17 on Chromosorb® W HP (80 — 100 mesh). = 1% diethylene glycol adipate on Chromosorb® W HP (100 — 120 mesh). = 3% OV-225 on Gas-Chrom® Q (120 — 120 mesh). = OV-1, 0.11 ^m film. = OV-17 = OV-73 = 140 —> 250°C at 3°C/min. = 130 —> 300°C at 5°C/min. = 170°C for 2 min; 170 -► 240°C at 10°C/min. = 170 180°C at l°C/min; 180 200°C at 2°C/min; held at 200°C for 10 min. = 175°C for 4 min; 175 260°C at 4°C/min; held at 260°C for 5 min. = 120°C for 3 min; 120 210°C at 12°C/min. REFERENCES

1. Seymour, F. R., Chen, E. C. M., and Bishop, S. H., Identification of aldoses by use of their peracetylated aldononitrile derivatives: a g.l.c.-m.s. approach, Carbohydr. R es., 73, 19, 1979. 2. Mawhinney, T. P., Feather, M. S., Barbero, G. J., and Martinez, J. R., The rapid quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography, Anal. Biochem ., 101, 112, 1980. 3. Turner, S. H. and Cherniak, R., Total characterization of polysaccharides by gas-liquid chromatography, Carbohydr. R es., 95, 137, 1981. 4. Guerrant, G. O. and Moss, C. W ., Determination of monosaccharides as aldononitrile, O-methyloxime, alditol, and cyclitol acetate derivatives by gas chromatography, Anal. C hem ., 56, 633, 1984. 5. Li, H.-P. Capillary gas chromatography of neutral sugars as their aldononitrile acetates from the hydrolysate of com bran residues, J. C hrom atogr., 410, 484, 1987.

TABLE GC 5 Acetylated Aldononitriles of Methylated Sugars P2 165 N2; 6

P3 180 n.a.

P4 T1 n.a.

P4 T2 n.a.

P5 T3 n.a.

P6 T4 n.a.

P7 150 He; n.a.a

P8 T5 He; n.a.a

P9 T6 He; n.a.a

300 0.32

300 0.32

180 0.2

180 0.2

180 0.2

200 0.4

200 0.4

2500 0.02

2500 0.02

3000 0.025

Packed SS FI 1

Packed SS FI 1

Packed Glass FI 2

Packed Glass FI 2

Packed Glass FI 2

Packed Packed Glass Glass FI FI 3 3

Capillary Fused silica FI 4

Capillary Fused silica FI 4

Capillary Fused silica FI 4

Parent sugar

rb

rb

r*

r*

r*

r*

r*

r*

r*

r*

L-Rhamnose 2-0-methyl 3-0-methyl 4-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 3,4-di-0-methyl 2,3,4-tri-0-methyl D-Galactose 2-0-methyl 3-0-methyl 4-0-methyl 6-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,6-di-0-methyl 3,4-di-0-methyl 3,6-di-0-methyl 4,6-di-0-methyl 2,3,4-tri-0-methyl

4.73 3.03 3.94 3.85 1.82 1.94 2.79 1.00

3.56 2.56 3.28 3.10 1.64 1.79 2.38 1.00

_ — — — — — — —

_ — — — — — — —

_ — — — — — — —

— — — — — — — — — — — —

— — — — — — — — — — — —

8.18 5.09 6.36 7.00 3.45 3.45 3.45 2.27 3.45 3.45 2.45 1.73

2.22 1.97 2.06 2.06 1.84 1.84 1.87 1.64

2.70 2.33 2.48 2.48 2.11 2.11 1.92 1.78

—

—

1.84 1.64

2.11 1.85

—

—

_ — — — — — — — — — — — — — — — — — — —

_ — — — — — — — — — — — — — — — — — — —

_ — — — — — — — — — — — — — — — — — — —

_ — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — —

21

PI 165 N2; 8

Carbohydrates

Packing Temperature (°C) Gas; flow rate (ml/ min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

K>

TABLE GC 5 (continued) Acetylated Aldononitriles of Methylated Sugars rb

rb

r*

2,3,6-tri-O-methyl 2,4,6-tri-O-methyl 3,4,6-tri-O-methyl 2,3,4,6-tetra-Omethyl D-Glucose 2-0-methyl 3-0-methyl 4-0-methyl 6-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,6-di-0-methyl 3,4-di-0-methyl 3,6-di-0-methyl 4,6-di-0-methyl 2,3,4-tri-0-methyl 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 3,4,6-tri-0-methyl 2,3,4,6-tetra-0methyl

— — — —

— — — —

1.45 1.27 1.82 1.00

— — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — —

r*

r*

r-

r*

r*

r*

1.45 1.42 1.52 1.00

1.48 1.37 1.63 1.00

— — — —

— — — —

— — — —

— — — —

— — — —

— — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — —

2.85 2.26 2.69 2.44 2.36 2.14 1.92 1.74 1.74 2.05 1.92 1.53 1.59 1.27 1.43 1.00

— 1.89 2.37 — — 1.75 1.62 1.51 1.47 1.72 1.62 1.34 1.38 1.22 1.24 1.00

— — — — — 2.49 2.27 — 2.77 — — 1.64 1.53 1.36 1.59 1.00

— — — — — 3.31 2.83 — 3.19 — — 2.26 2.38 1.71 1.96 1.00

— — — — — 1.38 1.36 — 1.42 — — 1.23 1.19 1.15 — 1.00

N ote: This table supplements Table GC 6 in CRC H andbook o f Chrom atography: Carbohydrates, Volume I.

a b c d e

Split ratio 1:100. t, relative to 5-0-acetyl-2,3,4-tri-0-methyl-L-rhamnononitrile (9.35 min on PI, 16.8 min on P2). t, relative to 5-0-acetyl-2,3,4,6-tetra-0-methyl-D-galactononitrile; all t, values compared with those of two widely separated standards (5-0-acety 1-2,3,4,6tetra-0-methyl-D-galactononitrile and penta-0-acetyl-D-galactononitrile) to lessen aging effect, i.e., decrease in tj with aging of column. tr relative to 5-0-acety 1-2,3,4,6-tetra-0-methyl-D-glucononitrile (14 min on P5, 18.5 min on P6); all t, values compared with that of myo-inositol hexaacetate (48 min). tr relative to 5-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucononitrile (4.9 min on P7, 6.4 min on P8, 16.3 min on P9); no internal standard.

CRC Handbook of Chromatography

Parent sugar

N>

Packing

Temperature

P1 P2 P3 P4 P5 P6 P7 P8 P9 TI T2 T3 T4 T5 T6

= 3 % QF-1 on Gas-Chrom® Q (100 — 120 mesh). = 1% XE-60 on Gas-Chrom® Q (100 — 120 mesh). = 3% ECNSS-M on Gas-Chrom® Q (100 — 120 mesh). = 3% OV-225 on Gas-Chrom® Q (100 — 120 mesh). = 5% OV-225 on Chromosorb® W AW DMCS (100 — 120 mesh). = 3% SP-2340 on Supelcoport® (100— 120 mesh). = SP-2100. = Carbowax 20M. = SE-54. = 150 —^ 245°C at 8°C/min; held at 245°C. = 185 —►245°C at 4°C/min; held at 245°C. = 160 210°C at l°C/min. = 150 225°C at 2°C/min. = 135°C for 5 min; 135 -► 165°C at 4°C/min. = 120°C for 5 min; 120 250°C at 4°C/min. REFERENCES

1. Janecek, F., Toman, R., Karàcsonyi, S., and Änderte, D., Gas-liquid chromatographic separation o f methyl ethers o f L-rhamnose as their methyl glycosides, trifluoroacetylated L-rhamnitols, and acetylated L-rhamnononitriles, J. Chrom atogr . ., 173, 408, 1979. 2. Stortz, C. A., Matulewicz, M. C., and Cerezo, A. S., Separation and identification of 0-acetyl-O-methyl galactononitriles by gas-liquid chromatography and mass spectrometry, Carbohydr. R es., I l l , 31, 1982. 3. Tanner, G. R. and Morrison, I. M., Gas chromatography-mass spectrometry of partially methylated glycoses as their aldononitrile peracetates, J. C hrom atogr., 299, 252, 1984. 4. Slodki, M. E., England, R. E., Plattner, R. D., and Dick, W. E., Jr., Méthylation analyses of NRRL dextrans by capillary gas-liquid chromatography, Carbohydr. R es., 156, 199, 1986.

Carbohydrates 23

24

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE GC 6 Acetylated A-Alkylaldonamides Derived from Aldonic and Alduronic Acids* Packing Temperature Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 He; 20

PI T2 He; 20

P2 T3 He; 10

P3 T4 He; 12b

100 0.2 Packed

100 0.2 Packed

2400 0.075 Capillary

Glass

Glass

Glass

3000 0.025 Bonded phase Fused silica

FI 1

FI 2

FI 3

4

r*

r°

r*

1.42

—

iV-AIkylaldonamide D-Ribonamide,

D-Xylonamide,

L-Mannonamide,

D-Gluconamide,

D-Galactonamide,

L-Gulonamide, L-Idonamide,

N -{\ -propyl) N -(l -butyl) N -(\ -pentyl) N-( 1-hexyl) N -(\ -propyl) N -(\ -butyl) N -( 1-pentyl) N -(\ -hexyl) N-( 1-propyl) N -(\ -butyl)

N-(l-pentyl) N-( 1-hexyl) N-(l-propyl) N-( 1-butyl) N -(l -pentyl) N -(\ -hexyl) N-( 1-propyl) N -(\ -butyl) N-( 1-pentyl) N-( 1-hexyl) N -(\ -propyl) N-( 1-hexyl) N-(( 1-hexyl)

1.25 1.31 1.37 1.44 1.47 1.52 1.57 1.64 1.68 1.73 1.81 1.85 1.77 1.81 1.88 1.92 1.87 1.93 2.00 2.01 — — —

1.70

FI

—

—

—

—

—

—

—

—

—

2.17

—

—

—

—

—

— —

2.75 2.87 3.07 — 3.01 3.15 3.40 — 3.44 3.63 3.90 — — — —

— — 2.06f

— — —

—

—

— — 2.24f — — — 2.46f — — — 2.35f — —

— 3.00*

— — — 3 .W

— — — 3.30f — 3 .17f 3 .19f

Simultaneous analysis of neutral sugars (as alditol acetates) and sugar acids is possible by this method; see data for sugars in Tables GC 1 (Reference 2) and GC 2 (References 9 and 11). b Split ratio 1:50. c t, relative to myo-inositol hexaacetate (88.3 min,1 7.8 min3). d f relative to mannitol hexaacetate (3.1 min). e tj relative to glucitol hexaacetate (11.3 min). a

f

JV-alkyl-D-mannonamide, -L-gulonamide, -D-gluconamide, -L-galactonamide, and -L-idonamide produced from D-mannuronic acid, D-glucuronic acid, L-guluronic acid, D-galacturonic acid, and L-iduronic acid, respectively, by NaBH4 reduction of uronates prior to lactonization and treatment with alkylamine.

Packing

Temperature

PI P2 P3 T1 T2 T3 T4

= 3 % SP-2340 on Supelcoport® (100 — 120 mesh). = SP-2330, 1.0-p.m film. = DB-1701 (J and W Scientific). = 190 —►260°C at 5°C/min. = 220°C for 2 min, 220 -► 250°C at 32°C/min. = 200°C for 2 min; 200 -► 235°C at 3°C/min; held at 235°C. = 220 -► 270°C at l°C/min.

C a r b o h y d ra te s

TABLE GC 6 (continued) Acetylated A-Alkylaldonamides Derived from Aldonic and Alduronic Acids* REFERENCES 1. Lehr field, J ., Gas chromatographic analysis of mixtures containing aldonic acids, alditols, and glucose, Anal. C hem ., 56, 1803, 1984. 2. Lehrfield, J., G. 1. c. determination of aldonic acids as acetylated aldonamides, Carbohydr. R es., 135, 179, 1985. 3. Lehrfield, J., Simultaneous gas-liquid chromatographic determination of aldoses and alduronic acids, J. C hrom atogr., 408, 245, 1987. 4. Walters, J. S. and Hedges, J. L, Simultaneous determination of uronic acids and aldoses in plankton, plant tissues, and sediment by capillary gas chromatography of N-hexyaldonamide and alditol acetates, Anal. C hem ., 60, 988, 1988.

25

26

TABLE GC 7 Acetylated Oxime Derivatives of Neutral and Amino Sugars

Parent sugar

L-Rhamnose L-Fucose 2-Deoxy-D-erythro-pentose

D-Ribose D-Arabinose D-Xylose 2-Deoxy-D-arabino-hexose

D-Mannose D-Glucose D-Galactose D-erythro-Pentulose

D-Fructose 1,3,4-tri-C>-methyl 3,4,6-tri-O-methyl 1,3,4,6-tetra-O-methyl D-Tagatose L-Sorbose D-g/wco-Heptulose D-manno-Heptulose 2-Amino-2-deoxy-D-glucose

PI T1 N2; 22

P2 T2 N2; 24

P3 T3 N2; 20

P4 T4 N2; 0.55“

P5 T5 He; 0.4

P6 T6 He; 0.5“

P7 T7 H2; n.a.

P7 T7 H2; n.a.

P8 T8 N2; 35

P9 T9 He; 3

123 0.2 Packed Glass FI 1

183 0.18 Packed Ni FI 2

183 0.18 Packed Glass FI 3

1000 0.021 Capillary Fused silica

1100 0.021 Capillary Fused silica

FI

FI

4

2500 0.03 Capillary Fused silica FI 6

2400 0.03 Capillary Fused silica

4

5000 0.02 Capillary Fused silica FI 5

200 0.2 Packed Glass FI 8

1500 0.032 WCOT Fused silica FI 8

Ob r*

MOb

MO

MO

MO r*

MO rf

MO rf

MO rf

— — — — — — — — — —

1.52; 1.56h

— — — — — — — — — — — — — — — — — — —

_ — — — — — — — — — — — — — — — — — —

_ — — — — — — — — — — — — — — — — — —

—

1.57'

0.77

0.75

1.07; l.KP 1.31 0.96; 0.97h 0.98 0.66;h 1.70 1.31; 1.35h 1.35

—

r*

— — — — — — — — — — — — — — — — — — — —

0.59; 0.58; 0.46 0.60; 0.59; 0.65 0.76 0.90; 0.92; 0.89;

—

fi

7

0.62h 0.45; 0.48h 0.45;0.47h 0.63h 0.46; 0.51h 0.46; 0.50*

— —

— —

PFBO rg

_ 0.50

— —

0.61h 0.63h 0.52; 0.55h 0.50; 0.54h 0.67 0.57; 0.59h 0.56; 0.59h 0.83

—

—

—

0.94h 0.81; 0.86h 0.81; 0.86h 1.22 0.95h 0.88; 0.89h 0.89; 0.90* 1.50 0.96h 0.84; 0.92h 0.83; 0.92h 1.43

0.97;h 0.98

— — — — —

1.21; 1.23h 1.24; 1.26h 1.12; 1.15h

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

PFBO rg

_ 0.65; 0.73h

— —

0.78; 0.86h 0.92; 0.95h

—

1.05; 1.12h 1.16; 1.24h 1.05; 1.22h

— — — _ — — — — — —

CRC Handbook of Chromatography

Packing Temperature Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

2-Amino-2-deoxy-D-galac— tose 2-Amino-2-deoxy-D-man— nose 2-Acetamido-2-deoxy-D— glucose 2-Acetamido-2-deoxy-D— galactose 2-Acetamido-2-deoxy-D— mannose 2- Deoxy-2-glycolyl-amino-— D-mannosek A-Acetylneuraminic acid — — Muramic acid A-Acetylmuramic acid — 3- Deoxy-D-mawio-2-octu- — losonic acid1

—

0.80

0.78

1.14;

1.16h —

—

—

—

1.86'

—

0.80

0.78

1.14;

1.17h —

—

—

—

—

1.14s

—

—

—

1.29;h 1.30

1.33;h 1.34

—

—

—

1.24s

—

—

—

1.36

1.40

—

—

—

1.29s

—

—

—

1.36;

l^O11 1.41;1.45h

—

—

—

—

—

—

—

—

1.88;1.98h

—

—

— — — —

— — — —

— 0.94 — —

— 0.89 — —

1.70 1.25 1.29; 1.30*1 1.35;h 1.36

— 1.64; — —

— — — —

— — — —

Split ratios 1:50,4 1:35.5 O = oxime, MO = 0-methyloxime, PFBO = O-pentafluorobenzyloxime. ^ relative to peracetylated glucononitrile (7.2 min,1 6.7 min2). t, relative to peracetylated D-g/wco-heptitol (10.5 min). t, relative to A-methylglucamine O-methyloxime (15.57 min on P4, 9.96 min on P5). ^ relative to myo-inositol hexaacetate (20.7 min,5 21.3 min,6 18.6 min7). ^ relative to meso-inositol hexaacetate (18 min on P8, 18.5 min on P9). Major peak. For simultaneous analysis of neutral sugars as aldononitrile acetates see Table GC 4, Reference 2. For simultaneous analysis of acetamidodeoxyhexitol acetates see Table GC 1, Reference 3. Product of cleavage of A-glycolylneuraminic acid by neuraminic acid aldolase. KDO.

Packing

= = = = = = =

2% OV-17 on Chromosorb® W HP (80 — 100 mesh). 1% diethylene glycol adipate on Chromosorb® W HP (100 — 120 mesh). 3% diethylene glycol adipate on Chromosorb® W HP (100 — 120 mesh). OV-101. SE-54. OV-1, 0.11-p.m film. Carbowax 20M.

