Handbook of Chromatography Vol I (1982): Carbohydrates [1 ed.] 9781138506213, 9781138559400, 9780203712740, 9781351363419, 9781351363402, 9781351363426

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Handbook of Chromatography Vol I (1982): Carbohydrates [1 ed.]
 9781138506213, 9781138559400, 9780203712740, 9781351363419, 9781351363402, 9781351363426

Table of contents :

Section 1: CHROMATOGRAPHIC DATA 1.Gas Chromatography Tables 2.Liquid Chromatography Tables 3.Paper Chromatography Tables 4.Thin-Layer Chromatography Tables 5. Electrophoretic Section 2: DETECTION TECHNIQUES 1. Detection and Identification Carbohydrates in Gas Chromatography 2. Detection methods for Liquid Chromatography 3.Detection Reagents for Paper and/or Thin-Layer Chromatography Section 3: SAMPLE PREPARATION AND DERIVATIZATION Section 4: PRODUCTS AND SOURCES OF CHROMATOGRAPHIC MATERIALS. Section 5: LITERATURE REFERENCES

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CRC HANDBOOK OF CHROM ATOGRAPHY G u n te r Z w eig a n d J o s e p h S h e rm a E d ito rs-in -C h ie f

GENERAL DATA AND PRINCIPLES Editors Gunter Zweig, Ph.D . U.S. Environmental Protection Agency Washington, D.C. Joseph Sherma, Ph.D . Lafayette College Easton, Pennsylvania

CARBOHYDRATES Editor Shirley C. Churms, Ph.D . Research Associate C .S .l.R . Carbohydate Chemistry Research Unit Department of Organic Chemistry University of Cape Town, South Africa

DRUGS Editor Ram Gupta, Ph.D . Department of Laboratory Medicine St. Joseph’s Hospital Hamilton, Ontario, Canada

CATECHOLAMINES TERPENOIDS Editor Carmine J. Cosica, Ph.D. Professor of Biochemistry St. Louis University School of Medicine St. Louis, Missouri

LIPIDS AND TECHNICAL LIPID DERIVATIVES Editor H. K. Mangold Executive Director and Professor Federal Center for Lipid Research 4400 Munster, Germany

HYDROCARBONS Editors Willie E. May, Ph.D . Center for Analytical Chemistry U .S. Department of Commerce Washington, D.C. Walter L. Zielinski, Jr., Ph.D . Air Program Manager National Bureau of Standards Washington, D.C.

INORGANICS Editor M. Qureshi, Ph. D. Professor, Chemistry Section Zakir Husain College of Engineering and Tech­ nology Aligarh Muslim University India

PHENOLS AND ORGANIC ACIDS Editor Toshihiko Hanai, Ph.D . University of Montreal Quebec, Canada

STEROIDS

AMINO ACIDS AND AMINES

Editor Joseph C. Touchstone, B .S., M .S., Ph.D . Professor School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

Editor Dr. S. Blackburn Leeds, England

PESTICIDES Editors Joseph Sherma, Ph.D . Lafayette College Easton, Pennsylvania Joanne M. Follweiler, Ph.D . Lafayette College Easton, Pennsylvania

POLYMERS Editor Charles G. Smith Dow Chemical, USA Midland, Michigan

PLANT PIGMENTS Editor Dr. Hand-Peter Kost Botanisches Institut der Universitat Munchen Munchen, West Germany

CRC Handbook of Chromatography Carbohydrates Volume I Editor

Shirley C. Churms, Ph.D. Research Associate C .S.I.R . Carbohydrate Chemistry Research Unit Department of Organic Chemistry University of Cape Town Republic of South Africa

Editors-in-Chief

Gunter Zweig, Ph.D. School of Public Health University of California Berkeley, California

Joseph Sherma, Ph.D. Professor of Chemistry Chemistry Department Lafayette College Easton, Pennsylvania

First published 1982 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1982 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 (http://www.copyright.com/) or contact the Copyright Clearance 01923, 978-750-8400. CCC is a not-for-profit organiza-tion that For organizations that have been granted a photocopy license arranged.

from this work, please access w w w .copyright.com Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA provides licenses and registration for a variety of users. by the CCC, a separate system of payment has been

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: 978-1-138-50621-3 (hbk) ISBN 13: 978-0-203-71274-0 (ebk) Visit the Taylor & Francis W eb site at http://www.taylorandfrancis.com and the CRC Press W eb site at http://www.crcpress.com

CRC HANDBOOK OF CHROM ATOGRAPHY

S E R IE S P R E F A C E T h is

Handbook of Chromatography, Carbohydrates

b y S h i r l e y C . C h u r m s , is o n e

in a s e r i e s o f s e p a r a t e v o l u m e s d e v o t e d t o a s i n g l e c la s s o f c h e m i c a l c o m p o u n d s o r t o c o m p o u n d s w i t h a s i m i l a r u s e p a t t e r n , li k e t h e p r o s p e c t i v e v o l u m e s o n p e s t i c i d e s a n d d r u g s . W h e n V o l u m e s I a n d II o f t h e

Handbook of Chromatography were

first p u b ­

l i s h e d in 1 9 7 2 , t h e e d i t o r s m a d e a n a t t e m p t t o s e l e c t t h e d a t a t h a t w o u l d c o v e r m o s t o r g a n i c a n d i n o r g a n i c c o m p o u n d s in a v o l u m e o f a b o u t o n e t h o u s a n d p a g e s . H o w e v e r , d u r i n g t h e e n s u i n g 10 y e a r s , t h e l i t e r a t u r e o f c h r o m a t o g r a p h y , e s p e c i a l l y h i g h - p e r f o r m ­ a n c e liq u id c h r o m a t o g r a p h y ( H P L C ) , h a s g r o w n to su c h a n e x te n t t h a t , a f t e r a n in itial i n t e n t t o u p d a t e V o l u m e s I a n d I I , it w a s d e c i d e d t o p u b l i s h s e p a r a t e v o l u m e s d e v o t e d t o s p e c i f i c s u b j e c t s . T h e p r e s e n t v o l u m e o n t h e c h r o m a t o g r a p h y o f c a r b o h y d r a t e s is a n e x a m p l e o f th e e x p a n d e d h a n d b o o k series. In s e le c tin g V o l u m e E d i t o r s , th e E d it o r s i n - C h i e f e n d e a v o r e d t o s e l e c t s c i e n t i s t s w i t h e x t e n s i v e k n o w l e d g e a n d e x p e r t i s e in t h e c h r o m a t o g r a p h y o f sp e c ific c o m p o u n d s . T h e E d i t o r o f th is v o lu m e , D r. S h irley C . C h u r m s , is r e n o w n e d in t h e f i e l d o f c h r o m a t o g r a p h y o f c a r b o h y d r a t e s , w h i c h is e v i ­ d e n t f r o m t h e c o m p r e h e n s i v e a n d a u t h o r i t a t i v e t r e a t m e n t o f t h e s u b j e c t f o u n d in t h i s v o l u m e . W e h a v e g i v e n e a c h V o l u m e E d i t o r w i d e l a t i t u d e in d e s i g n i n g a f o r m a t t h a t w o u ld be m o s t u se fu l to th e re a d e r a n d d o ju s tic e to th e p a r tic u la r su b je c t b ein g c o v ­ e r e d . S u b s e q u e n t v o l u m e s o f t h i s s e r i e s w ill i n c l u d e t h e c h r o m a t o g r a p h y o f d r u g s , s te ro id s , lip id s a n d f a tty a cid s, te r p e n o id s , p la n t p ig m e n ts , h y d r o c a r b o n s , a m in o acid s, in o r g a n ic c o m p o u n d s , p o ly m e rs , a n d nu cleic acid s a n d a s s o c ia te d c o m p o u n d s . W e in v ite re a d e r s to c o m m u n ic a t e w ith th e V o lu m e E d ito r fo r c o m m e n ts a n d c o r ­ re c tio n s a n d to th e E d ito r s - in - C h ie f fo r s u g g e s tio n s fo r fu tu r e v o lu m e s . T h e E d ito rs in - C h ie f w a n t to th a n k D r. S h irley C . C h u r m s fo r h er o u ts ta n d in g e ff o rt a n d th e c o ­ o p e r a tio n o f h e r a sso ciate s. G u n te r Z w eig , P h .D . Joseph S herm a, P h .D . S p r i n g 1981

PREFACE T h i s h a n d b o o k is i n t e n d e d t o s e r v e a s a w o r k i n g m a n u a l a n d r e f e r e n c e b o o k f o r c a r b o h y d r a te c h e m is ts a n d b io c h e m is ts u sin g th e c h r o m a to g r a p h ic m e th o d s th a t a re i n d i s p e n s a b l e in t h i s f i e l d . E m p h a s i s is o n n e w e r m e t h o d s , s u c h a s h i g h - p e r f o r m a n c e liq u id c h r o m a t o g r a p h y ( H P L C ) a n d o t h e r a u t o m a t e d liq u id c h r o m a t o g r a p h y sy ste m s; a n d th e m a te r ia l in c lu d e d w as c o m p ile d m a in ly f r o m lite ra tu re p u b lis h e d d u rin g th e y e a r s 1 9 7 0 t o 1 9 7 8 . D a t a a p p e a r i n g in V o l u m e s I a n d II o f t h e

tography are

Handbook of Chroma­

n o t r e p e a t e d h e r e , b u t r e f e r e n c e s t o r e l e v a n t t a b l e s in V o l u m e s I a n d II

a r e g iv e n a t th e s t a r t o f c o r r e s p o n d i n g s e c tio n s o f th is h a n d b o o k . In s o m e cases m a t e ­ r i a l p u b l i s h e d b e f o r e 1 9 7 0 t h a t w a s o m i t t e d f r o m V o l u m e s I a n d II o f t h e s e r i e s is in c lu d e d h ere : th is a p p lie s p a rtic u la rly to th e se c tio n s d e a lin g w ith p a p e r c h r o m a to g ­ ra p h y a n d elec tro p h o resis.