27

PI P2 P3 P4 P5 P6 P7

— 1.69h — — —

Carbohydrates

a b c d e f g h 1 j k 1

1.721

TABLE GC 7 (continued) Acetylated Oxime Derivatives of Neutral and Amino Sugars = = = = = = = = =

3% SP-2340 on Supelcoport® (100 — 120 mesh). CP-Sil 88, 0.2-p.m film. 130 —> 300°C at 20°C/min. 170°Cc for 2 min; 170 -* 240°C at 10°C/min. 210 -» 240°C at 2°C/min. 190 —►230°C at 4°C/min. 160 —> 200°C at 4°C/min. 175°C for 4 min; 175 —» 260°C at 4°C/min; held at 260°C for 5 min. 80 -> 180°C at 20°C/min; held at 180°C for 1 min; 180 -» 210°C at 2°C/min; held at 210°C for 1 min; 210 -> 230°C at 10°C/min; held at 230°C. T8 = 240 260°C at 4°C/min; held at 260°C. T9 = 180°C for 4 min; 180 240°C at 5°C/min; held at 240°C for 10 min. REFERENCES

1. Seymour, F. R., Chen, E. C. M., and StoufTer, J. E., Identification of ketoses by use of their peracetylated oxime derivatives: a g.l.c.-m.s. approach, Carbohydr. R es., 83, 201, 1980. 2. Mawhinney, T. P., Feather, M. S., Barbero, G. J., and Martinez, J. R., The rapid, quantitative determination of neutral sugars (as aldononitrile acetates) and amine sugars (as 0-methyloxime acetates) in glycoproteins by gas-liquid chromatography, Anal. B iochem ., 101, 112, 1980. 3. Mawhinney, T. P., Simultaneous determination of A-acetylglucosamine, jV-acetylgalactosamine, N-acetylglucosaminitol and iV-acetylgalactosaminitol by gasliquid chromatography, J. Chrom atogr ., 351, 91, 1986. 4. Hicks, R. E. and Newell, S. Y., An improved gas chromatographic method for measuring glucosamine and muramic acid concentrations, Anal. B iochem ., 128, 438, 1983. 5. Guerrant, G. O. and Moss, C. W., Determination of monosaccharides as aldononitrile, O-methyloxime, alditol and cyclitol acetate derivatives by gas chro­ matography, Anal. C hem ., 56, 633, 1984. 6. Neeser, J.-R. and Schweizer, T. F., A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries, Anal. B iochem ., 142, 58, 1984. 7. Neeser, J.-R., G.l.c. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines, and sialic acid: “ one-pot” quantitative determination of the carbohydrate constituents of glycoproteins and a study of the selectivity of alkaline borohydride reductions, Carbohydr. R es., 138, 189, 1985. 8. Biondi, P. A., Manca, F., Negri, A., Seechi, C., and Montana, M., Gas chromatographic analysis of neutral monosaccharides as their 0-pentafluorobenzyloxime acetates, J. Chrom atogr ., 411, 275, 1987.

CRC Handbook of Chromatography

Temperature

P8 P9 T1 T2 T3 T4 T5 T6 T7

oc

C a rb o h ydra tes

29

TABLE GC 8

Deoxy(methoxyamino)alditol Derivatives Phase Temperature (°C) Gas; pressure (kPa) Column Length, m Diameter (I D.), mm Form Material Detector Reference Parent sugar Arabinose Xylose Ribose Rhamnose Fucose Altrose Alióse Idose Mannose Glucose Guióse Talóse Galactose Cellobiose Maltose Lactose Gentiobiose Melibiose

PI T1 H2; 90a

P2 T2 H2; 50a

P3 T2 H2; 50a

P3 T3 H2; 50a

P4 T4 H2; 70a

P4 260 H2; 70a

25 0.18

25 0.25

50 0.5

25 0.25

25 0.20

25 0.20

Capillary Glass FI 1

Capillary Glass FI 1

Capillary Glass FI 2

Capillary Glass NPDb 2

Capillary Glass MS 3

Capillary Glass FI,NPDb 3

TMSC

Acc

Mec

Va

Va

Va

Me r-

Me r*

Me r*

0.43 0.41 0.44 0.55 0.57

0.65 0.69 0.71 0.63 0.66

—

—

0.63 0.65 0.53 0.62 0.71 0.75 0.78 0.95 0.97 1.00 1.06 1.09 1.13

—

—

—

—

0.98 1.00

0.98 1.00

—

—

—

—

1.02

1.03

0.55 0.56 0.36 0.53 0.65 —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

0.96 1.00

—

—

—

—

—

—

—

—

—

—

—

—

0.94 1.00 1.04 1.58 1.62

0.95 1.00 1.05 1.52 1.54

1.17

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

a Split ratios 1:40,* 1:50,2 splitless.3 b Nitrogen-phosphorus selective detector. c TMS and Ac denote O-trimethylsilylated and 0-acetylated deoxy(methoxyamino)alditols, respectively; Me denotes permethylated deoxy(methylmethoxyamino)alditols, or glycosides from disaccharides. d ^ relative to 1-deoxy-1-methoxyaminoglucitol derivative (28.8 min on P I,1 28.2 min on P2,1 27.3 min on P3 with T2,2 37.0 min with T3.2) c L relative to permethylated deoxy(methylmethoxyamino)alditol glycoside derived from maltose (9.8 min for P4 with T4, 9.2 min at 260°C). Phase

Temperature

PI P2 P3 P4 T1 T2 T3 T4

= = = = = = = =

SE-30. OV-101. Carbowax 20M. Superox 0.1. 160°C for 10 min; 160 —> 170°C at 0.5°C/min. 170°C for 10 min; 170 210°C at 2°C/min. 120°C for 5 min; 120 160°C at TC/min. 240°C for 5 min; 240 —> 260°C at l°C/min. REFERENCES

1. Das Neves, H. J. C., Riscado, A. M. V., and Frank, H., Derivatives for the analysis of monosaccharides by capillary g.l.c.: trimethylsilylated deoxy(methoxyamino)alditols, Carbohydr. Res., 152, 1, 1986. 2. Das Neves, H. J. C., Riscado, A. M. V., and Frank, H., Single derivatives for GC-MS assay of reducing sugars and selective detection by the nitrogen-phosphorus detector (NPD), J. High Resolut. Chromatogr. Chromatogr. Commun., 9, 662, 1986. 3. Das Neves, H. J. C. and Riscado, A. M. V., Capillary gas chromatography of reducing disaccharides with nitrogen-selective detection and selected-ion monitoring of permethylated deoxy(methylmethoxyamino)alditol glycosides, J. Chromatogr., 367, 135, 1986.

30

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE GC 9 O-Isopropylidene Acetals of Aldoses, Ketoses, and Naturally Occurring 0-Methylaldoses Packing Temperature Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI TI N2; 20

PI T2 N2; 20

P2 T2 N2; 20

P3 T2 N2; 20

183 0.15 Packed Glass FI 1

183 0.15 Packed Glass FI 2

183 0.15 Packed Glass FI 2

183 0.15 Packed Glass FI 2

Parent sugar

Main acetal

r*

i-

i-

r*

L-Arabinose D-Arabinose, 2-0-methyl D-Xylose 2-0-methyl 3-0-methyl L-Rhamnose L-Fucose 2-0-methyl D-Galactose 3-0-methyl 6-0-methyl D-Glucose 3-0-methyl D-Mannose L -£0^rc>"Pentulose D-r/ireo-Pentulose D-Psicose D-Tagatose D-Fructose

l,2:3,4(ß-L-Arap) 3,4(D-Arap) l,2:3,5(a-D-Xyl/) 3,5(D-Xylf) 1,2(a-D-Xyiy) 2,3(L-Rha^) l,2:3,4(a-L-Fucp 3,4(L-Fucp) l,2:3,4(a-D-Galp) l,2:5,6(a-D-Gal/) l,2:3,4(a-D-Galp) 1,2:5,6(«-d-G1c/) l,2:5,6(a-D-Glcf) 2,3:5,6(D-Man/) 1,2:3,4(ß-L-Ribul/) 2,3(ß-D-Xylul/) l,2:3,4(ß-D-Psi/) l,2:3,4(ot-D-Tag/) 1,2:4,5(ß-D-Frup) 2,3:4,5(ß-D-Frup) 2,3:4,6(a-L-Sorb/)

— — — — — — — — — — — — — 1.00 0.23 0.85 0.65 0.75 0.80 0.87 0.93

— 0.71 — 0.72 0.74 — — 0.69 — 0.82 0.72 — 0.69 — — — — — — — —

— 0.73 — 0.68 0.74 — — 0.65 — 0.68 0.55 — 0.52 — — — — — — — —

0.45 0.72 0.57 0.70 0.74 0.85 0.40 0.68 0.91 0.79 0.68 0.94 0.65 1.00 — — — — — — —

L-Sorbose

Note: This table supplements Table GC 9 in CRC Handbook o f Chrom atography: Carbohydrates,

Volume I. a

t, relative to 2,3:5,6-di-0-isopropylidene-D-Man/(41 min,1 22 min on P32).

Packing

Temperature

PI P2 P3 T1 T2

= 3 % OV-225 on Supelcoport® (100 — 120 mesh). = 3% ECNSS-M on Gas-Chrom® Q (100 — 120 mesh). = PI + P2 (7:4). = 80 —> 160°C at 2°C/min. = 90 —> 190°C at 4°C/min. REFERENCES

1. Morgenlie, S., Gas chromatography-mass spectrometry of hexuloses and pentuloses as their 0isopropylidene derivatives: analysis of product mixtures from trióse aldol-condensations, Carbohydr. R es., 80, 215, 1980. 2. Aamlid, K. H. and Morgenlie, S., Analysis of mixtures of some mono-0-methylaldoses with the common aldoses by g.l.c.-m.s. after isopropylidenation, Carbohydr. R es., 124, 1, 1983.

C a rb o h y d ra te s

TABLE GC 10 Methyl Glycosides, Methylated and Acetylated Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 Ar; 60

P2 T1 Ar; 60

P3 T2 N2; 25

P3 170 N2; 20

P4 170 N2; 30

200 0.4 Packed Glass FI 1

200 0.4 Packed Glass FI 1

200 0.32 Packed SS

180 0.3 Packed Glass

FI

FI

2

3

300 0.3 Packed Glass FI 4

Methyl glycoside

AcPb

Ac Pb

Ac r“

r*

a-L-Arabinopyranoside 2-0-methyl 3-O-methyl 4- 350°C at 4°C/min. REFERENCES

1. Fournet, B., Dhalluin, J.-M., Strecker, G., Montreuil, J., Bosso, C., and Defaye, J., Gas-liquid chromatography and mass spectrometry of oligosaccharides obtained by partial acetolysis of glycans of glycoproteins, Anal. Biochem ., 108, 35, 1980. 2. Nilsson, B. and Zopf, D., Gas chromatography and mass spectrometry of hexosamine-containing oligo­ saccharide alditols as their permethylated N-trifluoroacetyl derivatives, M ethods Enzymol, 86, 46, 1982. 3. Mononen, I., Structural analysis of the neuraminic acid linkages in deaminated trisaccharide-alditols by gas-liquid chromatography-mass spectrometry, Carbohydr. R es., 104, 1, 1982.

TABLE GC 12 Trifluoroacetylated Alditols, Aminodeoxyalditols, and Cyclitols Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI 120 N2; 50

P2 T1 N2; 50

P3 T2 N2; 20

P4 T3 N2; n.a.

P5 T4 N2; 1.5*

P6 150 He; 2*

200 0.3 Packed Glass FI, NPDb 1

150 0.3 Packed Glass FI 1

183 0.2 Packed Glass FI 2

400 0.3 Packed Glass

5000 n.a. WCOT Glass

FI

FI 4

2500 0.02 Bonded phase Fused silica FI 5

3

CRC Handbook of Chromatography

36

TABLE GC 12 (continued) Trifluoroacetylated Alditols, Aminodeoxyalditols, and Cyclitols Parent compound

r*

r*

Glycerol Erythrose Threose Rhamnose Fucose 6-Deoxyglucose Ribose Arabinosed Lyxosed Xylose Allose Mannose Altrosed Talosed 2-Deoxy-D-ara6wo-hexose Glucose Idose 2-Deoxy-D-/yxo-hexose Galactose 2-Amino-2-deoxyglucose 2-Amino-2-deoxygalactose 2-Amino-2-deoxymannose c/u'ro-Inositol Pinitol Quebrachitol Leucanthemitol Bomesitol Vibumitol myo-Inositol scy//o-Inositol

—

—

—

—

— 0.34

—

—

—

r*

— —

— — —

— 0.40 0.47 0.48 0.55 0.62 0.63 0.74 0.74 0.84 0.89 1.00 1.10 1.10 1.13 1.27 1.32 1.40 1.40 — — — —

—

0.41 0.66 0.74 —

—

—

—

—

—

—

0.86 0.92

0.88 0.92

—

—

0.79

0.54 0.60

—

—

—

—

0.74

0.78

—

—

—

0.85

0.64 0.73 — —

—

—

—

—

r*

— —

—

—

—

0.96 0.98 1.00 1.03

—

—

—

—

—

—

—

—

—

—

1.00

— — —

1.09 1.85 2.09 2.03 — —

1.00

1.13 —

1.00

1.16 —

— 1.18 — —

— — —

—

—

—

—

—

—

—

—

—

—

—

—

—

1.25 — — — 0.52 0.60 0.70 0.77 1.32 1.35 1.36 1.87

1.07 1.08 — 1.11 — — — —

0.97 —

1.00 —

1.09 — —

1.14 —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.52 —

Note: This table supplements Table GC 15 in CRC Handbook o f Chromatography: Carbohydrates, Volume I.

Data for GLC of trifluoroacetylated alditols on chiral phases will be found in Table GC 24. a b

Split ratios 1:15,4 1:20.5 Nitrogen-phosphorus selective detector used for aminodeoxy sugars. c t, relative to hexa-O-trifluoroacetyl-D-mannitol (3.9 min on P I,1 18.1 min on P2,1 5.6 min,2 14.6 min,3 33.2 min,4 9.1 min5). d Pairs giving identical alditol. Packing

’emperature

PI P2 P3 P4 P5 P6 T1 T2

= = = = = = = =

5% OV-101 on Chromosorb® W AW DMCS (60 — 80 mesh). 2% XF-1105 on Gas-Chrom® P (60 — 80 mesh). 3% Dexsil 410 on Chromosorb W HP (80 — 100 mesh). 1% OV-225 on Chromosorb W HP (80 — 100 mesh). OV-225. Shimadzu CBP10 (cyanopropyl bonded phase), 0.25-pm film. 100 ^ 160°C at 2°C/min. 100°C for 1.5 min; 100 —* 310°C at 3.5°C/min for 3.5 min, 6°C/min for 5 min, 15°C/min for 5 min, 25°C/min for 3.7 min; held at 310°C for 6.3 min. T3 = 100°C for 3 min; 100 180°C at 5°C/min. T4 = 70°C for 2 min; 70 -> 180°C at 5°C/min; held at 180°C for 15 min. REFERENCES

1. Shinohara, T., Use of a flame thermionic detector in the determination of glucosamine and galactosamine in glycoconjugates by gas chromatography, J. Chromatogr., 207, 262, 1981. 2. Englmaier, P., Trifluoroacetylation of carbohydrates for g.l.c. using V-methylbis(trifluoroacetamide), Carbohydr. R es., 144, 177, 1985.

Carbohydrates

37

3. Decker, P. and Schweer, H., Gas-liquid chromatography on OV-225 of tetroses and aldopentoses as their 0-methoxime and 0-H-butoxime pertrifluoroacetyl derivatives and of C3-C6 alditol pertrifluoroacetates, J. C hrom atogr., 236, 369, 1982. 4. Decker, P. and Schweer, H., G.l.c. of the oxidation products of pentitols and hexitols as trifluoroacetylated O-methyloxime and 0-butyloxime derivatives, Carbohydr. R es., 107, 1, 1982. 5. Haga, H. and Nakajima, T., Analysis of aldoses and alditols by capillary gas chromatography as alditol trifluoroacetates, Chem. Pharm. B ull., 36, 1562, 1988.

TABLE GC 13 Trifluoroacetylated Methyl Glycosides: Capillary GLC of Methanolysates of Lipopolysaccharides and Gangliosides Phase Temperature Gas; flow rate ((ml/min) Column Length, m Diameter (I D.), mm Form Material Detector Reference

PI T1 He; 1.5

P2 T2 He; 2

P3 T3 n.a.

25 0.2 Coated Fused silica FI 1

25 0.22 Coated Glass 2,3

25 0.2 Bonded phase Fused silica FI 4

Parent sugar

r*

r*

r*

3,6-Dideoxy-D-ara6mo-hexoseb 3,6-Dideoxy-D-;cy/o-hexoseb L-Rhamnose L-Fucose D-Ribose D-Galactose D-Glucose D-Mannose

0.57;c 0.67; 0.69 0.65;c 0.70; 0.71 0.68;c 0.76 0.70; 0.73;c 0.79 0.73; 0.75; 0.76; 0.88c 0.92; 0.96;c 1.01 0.94; 1.00;c 1.02 0.99;c 1.05 1.15 1.20 1.29 1.24; 1.3 Ie 1.29; 1.41 ;c 1.45c 1.64

_

_

— 0.67;c 0.80 0.77; 0.83c —

— — — —

0.92; 0.97;c 1.00;c 1.05

0.86;c 1.07 ;c 1.00;c 1.03

—

—

D -g ly c e r o -D -m a n n o -H e p to s e L -g ly c e r o -D -m a n n o -H e p to s c

2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 3-Deoxy-D-ma/w6>-2-octulosonic aciddc N-Acetylneuraminic acidc

FI

1.25 1.33 1.42 1.42 1.67;c 1.70e —

— —

— — —

1.42

Note: This table supplements Table GC 14 in CRC Handbook o f Chromatography: Carbohydrates ,

Volume I. Data for GLC of trifluoroacetylated methyl glycosides on chiral phases will be found in Table GC 24. a b c d e

tr relative to major peak for trifluoroacetylated methyl glucoside (9.47 min,1 6.0 min,23 10.3 min4). 3,6-Dideoxy-D-arabmo-hexose = tyvelose, -D-ry/o-hexose = abequose. Major peak. KDO. Present in methanolysate as methyl ester methyl glycoside.

Phase

Temperature

PI P2 P3 T1 T2 T3

= = = = = =

SE-30. CP-Sil 5. BP-5 (5% phenyl-methyl silicone; S.G.E., Melbourne, Australia). 90°C for 4 min; 90 —» 250°C at 8°C/min. 90°C for 2 min; 90 260°C at 9°C/min. 90°C for 2 min; 90 -h> 290°C at 8°C/min. REFERENCES

1. Bryn, K. and Jantzen, E., Analysis of lipopolysaccharides by methanolysis, trifluoroacetylation, and gas chromatography on a fused-silica capillary column, J. C hrom atogr., 240, 405, 1982.

38

CRC Handbook of Chromatography 2. Brondz,

I. and Olsen, I., Differentiation between Actinobacillus actinomycetem comitans and H ae­ mophilus aphrophilus based on carbohydrates in lipopolysaccharide, J. C hrom atogr., 310, 261, 1984.

3. Brondz,

I.

and Olsen,

I.,

Whole-cell methanolysis as a rapid method for differentiation betweeen

A ctinobacillus actinomycetem comitans and Haemophilus aphrophilus ,, J. Chrom atogr ., 311, 347,

1984. 4. Laegreid, A., Otnaess, A.-B. K., and Bryn, K., Purification of human milk gangliosides by silica

gel chromatography and analysis of trifluoroacetate derivatives by gas chromatography, 7. C hro­ matogr., 377, 59, 1986.

TABLE GC 14 Trifluoroacetylated Mono- and Oligosaccharides Packing Temperature Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 N2; 20

P2 T2 He; 0.6a

183 0.2 Packed Glass FI 1

3000 0.026 Bonded phase Fused silica FI 2

Parent sugar

rb

r*

D-Glucose D-Fructose Sucrose Maltose 1-Kestose Melezitose Fructotriose Raffinose Fructotetraose Stachyose Fructopentaose Fructohexaose Glc(31-3Glc(31-3Glc GlcP 1-3Glc(31-4Glc Glc(31-4Glc(31-3Glc Glc(31-4Glc(31-4Glc Glca 1-4Glca 1-4Glc Glcal-4Glcal-6Glc Glca 1-6Glca 1-3Glc Glca 1-6Glca 1-4Glc Glca 1-6Glca 1-6Glc Xyl(31-4Xyip 1-4Xyl

0.79; 1.00 0.79 2.02 2.07; 2.12 — —

2.28 2.31 2.44 2.47 2.54 2.67 — — — — — — — — — —

_ — — —

1.04 1.13 —

1.00 — — — —

1.48; 1.49 1.35;d 1.42 1.54;d 1.57 1.42;d 1.49 1.32;d 1.34 1.37;d 1.64 1.29;d 1.39 1.18;d 1.21 1.06;d 1.31 1.99;d 2.02

Note: This table supplements Table GC 12 in CRC Handbook o f Chrom atography: Carbohydrates , Volume I. Data

for GLC of trifluoroacetylated sugars on chiral phases will be found in Table GC 24. a b c d

Split ratio 1:100. f relative to trifluoroacetylated (J-D-glucose (7.2 min). ^ relative to trifluoroacetylated raffinose (7.63 min). Major peak.

Carbohydrates

TABLE GC 14 (continued) Trifluoroacetylated Mono- and Oligosaccharides Packing

Temperature

PI = 3 % Dexsil 410 on Chromosorb® W HP (80— 100 mesh). P2 = DB-5 (J and W Scientific), 0.1-p.m film. T1 = 100°C for 1.5 min; 100—» 310°C at 3.5°C/min for 3.5 min, 6°C/min for 5 min, 15°C/min for 5 min, 25°C/min for 3.7 min; held at 310°C for 6.3 min. T2 = 180°C for 5 min; 180 200°C at 5°C/min; held at 200°C. REFERENCES

1. Englmaier, P., Trifluoroacetylation of carbohydrates for g.l.c. using yV-methylbis(trifluoroacetamide), Carbohydr. R es., 144, 177, 1985. 2. Selosse, E. J.-M . and Reilly, P. J., Capillary column gas chromatography of triifluoroacetyl trisaccharides, J. Chrom atogr ., 328, 253, 1985.

39

4-

TABLE GC 15 Trifluoroacetylated 0-Alkyloximes of Tri- to Hexoses: Capillary GLC OV-225 T1 He; 1.5a

OV-225 T1 He; 1.5a

OV-225 T1 He; 1.5a

OV-225 T2 N2; 1.5“

OV-225 T2 N2; 1.5a

OV-225 T3 He; 1.5a

OV-225 T4 N2; 2a

OV-225 T4 He; 1.5a

50 n.a. WCOT Glass MS 1,2

50 n.a. WCOT Glass MS 1,2

50 n.a. WCOT Glass MS 1,2

50 n.a. WCOT Glass FI 3,4

50 n.a. WCOT Glass FI 3,4

50 n.a. WCOT Glass MS 5

50 n.a. WCOT Glass FI 6

50 n.a. WCOT Glass MS 6

DL-Glyceraldehyde Dihydroxyacetone D-Erythrose 2-C-(hydroxymethyl)-DLglyceraldehyde D-Threose D-glycero-T etrulose D-Ribose D-Arabinose 3-C-(hydroxymethyl)-DLglycero- Tetrose D-Lyxose D-Xylose DL-r/îra?-2-Pentulose DL-^ryrAjro-2-Pentulose erythro-3-Pentu\ose

D-rAr^ö-3-Pentulose D-Allose D-Altrose D-Mannose D-Gulose D-Talose

MPOb r*

MOb r'

Parent compound — — — —

BuOb r*

—

—

—

—

— —

— 0.70; 0.77f 0.72; 0.84f — 0.80; 0.85f 0.82; 0.89f

—

BuO

BuO

BuO

BuO

r*

r*

r*

r*

r*

— —

— — 0.77; 0.80* —

— — 0.74; 0.81f —

— — — —

0.56; 0.64 0.64 0.73; 0.79f 0.73

0.56; 0.63 — 0.73; 0.80f —

— — 0.70; 0.81f 0.71; 0.88f —

— — 0.70; 0.79f 0.71; 0.86f —

0.78; 0.83; 0.86; 0.88; —

0.75; 0.80; 0.84; 0.85; —

— — — — —

0.74; 0.78; 0.82; 0.83; 0.88;

0.75; 0.79; 0.83; 0.84; 0.89;

0.78; 0.89f 0.80; 0.90f

0.77; 0.87f 0.79; 0.89f

0.93; 0.97f 0.95; 1.00f 0.96;f 1.03 0.97; 0.98f 1.01 1.03 — — — — —

— — 0.87;f 1.01 0.90; 0.93f 0.96 0.98 — — — — —

0.89; 0.98f 0.91; 1.00f 0.91;f 1.01 0.94; 0.95f — — — — — — —

—

—

—

—

—

—

— — 0.81; 0.85; 0.88; 0.91; 0.90;

— — 0.79; 0.83; 0.88; 0.89; 0.90;

— — 0.77; 0.81; 0.85; 0.86; 0.86;

0.90* 0.94f 0.96f 1.00f 0.97f

MO

0.92f 0.96f 0.98f

0.99f 1.01f

0.89f 0.94f 0.95f 1.00f 0.98f

0.85f 0.86f 0.92f 0.96f

0.85f 0.85f 0.91f 0.97f

0.90; 0.98f 0.91; 1.00f 0.92;f 1.01 0.94; 0.96f 0.98 0.99 — — — — —

0.84f 0.84f 0.90f 0.96f 0.92

0.85f 0.84f 0.91f 0.98f 0.93

0.90; 0.99f 0.91; 1.00f 0.92;f 1.01 0.94; 0.97f — — 0.91; 1.02f 0.94; 1.05f 0.97; 1.08f 0.98; 1.1 lf 0.99; 1.09f

CRC Handbook of Chromatography

Phase Temperature Gas; flow rate (ml/min) Column Length, m Diameter (I.D.), mm Form Material Detector Reference

o

D -a r a b in o -3 -H e x u \o s e

DL-ri7?o-3-Hexulose DL-Tagatose DL-xy/o-3-Hexulose D-/y;c0-3-Hexulose eryifcro-2,5-Hexodiulose D L -th reo -2 ,5 -Hexodiulose

a b c d e f

0.89; 1.07f 0.90; 1.00f 0.93; 1.06f

0.88; 1.06f 0.89; 1.00f 0.91; 1.06f

— — —

—

—

—

—

—

—

—

—

—

—

—

—

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— —

— 1.01 ;f 1.09 1.01; 1.04f 1.06;f 1.12 1.09;f 1.16 1.11; 1.12 1.13; 1.16f 1.13;f 1.18 1.15;f 1.17 1.21; 1.26;f 1.35 1.24; 1.30;f 1.39

1.03; 1.05f 1.07;f 1.13 1.09;f 1.17 1.12; 1.14 1.14; 1.17 1.14;f 1.20 1.16;f 1.18 —

1.00; 1.17f 1.00; l . l l f 1.03; 1.17f 1.02;f 1.09 1.02; 1.05f 1.06;f 1.12 — 1.11; 1.13 1.13; 1.16 1.13;f 1.17 1.15;f 1.17 —

—

—

—

—

— — 1.00;f 1.13 1.02; 1.07f 1.09;f 1.13 1.14;f 1.27 1.17; 1.21 1.20; 1.26f 1.21 ;f 1.30 1.24;f 1.28 1.36; 1.44;f 1.58 1.41; 1.51 ;f 1.64

— — ©

0.92; 1.06f 0.93; 1.00f 0.95; 1.06f

o

D-Galactose D-Glucose D-Idose D-Fructose DL-Psicose DL-Sorbose

Split ratios 1:10 (References 1, 2, 5, 6, MS detection), 1:15 (References 3, 4), 1:12 (Reference 6, FI detection). MO = 0-methyloxime, MPO = 0-2-methyl-2-propyloxime, BuO = 0-n-butyloxime. t,. relative to major peak for trifluoroacetylated glucose alkoxime (15.62 min for MO, 15.78 min for MPO,19.55 min forBuO).2 tj relative to major peak for trifluoroacetylated xylose alkoxime (26.41 min for MO, 31.67minfor BuO,3 23.05 and20.82 min for FI and MS detection6). t,. relative to major peak for trifluoroacetylated O-butyloxime of fructose (12.67 min). Major peak.

Temperature

T1 T2 T3 T4

= = = =

120°C for 2 min; 120 —> 180°C at 5°C/min; held at 180°C for 10 min. 70°C for 2 min; 70 -> 180°C at 5°C/min; held at 180°C for 15 min 150°C for 2 min; 150 -► 180°C at 5°C/min; held at 180°C for 20 min. 100°C for 2 min; 100 —» 180°C at 5°C/min; held at 180°C for 15 min. REFERENCES

Carbohydrates

1. Schweer, H ., Gas chromatography-mass spectrometry of aldoses as O-methoxime, 0-2-methyl-2-propoxime, and O-n-butoxime pertrifluoroacetyl derivatives on OV-225 with methylpropane as ionization agent. I. Pentoses, J. C hrom atogr. , 236, 355, 1982. 2. Schweer, H ., Gas chromatography-mass spectrometry of aldoses as O-methoxime, 0-2-methyl-2-propoxime, and O-n-butoxime pertrifluoroacetyl derivatives on OV-225 with methylpropane as ionization agent. II. Hexoses, J. Chrom atogr., 236, 361, 1982. 3. Decker, P. and Schweer, H ., Gas-liquid chromatography on OV-225 of tetroses and aldopentoses as O-methoxime and O-n-butoxime pertrifluoroacetyl derivatives and of C3-C6 alditol pertrifluoroacetates., J. Chrom atogr., 236, 369, 1982. 4. Decker, P. and Schweer, H ., G.l.c. of the oxidation products of pentitols and hexitols as trifluoroacetylated O-methyloxime and O-butyloxime derivatives, Carbohydr. R es., 107, 1, 1982. 5. Schweer, H ., G.l.c.-m.s. of the oxidation products of pentitols and hexitols as O-butyloxime trifluoroacetates, Carbohydr. R es., I l l , 1, 1982. 6. Decker, P ., Schweer, H ., and Pohlm an, R ., Bioids. X. Identification of formose sugars, presumable prebiotic metabolites, using capillary gas chromatographymass spectrometry of w-butoxime trifluoroacetates on OV-225, J. C hrom atogr., 244, 281, 1982.

41

42

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE GC 16 Trimethylsilylated Alditols, Aminodeoxyalditols, and Cyclitols Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI 190 n.a.a

PI T1 n.a.

P2 T2 N2; 15a

P3 T3 N2; 18

P4 T4 N2; 25

P5 T4 N2; 25

2500 0.023 Capillary Fused silica FI 1

3000 0.025 Capillary Glass MS 2,3

2500 0.032 WCOT Fused silica FI 4

183 0.18 Packed Glass FI 5

180 0.2 Packed SS FI 6

180 0.2 Packed SS FI 6

Parent compound

rb

rb

rb

—

—

—

—

—

— — —

— —

— —

— —

— — — — —

— — —

— — — — — —

— — — — —

— — — — — —

0.97 0.52 — 1.00 — 1.02 — 0.55 — —

0.32 0.54 0.34 0.56 0.35 0.77 0.60 — 0.78 0.61 — 0.80 0.66 0.82 0.98 0.83 0.84 1.00 — — — 0.85 — —

— — —

Glycerol Threitol 4-deoxy Erythritol 4-deoxy Xylitol 5-deoxy l,4/2,5-anhydrod Arabinitol 5-deoxy 1,4-anhydrod Ribitol 2-deoxy Allitol, 6-deoxy Mannitol 6-deoxy Gulitol, 6-deoxy Glucitol 1,4-anhydrod Galactitol 1,4/3,6-anhydrod 6-deoxy 1,4-anhydro-6-deoxyd 2,5 -anhydro-6-deoxyd 2-Amino-2-deoxyglucitol 2-Amino-2-deoxygalactitol 2-Acetamido-2-deoxyglucitol 2-Acetamido-2-deoxymannitol 2-Acetamido-2-deoxygalactitol 1,4-anhydrod 3,6-anhydrod l,4:3,6-dianhydrod mwco-Inositol O-methyl c/izro-Inositol Pinitol Quebrachitol scy//i>-Inositol D-methyl myo-Inositol Ononitol Sequoyitol

— —

0.23 — 0.42 — — 0.44 — —

0.45 — —

— 0.59 — 0.32 0.61 —

0.29 —

— —

— — — — — — — — — —

— — —

0.99 — — 1.00 0.64 1.01 0.64 0.75 0.36 0.40 0.96 0.95 1.22

—

—

— — — — — — — — — — — — — —

—

—

— — — — — — — — — — —

—

— — —

— — — — — — — — — —

1.08c

— — —

— — — —

—

1.13*

—

—

—

1.23

1.13*

—

—

— — — — —

0.95 0.89 0.50

— — — — — — — — — —

— — —

— —

0.76 0.59 0.82 0.61 0.67 0.92 0.82 1.00 0.78 0.79

0.45 0.46 0.53 0.45 0.53 0.87 0.99 1.00 0.83 0.84

1.01 — —

1.05 —

1.12 — —

— — — — — — — — — —

— —

1.00 — —

43

C a rb o h y d ra te s

TABLE GC 16 (continued) Trimethylsilylated Alditols, Aminodeoxyalditols, and Cyclitols Parent compound

rb

rb

rb

Ve

Bomesitol Dambonitol

— —

— —

_

_

—

—

Ve

0.86 0.74

1.07 1.21

Note: This table supplements Table GC 21 in CRC Handbook o f Chromatography: Carbohydrates, Volume I.

3

Split ratios L150,1 1:10.4

b

t, relative to trimethylsilylated D-glucitol (20.93 min,2 40.8 min3).

c d e

^ relative to trimethylsilylated myo-inositol (13.5 min,5 25.5 min on P46). Formed during methanolysis of oligosaccharide-alditols. For simultaneous analysis of acetamidodeoxyhexoses as trimethylsilylated O-methyloximes see Table GC 22, Reference 3.