THE EDITORS-IN-CHIEF G u n t e r Z w e i g , P h . D . , r e c e i v e d h is u n d e r g r a d u a t e a n d g r a d u a t e t r a i n i n g a t t h e U n i ­ v e r s i t y o f M a r y l a n d , C o l l e g e P a r k , w h e r e h e w a s a w a r d e d t h e P h . D . in b i o c h e m i s t r y in 1 9 5 2 . T w o y e a r s f o l l o w i n g h i s g r a d u a t i o n , D r . Z w e i g w a s a f f i l i a t e d w i t h t h e l a t e R . J . B l o c k , p i o n e e r in p a p e r c h r o m a t o g r a p h y o f a m i n o a c i d s . Z w e i g , B l o c k , a n d L e S tr a n g e w ro te o n e o f th e first b o o k s o n p a p e r c h r o m a to g r a p h y w h ich w as p u b lis h e d in 1 9 5 2 b y A c a d e m i c P r e s s a n d w e n t i n t o t h r e e e d i t i o n s , t h e l a s t o n e a u t h o r e d b y G u n t e r Z w e ig a n d D r . J o e S h e r m a , th e c o - E d i t o r - i n - C h i e f o f th is series.

matography ( 1 9 5 2 )

Paper Chro­

w as also tr a n s la te d in to R u ssia n .

F r o m 1 9 5 3 till 1 9 5 7 , D r . Z w e i g w a s r e s e a r c h b i o c h e m i s t a t t h e C . F . K e t t e r i n g F o u n ­ d a tio n , A n tio c h C o lle g e , Y ello w S p rin g s, O h io , w h e re he p u rs u e d res e a rc h o n th e p a th o f c a r b o n a n d s u l f u r in p l a n t s , u s i n g t h e t h e n n e w l y d e v e l o p e d t e c h n i q u e s o f a u t o r a ­ d i o g r a p h y a n d p a p e r c h r o m a t o g r a p h y . F r o m 195 7 till 1 9 6 5 , D r . Z w e i g s e r v e d a s le c ­ tu r e r a n d c h e m is t, U n iv e rs ity o f C a lifo rn ia , D av is a n d w o rk e d o n a n a ly tic a l m e th o d s f o r p e s t i c i d e r e s i d u e s , m a i n l y b y c h r o m a t o g r a p h i c t e c h n i q u e s . In 1 9 6 5 , D r . Z w e i g b e ­ c a m e D ire c to r o f L ife S cien ces, S y ra c u s e U n iv e rs ity R e se a rc h C o r p o r a t io n , N ew Y o rk ( r e s e a r c h o n e n v i r o n m e n t a l p o l l u t i o n ) , a n d in 1973 h e b e c a m e C h i e f , E n v i r o n m e n t a l F a t e B r a n c h , E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ( E P A ) in W a s h i n g t o n , D . C . D u r i n g h is g o v e r n m e n t c a r e e r , D r . Z w e ig c o n t i n u e d h is sc ie n tific w r itin g a n d e d itin g . A m o n g h i s w o r k s a r e ( m a n y in c o l l a b o r a t i o n w i t h D r . S h e r m a ) t h e n o w 1 1 - v o l u m e

Analytical Methods for Pesticides and Plant Growth Regulators ( p u b l i s h e d Journal of Toxicology and Environmental Health; c o - a u t h o r o f b a s i c r e v i e w o n p a p e r a n d t h i n l a y e r c h r o m a t o g r a p h y f o r Analytical Chemistry f r o m 196 8 t o 1 9 80; c o - a u t h o r o f a p ­ p l i e d c h r o m a t o g r a p h y r e v i e w o n p e s t i c i d e a n a l y s i s f o r Analytical Chemistry, b e g i n n i n g series o n

b y A c a d e m i c P re s s ); th e p e s tic id e b o o k series f o r C R C P r e s s ; c o - e d i to r o f

in 1 9 8 1 . A m o n g t h e s c i e n t i f i c h o n o r s a w a r d e d t o D r . Z w e i g d u r i n g h is d i s t i n g u i s h e d c a r e e r a r e t h e W i l e y A w a r d in 1 9 7 7 , R o t h s c h i l d F e l l o w s h i p t o t h e W e i z m a n n I n s t i t u t e in 1 9 6 3 / 6 4 ; t h e B r o n z e M e d a l b y t h e E P A in 1 9 8 0 . D r . Z w e i g h a s a u t h o r e d o r c o - a u t h o r e d o v e r 75 s c i e n t i f i c p a p e r s o n d i v e r s e s u b j e c t s in c h r o m a t o g r a p h y a n d b i o c h e m i s t r y , b e s i d e s b e i n g t h e h o l d e r o f t h r e e U . S . p a t e n t s . A t t h e p r e s e n t t i m e ( 1 9 8 0 / 8 1 ) , D r . Z w e i g is V i s i t i n g S c h o l a r in t h e S c h o o l o f P u b l i c H e a l t h , U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , w h e r e h e is d o i n g r e s e a r c h o n f a r m w o r k e r sa fe ty as re la te d to p e sticid e e x p o su re . J o s e p h S h e r m a , P h . D . , r e c e i v e d a B . S . in c h e m i s t r y f r o m U p s a l a C o l l e g e , E a s t O r ­ a n g e , N . J . in 1955 a n d a P h . D . in a n a l y t i c a l c h e m i s t r y f r o m R u t g e r s U n i v e r s i t y in 1 9 5 8 . H i s t h e s i s r e s e a r c h in i o n e x c h a n g e c h r o m a t o g r a p h y w a s u n d e r t h e d i r e c t i o n o f t h e l a t e W i l l i a m R i e m a n I I I . D r . S h e r m a j o i n e d t h e f a c u l t y o f L a f a y e t t e C o l l e g e in S e p t e m b e r 1 9 5 8 , a n d is p r e s e n t l y f u l l p r o f e s s o r t h e r e in c h a r g e o f t w o c o u r s e s in a n a ­ l y t i c a l c h e m i s t r y . A t L a f a y e t t e h e h a s c o n t i n u e d r e s e a r c h in c h r o m a t o g r a p h y a n d h a s a d d i t i o n a l l y w o r k e d a t o t a l o f 12 s u m m e r s in t h e f i e l d w i t h H a r o l d S t r a i n a t t h e A r g o n n e N a tio n a l L a b o r a t o r y , Illin o is, J a m e s F ritz a t Io w a S ta te U n iv e rs ity , A m e s , G u n ­ te r Z w eig a t S y ra c u s e U n iv e rs ity R e s e a rc h C o r p o r a t io n , N e w Y o rk , J o s e p h T o u c h ­ sto n e

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( E P A ) ] c o - a u t h o r e d V o l u m e s I a n d II o f t h e

b o o k o n p a p e r c h r o m a t o g r a p h y , a n d 6 v o lu m e s o f th e series

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b e in g e d ite d w ith D r. Z w e ig ,

a n d D r . S h e r m a w ill c o - a u t h o r t h e h a n d b o o k o n p e s t i c i d e c h r o m a t o g r a p h y . A b o o k o n q u a n tita tiv e T L C (p u b lish e d by W ile y -ln te rsc ie n c e , N ew Y o rk ) w as ed ited jo in tly w ith D r. T o u c h s t o n e . D r. S h e r m a h a s b e e n c o - a u t h o r o f 7 b ie n n ia l review s o f liq u id c h r o m a t o g r a p h y ( 1 9 6 8 t o 1 9 8 0 ) a n d t h e 1981 r e v i e w o f p e s t i c i d e a n a l y s i s f o r t h e j o u r ­ nal

Analytical Chemistry.

D r. S h e r m a h a s a u t h o r e d m a j o r in v ite d c h a p te r s a n d review

Chromatographic Reviews ( a n a l y s i s o f Advances in Chromatography ( a n a l y s i s o f f u n g i c i d e s ) , Advances in Chro­ matography ( a n a l y s i s o f n o n p e s t i c i d e p o l l u t a n t s ) , H e f t m a n n ’s Chromatography(c h r o ­ m a t o g r a p h y o f p e s t i c i d e s ) , R a c e ’s Laboratory Medicine ( c h r o m a t o g r a p h y in c l i n i c a l a n a l y s i s ) , Food Analysis: Principles and Techniques ( T L C f o r f o o d a n a l y s i s ) , Treatise on Analytical Chemistry ( p a p e r a n d t h i n l a y e r c h r o m a t o g r a p h y ) , a n d CRC Critical Reviews in Analytical Chemistry ( p e s t i c i d e r e s i d u e a n a l y s i s ) . A g e n e r a l b o o k o n t h i n p a p e r s o n c h r o m a t o g r a p h y a n d p e s t i c i d e s in fu n g ic id e s),