Packing

Temperature

PI P2 P3 P4 P5 T1 T2 T3 T4

= = = = = = = = =

OV-101. CP-Sil 5. 3% SP-2250 on Supelcoport® (100 — 120 mesh). 3% SE-30 on Gas-Chrom® Q (80 — 100 mesh). 10% Carbowax 20M on Gas-Chrom Q (80 — 100 mesh). 120 —> 260°C at 3°C/min. 130—» 220°C at 2°C/min. 120 220°C at 5°C/min. 130 -> 190°C at 2°C/min. REFERENCES

1. Bradbury, A. G. W., Halliday, D. J., and Medcalf, D. G., Separation of monosaccharides as trime­ thylsilylated alditols on fused-silica capillary columns, J. Chrom atogr., 213, 146, 1981. 2. Niwa, T., Yamamoto, N., Maeda, K., Yamada, K., Ohki, T., and Mori, M., Gas chromatographicmass spectrometric analysis of polyols in urine and serum of uremic patients. Identification of new deoxyalditols and inositol isomers, J. Chrom atogr., 277, 25, 1983. 3. Niwa, T., Yamada, K., Ohki, T., Saito, A., and Mori, M., Identification of 6-deoxyallitol and 6deoxygulitol in human urine. Electron-impact mass spectra of eight isomers of 6-deoxyhexitol, J. Chro­ matogr., 336, 345, 1984. 4. Gerwig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G., Anhydroalditols in the sugar analysis of methanolysates of alditols and oligosaccharide-alditols, Carbohydr. R es., 129, 149, 1984. 5. Mawhinney, T. P., Simultaneous determination of N-acetylglucosamine, N-acetylgalactosamine, N - acetylglucosaminitol and W-acetylgalactosaminitol by gas-liquid chromatography, J. Chrom atogr., 351, 167, 1986. 6. Ford, C. W. Identification of inositols and their mono-O-methyl ethers by gas-liquid chromatography, J. Chrom atogr., 333, 167, 1985.

TABLE GC 17 Trimethylsilylated Alditols from Partially Methylated Sugars Packing Temperature Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI T1 He; n.a.

PI T2 He; n.a.

PI T3 He; 40

300 0.25 Packed3 Glass FI 1

240 0.25 Packed3 Glass FI 1

300 0.25 Packed3 Glass FI 2

44

CRC Handbook o f Chromatography

TABLE GC 17 (continued) Trimethylsilylated Alditols from Partially Methylated Sugars rb

Parent sugar D-Mannose 2-O-methyl 6-O-methyl 2,4-di-O-methyl 2,6-di-O-methyl 3,4-di-O-methyl 3,6-di-O-methyl 4,6-di-O-methyl 2,3,4-tri-O-methyl 2,3,6-tri-O-methyl 2,4,6-tri-O-methyl 3,4,6-tri-O-methyl 2,3,4,6-tetra-O-methyl D-Galactose 2,3,4-tri-O-methyl 2,3,6-tri-O-methyl 2,4,6-tri-O-methyl 3,4,6-tri-O-methyl 2,3,4,6-tetra-O-methy 1 2-Acetamido-2-deoxy-D-glucose 3-O-methyl 3,4-di-O-methyl 3,6-di-O-methyl 3,4,6-tri-O-methyl

2.47 2.23 1.92 2.06 1.86 1.68 1.74 1.64 1.21 1.17 1.39 1.00

r*

—

— —

— — — —

— — — — —

rb

2.44 2.16 1.89 2.00 1.80 1.65 1.68 1.60 1.22 1.18 1.40 1.00

1.48 1.27 1.24 1.32 1.04

— — — —

1.49 1.30 1.21 1.33 1.06

— — — —

1.22 1.12 1.06 1.00

3.40 3.25 3.17 3.10

—

Note: This table supplements Table GC 22 in CRC H andbook o f C hrom atog­ raphy: C arbohydrates , Volume I.

a

Surface of Chromosorb® support was modified by treatment with polyeth­ ylene glycol (mol. wt. 20,000) at 280°C overnight, followed by extraction with methanol, leaving thin film of nonextractable polymer as surface coat­ ing. b f relative to l,5-di-0-trimethylsilyl-2,3,4,6-tetra-0-methyl-D-mannitol (9.2 min). c t, relative to 2-deoxy-2-V-methylacetamido-1,5-di-0-trimethylsilyl-3,4,6-triO-methylglucitol (13.7 min).

Packing Temperature

PI = 0.4% OV-225 on surface-modified Chromosorb® W AW (80 — 100 mesh). T1 = 130°C for 2 min; 130 —> 150°C at l°C/min. T2 = 130°C for 2 min; 130 -► 205°C at 4°C/min. T3 = 130°C for 2 min; 130 200°C at l°C/min. REFERENCES

1. Akhrem, A. A., Avvakumov, G. V., and Strel’chyonek, O. A., Methylation analysis in glycoprotein chemistry: low-bleeding columns for the gas chromatographic analysis of methylated sugar derivatives, J. Chrom atogr . , 176, 207, 1979. 2. Akhrem, A. A., Avvakumov, G. V., Sidorova, I. V., and Strel’chyonok, O. A., Methylation analysis in glycoprotein chemistry. General procedure for quantification of the products of solvolysis of permethylated glycopeptides and glycoproteins, J. C hrom atogr., 180, 69, 1979.

Carbohydrates

TABLE GC 18 Trimethylsilylated Aldonic and Aldaric Acids: Capillary GLC of Products of Alkaline Degradation of Carbohydrates Phase Temperature Gas; flow rate (ml/min) Column Length, m Diameter (I.D.), mm Form Material Detector Reference

PI T1 H2 2a

P2 T2 H2; 2a

25 0.32 Coated Fused silica 1-4

25 0.32 Coated Fused silica FI 5

P arent compound

rb

rb

Glyceric acid 2-C-methyl 2-Deoxytetronic acid 3-Deoxytetronic acid Erythronic acid Threonic acid 3,4-Dideoxypentonic acid 3-Deoxy-^ryi/iro-pentonic acid 3-Deoxy-r/2reo-pentonic acid í/íreo-2-Pentulosonic acid Xylonic acid Ribonic acid Lyxonic acid Arabinonic acid 3,6-Dideoxy-rifco-hexonic acid 3,6-Dideoxy-ara¿?mo-hexonic acid 3,4-Dideoxyhexonic acid Mannonic acid Gluconic acid L-r/ir^o-2-Hexenono-1,4-lactonec Erythraric acid Threaric acid 2,3-Dideoxypentaric acid 2,4-Dideoxypentaric acid 2-Deoxy-eryr/zro-pentaric acid 2-Deoxy-r/2ra?-pentaric acid 3-Deoxy-eo>f/iro-pentaric acid 3-Deoxy-r/tra?-pentaric acid Arabinaric acid Xylaric acid 3,4-Dideoxy-tfireo-hexaric acid 3,4-Dideoxy-eryi/iro-hexaric acid 3-Deoxy-jty/o-hexaric acid 3-Deoxy-/yjco-hexaric acid Altraric acid Galactaric acid

0.73 0.72 0.87 0.84 1.00

0.72 0.71

fi

—

0.95 1.12 1.14 — —

1.26 —

1.28 1.15 1.16 1.25 1.58 1.64 —

1.10 1.11 1.02 1.03 1.15 1.16 1.18 1.20 1.32 1.34 1.28 1.29 1.42 1.44 1.70 1.73

—

0.83 1.00 1.02 — —

1.09 1.22 —

1.24 — — — — — —

1.41 1.05 1.07 — — —

— — — — — — — — — — —

Note: This table supplements Table GC 24 in CRC Handbook o f Chromatog­ raphy: Carbohydrates, Volume I. a b c

Split ratio 1:20. ^ relative to trimethylsilated erythronic acid (6.25 m in,1^ 6.33 min5). L-Ascorbic acid.

45

46

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE GC 18 (continued) Trimethylsilylated Aldonic and Aldaric Acids: Capillary GLC of Products of Alkaline Degradation of Carbohydrates Packing Temperature

PI = OV-101 P2 = SE-54. T1 = 100°C for 2 min, 100 —» 200°C at 20°C/min; held at 200°C for 5 min. T2 = 110°C for 2 min; 100 230°C at 20°C/min; held at 230°C for 5 min. REFERENCES

1. Hyppänen, T., Sjöström, E., and Vuorinen, T., Gas-liquid chromato­ graphic determination of hydroxy carboxylic acids on a fused-silica capillary column, J. C hrom atogr., 261, 320, 1983. 2. Alen, R., Niemelä, K., and Sjöström, E., Gas-liquid chromatographic separation of hydroxyl monocarboxylic acids and dicarboxylic acids on a fused-silica capillary column, J. Chrom atogr., 301, 273, 1984. 3. Niemelä, K. and Sjöström, E., Non-oxidative and oxidative alkaline deg­ radation of pectic acid, Carbohydr. R es., 144, 87, 1985. 4. Niemelä, K. and Sjöström, E., Non-oxidative and oxidative degradation o f D-galacturonic acid with alkali, Carbohydr R es., 144, 93, 1985. 5. Niemelä, K., Oxidative and non-oxidative alkali-catalysed degradation of L-ascorbic acid, J. C hrom atogr., 399, 235, 1987.

TABLE GC 19 Trimethylsilylated Diethyl Dithioacetals: GLC of Neutral, Acidic, and Amino Sugars, 0-Methyl Ethers, and PeriodateOxidation Products Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

PI 225 N2 l-2a

P2 190 N2; 50

P3 T1 N2; 50

5000 0.028 SCOT Glass FI 1-5

100 0.3 Packed Glass FI 4

200 0.3 Packed Glass FI 5

Parent compound

rb

rb

r*

Glycolaldehyded D-Glyceraldehyded Glyoxald L-Lactaldehyded 2-Hydroxymalonaldehyded D-Threosed D-Erythrose D-Ribose D-Xylose 2,3-di-O-methyl 3,4-di-O-methyl 2,3,4-tri-O-methyl L-Arabinose L-Rhamnose 2,3,4-tri-G-methyl

—

_

0.24

— —

0.65 1.00 1.24 0.69 1.48

— — — 0.37

0.38 0.67 0.67 0.49 0.56 0.43 0.69 0.85 0.47

—

— —

— —

— — — — — —

—

— — — — — — — — — —

C a rb o h y d ra te s

TABLE GC 19 (continued) Trimethylsilylated Diethyl Dithioacetals: GLC of Neutral, Acidic, and Amino Sugars, 0-Methyl Ethers, and PeriodateOxidation Products Parent compound

rb

rb

r*

L-Fucose 2,3,4-tri-O-methy 1 D-Glucose 3- 250°C at 4°C/min. 140°C for 2 min; 140 ^ 260°C at 8°C/min. 150 -► 220°C at 2°C/min. REFERENCES

1. Aluyi, H. A. S. and Drucker, D. B., Fingerprinting of carbohydrates of Streptococcus mutans by combined gas-liquid chromatography-mass spectrometry, J. Chrom atogr., 178, 209, 1979. 2. Mononen, I., Quantitative analysis, by gas-liquid chromatography and mass fragmentography, of mono­ saccharides after methanolysis and deamination, Carbohydr. R es., 88, 39, 1981. 3. Chaplin, M. F., A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography, Anal. B iochem ., 123, 336, 1982. 4. Ha, Y. W. and Thomas, R. L., Simultaneous determination of neutral sugars and uronic acids in hydro­ colloids, J. F o o d S c i., 53, 574, 1988.

50

TABLE GC 21 Trimethylsilylated Mono- and Oligosaccharides PI T1 N2; 25

P2 255 Ar; 55

P3 220 Ar; 55

P4 T2 H2; na

P5 240 He; 0.8a

P5 180 N2; n.a.

P6 170 N2; n.a.

P7 T3 N2; 0.4

183 0.4

270 0.6

150 0.6

1000 0.032

3000 0.026

4000 0.018

2500 0.018

1000 0.022

Packed Glass FI 1

Packed Glass FI 2

Packed Glass FI 2

Capillary Glass FI 3

Capillary Fused silica FI 4

Capillary Glass FI 5,6

Capillary Glass FI 6

WCOT Fused silica FI 7

Parent sugar

rb

i*

r*

r*

r*

r*

r*

rb

2-Deoxy-D-erythropentose D-Lyxose

0.42; 0.48f

—

—

_

—

—

_

__

—

—

—

—

—

—

—

D-Arabinose

0.63; 0.72'

—

—

0.35; 0.37f

—

—

D-Ribose

0.66;' 0.68

—

—

—

—

0 34(ß); 0.37(a)f —

D-Xylose

0.71; 0.77'

—

—

0.42; 0.48f

—

L-Rhamnose

0.64;' 0.69

—

—

—

—

0.90;f g 0.9l;j 0.92;‘ 0.93h 0.90;* 0.91 ;fg 0.92;f h 0.94> 0.91;' 0.92;j 0.94;g 0.95f h 0.91;' 0.92;j 0.99;fg 1.00fh —

—

L-Fucose

—

—

—

—

—

—

D-Altrose

0.65; 0.68; 0.71' —

—

—

—

—

—

D-Mannose

0.83;'0.92

0.85;f,h 0.86;g 0.88;' 0.93j 0.87;f g 0.94;' 0.95;h 0.98;j 0.88;8 0.89;' 0.90;f,h 0.93j

D-Allose

—

—

—

—

—

— —

0.45(a); 0.54(ß)f 0.35(a);f 0.44(ß) — — 0.64(a); 0.82(ß)

CRC Handbook of Chromatography

Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

0.88;f h 0.89;8 0.92;! 0.93' 0.88;fg 0.89;! 0.91;' 0.93h 0.90;! o.91;f g 0.92;' 0.96h 0.89!; 0.92g; 0.95'; 0.96f,h 0.90;' 0.91;! 0.93;fg 1.00fh

—

D-Gulose

—

—

—

—

—

—

D-Idose

—

—

—

—

—

—

D-Talose

—

—

—

—

—

—

D-Galactose

—

—

0.59; 0.63f

—

—

D-Glucose

0.88; 0.90; 0.95f 0.92; 1.00

—

—

0.61; 0.72f

—

—

D-Fructose

0.95

L-Sorbose

0.97

—

0.98;' 0.99;j 1.00;h 1.03;g 1.06;k 0.97;' 0.98;j 1.01;« 1.02;h 1.05k 0.98;' 0.99;j LOO11 1.02;g 1.03k 0.98;h 0.99^ 1.00;g 1.01;! 1.03;k —

—

—

— —

— —

— —

—

—

—

—

—

—

— — — —

— — — —

— — — —

D-Tagatose

D-Psicose

2-Acetamido-2deoxy-D-galactose Xylobiose Lactulose Lactose

Cellobiose

—

—

— 0.90 1.06; 1.39

0.96 0.96; 1.49

— 1.00; 1.05

—

1.00 1.16; 1.51

1.00 1.13; 1.30

1.00 1.02; 1.04f

—

1.16; 1.51

1.16; 1.67

—

— —

— —

— —

—

—

—

1.23 — 1.36

1.26 — 1.32

— — —

0.58(a); 0.93(ß)f 0.93; 0.96f 1.00(a); 1.44(ß) 1.00 1.12(a); 1.27(ß)f 1.14(a); 1.58(ß)f 1.19; 1.21f 1.24 1.24;f 1.34 1.33

0.73(a); 0.82(ß) 0.78(a); l.OO(ß)1

51

Maltulose Turanose Nigerose aa-Trehalose

—

—

Carbohydrates

Sucrose Maltose

1.04; 1.06f

—

52

TABLE GC 21 (continued) Trimethylsilylated Mono- and Oligosaccharides rb

r*

Koj ibiose

—

—

Palatinose Laminaribiose Sophorose

—

1.38

— 1.35; 1.43

r*

r*

r*

r**

rb

—

1.33(ß);f 1.64(a) 1.38;f 1.63 1.51; 1.67f 1.52(ß); 1.76(a)f 1.90(a); 2.05(ß)f 1.98(a); 2.35(ß)f 2.24((a); 2.25(ß)f — — — —

—

—

—

— — —

— —

— — —

—

—

—

—

—

—

—

—

—

Melibiose

—

2.01

2.00; 2.20

1.09; l . l l f

Isomaltose

—

—

—

—

Gentiobiose

—

2.21

2.38

—

Raffinose Maltotriose Maltotetraose Stachyose

—

—

—

—

—

—

—

—

—

—

—

—

1.31 1.32; 1.34f 1.53 1.59

—

—

—

—

—

—

—

—

—

—

—

Note: This table supplements Tables GC 16 and 17 in CRC H andbook o f Chromatography: C arbohydrates , Volume I.

Split ratio 1:100. ^ relative to trimethylsilylated (3-D-glucopyranose (18.8 min7). t,. relative to trimethylsilylated sucrose (17 min on P2,2 12.6 min on P3,2 20 min,3 26 min4). Calculated from ratio of retention index to that for trimethylsilylated (3-D-xylopyranose. Calculated from ratio of retention index to that for trimethylsilylated (3-D-glucopyranose. Major peak, a , pyranose. (3, pyranose. a , furanose. (3, furanose. Open chain.

—

— —

CRC Handbook of Chromatography

Parent sugar

Packing

Temperature

3% SE-30 on Chromosorb® W HP (100 — 120 mesh). 10% OV-17 on Gas-Chrom® Q (80 — 100 mesh). 3% OV-1 on Gas-Chrom® Q (80 — 100 mesh). OV-1 (0.1-to 0.12-^m film). SE-54. OV-225. CP-Sil 5 CB (Chrompack, Middelburg, The Netherlands), 0.12-p,m film. 80 —> 250°C at 2°C/min. on-column injection temperature 80°C; held for 1 min; 80 —> 140°C at 5°C/min; held for 1 min; 140 —» 190°C at 20°C/min; held for 1 min; 190 340°C at 8°C/min. T3 = 140 182°C at 2°C/min; 182 -► 290°C at 3.5°C/min.

PI P2 P3 P4 P5 P6 P7 T1 T2

= = = = = = = = =

REFERENCES 1. Aluyi, H. A. S. and Drucker, D. B . , Fingerprinting of carbohydrates of Streptococcus m utans by combined gas-liquid chromatography-mass spectrometry, J. C hrom atogr., 178, 209, 1979. 2. Laker, M. F., Estimation of disaccharides in plasma and urine by gas-liquid chromatography, J. C arbohydr ., 163, 9, 1979. 3. Traitler, H., Del Vedovo, S., and Schweizer, T. F., Gas chromatographic separation of sugars by on-column injection on glass capillary columns, J. High Resolut. Chromatogr. Chromatogr. C om m un., 7, 558, 1984. 4. Nikolov, Z. L. and Reilly, P. J., Isothermal capillary column gas chromatography of trimethylsilyl disaccharides, J. C hrom atogr. , 254, 157, 1983. 5. García-Raso, A., Martínez-Castro, I., Páez, M. I., Sanz, J., García-Raso, J., and Saura-Calixto, F., Gas chromatographic behaviour of carbohydrate trimethylsilyl ethers. I. Aldopentoses, J. C hrom atogr. , 398, 9, 1987. 6. Páez, M., Martínez-Castro, I. Sanz, J., Olano, A., García-Raso, A., and Saura-Calixto, F., Identification of the components of aldoses in a tautomeric equilibrium mixture as their trimethylsilyl ethers by capillary gas chromatography, C hr ornato graphia, 23, 43, 1987. 7. Ford, C. W. and Hartley, R. D., Identification of phenols, phenolic acid dimers, and monosaccharides by gas-liquid chromatography on a capillary column, J. C hrom atogr ., 436, 484, 1988.

Carbohydrates 53

in

TABLE GC 22 Trimethylsilylated Oxime and 0-Methyloxime Derivatives of Mono- and Oligosaccharides

Parent compound DL-Glyceraldehyde Dihydroxyacetone 2-C-(hydroxymethyl)DL-Glyceraldehyde D-glycero-T etrulose D-Erythrose D-Threose 2-Deoxy-D-erythropentose D-Lyxose D-Xylose D-Arabinose D-Ribose D-eo’i/iro-2-Pentulose D-r/ireo-2-Pentulose L-Rhamnose L-Fucose 2-Deoxy-D-arabinohexose

PI T1 He; 30

P2 T2 N2; 24

P3 T2 N2; 24

P3 T3 N2; 18

P3 T4 N2; 60

P4 T5 He; 2

P5 180 He; 1

P5 T6 He; 0.8a

P6 T6 He; 5

P7 165 Ar; 0.7

P8 175 Ar; 0.7

P8 T7 H2; 0.35a

183 0.32 Packed SS FI,NPDb 1

183 0.32 Packed Ni FI 2

183 0.32 Packed Ni FI 2

183 0.32 Packed Glass FI 3

50 0.3 Packed SS FI 4

3000 0.025 Capillary Glass MS 5

5000 0.02 Capillary Fused silica MS 5

6000 0.025 Capillary Glass FI 6

4000 0.05 Capillary Glass FI 6

3500 0.03 Capillary Glass FI 7

5000 0.03 Capillary Glass FI 7

2500 0.023 Capillary Fused silica FI 8

Oc rd

0/M O c O/MO r* r*

MO rf

O

MO r8

MO r8

O rh

O rh

MO r8

MO r8

O rd

0.76; 0.80' 0.75; 0.80* 0.78 0.78 1.01; 1.04* 1.01; 1.04*

—

—

—

— —

— —

—

—

—

— —

— —

—

i*

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

0.37

—

—

—

0.48; 0.50* 0.49; 0.51* 0.51 0.52 0.53 0.54 0.56 0.58;* 0.60 0.77; 0.78*

_ 0.47 0.45; 0.46* — — 0.46 — 0.48 — 0.48 — 0.48 0.55;* 0.59 — 0.57;* 0.60 — 0.69

_

_

_

_

_

—

—

—

—

—

—

—

0.68

—

—

0.48

—

—

—

—

—

—

—

—

—

—

—

0.86

—

—

—

0.75

—

—

—

0.81 0.82 0.84

"

"

"

0.52 0.67

—

"

1.02; 1.05* 1.02; 1.05* 1.05; 1.06* 1.05; 1.07* 1.05; 1.07* 1.05; 1.08' — —

"

_

—

—

_

_

—

—

—

—

—

—

0.34

0.37

—

_

_

_

—

—

—

— 0.43

0.46

—

—

—

—

—

—

—

—

—

—

—

—

—

0.52;* 0.53 0.55;* 0.57

—

—

—

0.65

0.68

“

— — —

CRC Handbook of Chromatography

Packing Temperature (°C) Gas; flow rate (ml/min) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

D-Allose D-Mannose D-Talose D-Galactose D-Glucose D-Fructose D-Tagatose L-Sorbose D-a/iro-Heptulose D-ma/wo-Heptulose D-g/wco-Heptulose D-Gluconic acid D-Glucuronic acid

— — — 0.99 1.00 — — — — — — — —

2-Acetamido-2-deoxy- — D-glucose 2-Acetamido-2-deoxy- — D-galactose 2-Acetamido-2-deoxy- — D-mannose N-Acety lneuraminic acid, methyl ester ethyl ester

ethyl ester

— — — — —

— — — — 0.78 0.74 — — — — — — 0.87

— — — — 1.00 0.90 — — — — — — —

—

—

1.20*

—

—

—

—

1.49 (MO); 1.57(0) 1.50 (MO); 1.58(0) 1.90 (MO); 2.06(0) 1.90 (MO); 2.04(0) — — — — —

1.36 — (MO = O)

0.95;* 1.02 0.97;* 1.02 0.98;' 1.04 0.99;* 1.06 1.00;* 1.08 0.93;* 0.97 0.84; 0.90* 0.90; 0.94 — 2.15; 2.19' 2.30 0.88 0.84; 0.94; 1.00; 1.11* 1.70; 1.75*

— — — — — — — — — — — — —

— — — — — — — — — — — — —

— — — 0.97;* 1.05 1.00;* 1.06 0.92;* 0.95 — — — — — — —

— — — 0.98;* 1.05 1.00;* 1.06 0.92;* 0.95 — — — — — — —

— — — — 1.00;* 1.06 0.91; 0.93 — — — — — — —

—

0.91;* 0.95 0.94;' 0.95 0.97;' 0.99 0.97;' 1.02 1.00;' 1.04 0.91;' 0.96 0.86; 0.94' 0.94; 0.96 1.17 1.32 1.34' 1.39 1.11 1.13; 1.20;' 1.23; 1.25 1.45; 1.48*

—

—

—

—

—

1.24*

—

1.55

1.89

—

—

—

—

—

1.25*

—

1.50;* 1.52

1.77;* 1.85 —

—

—

—

—

— — — — 1.70

— — — — —

— — — — —

— — — — —

— — — —' —

— — — — —

3.11; 3.14 3.18;* 3.26 3.19; 3.21 3.20;* 3.24 —

1.43(MO); — 1.45(0) 1.72(MO); — 1.74(0) 1.80(MO); — 1.88(0) — — — — —

— — — — —

— — — — —

55

Maltulose Nigerose Turanose Maltose Cellobiose

— — — — — — — — — — — — —

Carbohydrates

N-Glycolylneuraminic acid, methyl ester

— — — — — — — — — — — — —

in os

TABLE GC 22 (continued) Trimethylsilylated Oxime and O-Methyloxime Derivatives of Mono- and Oligosaccharides 0/M Oc O/MO r* r*

Kojibiose Palatinose Galactobiosek Gentiobiose

—

—

—

—

Melibiose

—

—

Isomaltose

—

Maltotriose

—

Cellotriose Galactotriosek Manninotriosek Verbascotetraosek

MO i**

O

MO r8

r*

MO r8

O rh

O rh

MO r8

MO r8

—

—

—

—

—

—

—

____

____

____

____

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.73 —

—

—

—

—

—

—

—

—

—

—

1.81

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

2.12 2.21 2.28 2.59

O r*

3.22;' 3.24 3.32; 3.36

_

3.10; 3.34;' 3.42 3.32; 3.44;' 3.54 3.24; 3.45;' 3.54 5.35; 6.00;' 6.17

—

—

—

—

—

—

—

—

—

____

—

____

____

_

—

—

—

—

—

____

____

—

____

____

—

____

_

_

Note: This table supplements Table GC 20 in CRC Handbook o f Chrom atography: Carbohydrates, Volume I.

a Split ratios 1:60,6 1:75.8 b Nitrogen-phosphorus selective detector. c O = oxime, MO = O-methyloxime. d t, relative to major peak for trimethylsilylated glucose oxime (17.5 min,1 6.7 min,4 9.6 min8). e t, relative to trimethylsilylated N-acetylneuraminic acid methyl ester (5.41 min on P2, 7.00 min on P3). f ^ relative to trimethylsilylated myo-inositol (13.5 min). g t, relative to major peak for trimethylsilylated O-methyloxime of glucose (29 min on P4, 23.7 min on P5,5, 37.6 min on P87). h t,. relative to trimethylsilylated erythritol (27.3 min on P5, 25.4 min on P6). * Major peak. j For simultaneous analysis of trimethylsilylated acetamidodeoxyalditols see Table GC 16, Reference 5. k Galactobiose and -triose are a-(I —> 6)-Iinked, manninotriose is Galal-6Galal-6Glc, verbascotetraose is Gala-6Galal-6GaIal-6Glc; related non-reducing oli­ gosaccharides of raffinose family (sucrose, raffinose, stachyose, and verbascose) can be analyzed simultaneously as trimethylsilyl ethers (t, = 1.61, 2.06, 2.49, 2.78, respectively.)

CRC Handbook of Chromatography

Oc Parent compound

Packing

Temperature

PI P2 P3 P4 P5 P6 P7 P8 T1 T2 T3 T4 T5 T6 T7

= = = = = = = = = = = = = = =

2% OV-17 on Chromosorb® W HP (800 — 100 mesh).

1.5% SE-52 on Chromosorb® W HP (100 — 120 mesh). 3% SP-2250 on Supelcoport® (100 — 120 mesh,2 3 80 — 100 mesh4).

SP-2250. SP-2100. OV-1. SE-30 containing Silanox 101. OV-101 120°C for 8 min; 120 —> 300°C at 8°C/min. 200°C for 2 min; 200 260°C at 6°C/min. 120 220°C at 5°C/min. 80 -> 380°C at 16°C/min. 75°C for 30 sec; 75 -> 150°C at 10°C/min; held at 150°C for 23 min; 150 -* 180°C at 20°C/min; held at 180°C. 80 —►275°C at 4°C/min. 180—» 280°C at 3°C/min; held at 280°C for 4 min; 280 -> 290°C at 2°C/min; held at 290°C. REFERENCES

Carbohydrates

1. Morita, H. and Montgomery, W. G., Gas chromatography of silylated oxime derivatives of peat monosaccharides, J. Chrom atogr., 155, 195, 1978. 2. Mawhinney, T. P., Madson, M. A., Rice, R. H., Feather, M. S., and Barbero, G. J., Gas-liquid chromatography and mass-spectral analysis of per-Otrimethylsilyl acyclic ketoxime derivatives of neuraminic acid, Carbohydr. R es., 104, 169, 1982. 3. Mawhinney, T. P., Simultaneous determination of N-acetylglucosamine, ^-acetylgalactosamine, N-acetylglucosaminitol and N-acetylgalactosaminitol by gasliquid chromatography, J. Chrom atogr ., 351, 91, 1986. 4. Molnar-Perl, I., Pinter-Szakacs, M., Kovago, A., and Petroczy, J., Gas-liquid chroomatographic determination of the raffinose family of oligosaccharides and their metabolites present in soy beans, J. Chrom atogr., 295, 433, 1984. 5. Pelletier, O. and Cadieux, S., Glass capillary or fused-silica gas chromatography-mass spectrometry of several monosaccharides and related sugars: improved resolution, J. C hrom atogr., 231, 225, 1982. 6. Willis, D. E., GC analysis of C2-C7 carbohydrates as the trimethylsilyl-oxime derivatives on packed and capillary columns, J. Chromatogr. S c i ., 21, 132, 1983. 7. Zegota, H., Separation and quantitative determination of fructose as the O-methyloxime by gas-liquid chromatography using glass capillary columns, J. C hro­ m atogr., 192, 446, 1980. 8. Mateo, R., Bosch, F., Pastor, A., and Jimenez, M., Capillary column gas chromatographic identification of sugars in honey as trimethylsilyl derivatives, J. Chrom atogr ., 410, 319, 1987.

62.5% acetonitrile in water in 30 min. acetonitrile-water (57:43). acetonitrile-water (1:1). acetonitrile-water (53:47). acetonitrile-water (29:21).

CRC Handbook of Chromatography

(24 min) 4.00 13.2 4.34 15.2 4.71 17.6 5.14 20.0 5.57 22.8 6.00 (40.5 min) 6.57 7.05 7.65 8.26 (32.5 min)

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

rb

Detection

56 57 58 59 S10 D1 D2

= = = =

acetonitrile-water (3:2). acetonitrile-water (11:9). acetonitrile-water (14:11). linear gradient, acetonitrile initially 65% in water, decreased at 0.5% per minute. = linear gradient, 66 —» 57% acetonitrile in water in 40 min. = automated tetrazolium blue colorimetricmethod. = scintillation counting of samples reduced with sodium borotritiide. REFERENCES

1. Noël, D., Hanai, T., and D’Ambroise, M., Systematic liquid chromatographic separation of poly-, oligo-, and monosaccharides, J. Liq. C hrom atogr ., 2, 1325, 1979. 2. Koizumi, K., Utamura, T., and Okada, Y., Analyses of homogeneous D-gluco-oligosaccharides and -polysaccharides (degree of polymerization up to about 35) by high-performance liquid chromatography and thin-layer chromatography, J. Chrom atogr ., 321, 145, 1985. 3. Koizumi, K., Utamura, T., Kubota, Y., and Hizukuri, S., Two high-performance liquid chromatographic columns for analyses of malto-oligosaccharides, J. Chrom atogr. 409, 396, 1987. 4. Mellis, S. J. and Baenziger, J. U., Separation of neutral oligosaccharides by high-performance liquid chromatography, Anal. B iochem ., 114, 276, 1981. 5. D’Amboise, M., Noël, D., and Hanai, T., Characterization of bonded-amine packing for liquid chromatography and high-sensitivity determination of carbohydrates, Carbohydr. R es., 79, 1, 1980.

TABLE LC 4 HPLC of Cyclic Oligosaccharides on Amine- and Amide-Bonded Silica Packings PI

P2

P2

P3

P4

P5

P5

P5

P6

P3

30 0.39 SS SI 2 25 RI 1

25 0.4 SS S2 1 ambient RI 2

25 0.4 SS S3 1 ambient RI 3

20 0.6 SS S3 1 ambient RI 3

25 0.46 SS S4 1 ambient RI 3

25 0.46 SS S3 1 ambient RI 3

25 0.46 SS S2 1 50 RI 3

25 0.46 SS S5 1 70 RI 3

16 0.46 SS S6 1 ambient RI 4

20 0.6 SS S7 1 ambient RI 5

Carbohydrates

Packing Column Length, cm Diameter (I.D.), cm Material Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

00

QC N>

TABLE LC 4 (continued) HPLC of Cyclic Oligosaccharides on Amine- and Amide-Bonded Silica Packings

Cyclomaltohexaose3 6-O-a-D-glucosyl6-0-a-maltosyl6-0-a-maltotriosyl6-O-a-maltotetraosyl6-O-a-maltopentaosyldi-O-a-D-glucosyldi-O-a-maltosylCyclomaltoheptaose3 6-O-a-D-glucosyl6-O-a-maltosyl6-0-a-maltotriosyl6-O-a-maltotetraosyldi-O-a-D-glucosyldi-O-a-maltosyl6A, 6D-di-0-a-maltotriosyl6-0-a-(6'-0-a-maltotriosyl)maltotriosyl6-0-a-(6"-0-a-maltotriosyl)maltotriosylCyclomaltooctaose3 6-O-a-D-glucosyl6-0-a-maltosyl6-O-a-maltotriosyl6-0-a-maltotetraosyldi-O-a-D-glucosyldi-O-ot-maltosylCyclosophoraosesb DP 17 18 19

4.16 — — — — — — — 5.75 — — — — — — — —

1.50 2.10 2.80 3.48 — — — — 2.00 2.52 3.20 4.28 — 3.40 — — —

1.59 2.27 2.88 3.58 4.39 5.34 3.09 4.60 2.02 2.86 3.64 4.56 5.57 3.94 6.06 9.53 9.34

1.03 1.41 1.71 2.05 2.42 2.84 1.82 2.60 1.45 1.91 2.33 2.79 3.28 2.39 3.42 4.63 4.60

1.52 2.14 2.27 3.26 3.94 4.68 2.86 4.28 1.89 2.64 3.29 4.03 4.84 3.51 5.24 7.88

7.77

2.22 2.97 3.59 4.34 5.18 6.21 3.75 5.31 2.61 3.50 4.22 5.07 6.04 4.43 6.20 8.94 9.00

1.97 2.72 3.42 4.27 5.22 6.43 3.58 5.39 2.39 3.30 4.12 5.09 6.24 4.34 6.52 9.88 10.09

2.23 3.16 4.08 5.21 6.56 8.25 4.23 6.76 2.81 3.97 5.06 6.44 8.07 5.27 8.33 13.30 13.73

1.19 — — — — — — — 1.42 — — — — — — — —

— — — — — — — — — — — — — — — — —

—

—

8.86

4.47

7.51

9.32

10.36

14.10

—

—

7.90 — — — — — —

2.28 3.20 4.28 5.40 — — —

2.57 3.62 4.60 5.74 6.99 5.01 7.69

1.69 2.23 2.71 3.25 3.81 2.83 4.05

2.38 3.30 4.14 5.07 6.06 4.40 6.63

3.20 3.67 5.10 6.09 7.22 5.44 7.55

3.04 4.18 5.17 6.36 7.78 5.50 8.14

3.60 5.07 6.46 8.15 10.21 6.87 10.68

1.69 — — — — — —

— — — — — — —

— — —

— — —

— — —

— — —

— — —

— — —

— — —

— — —

3.16 3.76 4.34

1.94 2.12 2.19

CRC Handbook of Chromatography

k'

Compound

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

_

—

_

____

—

____

____

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

4.98 5.72 6.57 7.30 8.11 8.90 10.0 11.1 12.2 13.4 14.6 15.8 17.6 20.1

—

—

— —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

— —

—

—

— —

— —

—

____

—

2.44 2.56 2.88 3.12 3.38 3.62 3.94 4.25 4.50 4.88 5.12 5.56 5.94 6.31 6.87 7.19 7.87 8.37 8.88 9.62 10.2

N ote: Further HPLC data for these oligosaccharides will be found in Table LC 13, ion chromatography in Table LC 47, and TLC data

a b

for cyclodextrins in TLC 6. Cyclomaltohexaose, -heptaose, and -octaose are generally known, respectively as a-, (3, and -y-cyclodextrin. Produced by Rhizobium and Aqrobacterium strains.