l a y e r c h r o m a t o g r a p h y c o - a u t h o r e d b y D r . S h e r m a is n o w in p r e s s a t M a r c e l D e k k e r . D r . S h e r m a s p e n t 6 m o n t h s in 197 2 o n s a b b a t i c a l l e a v e a t t h e E P A P e r r i n e P r i m a t e L a b o r a t o r y , P e r r i n e , F l a . , w ith D r. T . M . S h a f i k , a n d tw o a d d i t i o n a l s u m m e r s (19 7 5 , 1 9 7 6 ) a t t h e U . S . D e p a r t m e n t o f A g r i c u l t u r e ( U S D A ) in B e l t s v i l l e , M d . , w i t h M e l v i n G e tz d o in g re se a rc h o n p esticid e re sid u e an aly sis m e th o d s d e v e lo p m e n t. H e sp en t 3 m o n t h s in 1 9 7 9 o n s a b b a t i c a l l e a v e w i t h D r . T o u c h s t o n e d e v e l o p i n g c l i n i c a l a n a l y t i c a l m e t h o d s . A to ta l o f m o r e th a n 200 p a p e rs , b o o k s , b o o k c h a p te rs , a n d o ra l p re s e n ta ­ ti o n s c o n c e r n e d w ith c o l u m n , p a p e r , a n d th in la y e r c h r o m a t o g r a p h y o f m e ta l io n s , p la n t p ig m e n ts , a n d o th e r o rg a n ic a n d b io lo g ic a l c o m p o u n d s ; th e c h r o m a to g r a p h ic a n a ly s is o f p esticid es; a n d th e h is to ry o f c h r o m a to g r a p h y h a v e b een a u th o r e d by D r. S h e r m a , m a n y in c o l l a b o r a t i o n w i t h v a r i o u s c o - w o r k e r s a n d s t u d e n t s . H i s m a j o r r e ­ s e a r c h a r e a a t L a f a y e t t e is c u r r e n t l y q u a n t i t a t i v e T L C ( d e n s i t o m e t r y ) , a p p l i e d m a i n l y to c lin ic a l a n a ly s is a n d p e s tic id e re s id u e d e te r m i n a ti o n s . D r. S h e r m a h a s w ritte n a n a n a ly tic a l q u a lity c o n tr o l m a n u a l fo r p esticid e an aly sis u n d e r c o n t r a c t w ith th e U .S . E P A a n d h a s re v ise d this a n d th e E P A P e stic id e A n a l y t ­ ical M e t h o d s M a n u a l u n d e r a 4 - y e a r c o n t r a c t ( E P A ) j o i n tl y w ith D r . M . B e r o z a o f th e A s s o c ia tio n o f O ffic ia l A n a ly tic a l C h e m is ts ( A O A C ) . D r. S h e r m a h as also w ritte n an in s tr u m e n ta l a n a ly s is q u a lity a s s u ra n c e m a n u a l a n d o th e r a n a ly tic a l re p o r ts fo r th e U . S . C o n s u m e r P r o d u c t S a f e t y C o m m i s s i o n , a n d is c u r r e n t l y p r e p a r i n g a m a n u a l o n th e a n a ly s is o f fo o d a d d itiv e s fo r th e U .S . F o o d a n d D ru g A d m in is tr a tio n , b o th o f t h e s e p r o j e c t s a l s o in c o l l a b o r a t i o n w i t h D r . B e r o z a o f t h e A O A C . D r. S h e r m a t a u g h t th e first p r o t o t y p e s h o r t c o u rs e o n p e stic id e a n a ly s is w ith H e n ry E n o s o f t h e E P A f o r t h e C e n t e r f o r P r o f e s s i o n a l A d v a n c e m e n t . H e is e d i t o r o f t h e K o n te s T L C q u a r te r ly n e w s le tte r a n d also te a c h e s s h o r t c o u rs e s o n T L C fo r K o n te s a n d t h e C e n t e r f o r P r o f e s s i o n a l A d v a n c e m e n t . H e is a c o n s u l t a n t f o r s e v e r a l i n d u s t r i a l c o m p a n ie s a n d fe d eral ag en cies o n c h e m ic a l an aly sis a n d c h r o m a to g r a p h y a n d re g u ­ larly refe re e s p a p e r s fo r a n a ly tic a l jo u r n a ls a n d re s e a rc h p ro p o s a ls fo r g o v e rn m e n t agencies. D r. S h e r m a h a s receiv ed tw o a w a r d s fo r s u p e r io r te a c h in g at L a fa y e tte C o lleg e a n d th e 1979 D is tin g u is h e d A l u m n u s A w a r d f r o m U p s a la C o lle g e fo r o u ts ta n d i n g a c h ie v e ­ m e n t s a s a n e d u c a t o r , r e s e a r c h e r , a u t h o r , a n d e d i t o r . H e is a m e m b e r o f t h e A m e r i c a n C h e m ic a l S o c ie ty , S ig m a X i, P h i L a m b d a U p s ilo n , S o ciety fo r A p p lie d S p e c tro s c o p y , a n d th e A m e ric a n In s titu te o f C h e m ists.

THE EDITOR Shirley C. Churms, Ph.D.,

is R e s e a r c h A s s o c i a t e in t h e C a r b o h y d r a t e C h e m i s t r y

R e s e a r c h U n it ( s p o n s o r e d by th e S o u t h A f r i c a n C o u n c il fo r S c ie n tific a n d In d u s tria l R e se a rc h ) o f th e D e p a r tm e n t o f O rg a n ic C h e m is try at th e U n iv ersity o f C a p e T o w n , R e p u b lic o f S o u th A fric a . D r. C h u r m s (b o r n M a c in to s h ) w as e d u c a te d at R u s te n b u r g S ch o o l, R o n d e b o s c h , C a p e T o w n a n d th e U n iv e rs ity o f C a p e T o w n , w h e re she o b ta in e d th e d e g re e s o f B .S c. ( w i t h d i s t i n c t i o n in c h e m i s t r y ) in 19 5 7 a n d B . S c . ( H o n s ) in 1958. S h e t h e n u n d e r t o o k r e s e a r c h , a t t h e s a m e U n i v e r s i t y , o n c a t i o n - e x c h a n g e p r o c e s s e s in a q u e o u s m o n o e t h a n o l a m i n e , f o r w h i c h t h e d e g r e e o f P h . D . w a s a w a r d e d in 1 9 6 2 . D u r i n g t h i s p e r i o d s h e s e r v e d a s a J u n i o r L e c t u r e r , a n d in 1961 a s L e c t u r e r in a t e m p o r a r y c a p a c i t y , in t h e D e p a r t m e n t o f C h e m i s tr y . In 1962 sh e w a s a w a r d e d th e O h io S ta te F e llo w s h ip b y th e I n t e r n a t i o n a l F e d e r a t i o n o f U n iv e rs ity W o m e n , w h ic h e n a b le d h e r to s p e n d h e r p o s t ­ d o c t o r a l y e a r a t t h e I m p e r i a l C o l l e g e o f S c i e n c e a n d T e c h n o l o g y in L o n d o n , E n g l a n d . W h il e th e r e sh e c a r r i e d o u t a n e x te n s iv e s u r v e y o f th e p r o p e r tie s o f in o r g a n ic io n e x c h a n g e r s in g e n e r a l a n d c o m m e n c e d a n i n v e s t i g a t i o n o f t h e i o n - e x c h a n g e p r o p e r t i e s o f h y d r a t e d a l u m i n a in p a r t i c u l a r . T h i s w o r k w a s c o n t i n u e d a f t e r h e r r e t u r n t o t h e U n i v e r s i t y o f C a p e T o w n in 1 9 6 4 , a n d d u r i n g t h e p e r i o d 1 9 6 4 t o 1968 D r . C h u r m s w a s a l s o i n v o l v e d in t h e s u p e r v i s i o n o f r e s e a r c h s t u d e n t s w h o w e r e e x t e n d i n g h e r o w n e a r l i e r w o r k o n i o n - e x c h a n g e in n o n a q u e o u s s o l v e n t s . A n o t h e r r e s e a r c h p r o j e c t , c o n ­ c e rn e d w ith f u n d a m e n ta l a sp e c ts o f c a tio n e x c h a n g e o n th e h ig h -c a p a c ity c a rb o x y lic a c i d r e s i n s , t o w h i c h s h e m a d e a m a j o r c o n t r i b u t i o n r e s u l t e d in t h e p u b l i c a t i o n , in 1967, o f a p a p e r fo r w h ic h she w as a w a rd e d , w ith tw o c o -a u th o rs , th e A fric a n E x p lo ­ sives a n d C h e m i c a l I n d u s t r y m e d a l c o n f e r r e d a n n u a l l y b y th e S o u t h A f r i c a n C h e m i c a l In stitu te . I n 1 9 6 5 a n d 1 9 6 7 D r . C h u r m s h e l d t e m p o r a r y l e c t u r i n g p o s t s in t h e D e p a r t m e n t o f C h e m i s t r y a t t h e U n i v e r s i t y o f C a p e T o w n , w h i l e in 1 9 6 4 a n d

196 6 h e r w o r k w a s

s u p p o r te d b y g ra n ts a d m in is te r e d by th e S o u th A fr ic a n C o u n c il fo r S cien tific a n d I n ­ d u s tria l R e s e a rc h . In 1968, s h o rtly a f te r th e la tte r b o d y h a d c o m m e n c e d s u p p o r t o f a C a r b o h y d r a t e C h e m is tr y R e s e a rc h U n it, u n d e r th e d ire c tio n o f P r o f e s s o r A lis ta ir M . S t e p h e n , a t t h e U n i v e r s i t y o f C a p e T o w n , s h e a s s u m e d h e r p r e s e n t p o s i t i o n in t h i s U n it. H e r i n v o l v e m e n t i n c a r b o h y d r a t e s t u d i e s r e s u l t e d in a c h a n g e o f r e s e a r c h i n t e r e s t fro m ion e x c h a n g e

per se t o

t h e a p p l i c a t i o n o f c h r o m a t o g r a p h i c m e t h o d s , a n d in p a r ­

tic u la r g e l- p e rm e a tio n c h r o m a to g r a p h y , to th e e x a m in a tio n o f p o ly sa c c h a rid e s a n d o f t h e c o m p l e x m i x t u r e s o f p r o d u c t s o b t a i n e d in t h e d e g r a d a t i o n p r o c e s s e s u s e d f o r i n ­ v e stig a tio n o f th e m o le c u la r stru c tu re s o f p o ly s a c c h a rid e s. D r. C h u rm s has c o n trib u te d c h a p t e r s o n t h i s t o p i c t o t w o b o o k s a l r e a d y p u b l i s h e d , a n d a f u r t h e r t w o a t p r e s e n t in th e c o u r s e o f p u b l i c a t i o n . S h e w a s a ls o th e a u t h o r o f a series o f five re v ie w a rtic le s o n i n o r g a n i c i o n - e x c h a n g e r s , p u b l i s h e d in 1 9 6 5 , a n d t h e r e s e a r c h p a p e r s o f w h i c h s h e h a s b een a u th o r o r c o - a u th o r n u m b e r a p p ro x im a te ly 40. D r . C h u r m s is a M e m b e r o f t h e R o y a l S o c i e t y o f C h e m i s t r y ( L o n d o n ) a n d is t h u s a C h a r t e r e d C h e m i s t . S h e is a l s o a m e m b e r o f t h e S o u t h A f r i c a n C h e m i c a l I n s t i t u t e a n d o f th e in te rd is c ip lin a r y E x p e r im e n ta l B io lo g y G r o u p o f th e C a p e , a n d h as serv ed as S e c re ta ry o f th e la tte r a ss o c ia tio n .