Packing

= = = = = =

|x-Bondapak® Carbohydrate Analysis Column (Waters; 10-p,m particles). Hibar LiChrosorb®-NH2 (Merck). ERC®-NH-1171 (Erma Optical Works, Tokyo; 3-p.m particles). YMC-Pack® PA-03 (Yamamura Chemical Company, Kyoto). TSK® gel Amide-80 (Toyo Soda, Tokyo). 5-p,m silica with bonded aminopropyl groups (0.6 mmol/g); supplied by Phase Separations (Queensferry, U.K.), packed in authors’ laboratory.

Carbohydrates

PI P2 P3 P4 P5 P6

83

TABLE LC 4 (continued) HPLC of Cyclic Oligosaccharides on Amine- and Amide-Bonded Silica Packings SI 52 53 54 55 56 57

= = = = = = =

acetonitrile-water acetonitrile-water acetonitrile-water acetonitrile-water acetonitrile-water acetonitrile-water acetonitrile-water

(7:3). (3:2). (29:21) (11:9). (31:19), (16:9). (57:43), REFERENCES

1. Zsadon, B., Otta, K. H., Tüdôs, F., and Szejtli, J., Separation of cyclodextrins by high-performance liquid chromatography, J. Chrom atogr., 172, 490, 1979. 2. Koizumi, K., Utamura, T., Kuroyanagi, T., Hizukuri, S., and Abe, J.-I., Analysis of branched cyclodextrins by high-perfor­ mance liquid and thin-layer chromatography, J. C hrom atogr., 360, 397, 1986. 3. Koizumi, K., Utamara, T., Kubota, Y., and Hizukuri, S., Two high-performance liquid chromatographic columns for analyses of malto-oligosaccharides, J. Chrom atogr., 409, 396, 1987. 4. Benincasa, M., Cartoni, G. P., Coccioli, F., Rizzo, R., and Zevenhuizen, L. P. T. M., High-performance liquid chromatography of cyclic (3 (1 —> 2)-D-glucans (cyclosophoraoses) produced by Rhizobium meliloti and Rhizobium trifolii, J. C hrom atogr., 393, 263, 1987. 5. Koizumi, K., Okada, Y., Utamura, T., Hisamatsu, M., and Amemura, A., Further studies on the separation of cyclic (1 —► 2)-(3-D-glucans (cyclosophoraoses) produced by Rhizobium meliloti IFO 13336, and determination of their degrees of polymerization by high-performance liquid chromatography, J. Chrom atogr., 299, 215, 1984. Data from Reference 3 reprinted with permission of authors and Elsevier Science Publishers.

CRC Handbook of Chromatography

Solvent

00

85

C a rb o h y d ra te s

TABLE LC 5 HPLC of Mono- and Oligosaccharides Derived from Glycoproteins, and of Milk Oligosaccharides, on Amino Phases Packing Column Length, cm Diameter (I.D.), cm Material Solvent Flow rate (ml/min) Detection Reference

PI

PI

P2

P3

P3

25 0.4 SS SI 2 UV, D1 1

25 0.4 SS S2 2 UV 1

25 0.46 SS S3 1 UV 2

30 0.4 SS S4 1.3 UV 2

30 0.4 SS S5 1 UV 3

Compound

r*

k 'b

k'

k 'b

k 'b

0.59 0.86 1.00 1.08 1.05 0.65 0.87 0.66 0.79

— — — — — — — — —

— — — — — — — 1.7

— — — — — — — — 2.0

— — — — — — — — —

1.53 1.60 2.41 2.14 2.01 2.27 2.92 2.55 2.31 3.40 — — 3.19 4.12 5.43 3.55 5.00 5.52 5.32 5.85 5.78 4.81 5.52 5.64 6.50 6.67 6.52 5.93 6.66 —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — 2.6 — — — — — 2.3 2.7 — — — — — — — — — — — — — — — — — 4.6 4.0 3.6 4.8 6.2

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — —

— — — — 2.3 — — — — — 2.1 2.4 — — — — — — — — — — — — — — — — — 3.7 3.1 2.9 4.0 4.4

Sugars and alditols L-Fucose D-Mannose D-Glucose D-Galactose Galactitol 2-Acetamido-2-deoxy-D-glucose 2-Acetamido-2-deoxy-D-glucitol 2-Acetamido-2-deoxy-D-galactose 2-Acetamido-2-deoxy-D-galactitol Oligosaccharides and oligosaccharide-alditols Gaipi-3GlcNac Gaipi-4GlcNAc Gal(31-6GlcNAc Gaipi-3GalNAc Gaip 1-3GalNAc-ol GlcNAc(31-2Man GlcNAc|31-6Man GlcNAc|31-3Gal GlcNAc(31-4Gal GlcNAc01-6Gal GlcNAcP1-3GalNAc-ol Fuca 1-2Gaip 1-3GalN Ac-ol Fuca 1-2Gaip 1-4GlcNAc Gala 1-3Gaip 1-4GlcNAc Gaip 1-6Gaip 1-6GlcNAc Gaip 1-4GlcNAc(J 1-4GlcN Ac Gaip 1-4GlcNAc|31-2Man Gaip 1-4GlcNAc(31-6Man Gaip 1-4GlcNAc(31-3Gal Gal(31-4GlcNAc(31-6Gal Mana 1-3(Mana 1-6)Man GlcNAc|31-2(GlcNAc(31-4)Man GlcNAcP 1-2(GlcNAcp 1-6)Man GlcNAc(31-3(GlcNAc(31-6)Gal Gaip 1-3GlcNAc(31-3Gaip 1-4Glc Galf3 1-3GlcN Ac0 1-3Gal(31-4Glc-ol Gal (31-4GlcNAc(31-3Gal(31-4Glc Gal pi -4GlcN Acp 1-3Gal(3 1-4GlcN Ac Gaip 1-4GlcN Ac(3 1-6(GlcN Ac(3 1-3)Gal Gal (31-4GlcNAc(3 1-6(Gal(3 1-3)GalNAc-ol Fuca 1-2Gal(3 1-3GlcNAc(3 1-3GalN Ac-ol Fuca 1-2Gal(3 1-3(GlcNAc|3 1-6)GalN Ac-ol Fuca 1-2Gal(31-3(Gal(3 1-4GlcNAc(3 1-6)GalNAc-ol Fuca 1-2Gal(3 1-4GlcN Ac(31-6(Gal|31-3)GalN Ac-ol

— — —

86

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE LC 5 (continued) HPLC of Mono- and Oligosaccharides Derived from Glycoproteins, and of Milk Oligosaccharides, on Amino Phases r“

Compound Galß 1-4GlcNAcß 1-2Mana 1-3Manß 1-4GlcNAc Galß 1-4GlcNAcß 1-2(Galß 1-4GlcNAcß 1-4)Man Galß 1-4GlcN Acß 1-2(Galß 1-4GlcNAcß 1-6)Man Galß 1-4GlcN Acß 1-3(Galß 1-4GlcNAcß 1-6)Gal Galß 1-3GlcNAcß 1-3(Galß 1-4GlcNAcß 1-6)Gal Galß 1-3GlcNAcß 1-3(Galß 1-4GlcNAcß 1-6)Galß 14Glc Galß 1-3GlcNAcß 1-3 [Gal ß 1-4(Fuca 1-3)GlcN Acß 16]Galßl-4Glc Galß 1-3(Fuca 1-4)GlcN Acß 1-3[Galß 1-4(Fuca 13)GlcNAcß 1-6]Galß 1-4Glc Fuca 1-2Galß 1-3(Fuca 1-4)GlcN Acß 1-3Galß 13GalNAc-ol Fuca 1-2Galß 1-3(Fuca 1-2Galß 1-4GlcNAcß 16)GalNAc-ol Fuca 1-2Galß 1-3 [Fuca 1-2Galß 1-4(Fuca 13)GlcNAcß 1-6]GalNAc-ol Galß 1-4GlcN Acß 1-2Mana 1-3(Gal ß 1-4GlcNAcß 12Manal-6)Man Biantennary octasaccharide Ie Triantennary decasaccharide IIe Triantennary decasaccharide IIIe Tetraantennary dodecasaccharide IVe

k'

k 'b

k 'b

k 'b

11.3

—

—

—

—

—

—

—

—

—

—

—

— — —

— — —

—

—

— — —

—

—

—

—

2.15

—

—

—

—

3.00

—

—

5.1

9.2

—

—

—

4.8

8.8

—

—

—

5.5

9.3

—

8.45

—

—

—

—

—

— 17.7 — 20.0 — 21.0 22.7 — (71 min)

—

—

—

7.02 7.28 7.40 7.40

— —

—

—

1.70

—

—

—

—

(18 min) (8 min)

Note: Data on reversed-phase HPLC of reduced oligosaccharides in Reference 2 will be found in Table LC 9.

a t, relative to D-glucose (8.6 min). b t, of last-eluting oligosaccharide in series is given in brackets at foot of column. c Structures of oligosaccharides I—IV appended. Packing

Solvent

PI P2 P3 SI

= = = =

52 =

Detection

53 54 55 D1

= = = =

LiChrosorb®-NH2 (Merck; 5-|xm particles). 605 NH(Alltech; 5-|im particles). MicroPak® AX-5 (Varian; 5-|xm particles). acetonitrile-15 m M aqueous phosphate buffer, pH 5.2; 4:1 for 30 min, then linear gra­ dient with buffer content increasing at 0.5%/min. as SI, but linear gradient with buffer content increasing at 0.3%/min throughout elu­ tion. acetonitrile-water (3:2). acetonitrile-1 m M aqueous phosphate buffer, pH 5.4 (3:2). acetonitrile-1 m M aqueous phosphate buffer, pH 5.4 (1:1). compounds without UV absorbance detected by phenol-H2S04 colorimetric assay or by scintillation counting after labeling with UDP-[14C]Gal. REFERENCES

1. Blanken, W. M., Bergh, M. L.-E., Koppen, P. L., and Van den Eijnden, D. H., High-pressure liquid chromatography of neutral oligosaccharides: effects of structural parameters, Anal. B iochem ., 145, 322, 1985. 2. Dua, V. K., Dube, V. E., and Bush, C. A., The combination of normal-phase and reverse-phase highpressure liquid chromatography with NMR for the isolation and characterization of oligosaccharide alditols from ovarian cyst mucins, Biochim. Biophys. A cta , 802, 29, 1984. 3. Dua, V. K., Goso, K., Dube, V. E., and Bush, C. A., Characterization of lacto-W-hexaose and two fucosylated derivatives from human milk by high-performance liquid chromatography and proton NMR spectroscopy, J. Chrom atogr ., 328, 259, 1985.

C arbohydrates

87

APPENDIX TO TABLE LC 5: STRUCTURES OF NUMBERED COMPOUNDS

I

II

Ill

IV

88

C R C H a n d b o o k o f C h ro m a to g ra p h y

TABLE LC 6 HPLC of Gangliosides on Amino Phases Packing Column Length, cm Diameter (I.D.), cm Material Solvent Flow rate (ml/min) Detection Reference

PI

P2

P2

25 0.4 SS SI 1 uv 1

20 0.6 SS S2 2 UV 2

20 0.6 SS S3 2 UV 2

Compound

RRT*

RRT"

RRTb

g M3

0.20 0.42 0.66 0.90 1.00 1.18 1.35 1.50 1.87 — — —

—

—

GM2 GM1 F u c - G m1

GDla GDib Fuc-GDlb GTib GQib Gqic Gpi GHi

—

—

0.55

—

—

—

1.00 1.22

— —

—

—

1.57 2.29 — — —

— —

1.00 1.56 2.34

Note: TLC data for gangliosides will be found in Table LC 7.

a b

RRT = L - to for compound, relative to L - to for GDla (38 min,1 13.4 min.2 RRT relative to ^ — to for GQlc (12.8 min).

Packing

Solvent

PI = LiChrosorb®-NH2 (Merck; 7-pm particles) P2 = SSC® NH2-2201N (5-|xm aminopropyl-silica gel; Senshu Science, Tokyo). 51 = (A) acetonitrile-5 mM phosphate buffer, pH 5.6 (83:17); (B) acetonitrile-20 mAf phos­ phate buffer, pH 5.6 (1:1); elution program: solvent A for 7 min; linear gradient A —> AB (33:17) in 53 min; linear gradient to A-B (9:16) in 20 min. 52 = (A) acetonitrile-5 mAf phosphate buffer, pH 5.5 (4:1); (B) acetonitrile-20 mAf phosphate buffer, pH 5.5 (1:1); elution program: solvent A for 3.5 min; linear gradient A —» A-B (9:16) in 26.5 min; isocratic for 10 min. 53 = (A) acetonitrile-30 mAf phosphate buffer, pH 5.5 (7:3); (B) acetonitrile-50 mAf phosphate buffer, pH 5.5 (3:7); elution program: solvent A for 5 min; linear gradient A —> A-B (2:3) in 30 min. REFERENCES

1. Gazzotti, G., Sonnino, S., and Ghidoni, R., Normal-phase highperformance liquid chromatographic separation of non-derivatized ganglioside mixtures, J. Chrom atogr ., 348, 371, 1985. 2. Ando, S., Waki, H., and Kon, K., High-performance liquid chro­ matography of underivatized gangliosides, J. Chrom atogr ., 408, 285, 1987.

C a rb o h y d ra te s

89

TABLE LC 7 HPLC of Methyl Glycosides on Reversed-Phase Packings Packing Column Length, cm Diameter (I.D.), cm Form Solvent Flow rate (ml/min) Detection Reference

PI

PI

PI

P2

P3

10 0.8 Cartridge h 2o 2 RI 1

10 0.8 Cartridge h 2o 2 RI 2

10 0.8 Cartridge h 2o 2.5 RI 3

10 0.46 Column h 2o 0.7 RI 4

25 0.46 Column SI 0.5 RI,D1 5

r*

k 'b

Sugar

Methyl glycoside

i-

r*

k 'b

D-Ribose

a-Furanoside (3-Furanoside a-Pyranoside p-Pyranoside a , (3-Furanoside a-Pyranoside P-Pyranoside a, (3-Furanoside a , p-Pyranoside a , P-Pyranoside a,p~Furanoside a-Pyranoside (3-Pyranoside a-Furanoside P-Furanoside a-Pyranosidel (3-Pyranoside J a , (3-Furanoside P-Furanoside a-Pyranoside a-Furanoside (3-Furanoside Not assigned Not assigned

1.23 1.47 2.03 2.47 1.17; 1.50 1.77 1.10; 1.50 0.83 1.03; 1.00 1.07 1.37 1.47 1.40 1.67;

1.25 1.42 1.93 2.32 1.05; 1.15 1.40 1.62 1.07; 1.21 1.39 0.78 0.96; 1.10 1.00 — 1.35 — 1.31 1.51

_

_

_

— — — — — — — — — — — — — — — —

— — — — 1.46 1.63 — — 0.87 1.20 1.00 1.06 — — 1.33 1.53

— — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — 7.6;d 11.7 0.7; 1.2

— — — — — 2.17; 2.97; 3.27 — — — — — — — — — —

— — — — — —

— — — — — — — — — —

— — — — 3.02; 3.32 1.70; 2.17; 3.15; 3.54 4.54 5.22 4.83; 6.25 1.06 1.16 2.77d 3.94 4.32; 4.60d — —

— —

— —

6.8; 7.9 3.0; 4.6

— —

— —

D-Xylose

L-Arabinose D-Galactose D-Glucose

D-Mannose

D-Fructose

L-Rhamnose L-Fucose

(3-Pyranosidec a-Pyranosidec a,p-Furanosidec D-Glucurono-6,3P-Furanoside a-Furanoside lactone a,p-Pyranoside D-Galacturonic acid a,p-Furanosidec a,p-Pyranosidec D-Mannuronic acidc Not assigned D-Mannurono-6,3-lac- Not assigned tone L-Guluronic acidc Not assigned L-Gulurono-6,3-lacNot assigned tone L-Iduronic acidc Not assigned D-Glucuronic acid

0.93 1.13 1.27 1.83 — —

1.25

1.23

1.17

1.77

(13 min) a b c d

t, relative to methyl a-D-glucopyranoside (3.0 min,1 3.2 min,2 4.2 min4). L of latest-eluting peak given in brackets at foot of column. Methyl ester. Minor peak.