ADVISORY BOARD Kirsti Granath, D.Sc.

Michele Lato, Ph.D.

D e p a rtm e n t o f P o ly m e r P hysical

D irecto r

C h e m istry

Istitu to C lin ic a P e d ia tr ic a

P h a r m a c ia F in e C h e m ic a ls

U n i v e r s i t a di P e r u g i a

U p p sala, S w eden

P e r u g i a , Italy

Leslie Hough, D.Sc., F.R.I.C.

Frederic M. Rabel, Ph.D.

D e p a rtm e n t o f C h em istry

S ta f f S c ien tist

Q u e e n E liz a b e th C o lleg e

W h a t m a n Inc.

L o n d o n , E n g lan d

C lifto n , N ew Jersey

CONTRIBUTORS John E. Brewer, Ph.D.

Edwin H. Merrifield, Ph.D.

P roduct G ro u p M anager

R esearch A ssista n t

C h ro m a to g ra p h ic M ed ia

C .S .I.R . C a rb o h y d ra te C h e m istry

P h a r m a c ia F in e C h e m ic a ls U p p sala, S w eden

R e se a rc h U n it D e p a rtm e n t o f O rg a n ic C h em istry U n iv ersity o f C a p e T o w n

Michele Ghebregzabher, Ph.D.

R ep u b lic o f S o u th A fric a

D ietitian A ssista n t Is titu to di C lin ica P e d ia tric a

Frederic M. Rabel, Ph.D.

U n iv ersita di P e ru g ia

S ta f f S cien tist

P e ru g ia , Italy

W h a tm a n Inc. C lifto n , N ew Jersey

Kirsti Granath, D.Sc. D e p a rtm e n t o f P o ly m e r P hysical C h e m istry P h a r m a c ia F in e C h e m ic a ls U p p sa la , S w eden

Stefano Rufini, Ph.D. A ssista n t L e c tu re r I s titu to di C lin ic a P e d ia tric a U n iv e rs ita di P e ru g ia P e ru g ia , Italy

Leslie Hough, D.Sc., F.R.I.C. D e p a rtm e n t o f C h em istry

Ramon L. Sidebotham, Ph.D.

Q u e e n E liz a b e th C o lleg e

D e p a rtm e n t o f C h e m istry

L o n d o n , E n g la n d

Q u e e n E liz a b e th C o lleg e L o n d o n , E n g lan d

Michele Lato, Ph.D. D ire c to r

Helmut Weigel, Ph.D.

Is titu to di C lin ic a P e d ia tric a

D e p a rtm e n t o f C h e m istry

U n iv ersita di P eru g ia

R o y a l H o llo w a y C o lleg e

P e ru g ia , Italy

S urrey, E n g la n d

ACKNOWLEDGMENTS I w o u l d l i k e t o t h a n k t h e m e m b e r s o f t h e A d v i s o r y B o a r d a n d a ll o t h e r c o n t r i b u t o r s fo r th e ir in v a l u a b le h e lp a n d a d v ic e d u r i n g th e c o m p i l a t i o n o f th is h a n d b o o k . T h e k in d n e s s o f D r. D . A n d e r le (I n s ti tu t e o f C h e m i s tr y , S lo v a k A c a d e m y o f S cience s, B r a ­ tisla v a , C z e c h o s la v a k ia ) , P r o f e s s o r N . K. K o c h e tk o v (N . D . Z e lin sk y In s titu te o f O r ­ g a n ic C h e m i s tr y , U S S R A c a d e m y o f S cien ce s, M o s c o w ) a n d P r o f e s s o r M . T o rii ( R e ­ s e a r c h I n s t i t u t e f o r M i c r o b i a l D i s e a s e s , O s a k a , J a p a n ) in m a k i n g a v a i l a b l e c o p i e s o f c e r t a i n m a t e r i a l f r o m t h e i r p u b l i c a t i o n s n o t o t h e r w i s e a c c e s s i b l e t o m e , is a p p r e c i a t e d . S p e c i a l t h a n k s a r e d u e t o M i s s M a r g a r e t D r e y e r f o r h e r s t e r l i n g e f f o r t s in t y p i n g m o s t o f th e m a n u s c r ip t o f this h a n d b o o k . T h e a ss is ta n c e o f M s. E le a n o r S te n to n a n d M r s . J e a n G o o d e in t h e t y p i n g is a l s o g r a t e f u l l y a c k n o w l e d g e d , a s is t h e c o o p e r a t i o n o f th e e d ito ria l sta ff o f C R C P ress.

D E D IC A T IO N M y i n t e r e s t in c a r b o h y d r a t e c h e m i s t r y in g e n e r a l , a n d in c a r b o h y d r a t e c h r o m a t o g ­ r a p h y in p a r t i c u l a r , h a s d e v e l o p e d u n d e r t h e i n s p i r i n g g u i d a n c e o f P r o f e s s o r A l i s t a i r M . S t e p h e n , t o w h o m t h i s v o l u m e is d e d i c a t e d . S h irle y C . C h u r m s A p r i l 1979

TABLE OF CONTENTS CARBOHYDRATES VOLUME I SECTION I — C H ROM A TO GRA PH IC DATA 1.1

G a s C h r o m a t o g r a p h y T a b l e s ................................................................................................................3

1.11

L i q u i d C h r o m a t o g r a p h y T a b l e s ......................................................................................................6 9

1.111

P a p e r C h r o m a t o g r a p h y T a b l e s .....................................................................................................129

I.IV

T h i n - L a y e r C h r o m a t o g r a p h y T a b l e s ..........................................................................................137

I.V

E l e c t r o p h o r e t i c D a t a .............................................................................................................................155

SECTION II — DETECTION TECHNIQUES 11.1

D e t e c t i o n a n d I d e n t i f i c a t i o n o f C a r b o h y d r a t e s in G a s C h r o m a t o g r a p h y . . . . 171

11.11

D e t e c t i o n M e t h o d s f o r L i q u i d C h r o m a t o g r a p h y ...............................................................175

11.111

D e t e c t i o n R e a g e n t s f o r P a p e r a n d / o r T h i n - L a y e r C h r o m a t o g r a p h y .................... 187

SECTION III — SAM PLE PREPARATION AND DERIVATIZATION SECTION IV — PRODUCTS AND SOURCES OF CH ROM A TO GRA PH IC MATERIALS SECTION V — LITERATURE REFERENCES IN D E X

267

Section I Chromatographic Data

Carbohydrates

3

S e c t i o n 1.1

GAS CHROM ATOGRAPHY TABLES T h e s e ta b le s a re g r o u p e d a c c o rd in g to th e ty p e o f v o latile d e riv a tiv e p re p a r e d fo r G C o f t h e c a r b o h y d r a t e s , w i t h t h e d e r i v a t i v e s a r r a n g e d in a l p h a b e t i c a l o r d e r . S o m e e a r l i e r G C d a t a f o r a l d i t o l a c e t a t e s , i s o p r o p y l i d e n e a c e t a l s , t r i f l u o r o a c e t a t e s , a n d tr im e t h y l s i l y l e t h e r s o f c a r b o h y d r a t e s w ill b e f o u n d in S e c t i o n A , V o l u m e I, S e c t i o n I I . I , T a b l e s G C 1 3 - 1 5 , 7 6 , a n d 124. T h e c o - o p e r a t i o n o f D r . E . H . M e r r i f i e l d ( U n i v e r s i t y o f C a p e T o w n ) in t h e c o m p i ­ l a t i o n o f T a b l e s G C 1 , G C 2 , a n d G C 2 2 is g r a t e f u l l y a c k n o w l e d g e d .

4

CRC Handbook of Chromatography Table GC 1 PERACETYLATED ALDITOLS, AMINODEOXYALDITOLS, AND REDUCED OLIGOSACCHARIDES PI 220 N 2, 40

P2 T1 N2, 34

P3 190 N2, 40

P4 240 He, 100

P5 260 N 2, 20

152 0.6 na glass FI 1

150 0.3 na glass FI 2

170 0 .6 (0 .D.) coiled glass FI 3

152 0.6 U-tube glass FI 4

183 0.3 na glass FI 5

Parent sugar

r“

r“

r“

Tb

rc

Rhamnose Fucose Ribose Arabinose Xylose Mannose Galactose Glucose 2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 2-Amino-2-deoxy-D-mannose 2-Amino-2-deoxy-D-gulose 2-Amino-2-deoxy-D-talose Sucrosed Trehalosed Maltose Lactose

_

_

0.72 0.80

_

_













Packing Tem perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

0.76



0.88 1.00 1.23 2.32 2.75 2.68 — —



1.00 1.24 2.11 2.27 2.43 4.7 5.1



1.00 1.32 2.76 3.04 3.24 — —













1.00 2.87 3.32 3.54 3.59 2.69



















































— — — — — —

0.83 1.00 1.34 1.65

Note: This table supplements Table GC 14 in Section A, Volume I, Section II.I. ° 6 c *

tr relative to arabinitol pentaacetate (absolute retention 6.4 min for Reference 1, 9.1 min for 2, 5 min for 3). tr relative to glucitol hexaacetate (8 min). t, relative to trehalose derivative (79 min). Peracetylated derivatives of sugars.

Packing

PI P2 P3 P4

P5 Tem perature T1

5% polyethylene glycol adipate) on acid-washed Diatomite C (85 to 100 mesh BSS). 1% OV-225 on Chrom osorb G-H P (80 to 100 mesh). 3% OV-225 on Chrom osorb W, acid-washed, DMCS (80 to 100 mesh). 10% neopentyl glycol sebacate polyester on acid-washed C hrom osorb W (80 to 100 mesh). 3% OV-225 on Gas-Chrom Q (100 to 120 mesh). tem perature program m ed, 170 -*■ 230°C, 1°/m in to end tem perature, 15 min isothermal.