3.5 4.5 — 1.0 — — — — — —

2.5;d 2.9 3.2; 4.8 (31.7 min)

90

C R C H an d b o o k o f C h ro m a to g ra p h y

TABLE LC 7 (continued) HPLC of Methyl Glycosides on Reversed-Phase Packings Packing

Solvent Detection

PI = Dextropak® (Waters); C18-bonded silica in plastic cartridge operated under radial compression (Waters module RCM 100). P2 = CP-Microspher® C18 (Chrompack, Middelburg, Netherlands); 3-p.m silica with C18 bonded phase. P3 = ODS-Hypersil® (Shandon, Runcorn, Cheshire, U.K.). SI = water-methanol (19:1). D1 = automated carbole colorimetric method. REFERENCES

1. Cheetham, N. W. H. and Sirimanne, P., High-performance liquid chromatographic separation of methyl glycosides, J. Chrom atogr., 208, 100, 1981. 2. Cheetham, N. W. H. and Sirimanne, P., Methanolysis studies of carbohydrates, using HPLC, Carbohydr. R es., 112, 1, 1983. 3. Annison, G., Cheetham, N. W. H., and Couperwhite, I., Determination of the uronic acid composition of alginates by high-performance liquid chromatography, J. Chrom atogr., 264, 137, 1983. 4. Hjerpe, A., Engfeldt, B., Tsegenidis, T., and Antonopoulos, C. A., Separation and determination of neutral monosaccharides using methanolysis and high-performance liquid chromatography, J. Chrom atogr., 259, 334, 1983. 5. Hjerpe, A., Antonopoulos, C. A., Classon, B., Engfeldt, B., and Nurminen, M., Uronic acid analysis by high-performance liquid chromatography after methanolysis of glycosaminoglycans, J. Chromatogr., 235, 221, 1982.

TABLE LC 8 HPLC of Partially Methylated Sugars and Polyols on Reversed-Phase Packings Packing Column Length, cm Diameter (I.D.), cm Form Solvent Flow rate (ml/min) Detection Reference

PI

P2

P2

P3

P3

P3

P3

P3

30 0.38 Column SI 2.5 RI 1

10 0.8 Cartridge S2 1 RI, D1 2

10 0.8 Cartridge S3 2 RI, D1 2

25 0.46 Column h 2o 1.8 RI 3

25 0.46 Column S4 1.8 RI 3

25 0.46 Column S5 1.8 RI 3

25 0.46 Column S6 1.8 D2 3

25 0.46 Column S7 1.8 D2 3

Compound

r-

RRTb

RRTb

r*

rd

rd

r-

r“

0.85; 1.18

—

—

—

—

—

—

—

0.85; 1.22(a) 0.81; 1.30(a)

—

—

—

—

—

—

—

—

—

—

—

1.04; 1.44(a) — — 0.81; 1.30(a)

0.22; 0.30 0.65; 1.02 0.65; 0.92 —

—

—

—

—

—

0.88; 1.00 0.88; 0.94 1.06; 0.94 2.00

— — —

— — —

— — —

— — —

— — —

0.55 0.55 0.55 0.85; — 1.30; 0.81; — 2.44;

— — — 0.40; 0.28; 2.32; 0.87; 1.16; —

— — — — — 1.50; 2.00 1.00 1.06; 1.25 4.31; 4.62

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

D-Xylose 2,3,4-tri-0-methyl D-Mannose 2,3,6-tri-0-methy 1 2,3,4,6-tetra-0-methyl D-Galactose 2,3-di-0-methyl

0.96(a) 1.52(a) 1.00(a) 2.67(a)

0.43 0.32 3.63 1.00 1.80

Carbohydrates

2,3,6-tri-0-methyl 2,4,6-tri-0-methy 1 2,3,4,6-tetra-0-methyl D-Glucose 2-0-methyl 3-0-methyl 6-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 2,3,4-tri-0-methyl 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 2,3,4,6-tetra-0-methyl

—

91

so

TABLE LC 8 (continued) HPLC of Partially Methylated Sugars and Polyols on Reversed-Phase Packings Compound

RRTb

RRTk

r*

r-

r*

r*

r-

— — — — — — — — — — —

— — — — 0.30 — — 0.70 0.75 1.38 —

— — — — — — — 0.94 0.94 1.12 1.69

1.41 1.18 1.35 1.59 2.76 — — — — — —

0.29 — — — 0.46 0.49 0.72 1.00 1.13 1.68 3.54

0.62 — — — 0.76 — — 1.00 1.00 1.31 2.48

0.29 — — — 0.46 — — 1.00 1.13 — 2.46

0.39 — — — 0.72 — — 1.00 1.05 — 1.94

— — —

— —

1.29 1.29

— — — — — —

— — — — — — —

— — — — —

— —

—

— — 0.43 0.80 0.40 0.70 1.01 1.16 2.68 1.33 —

— —

—

— — — —

— —

— —

— —

—

—

—

—

—

0.25 0.72 0.80 0.37

—

—

— — —

— —

—

—

— — — — — —

— — — — — —

— — — —

0.88 0.94 1.00 2.00

1.24 1.24 1.41

—

—

—

—

—

—

— — 1.93 — 3.00

— — — — — —

— — — — — —

— —

— —

— — —

—

—

—

—

—

—

—

—

—

—

—

—

— —

—

—

—

—

—

—

—

—

—

—

—

2.69

—

—

CRC Handbook of Chromatography

D-Glucitol 2-0-methyl 3-0-methyl 4-0-methyl 6-0-methyl 2,3-di-0-methyl 2,6-di-0-methyl 4,6-di-0-methyl 2,3,4-tri-0-methyl 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 2,3,4,6-tetra-0-methyl D-Mannitol 3-0-methyl 4-0-methyl 2,3-di-0-methyl 2,4-di-0-methyl 3,4-di-0-methyl 4,6-di-0-methyl 2,3,4-tri-0-methy 1 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 3,4,6-tri-0-methyl 2,3,4,6-tetra-0-methyl Galactitol 3-0-methyl 4-0-methyl 6-0-methyl 2,3-di-0-methyl 2,3,4-tri-0-methyl 2,3,6-tri-0-methyl 2,4,6-tri-0-methyl 2,3,4,6-tetra-0-methyl

r*

myo-Inositol 1-O-methyl 1,3-di-O-methyl 1,2,3,5-tetra-O-methyl 1,3,4,5-tetra-O-methyl 1,3,4,6-tetra-O-methyl 1,4,5,6-tetra-O-methyl 1,2,4,5,6-penta-O-methyl

— — — — — — —

—

—

—

—

1.06 1.65

—

—

—

— — — —

— — — —

— — — —

—

0.33 1.22 2.01 1.04 2.90 —

—

—

—

—

—

—

—

—

— — — —

— — — —

1.14 1.52 1.07 1.93 3.45

a t, relative to a-anomer of 2,3,6-tri-O-methyl-D-glucose (1.35 min). b RRT = Vt0 for compound relative to that for a-anomer of 2,3,6-tri-O-methyl-D-glucose (6.0 min with S2, 1.6 min with S3). c t, relative to D-glucitol (1.7 min). d t, relative to 2,3,4-tri-O-methyl-D-glucitol (6.9 min with S4 and S6, 2.9 min with S5, 6.6 min with S7). Packing

Solvent

Detection

|x-Bondapak® C18 (Waters). Dextropak® (Waters). Supelcosil® LC-18 (Supelco). 1% aqueous ammonium acetate, pH 6.9 - ethanol (9:1). water-methanol (49:1). water-methanol (4:1). water-acetonitrile (99:1). water-acetonitrile (19:1). S4 for 10 min, then S5. hyperbolic gradient, water —» S5 in 10 min (program no. 5, Waters Model 660 solvent programmer); isocratic with S5 for 5 min. D1 = polarimetrie detection; [a]350 recorded on spectropolarimeter with 10-p.l flow-through cell, operating simultane­ ously with refractometer. D2 = scintillation counting after labeling by sodium borotritiide reduction.

PI P2 P3 SI S2 S3 S4 S5 S6 S7

= = = = = = = = = =

93

1. Cheetham, N. W. H. and Sirimanne, P., Separation of partially methylated sugars by reversed-phase high-performance liquid chro­ matography, J. Chrom atogr., 196, 171, 1980. 2. Heyraud, A. and Salemis, P., Liquid chromatography in the méthylation analysis of carbohydrates, and the use of combined refractometricpolarimetric detection, Carbohydr. R es., 107, 123, 1982. 3. Saadat, S. and Ballou, C. E., Separation of O-methylhexitols and O-methyl-myo-inositols by reverse-phase high-performance liquid chromatography, Carbohydr. R es., 119, 248, 1983.

Carbohydrates

REFERENCES

TABLE LC 9 HPLC of Milk Oligosaccharides, Glycopeptides, and Glycoprotein-Derived Reduced Oligosaccharides on Reversed-Phase Packings PI

P2

P3

P2

P2

20 0.8 Cartridges3 H20 2 Ambient RI 1

25 0.46 Column h 2o 0.5 Ambient UV 2

25 0.46 Column H20 2 Ambient UV 3

25 0.46 Column SI 0.5 25 UV 4

25 0.46 Column SI 0.5 5 UV 4

Compound

k 'b

k 'b

k 'b

k 'b

k 'b

—

—

—

—

—

—

— —

—

—

—

—

—

—

—

—

—

—

—

—

—

2.1 2.5 9.0 2.3 3.5 11.1 12.5

— — — — — — —

— — — — — — —

Milk oligosaccharides Galßl -3GlcN Acß 1-3Galß 1-4Glc Galß 1-4GlcN Acß 1-3Galß 1-4Glc Fuca 1-2Galß 1-3GlcNAcß 1-3Galß 1-4Glc Galß 1-3(Fuca 1-4)GlcN Acß 1-3Galß 1-4Glc Galß 1-4(Fuca 1-3)GlcNAcß 1-3Galß 1-4Glc Fuca 1-2Galß 1-3(Fuca 1-4)GlcN Acß 1-3Galß 14Glc Galß 1-3(Fuca 1-4)GlcNAcß 1-3Galß 14(Fucal-3)Glc Reduced oligosaccharides Galß 1-3GalNAc-ol GlcNAcß 1-3GalNAc-ol Fuca 1-2Galß 1-3GalNAc-ol Galß 1-4GlcNAcß 1-6(Galß 1-3)GalNAc-oI Fuca 1-2Galß 1-3GlcNAcß 1-3GalNAc-ol Fuca 1-2Galß 1-3(GlcNAcß 1-6)GalNAc-ol Fuca 1-2Galß 1-3(Galß 1-4GlcN Acß 16)GalNAc-ol

1.40; 1.23; 2.51; 0.88; 0.88; 0.80;

1.91 1.46 3.74 1.06; 1.23 1.06; 1.23 0.91

1.14; 0.94; 1.80; 0.71; 0.63; 0.43;

1.48 1.04 2.65 0.86 0.71 0.57

0.80; 0.91 (16.6min)

(12.8min)

—

—

— — — — — —

— — — — — —

CRC Handbook of Chromatography

Packing Column Length, cm Diameter (I.D.), cm Form Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

S

3.8

Fuca 1-2Gal(3 1-4GlcNAc(31-6(Gal(3 13)GalNAc-ol Fuca 1-2Gal(3 1-3(Fuca 1-4)GlcNAc|3 1-3Gal(S 13GalNAc-ol Fuca 1-2Gal(3 1-3(Fuca 1-2Gal|31-4GlcN Ac(316)GalNAc-ol Fuca 1-2Gal(31 -3[Fuca 1-2Gal(3 1-4(Fuca 13)GlcN AcP 1-6]GalNAc-ol Ovalbumin glycopeptidesc E3 d3 c 3b

Note:

a b c

1.8

24.6 19.1 (25.5 min) 1.57 1.28 1.97 (10.2 min)

2.26 1.86

2.71 (13.0 min)

Data on normal-phase HPLC of reduced oligosaccharides in Reference 3 will be found in Table LC 5.

Two cartridges, each 10 x 0.8 cm, connected in series. t,. of last-eluting peak given in brackets at end of each column of data. Structures of glycopeptides appended.

Packing

Solvent

PI P2 P3 SI

= = = =

Dextropak®; under radial compression (Waters module RCM 100). 600 RP (Alltech). 605 RP (Alltech; 5-|xm particles). aqueous phosphate buffer (1 mAf, pH 6.0). REFERENCES

Carbohydrates

1. Cheetham, N. W. H. and Dube, V. E., Preparation of lacto-A-neotetraose from human milk by high-performance liquid chromatography, J. C hrom atogr., 262, 426, 1983. 2. Dua, V. K. and Bush, C. A., Identification and fractionation of human milk oligosaccharides by proton-nuclear magnetic resonance spectroscopy and reverse-phase high-performance liquid chromatography, Anal. Biochem ., 133, 1, 1983. 3. Dua, V. K., Dube, V. E., and Bush, C. A., The combination of normal-phase and reverse-phase high-pressure liquid chromatography with NMR for the isolation and characterization of oligosaccharide alditols from ovarian cyst mucins, Biochim. Biophys. A cta , 802, 29, 1984. 4. Dua, V. K. and Bush, C. A., Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography, Anal. B iochem ., 137, 33, 1984.

95

Appendix to Table LC 9: Structures of Glycopeptides

so

os

CRC Handbook of Chromatography

TABLE LC 10 HPLC of Malto-Oligosaccharides and Oligosaccharide-Alditols on Silica-Based Reversed-Phase Packings Packing Column Length, cm Diameter (I.D.), cm Material Form Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

PI

P2

P2

P2

P2

P2

P3

P2

30 0.39 SS Column h 2o 0.3 15 RI 1

10 0.8 Plastic Cartridge h 2o 1 Ambient RI 2

10 0.8 Plastic Cartridge h 2o 2 Ambient RI 3

10 0.8 Plastic Cartridge SI 2 Ambient RI 3

10 0.8 Plastic Cartridge S2 2 Ambient RI 3

10 0.8 Plastic Cartridge S3 2 Ambient RI 3

10 0.8 Plastic Cartridge H20 2 Ambient RI 3

10 0.8 Plastic Cartridge S4 2 Ambient RI 3

DP

r*

r*

rb

rb

rb

rb

rb

r*

0.89 1.00 1.17 1.40; 1.48c 1.76; 1.84c 2.43; 2.55c 3.05d 3.54d — — — — — — —

0.77 1.00 1.26; 1.64; 2.46; 3.41; 4.31;

— — 1.00 1.11 1.32 1.64 1.93 2.25 2.61 3.14 3.86 5.00 6.71 — —

— — 1.00 1.18 1.47 1.84 2.21 — — — — — — —

— — 1.00 1.24 1.64 2.17 2.67 — — — — — — — —

— — 1.00 1.16 1.37 1.58 2.00 2.37 2.74 3.26 4.00 5.21 6.42 8.37 10.2

— 1.00 1.55; 1.77 2.50; 3.14 4.59; 5.45 —

— — — — — —

— — 1.00 1.17; 1.30c 1.60; 1.80e 2.23; 2.40e 2.87; 3.03 3.37; 3.63 4.13; 4.70 5.47; 6.40 7.53; 8.83 10.4; 11.8 13.9; 15.7 — —

— —

0.77 0.79 1.23

— — —

— —

— —

— —

—

—

—

— — —

— — —

—

—

—

— — — — — — — —

97

—

—

1.36 1.90 2.72 3.74 4.54c

Carbohydrates

Oligosaccharides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Oligosaccharide-alditols 1 2 3

so

TABLE LC 10 (continued) HPLC of Malto-Oligosaccharides and Oligosaccharide-Alditols on Silica-Based Reversed-Phase Packings r*

rb

rb

4 5

____

—

—

—

—

6

—

—

—

7

—

1.69 2.41 3.31 4.31

—

—

—

rb

rb

rb

r*

Packing Column Length, cm Diameter (I.D.), cm Material Form Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

P4

P4

P4

P5

P6

P6

P7

P7

15 0.46 SS Column H20 1 20 RI 4

15 0.46 SS Column S5 1 20 RI 4

15 0.46 SS Column S6 1 20 RI 4

15 0.32 Glass Column h 2o 0.23 Ambient RI 5

25 0.6 SS Column h 2o 0.8 24 RI 6

25 0.6 SS Column h 2o 0.8 60 RI 6

25 0.46 SS Column h 2o 1 25 RI 7

25 0.46 SS Column h 2o 1 35 RI 7

DP

r*

V

r*

r*

r*

r-

r“

r*

0.89 1.00 1.17 1.36; 1.65; 2.12; 2.56; 3.31;

—

0.98 1.00 1.13 1.24 1.43 1.69d 1.92; 2.03c 2.29; 2.63

0.91 1.00 1.14 1.32 1.64 2.00 2.34 2.73

0.89 1.00 1.20; 1.24c 1.39; 1.47 1.82; 1.94 2.28; 2.40c 2.71d

0.83 1.00 1.17; 1.45; 1.90; 2.59; 3.17; 3.76;

0.85 1.00 1.13; 1.38; 1.73; 2.15; 2.54; 2.92;

—

—

—

1.00 1.00 1.08 1.17 1.26 1.35 1.44 1.53 1.80 1.98

Oligosaccharides 1 2 3 4 5 6

7 8 9 10 11

12

13

_

1.42 1.74 2.22 2.90 3.56

1.00 1.23 1.50; 1.96; 2.69; 3.50; — —

1.61 2.11 2.88 4.17

—

—

1.24 1.59 2.14 2.76 3.38 4.03

—

—

—

—

1.19 1.50 1.92 2.30 2.69 3.08

CRC Handbook of Chromatography

r*

DP

00

14 15 Oligosaccharide-alditols 1 2 3 4 5 6 7 a

t, relative to maltose (3.9 min,2 2.2 min;3 1.91 min in H20 and S64_, 2.06 min in S5;4 4.4 min;5 4.3 min at 24°C,6 3.7 min at 60°C6; 2.9 min at 25°C,7 2.6 min

at 35°C7). b f relative to maltotriose (3.0 min in H20 , 2.8 min in SI, 1.9 min in S2, and with P3, 2.1 min in S3)3. c Shoulder. d Broad peak due to unresolved anomers. Packing

Solvent

REFERENCES

99

1. Heyraud, A. and Rinaudo, M., Carbohydrate analysis by high pressure liquid chroamtography using water as the eluent, J. Liq. Chrom atogr., 3, 721, 1980. 2. Cheetham, N. W. H, Sirimanne, P., and Day, W. R., High-performance liquid chromatographic separation of carbohydrate oligomers, J. C hrom atogr., 207, 439, 1981.