REFERENCES 1. Holligan, P. M. and Drew, E. A., Routine analysis by gas-liquid chrom atography of soluble carbohydrates in extracts of plant tissues. II. Quantitative analysis of standard carbohydrates, and the separation and estimation of soluble sugars and polyols from a variety of plant tissues, New P hytol.,10, 271, 1971.

Carbohydrates Table GC 1 (continued) PERACETYLATED ALDITOLS, AMINODEOXYALDITOLS, AND REDUCED OLIGOSACCHARIDES 2. Metz, J., Ebert, W ., and Weicker, H ., Gas chrom atographic analysis of protein and carbohydrate portions of erythrocyte membrane glycoproteins, Clin. Chim. A cta , 34, 31, 1971. 3. Merrifield, E. H ., unpublished laboratory data, 1973. 4. Perry, M. B. and W ebb, A. C ., Analysis of 2-amino-2-deoxyhexoses by gas-liquid parti­ tion chrom atography, Can. J. Biochem., 46, 1163, 1968. 5. Schwind, H ., Scharbert, F ., Schmidt, R ., and K atterm an, R ., Gas chrom atographic de­ term ination of di- and trisaccharides, J. Clin. Chem. Clin. Biochem ., 16, 145, 1978.

5

6

Table GC 2 ALDITOL ACETATES DERIVED FROM PARTIALLY METHYLATED SUGARS

Parent sugar

P4 PI P2 P3 190 170 200 245 Ar, 20 Ar, 100 He, 100 N 2,2 0 —25

152 120 120 0.5 0.5 0.3 straight straight U-tube glass glass glass D1 D1 FI 2 ,4 2, 3 1,2

P5 170 n 2, 15—:

P10

P4

P6

P7

P8

P4

P4

P9

T1

T1

T2

T3

T4

175

180

170

H e, 60

H e, 60

H e, 6

N j, 50

na

na

N 2, 7 0 — 80

N 2, 4 0

150 0.4 na na FI 10

180 0.3 na glass FI 11

180 0.3 na glass FI 11

0.22 na glass FI 12

r*

Td

T d

rf

Td



1500 120 120 0.3 (O.D.) 0.3 (O.D.) 0.051 SCOT na na copper ss copper FI FI FI 9 9 9

180 0.2 na glass FI 5—7

180 0.15 na glass FI 7, 8

Td

Td

r'

r*

1.56

1.47

1.38

1.69







1.60

1.51

1.44

1.65







2 2 0

170 0 .6 ( 0 .D coiled glass FI 13



Tb

rc

2-O-methyl 3-O-methyl

4.70 4.95













1.39 1.48

4-O-methyl

4.90









5-O-methyl 2,3-di-O-methyl

3.23







2.56





— 1.07

1.14

1.10

1.03

1.19

-

-

0.74

-







— 2.76

— —

— —

— 1.40 1.10 1.38

1.10 0.84

1.00

1.00

0.91

1.06





0.61









0.77



1.95 1.32





0.91





1.00





r*

L-Arabinose

2,4-di-O-methyl 2,5-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 2,3,4-tri- O-methyl 2,3,5-tri-O-methyl D-Xylose





0.91

0.92

0.85

0.96





0.63

-

0.73

— 0.80 0.54

0.68

0.73

0.65

0.74





0.57



0.48

0.41

0.52

0.56

0.53

0.60





0.46





2-O-methyl







2.92

2.15

1.65

_

1.50

1.52

_

_

_



3-O-methyl







2.92

2.15

1.65

1.55

1.50

1.84









4-O-methyl 2,3-di-O-methyl







2.92

2.15

1.65



1.50

1.52















1.54

1.19

1.22

1.16

1.09

1.31





0.74

2,4-di-O m ethyl









1.02

1.12















1.06 1.19

1.16

3,4-di-O m ethyl

1.34 1.54

1.22

1.16

1.09

1.31









3,5-di-O m ethyl 2,3,4-tri- Omethyl













1.08 0.68

— 0.54

0.68

0.71

0.66

0.76

_

_

0.56

_

-

CRC Handbook of Chromatography

Phase Tem perature (°C) Gas; flow rate ( m l/ min) Colum n Length, cm D iam eter (I.D .), cm Form M aterial Detector Reference

D-Ribose 3,5-di-O-methyl 2,3,5-tri-O-methyl D-Glucose







0.77













_

__

__







0.40

















__

2-O-methyl



6.6 7.6

_

_

_



__

__

__



1.60 1.81



3-O-methyl





_

_

_

__

__

__ __

4-O m ethyl



6-O-methyl

— —

2,3-di-O-methyl

2.00 1.14 1.00



— — — —

7.9 9.6 11.5 5.62 5.39 5.10

2,4-di-O-methyl



1.00



2,6-di-O-methyl





3,4-diO-methyl



0.75 0.97

3,6-di-O-methyl 4,6-di-O-methyl



0.82







2 ,3 ,4-tri- O-methyl



0.80 0.47

4.40 4.02



2,3,6-tri-O-methyl 2 ,4,6-tri- O-methyl



0.48





0.41



— —

0.39 0.24

— —

— — — — —

— — — — —



3-O-methyl 4 -O-methyl 6-O-methyl 2,3-di-O-methyl 2,4-di-O-methyl



2 ,5-di-O-methyl

3,4,6-tri-O-methyl 2,3,4,6-tetra-O-methyl D-Galactose 2-O-methyl



3.83 5.27

8.4











_

_

5.0 4.50 4.21







2.45



_

2.14

2.01

_

1.98

2.09 1.98

_

2.10

1.90 1.87

— —

3.38

1.88

1.73

1.81

1.86

4.26



1.96 1.94

2.49

3.73 3.49 2.22

2.50

2.32

1.63

1.95 1.98

1.82

1.44

1.83 1.00

0.98

1.00 8.1

1.58



_

__

1.39



_

_

1.72



1.91





_

1.00

1.00





_



0.99

— —

— —



6.35

5.1

2.19 2.27







5.8

4.65



2,6-di-O-methyl







3.65

3.14

1.88

3,4-di-O-methyl

— —

— —

6.93 6.35

5.5 5.1

2.32

3,5-di O-methyl

— —

3,6-di-O-methyl









1.99

1.81









3.28

2.76

2,3,6-tri-O-methyl 2 ,4,6-tri- O-methyl







2.42

2.22

1.59







2.28

2.03

1.55

2 ,5,6-tri-O-methyl

— —

— —



2.25

1.95



2.50

2.15

3 ,4,6-tri-O-methyl





1.61



2.22

__

__

2.52 2.52

__

1.75 1.83 2.19 _

__

__

1.44 _

__

_ —

__

1.95

2.04

2.12

2.03 —

2.09 —

2.28 —

_

__



_

1.72

1.77

2.34

_

_

2.05 _

2.14 —

2.52 —

— _

_ __

__

__

1.87

2.56



__

1.57

__





1.67 _

1.70

1.48 1.47

1.52 1.47



1.49





1.52

__

__ __

__





1.54



_

1.61



_

1.59





1.17

__

1.56



_

1.98



1.57 —



1.82

_





1.35 —



__

__

__ __

__

_

1

2,3,5-tri-O-methyl



1.82

— — — — _

_

1.00

Carbohydrates

4.7



2.89

__

__



— — —

3.41

__



_ — —



_











— — —



__



1.65 1.18





__

__

1.54

— — —



__

_

1.52









1.56

1.50 1.38





2,3 ,4 ,-tri-O-methyl



2.22



4,6-di-O-methyl

_ _







_ __

1.88 1.84



4.35 3.64

— —



11.1 11.1 5.10 5.68



— —



1.76 1.47



_

3.24

L en gth , cm D ia m e te r ( I . D . ) , cm F or m

P2 PI P4 P3 P5 200 190 170 170 245 Ar, 20 Ar, 100 He, 100 N2, 20—25 N2, 15—20

120 0.5

120 0.5 straight straight

152

180

180

0.3

0.2

0.15

Material D e te ct o r

glass D1

glass

U -t u be glass

na glass

glass

D1

FI

R e fe re nce

1,2

2, 3

2 ,4

FI 5— 7

ra

r6

rc

_

_

_





— — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — —

P a r e n t sugar 2,3,4,6-tetra- O-methyl 2,3,5,6-tetra-O-methyl D-Mannose 2 -O-methyl 3-O-methyl 4 - O-methyl 5- O-methyl 6- O-methyl 2,3-di-Omethyl 2,4-di-Omethyl 2,5-di-Omethyl 2,6-di-Omethyl 3,4-di-Omethyl 3,5-di-Omethyl 3,6-di-Omethyl 4,6-di-Omethyl 2,3,4-tri-O-methyl 2,3,5-tri-O-methyl 2,3,6-tri-O-methyl 2,4,6-tri-O-methyl 2,5,6-tri-O-methyl

na

P4 T1 He, 60

P6 T1 He, 60

120 120 0.3 (O . D .)i 0.3 ( O . D .)

P7 T2 He, 6

P8 T3 N2, 50

P4 T4 na

1500

150 0.4

0.051 SCOT

na

P4 175 na

P9 180 N2, 70—80

P10 170 N2, 40

180

180

0.3

0.3

220 0.2 2

170 0 .6 ( O . D . )

na

na

coiled

glass FI

glass

glass

FI

FI

na copper

na copper

FI 7, 8

FI

FI

r“

Td

r'

r'

1.25 1.15

1.19 1.10

1.14

1.13

1.10

1.16

_













— — — — — — — — — — — — — — — — — —

7.0 8.8 8.8 — 4.48 4.83 5.44 — 3.35 5.37 5.44 4.15 3.29 2.48 — 2.20 2.09 —

5.64 6.8 6.8 — 4.1 3.69 4.51 — — 4.36 4.51 3.67 2.92 2.19 — 2.03 1.90 —

— — — — — 2.06 — — — — — — — 1.59 — 1.53 1.47 —

— — — — — — — — 1.67 1.87 — — — 1.49 — 1.44 1.40 —

— — — — — — — — — — —

— — — — — -— — 2.08 2.09 — — — 1.62 — 1.40 1.45 —

3.44 4.25 — 3.44 2.39 2.52 — 2.09 1.96 — 2.71 2.25 — — 1.74 1.50 — 1.35

9

ss FI 9

9 r'



— — — — — —

na

na glass

FI

FI

10

11

11

12

13

r'

Td

Td

r'

Td

_

1.00

1.14







7.26 9.58 — 7.26 4.45 4.80 — 3.66 3.35 — 5.30 4.12 — — 2.78 2.17 — 1.79

— — — — — — — — — — — — — — — — — —

— — — — 3.66 — — — — — — — 2.69 — — — 1.89 —

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

Phase Temperature (°C) Gas; flow rate ( m i / min) Column

8

Table GC 2 (continued) ALDITOL ACETATES DERIVED FROM PARTIALLY METHYLATED SUGARS

— — —

— — —

— — —

1.95 1.00 —

1.82 0.99 —

1.44 1.00 —

1.37 1.00 —

— 1.00 —

— 1.00 —

— — 1.03

— — 1.02

— — —

1.64 0.98 —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

1.67 2.05 2.08 — 1.18 1.12 — 0.65 0.62

1.43 — 1.71 1.33 — 1.02 0.96 0.58 0.47

1.33 — — — — 1.06 — 0.69 —

— — — — — — — 0.71 —

— — — — — — — 0.69 —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

— — — — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

1.52 1.94 1.72 0.98 0.99 0.92 0.46

1.37 1.67 1.57 0.92 0.94 0.87 0.35

1.26 1.38 — 0.97 — 0.92 0.50

1.18 1.33 — 0.97 — 0.92 0.55

1.21 1.33 — 0.95 — 0.90 0.55

— — — — — — —

— — — — — — —

— — — — — — —

0.84 0.96 — — 0.78 0.72 0.55

— — 1.33 0.83 0.89 0.77 0.46







0.32

























0.34

























0.29



















— — — — — — —

— — — — — — —

0.72 0.67 0.59 0.54 0.33 0.51 0.29

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

Carbohydrates

3,4,6-tri-O-methyl 2,3,4,6-tetra-O-methyl 2,3,5,6-tetra-O-methyl L-Fucose 2-O-methyl 3-O-methyl 4-O-methyl 5-O-methyl 2,3-di-O-methyl 2,4-di-O-methyl 3,5-di-O-methyl 2,3,4-tri-O-methyl 2,3,5-tri- O-methyl L-Rhamnose 2-O-methyl 3-O-methyl 4- O-methyl 2,3-di-Om ethyl 2,4-di- Omethyl 3,4-di-Om ethyl 2,3,4-tri-Om ethyl D-xy/o-Hexose,3,6dideoxy 2,4-di-Om ethyl D-r/6o-Hexose,3,6dideoxy 2,4-di- Omethyl D-arab/no-Hexose,3,6dideoxy 2,4-di- Omethyl 2-Amino-2-deoxy-D-glucose 3-O-methyl 4- Omethyl 6 -Omethyl 3,4-di-Om ethyl 3,6-di-Om ethyl 4 ,6-di-Om ethyl 3,4,6-di-O m ethyl

VO

Phase

Temperature

Detector

PI P2 P3 P4 P5 P6 P7 P8 P9 P10 T1 T2 T3 T4 D1

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

d r 1

tr relative to 1,5-di-O acetyl-2,3,4,6-tetra- Omethyl-D-glucitol (about II min in Reference 5, 10 min in Reference 12). tr relative to 1,5-di- O-acetyl-2,3,4,6-tetra- O-mcthyl-D-mannitol. tr relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-galactitol (14.8 min).

15% LAC-4R-886 on Chromosorb W (100 to 120 mesh). 10% LAC-4R-886 on Chromosorb W (60 to 80 mesh). 10% neopentyl glycol sebacate polyester on acid-washed Chromosorb W (80 to 100 mesh). 3% ECNSS-M on Gas-Chrom Q (100 to 120 mesh). 3% OV-225 on Gas-Chrom Q (100 to 120 mesh). mixture o f 0.2% polyethylene glycol adipate), 0.2% polyethylene glycol succinate) and 0.4% silicone XF-1150 on Gas-Chrom P (100 to 120 mesh). OV-225 SCOT capillary column (Perkin-Elmer) mixture o f 0.3% OV-275 and 0.4% XF-1150 on Gas-Chrom Q (100 to 120 mesh). 15% Apiezon T on Gas-Chrom Q (100 to 120 mesh). 3% OV-17 on acid-washed Chromosorb W (80 to 100 mesh). temperature programmed, 110 180°C at 1°C/m in. temperature programmed, 150 -* 190°C at0.5°C /m in. temperature 120°C for 5 min, then 120— 180°C at 1°C/m in. temperature programmed, 140-* 185°Cat 1.5°C/m in. /3-ionization (90Sr). REFERENCES

1. 2.

Williams, S. C. and Jones, J. K. N ., The synthesis, separation and identification of the methyl ethers of arabinose and their derivatives, Can. J. Chem., 45, 275, 1967. Jones, H. G ., Gas-liquid chromatography of methylated sugars, in M ethods in Carbohydrate Chemistry, Vol. 6, Whistler, R. L. and BeMiller, J. N ., Eds., Academic Press, New York, 1972, 25. 3. Jones, H. G. and Jones, J. K. N ., Separation and identification of methyl ethers of D-glucose and D-glucitol by gas-liquid chromatography, Can. J. Chem., 47, 3269, 1969. 4. Perry, M. B. and Webb, A . C ., Chromatographic analysis of methyl ethers of 2-amino-2-deoxy-D-glucopyranose, Can. J. Chem., 41, 4091, 1969. 5. Bjorndal, H ., Lindberg, B ., and Svensson, S., Gas-liquid chromatography of partially methylated alditols as their acetates, Acta Chem. Scand., 21, 1801, 1967. 6. Bjorndal, H ., Hellerqvist, C. G., Lindberg, B., and Svensson, S., Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides, Angew. Chem. Int. Ed. Engl.,9 , 610, 1970. 7. Jansson, P. E., Kenne, L., Liedgren, H ., Lindberg, B., and Lonngren, J., A practical guide to the methylation analysis of carbohydrates, Chem. Commun. Univ. S to ck h o lm ,8, 1976. 8. Lonngren, J. and Pilotti, A ., Gas-liquid chromatography of partially methylated alditols as their acetates. II, Acta Chem. Scand.,25, 1144, 1971. 9. Talmadge, K. W ., Keegstra, K., Bauer, W. D ., and Albersheim, P., The structure of plant cell walls. I. The macromolecular components of the walls of suspensioncultured sycamore cells with a detailed analysis of the pectic polysaccharides, Plant Physiol. ,51, 158, 1973. 10. Darvill, A. G., Roberts, D. P., and Hall, M. A ., Improved gas-liquid chromatographic separation of methylated acetylated alditols, J. Chromatogr., 115, 319, 1975. 11. W oolard, G. R. and Rathbone, E. B ., Gas-liquid chromatography of methylated cr-D-mannosides and their O-methyl ethers. I. Acetylated methyl glycosides and alditols derived from methyl a-D-mannofuranoside, 5. A fr. J. C hem .,30, 69, 1977. 12. Parolis, H. and McGarvie, D ., G.l.c. o f partially methylated alditol acetates, Carbohydr. Res.,62, 363, 1978. 13. Merrifield, E. H ., laboratory data, 1979.

CRC Handbook of Chromatography

tr relative to 1,4-di-O-acetyl-2,3,5-tri-Omethyl-l-arabinitol (4.4 min). tr relative to 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl-D-glucitol (29 min). tr relative to 2-acetamido-l ,3 ,4 ,5 ,6-penta-Oacetyl-2-deoxy-D-glucitol.

h r

10

Table GC 2 (continued) ALDITOL ACETATES DERIVED FROM PARTIALLY METHYLATED SUGARS

Carbohydrates Table GC 3 ALDITOL ACETATES DERIVED FROM PARTIALLY ETHYLATED SUGARS Phase Tem perature Gas; flow rate (m i/m in ) Column Length, cm Diameter (O .D .), cm Form

PI T1 He, 60

P2 T1 He, 60

P3 T2 He, 60

P4 T3 He, 60

120 0.3 na

120 0.3 na

120 0.3 na

M aterial Detector

copper FI

copper FI

copper FI

1520 0.051 (I.D .) open tubular SS FI

Parent sugar L-Arabinose 2-O ethyl 3-O-ethyl 4-O-ethyl 5-O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 2,5-di-O-ethyl 3,4-di-O-ethyl 3,5-di-O-ethyl 2,3,4-tri-O-ethyl 2,3,5-tri-O-ethyl D-Xylose 2- O-ethyl 3-O-ethyl 4-O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 3,4-di-O-ethyl 2,3,4-tri-O-ethyl D-Glucose 2 -O-ethyl 3-O-ethyl 4-O-ethyl 6 -O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 2,6-di-O-ethyl 3,4-di-O-ethyl 3,6-di-O-ethyl 4,6-di-O-ethyl 2,3,4-tri-O-ethyl 2,3,6-tri- O-ethyl 2,4,6-tri-O-ethyl 3,4,6-tri-O-ethyl 2,3,4,6-tetra- O-ethyl D-Galactose 2 -O-ethyl 3-O-ethyl 4-O-ethyl 6 -O-ethyl 2,3-di-O-ethyl