Carbohydrates

PI = |i-Bondapak®-C18 (Waters; 10-p,m particles). P2 = Dextropak® (Waters; 10-(xm particles), used under radial compression (Waters module RCM-100). P3 = Dextropak®, modified by coating with nonionic detergent Triton® X-100 (p-rm-octylphenoxypolyethoxyethanol oligomers); aqueous solution of Triton® X-100 (0.1% w/v) pumped through cartridge at 1 ml/min for 300 min before use of cartridge. P4 = Polygosil® RP-18 (Macherey-Nagel); irregularly shaped particles of average diameter 5 |xm. P5 = Separon® 6 RPS (Laboratory Instrument Works, Prague; 10-p.m particles), doped with primary amino groups (0.25 mmol/g). P6 = Separon® 6 RPS without prior treatment. P7 = Spherisorb® S 5 ODS2 (Phase Separations, Queensferry, U.K.; 5-p,m particles). 51 = water containing tetramethylurea (0.025%). 52 = 0.1 M urea in water. 53 = 0.1 M guanidine hydrochloride in water. 54 = 1 M ammonium sulfate. 55 = 1 M sodium chloride. 56 = water containing triethylamine (1 mA/).

©

o

TABLE LC 10 (continued) HPLC of Malto-Oligosaccharides and Oligosaccharide-Alditols on Silica-Based Reversed-Phase Packings

TABLE LC 11 H P L C Packing Column Length, cm Diameter (I.D.), cm Material Form Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

o f O lig o s a c c h a r id e s o f V a r io u s S e rie s o n S ilic a -B a s e d R e v e r s e d - P h a s e P a c k in g s

PI 10

P2 30

P3 15

P4 15

P4 15

P4 15

P5 15

PI 10

PI 10

P6 10

P7 25

PI 10

pi

10

PI 10

PI 10

0.8 Plastic Cartridge h 2o 1 Ambient RI 1

0.39 SS Column H20 FI 15 RI 2

0.32 Glass Column h 2o 0.52 70 RI 3

0.46 SS Column h 2o 1 20 RI 4

0.46 SS Column SI 1 20 RI 4

0.46 SS Column S2 1 20 RI 4

0.46 SS Columni S3 1 20 RI 4

0.8 Plastic Cartridge h 2o 1 Ambient RI 1

0.8 Plastic Cartridge S4 2 Ambient RI 5

0.8 Plastic Cartridge H20 2 Ambient RI 5

0.4 SS Column H20 2 Ambient RI 5

0.8 Plastic Cartridge S5 2 Ambient RI 5

0.8 Plastic Cartridge H20 2 Ambient RI 6

0.8 Plastic Cartridge h 2o 3 Ambient RI 6

0.8 Plastic Cartridge H20 1 Ambient RI 1

Cello-oligosaccharides Oligosaccharides Linear, DP 1 2 3 4 5 6 7 8

r*

r*

rb

0.88 1.00 1.42; 1.67 2.23; 2.37 4.58; 4.88

0.88 1.00 1.18 1.57 2.84 4.80 — —

0.51 0.63 1.00; 1.03 2.36; :2.58 7.08; 7.92 — — —

r*

— 1.00 2.50; 2.71 7.50; 8.36 — —

—

— —

— —

Isomaltorb

rb

1.00 1.60; 1.71 3.21; 3.47 7.33; 7.98 — —

— — 1.00 1.44; 1.52 2.52; 2.67 4.98; 5.40 — —

— —

rb

r
4)-a-D(Malto-)

(1 -»• 4)-p-D(Cello-)

(1 -► 6)-ot-D(Isomalto-)

(1 -► 6)-p-D(Gentio-)

rb(tr)c

DP 2 3 4 5 6 7 8 9 10

15 0.6 SS Column h 2o

1.00 1.04 1.06 1.09 (2.98 min)

1.00; 1.23; 1.51 1.71; 2.11; 2.80; 3.66; 4.80; 5.88;

1.06 1.00 1.28 1.17 1.44 1.88 1.69 2.34 2.14 3.14 2.83 4.23 3.78 5.31 5.05 6.94 6.61

1.00; 1.39; 2.61; 5.14; (25.0

1.06 1.64 3.03 6.94 min)

1.00 1.55 2.04 6.50 (23.4 min)

1.00 1.00 1.60; 1.70 1.68 4.43; 5.20 4.57 (15.6 min) (12.8 min)

1.00 1.10 1.20 1.33 1.43; 1.60; 1.77; 2.00; 2.30;

1.50 1.70 1.90 2.13 2.43

1.00 1.07 1.17 1.30 1.40 1.57 1.73 1.93 2.20

1.00 1.37 2.21; 4.63; 11.5; (48.0

2.29 5.05 12.6 min)

1.00 1.41 2.50 5.31 12.8 (41.0 min)

1.00 1.17 1.41 1.76; 2.31; 3.21; 4.55; 6.62; 9.59;

1.86 2.48 3.45 4.93 7.17 10.3

1.00 1.14 1.43 1.78 2.39 3.43 5.00 7.28 10.7

1.00; 1.24 1.48; 1.91; 2.61; 3.64; 5.12; 7.39; 10.7;

1.06 1.00 1.24 1.64 1.48 2.15 1.88 3.15 2.64 4.42 3.67 6.18 5.15 8.97 7.42 13.0 10.9

CRC Handbook of Chromatography

REFERENCES

11 12

7.77; 8.57 8.55 9.82; 11.8 11.6 (41.4 min) (42.0 min)

2.65; 2.80 2.50 3.00; 3.17 2.87

13 14 15 16 17 18 19 20 21 22 23

3.40; 3.63 3.93; 4.27 4.63; 5.03 5.53; 6.00 6.60; 7.07 7.80; 8.43 9.23; 10.0 11.0; 12.0 13.1; 14.2 15.6; 17.0 18.6; 20.2 (60.6 min)

Note:

14.1; 15.1 15.7 15.4; 18.5 15.9 (43.8 min) (44.0 min) (61.0 min) (52.5 min)

3.27 3.80 4.47 5.33 6.33 7.53 9.00 10.8 12.9 15.5 18.5 (55.6 min)

See data for HPLC on amino-bonded silica in Table LC 3.

a Conditions listed in center of column headings apply throughout except for solvent, which changes as indicated. b ^ relative to dimer of series or first anomer eluted ((3) where anomer resolution occurs. c t, of highest oligomer eluted is given in brackets at end of each column. Packing Solvent

PI SI

= =

Asahipak ODP-50 (Asahi Kasei, Tokyo); C18-bonded vinyl alcohol copolymer gel, 5-|xm particles. aqueous sodium hydroxide solution, pH 11. REFERENCE

1. Koizumi, K. and Utamura, T., High-performance liquid chromatography of gluco-oligomers on CI8-bonded vinyl alcohol copolymer gel with alkaline eluents, J. C hrom atogr., 436, 328, 1988.* *

Data reproduced with permission of authors and Elsevier Science Publishers

PI

PI

PI

PI

PI

PI

P2

P3

P4

P5

P5

P5

PI

P6

P7

25 0.4 SS

25 0.4 SS

25 0.4 SS

25 0.4 SS

25 0.4 SS

25 0.4 SS

15 0.46 SS

15 0.46 SS

15 0.46 SS

15 0.6 SS

15 0.6 SS

15 0.6 SS

25 0.4 SS

20 0.6 SS

15 0.6 SS

103

Packing Column Length, cm Diameter (I.D.), cm Material

Carbohydrates

TABLE LC 13 HPLC of Cyclic Oligosaccharides on Silica- and Polymer-Based Reversed-Phase Packings

104

TABLE LC 13 (continued) HPLC of Cyclic Oligosaccharides on Silica- and Polymer-Based Reversed-Phase Packings SI 1 28 RI 1

S2 1 28 Rl 1

S3 1 28 Rl 1

S4 1 28 RI 1

SI 1 33 RI 1

SI 1 38 RI 1

S5 1 25 RI 2

S5 1 25 Rl 2

di-O-a-maltosylCyc lomaltooctaose3 6-O-a-D-glucosyl6-0-a-maltosyl6-O-a-maltotriosyl6-0-a-maltotetraosyldi-O-a-D-glucosyldi-O-a-maltosyl-

S5 1 25 RI 2

S6 1 25 RI 2

—

—

2.38 1.41 1.48 1.77 1.96 1.47

—

—

S7 1 25 RI 2

S8 0.7 Ambient RI 3

S7 0.7 Ambient RI 3

S5 0.7 Ambient RI 3

k'

Compound Cyclomaltohexaose* 6-O-a-D-glucosyl6-0-a-maltosyl6-0-a-maltotriosyl6-O-a-maltotetraosyl6-0-a-maltopentaosyl6-O-a-D-glucosyldi-O-a-maltosylCyclomaltoheptaose* 6-0-a-D-glucosyl6-0-a-maltosyl6-0-a-maltotriosyl6-0-a-maltotetraosyldi-O-a-D-glucosyl-

S5 1 25 RI 2

3.54 2.35 2.29 2.91

2.64 1.72 1.57 1.99

2.01 1.20 1.07 1.36

1.85 1.81 0.70 0.87

2.64 1.72 1.64 2.08

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

12.26 7.17 7.01 8.75

9.27 5.21 4.94 6.05

6.75 3.75 3.43 4.13

5.41 2.52 2.29 2.70

8.95 5.30 5.11 6.28

6.01 3.83 3.59 4.40

—

—

—

—

—

—

3.19

2.33 —

4.17 2.88

1.97

1.30

1.74 1.19 1.16 1.42

—

—

—

—

—

3.17 1.95 1.88 2.30

2.37 1.41 1.32 1.57

1.72 1.00 0.91 1.08

1.68 0.68 0.59 0.68

2.36 1.45 1.40 1.67

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

1.59 1.05 1.00 1.14

3.89 3.05 3.57 5.13 5.83 4.75 2.14 3.25 11.6 7.85 9.22 12.8 14.4 4.88; 5.13b 7.29 2.83 2.01 2.38 3.21 3.62 1.45 2.14

3.61 2.83 3.30 4.75 5.35 4.25 — —

11.0 6.35 8.70 12.3 13.8 —

—

2.42 1.74 2.08 2.84 3.17 — —

3.80 2.98 3.47 4.98 5.60 4.48 2.21 3.23 11.8 7.95 9.36 13.3 14.9 5.07; 5.42b 6.90 2.55 1.83 2.17 2.95 3.30 1.38 1.99

— — — —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

— —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

3.07 1.91 2.05 2.48 2.73

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

9.27 5.00 4.87 6.18 6.44

CRC Handbook of Chromatography

Solvent Flow rate (ml/min) Temperature (°C) Detection Reference

Cyclosophoraosesc DP 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

33

_

_

_

_

_

_

_

_

_

_

34

_ _

_ _

_ _

_ _

_ _

_ _

_ _

_ _

_ _

_ _

35

Note:

a b c

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

1.20 2.60 3.80 3.40 6.01 4.41 6.61 7.41 7.41 n .8 10.6 14.2 16.2 17.0 25.4 24.2

0.87 1.50 1.87 1.87 2.78 2.37 3.99

_

_

31.4

16.5

20.0

_ _

_ _

38.6 38.6

20.6 20.6

24.0 24.0

4 .2 4

4.62 6.12 6.12 7.73 9.23 9.23 13.2 13.2

1.00 1.62 2.12 1.87 3.49 2.93 4.62 4.86 5.12 7.74 7.11 9.49 11.2 11.2 16.5 16.0

See data for HPLC of these oligosaccharides on amino phases (Table LC 4).

Cyclomaltohexaose, -heptaose, and -octaose are ot-, (3-, and y-cyclodextrins, respectively. Splitting of peak, due to positional isomers, seen with highly efficient columns. Produced by Rhizobium and Agrobacterium strains.

Solvent

= = = = = = = = = =

Hibar LiChrosorb® RP-18 (Merck); 5-(xm C18-bonded silica. YMC-Pack® AL-302 ODS (Yamamura Chemical Co., Kyoto); 5-|xm C18-bonded silica. YMC-Pack® A-302 ODS; as P2, but “ endcapped” by treatment with trimethylchlorosilane to reduce proportion of reactive silanol groups. YMC-Pack® AM-302 ODS; P3 treated further to free it completely from unreacted silanol groups. Asahipak® ODP-50 (Asahi Kasei, Tokyo); C18-bonded vinyl alcohol copolymer gel (5-|xm particles). ERC®-ODS-l 171 (Erma Optical Works, Tokyo); 3-p.m C18-bonded silica. YMC-Pack® AL-312 ODS; 5-jxm C18-bonded silica. water-methanol (23:2). water-methanol (91:9). water-methanol (9:1).

105

PI P2 P3 P4 P5 P6 P7 SI 52 53

Carbohydrates

Packing

106

TABLE LC 13 (continued) HPLC of Cyclic Oligosaccharides on Silica- and Polymer-Based Reversed-Phase Packings = = = = =

water-methanol water-methanol water-methanol water-methanol water-methanol

(89:11). (47:3). (93:7). (19:1). (94.5:5.5). REFERENCES

1. Koizumi, K., Utamura, T., Kuroyanagi, T., Hizukuri, S., and Abe, J.-I., Analysis of branched cyclodextrins by high-performance liquid and thin-layer chromatography, J. C hrom atogr., 360, 397, 1986. 2. Koizumi, K., Kubota, Y., Okada, Y., Utamura, T., Hizukuri, S., and Abe, J.-I., Retention behaviour of cyclodextrins and branched cyclodextrins on reversed-phase columns in high-performance liquid chromatography, J. C hrom atogr., 437, 47, 1988. 3. Koizumi, K., Okada, Y., Utamura, T., Hisamatsu, M., and Amemura, A., Further studies on the separation of cyclic (1 —» 2)-[3-D-glucans (cyclosophoraoses) produced by Rhizobium m eliloti IFO 13336, and determination of their degrees of polymerization by high-performance liquid chromatography, J. Chrom atogr., 299, 215, 1984. Data from References 1 and 2 reproduced with permission of authors and Elsevier Science Publishers.

TABLE LC 14 Reversed-Phase HPLC of Derivatized Sugars Packing Column Length, cm Diameter (I.D.), cm Material Solvent Flow rate (ml/min) Temperature (°C) Technique Detection Reference

PI

P2

P3

P3

P4

P5

P5

P6

P7

P8

P9

P9

P9

P10

P ll

30 0.39 SS SI 1 28 T1 UV, D1 1

25 0.46 SS S2 1 Ambient T2 D2 2

20 0.46 SS S3 1 Ambient T2 D2 2

25 0.46 SS S4 1 Ambient T3 D2 2

25 0.46 SS S5 1 Ambient T4 UV 3

5 0.46 SS S6 1.5 40 T5 D3 4

5 0.46 SS S7 1.5 40 T5 D3 4

15 0.39 SS S8 1.2 Ambient T6 D4 5

10 0.8 Plastic S9 2 Ambient T7 D5 6

25 0.46 SS S10 1 Ambient T6 UV 7

25 0.46 SS S ll 1.5 Ambient T6 D6 8

25 0.46 SS S ll FI Ambient T6 D6 8

50 0.46 SS S ll 0.5 Ambient T8 D6 8

25 0.46 SS S12 1 Ambient T6 D7 9

25 0.46 SS S13 1 Ambient T9 UV 10

CRC Handbook of Chromatography

54 55 56 57 58

Dansylhydrazones*

DDBC

MPGAd

k'

k'

__

_

__

_

— — —

— — — 1.45 1.65 1.92 2.58 2.77 — 2.78 3.14 3.09 3.47

— — —

— 6.6 7.6 8.6 — — 9.6 10.2 — 11.4

— — — — — — — — — — — — —

—

—

—

13.0

— — — —

— 5.17 4.75 5.30

— 1.0 1.0 1.2 — — — 1.2 — 1.4 — 3.2

Dabsylhydrazonesb k'

__

4.5 5.5 6.2 6.1 — 6.6 7.2 8.1 — 9.4 8.7 9.4 9.4 11.0 11.0 13.9

3.7 4.8 5.5 5.5 — 5.5 5.5 7.4 — 8.1; 10.1 7.4 8.1 8.7; 9.2 9.4; 10.9 10.7 8.1

k'

Pyridylamino-*

NBDGf

Benzoyl8

k'

k'

k'

—

_

—

—

— — —

— — —

— — —

— — —

— 1.0 1.1 1.3 — — — 1.3 — 1.5 — 2.1

— 1.2 1.4 1.6 — — — 1.8 — 2.0 — 4.7

— 5.7 6.0 6.6 — — — 7.6 — 8.3 —

— 12.2; 12.5 12.2; 12.6 10.5; 11.8 — — — 9.4

— 2.2 2.4

— 1.7 1.9

— 2.9 3.3

— — 9.3 —

— — 8.9; 9.7 —

— —

— —

— —

— —

—

—

—

—

— 5.9; 6.5 6.1; 6.8

2.8 4.0 4.4

2.0 2.3 2.5

3.8 5.4 6.0

— 7.0

— —

— —

— — — — —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

k'

Gentiobiose Lactose Maltose Cellobiose D-g/ycero-D-g/uc