r“

r*



r*



Tb



r*

0.43 0.45 0.41 0.34 0.26 0.27 0.23 0.26 0.20 0.11 0.09

1.64 1.73 1.56 1.31 1.00 1.04 0.88 1.00 0.76 0.40 0.32

0.47 0.49 0.47 0.38 0.30 0.32 0.27 0.30 0.24 0.15 0.11

1.57 1.62 1.54 1.29 1.00 1.04 0.89 1.00 0.80 0.48 0.36

0.55 0.57 0.56 0.47 0.41 0.41 0.37 0.41 0.34 0.23 0.20

1.36 1.42 1.39 1.17 1.00 1.00 0.92 1.01 0.85 0.56 0.49

0.53 0.55 0.54 0.47 0.41 0.42 0.38 0.41 0.36 0.27 0.24

1.32 1.36 1.34 1.17 1.01 1.03 0.94 1.01 0.89 0.67 0.59

0.48 0.48 0.48 0.29 0.27 0.29 0.11

1.83 1.82 1.83 1.09 1.00 1.09 0.43

0.51 0.51 0.51 0.33 0.31 0.33 0.15

1.68 1.68 1.68 1.06 1.00 1.06 0.48

0.60 0.60 0.60 0.43 0.41 0.43 0.24

1.49 1.49 1.49 1.07 1.00 1.07 0.59

0.57 0.57 0.57 0.43 0.40 0.43 0.25

1.45 1.45 1.45 1.08 1.01 1.08 0.65

0.76 0.80 0.82 0.70 0.61 0.60 0.55 0.61 0.58 0.55 0.40 0.42 0.36 0.36 0.19

2.68 2.82 2.94 2.47 2.17 2.15 1.94 2.15 2.04 1.97 1.38 1.49 1.28 1.28 0.69

0.77 0.80 0.82 0.71 0.62 0.62 0.57 0.62 0.59 0.57 0.41 0.44 0.39 0.39 0.22

2.58 2.66 2.76 2.39 2.09 2.08 1.90 2.09 1.98 1.92 1.39 1.48 1.29 1.29 0.75

0.81 0.83 0.87 0.75 0.69 0.69 0.63 0.68 0.66 0.63 0.48 0.53 0.45 0.46 0.30

1.99 2.05 2.14 1.85 1.69 1.69 1.56 1.69 1.63 1.56 1.20 1.30 1.11 1.13 0.75

0.76 0.78 0.81 0.71 0.65 0.65 0.61 0.66 0.63 0.62 0.50 0.52 0.48 0.48 0.35

1.88 1.94 2.02 1.76 1.76 1.61 1.52 1.62 1.57 1.53 1.24 1.29 1.18 1.18 0.85

0.75 0.81 0.82 0.66 0.62

2.79 2.98 2.82 2.34 2.31

0.76 0.82 0.83 0.68 0.63

2.67 2.81 2.82 2.35 2.23

0.79 0.85 0.86 0.71 0.69

1.94 2.09 2.13 1.73 1.71

0.74 0.80

1.85 1.98



0.69 0.65



1.69 1.63

11

12

CRC Handbook of Chromatography Table GC 3 (continued) ALDITOL ACETATES DERIVED FROM PARTIALLY ETHYLATED SUGARS Phase Tem perature Gas; flow rate (m i/m in ) Column Length, cm Diameter (O .D .), cm Form

PI T1 He, 60

P2 T1 He, 60

P3 T2 He, 60

P4 T3 He, 60

120 0.3 na

120 0.3 na

120 0.3 na

M aterial Detector

copper FI

copper FI

copper FI

1520 0.051 (l.D .) open tubular SS FI

Parent sugar 2,4-di-O-ethyl 2,6-di-O ethyl 3,4-di-O-ethyl 3,6-di-O-ethyl 4,6-di-O-ethyl 2,3,4-tri-O-ethyl 2,3,6-tri-O-ethyl 2,4,6-tri-O-ethyl 3,4,6-tri-O ethyl 2,3,4,6-tetra- O-ethyl D-Mannose 2 -O-ethyl 3 -O-ethyl 4 -O-ethyl 6 -O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 2,6-di-O-ethyl 3,4-di-O-ethyl 3,6-di-O-ethyl 4,6-di-O-ethyl 2,3,4-tri-O-ethyl 2,3,6-tri-O-ethyl 2,4,6-tri-O-ethyl 3,4,6-tri-O-ethyl 2,3,4,6-tetra- O-ethyl L-Fucose 2- O-ethyl 3-O-ethyl 4 -O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 3,4-di-O-ethyl 2,3,4-tri-O-ethyl L-Rhamnose 2 -O-ethyl 3 -O-ethyl 4 -O-ethyl 2,3-di-O-ethyl 2,4-di-O-ethyl 2,3,4-tri-O-ethyl “ b

ra

rb

r“

r*



Tb

0.63 0.53 0.63 0.57 0.53 0.44 0.39 0.40 0.40 0.21

2.32 1.94 2.32 2.10 1.92 1.66 1.41 1.43 1.43 0.78

0.67 0.55 0.67 0.59 0.56 0.47 0.42 0.42 0.42 0.25

2.29 1.90 2.26 2.03 1.90 1.58 1.43 1.42 1.42 0.84

0.70 0.62 0.71 0.65 0.62 0.54 0.49 0.50 0.50 0.33

0.70 0.79 0.79 0.62 0.55 0.55 0.50 0.60 0.55 0.49 0.35 0.36 0.35 0.34 0.17

2.64 2.95 2.95 2.31 2.09 2.09 1.88 2.24 2.08 1.85 1.31 1.34 1.32 1.30 0.62

0.72 0.80 0.80 0.65 0.58 0.58 0.54 0.62 0.57 0.53 0.40 0.40 0.40 0.39 0.22

2.40 2.60 2.60 2.15 1.94 1.94 1.78 2.03 1.90 1.77 1.28 1.31 1.29 1.26 0.72

0.76 0.83 0.83 0.69

0.59 0.67 0.65 0.59 0.46 0.47 0.46 0.46 0.29

0.35 0.40 0.38 0.25 0.25 0.25 0.11

1.24 1.42 1.35 0.88 0.88 0.88 0.40

0.39 0.43 0.43 0.28 0.29 0.30 0.14

1.23 1.37 1.37 0.89 0.91 0.93 0.46

0.34 0.39 0.36 0.21 0.22 0.07

1.18 1.34 1.25 0.75 0.77 0.28

0.36 0.40 0.38 0.23 0.25 0.09

1.18 1.32 1.24 0.76 0.81 0.30

r“

r*

1.70 1.51 1.72 1.66 1.50 1.32 1.21 1.22 1.22 0.81

0.68 0.61 0.68 0.63 0.60 0.55 0.50 0.50 0.50 0.36

1.67 1.53 1.68 1.55 1.48 1.36 1.24 1.23 1.24 0.90

1.86 2.02 2.02 1.70

1.45 1.65 1.60 1.44 1.13 1.15 1.14 1.13 0.70

0.77 0.82 0.82 0.69 0.64 0.64 0.59 0.68 0.62 0.59 0.49 0.49 0.48 0.47 0.32

1.97 2.11 2.11 1.79 1.66 1.65 1.50 1.74 1.56 1.53 1.21 1.22 1.21 1.20 0.81

0.45 0.50 0.50 0.36 0.36 0.36 0.22

1.11 1.22 1.22 0.89 0.89 0.89 0.53

0.46 0.50 0.50 0.39 0.39 0.40 0.26

1.15 1.24 1.24 0.95 0.95 0.97 0.64

0.44 0.50 0.43 0.32 0.33 0.16

1.06 1.21 1.06 0.79 0.81 0.38

0.45 0.49 0.45 0.35 0.36 0.21

1.13 1.23 1.14 0.87 0.90 0.53

— —

tr relative to m yoinositol hexaacetate. tr relative to 1,5-di-O-acetyl-2,3,4,6-tetra- O-methyl-D-mannitol.

— —

Carbohydrates Table GC 3 (continued) ALDITOL ACETATES DERIVED FROM PARTIALLY ETHYLATED SUGARS Phase

P1 P2

P3 P4

Temperature T1 T2 T3

3% ECNSS-M on Gas-Chrom Q (100 to 120 mesh), mixture of 0.2% poly(ethylene glycol succinate), 0.2% poly(ethylene glycol adipate) and 0.4% GE silicone XF-1150 on Gas-Chrom P (100 to 120 mesh). 2% Silar 10-C on Gas-Chrom Q (100 to 120 mesh), open tubular capillary column coated with mixture containing 1% polyethylene glycol adipate), 1% polyethylene glycol succinate) and 4% GE silicone XF-1150 (initially dissolved in chloroform ), tem perature program m ed: 110°C for 6 min after injection, then 110 -» 180°C at l°/m in ; isothermal at 180°C for 10 min. 115°C for 6 min after injection, then 115 -* 235°C at 1°/m in. 135°C for 6 min after injection, then 135 205°C at l°/m in ; isothermal at 205°C for 20 min.

From Sweet, D. P., Albersheim, P ., and Shapiro, R. H., Carbohydr. Res., 40, 199, 1975. With permission.

13

14

CRC Handbook of Chromatography Table GC4 PERMETHYLATED ALDITOLS AND REDUCED OLIGOSACCHARIDES Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference Parent sugar Erythrose Ribose Arabinose Xylose Glucose M annose Galactose 2-Deoxy-D-ribose Rhamnose Fucose 2-Deoxy-D-glucose Laminaribiose Cellobiose Maltose Gentiobiose Lactose Melibiose /3-D-Gal p N Ac-( 1-*4)-D-Gal /3-D-Gal p-( 1_>4)-D-Glc p N Ac a-D-Galp-( 1-*3)-D-Galp N Ac /3-D-Gal p-( 1-3 )-D -G al p NAc /3-D-Galp-(l-*3)-D-GlcpNAc a-D-Gal p-( 1-*6)-D-Glc p N Ac /3-D-Galp-( 1-*6)-D-Glc p N Ac /3-D-Glc p-( 1—2)-/3-D-Glc p-( 1—2)-D-G1 c /3-D-G1c p-( 1-*2)-/3-D-Glc p-( 1-*4)-D-Gal /3-D-Glc p-( 1—3)-/3-D-Glc p-( 1-^3)-D-Glc /3-D-Glc p-( 1-3)-/3-D-Glc p-( 1-*4)-D-Glc /3-D-Glcp-(l—3)-^-D-Glcp-(l—6)-D-Glc /3-D-Glc p-( 1—4)-/3-D-Glc p-( 1- 3)-D-Glc a-D-Glc p -(l_*'4)-a-D-Glcp-( 1~*4)-D-Glc a-D-Glc p-( 1-*4)-a-D-Glc p-( 1-*6)-D-Glc /3-D-Glcp-(l->6)-/3-D-Glcp-(l_^3)-D-Glc a-D-Glc p-( 1-*6)-a-D-Glc p-( 1-*4)-D-Glc /3-D-Glcp-(l-^6)-/3-D-Glcp-(l-^6)-D-Glc /3-D-Gal p-( 1-*6)-/3-D-Gal p-( 1-*3)-L-Ara /3-D-Gal p-( 1—3)-/3-D-Gal p-( 1-4)-D -G lc /3-D-Galp-( 1-*,4)-/3-D-Galp-( 1-»>4)-D-Glc /3-D-Gal p-(l-*6)-/3-D-Galp-(l-»'4)-D-Glc a-D-Gal p-( 1-6 )-a-D -G al p-( 1-6)-D -G lc /3-D-Gal p-( 1—6)-/3-D-Gal p-( 1-6)-D -G al a-D-Manp-(l~*,2)-a-D-Man p -(l- *,2)-D-Man /3-D-Manp-(l-^4)-/3-D-Manp-(l“^4)-D-Man

PI 110 N2, 60

P2 197 He, na

PI 220 He, na

P2 265 He, na

180 0.45 coiled aluminium FI 1

200 0.35 na glass FI 2

200 0.2 na na FI 3

200 0.35 na glass FI 4

r“

Tb

rc

rc

0.26 0.60 1.05 1.00 2.46 2.84 3.88 0.40 1.25 1.47 1.39

_

_

_







— — —

— — —

— — —













— — —

— — —

— — —











— — — —

0.61 0.55 0.61 0.92 0.70 1.00 2.52d 3.06d









— — — —











— — — — — — —













— — — 0.87 0.66 0.77 0.72 0.70 0.83







__



































_

_

_





























































— — —

0.83 0.80 1.01 0.95 1.56 1.03 1.00 1 A 1 1.41

0.87 1.39 1.33 0.97 1.23 1.04 1.39 1.69 2.42 0.67 1.52

Carbohydrates Table GC 4 (continued) PERMETHYLATED ALDITOLS AND REDUCED OLIGOSACCHARIDES Packing T em perature (°C) Gas; flow rate (mf /m in) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference Parent sugar /3-DXylp-(l-*,3)-/3-D-Xylp-(l-*,4)-D-Xyl p

PI 110 N2, 60

P2 197 He, na

PI 220 He, na

P2 265 He, na

180 0.45 coiled aluminium FI 1

200 0.35 na glass FI 2

200 0.2 na na FI 3

200 0.35 na glass FI 4

r“

r*

rc

rc







0.42





__

1.03

0-D-Glc

6 /3-D-Glcj>(l-*3)-D-GIc a h c d

tr relative to permethylated xylitol (5.7 min). tr relative to permethylated melibiitol. tr relative to permethylated m altotriitol (9 min in Reference 4). Column tem perature 221 °C.

Packing

PI = 3% QF-1 on Gas-Chrom Q (80 to 100 mesh). P2 = 1% OV-22 on Supelcoport (80 to 100 mesh). REFERENCES

1. W hyte, J. N. C., Gas-liquid chrom atographic analysis of permethylated alditols and aldonic acids, J. Chromatogr.,%1, 163, 1973. 2. Karkkainen, J., Analysis of disaccharides as permethylated disaccharide alditols by gas-liquid chrom atography-m ass spectrom etry, Carbohydr. Res., 14, 27, 1970. 3. M ononen, I., Finne, J ., and K arkkainen, J., Analysis of permethylated hexopyranosyl-2acetamido-2-deoxyhexitols by g.l.c.-m .s., Carbohydr. Res., 60, 371, 1978. 4. Karkkainen, J., Structural analysis of trisaccharides as permethylated trisaccharide alditols by gas-liquid chrom atography-m ass spectrometry, Carbohydr. Res., 17, 11, 1971.

15

16

CRC Handbook o f Chromatography

Table GC 5 ACETYLATED ALDONONITRILES DERIVED FROM NEUTRAL SUGARS AND DEAMINATED AMINODEOXYHEXOSES Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Reference Parent sugar Erythrose L-Arabinose D-Xylose D-Ribose D-Lyxose D-Glucose D-Galactose D-Mannose L-Rhamnose L-Fucose D-glycero-D-gu/oH eptose D-glycero-D- manno- Heptose 2,5-A nhydro-D-m annosef 2,5-Anhydro-D-talosed “ b c d

PI T1 N a, 50

P2 T2 N2, 40

P3 185 na

P4 210 na

P5 210 na

P6 190 N 2, 75

PI T3 N 2, 24

100 0.3 coiled glass FI 1

152 0.4 coiled na FI 2

150 0.4 coiled glass FI 3

200 0.4 coiled glass FI 3

150 0.4 coiled glass FI 3

152 0.3 na SS FI 4—6

274 0.3 na SS FI 7 ,8





rb

r*

Tb



r‘

— 0.73

— 0.23 0.28 0.17 0.19 0.65 0.70 0.49 0.11 0.15 — — — —

— 0.24 0.26 0.20 0.22 0.67 0.73 0.54 0.18 0.19 — — — —

0.28 0.31 0.23 0.25 0.72 0.78 0.61 0.19 0.24 — — — —



0.59 0.68 0.52 —

1.00 1.09 0.91 0.38 — 1.38 1.27 — —



0.69 —

1.00 1.06 0.93 0.65 — — — — —



0.08 0.33 0.42 0.27 0.31 1.00 1.14 0.78 0.17 0.25 —

— 0.39 0.67



0.70 0.76 —

— 1.00 1.05 0.93 0.56 0.64 — — 0.77 0.89

tr relative to peracetylated D-glucononitrile (22 min in Reference 1, 58 min in Reference 2, 47.2 min in Reference 4, 70.2 min in Reference 7). tr relative to m yoinositol hexaacetate. From deam ination of 2-amino-2-deoxy-D-glucose. From deam ination of 2-amino-2-deoxy-D-galactose.

Packing

PI P2 P3 P4 P5 P6 Tem perature T1 T2 T3

= = = = = = = = =

3% poly(neopentyl glycol succinate) on C hrom osorb W (60 to 80 mesh). 10% poly(neopentyl glycol succinate) on Gas-Chrom Q (100 to 120 mesh). 3% ECNSS-M on Gas-Chrom Q (100 to 120 mesh). 5% OV-225 on C hrom osorb W AW DMCS (100 to 120 mesh). 3% OV-17 on Supasorb AW DMCS (100 to 120 mesh). 10% LAC-4R-886 on acid-washed Chrom osorb W (100 to 200 mesh). tem perature program m ed, 160 230°C at 2°/m in. isotherm al at 160°C for 10 min, then tem perature program m ed, 160 -*■ 235°C at 4°/m in. tem perature program m ed, 130 — *• 195°C at l°/m in , then isothermal at 195°C for about 15 min.

REFERENCES 1.

2. 3. 4.

Dmitriev, B. A ., Backinowsky, L. V., Chizhov, O. S., Zolotarev, B. M ., and Kochetkov, N. K., Gas-liquid chrom atography and mass spectrom etry of aldononitrile acetates and partially methylated aldononitrile acetates, Carbohydr. Res., 19, 432, 1971. Baird, J. K., H olroyde, M. J., and Ellwood, D. C ., Analysis of the products of Smith degradation of polysaccharides by g.l.c. of the acetylated, derived aldononitriles and alditols, Carbohydr. Res., 27, 464, 1973. M orrison, I. M ., Determ ination of the degree of polymerisation of oligo- and polysaccharides by gas-liquid chrom atography, Carbohydr. Res., 108, 361, 1975. Varm a, R ., V arm a, R. S., and W ardi, A. H ., Separation of aldononitrile acetates of neutral sugars by gasliquid chrom atography and its application to polysaccharides, J. Chrom atogr.,1 1 , 222, 1973.

Carbohydrates

17

Table GC 5 (continued) ACETYLATED ALDONONITRILES DERIVED FROM NEUTRAL SUGARS AND DEAMINATED AMINODEOXYHEXOSES 5.

6. 7.

8.

Varm a, R ., V arm a, R. S., Allen, W. S., and W ardi, A. H ., Gas chrom atographic determination of neutral sugars from glycoproteins and acid m ucopolysaccharides as aldononitrile acetates, J. Chromatogr., 86, 205, 1973. Varm a, R. S., Varm a, R ., Allen, W. S., and W ardi, A. H ., A gas chrom atographic method for determ ination of hexosamines in glycoproteins and acid mucopolysaccharides, J. C hrom atogr.,93,221, 1974. Varm a, R. and Varm a, R. S., Simultaneous determ ination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides (glycosaminoglycans) by gas-liquid chrom atography, J. Chromatogr., 128, 45, 1976. Varm a, R. and Varm a, R. S., A simple procedure for combined gas chrom atographic analysis of neutral sugars, hexosamines and alditols. Determ ination of degree of polymerization of oligo- and polysaccharides and chain weights of glycosaminoglycans, J. Chromatogr., 139, 303, 1977.

18

CRC Handbook of Chromatography

Table GC 6 ACETYLATED ALDONONITRILES DERIVED FROM METHYLATED SUGARS Packing T em perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference Parent sugar D-Xylose 2-O-methyl 3-O-methyl 4-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 2,3,4-tri-O-m ethyl 2,3,5-tri-O-m ethyl D-Glucose 2-O-methyl 3-O-methyl 4-O-methyl 6-O-methyl 2,3-di-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-m ethyl 2,3,6-tri-O-m ethyl 2,4,6-tri-O-m ethyl 3,4,6-tri-O-m ethyl 2,3,4,6-tetra-O-m ethyl D-Mannose 2-O-methyl 3-O-methyl 4-O-methyl 5-O-methyl 6-O-methyl 2,3-di-O-methyl 2,4-di-O-methyl 2,5-di-O-methyl 2,6-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 3,6-di-O-methyl 4,6-di-O-methyl 5,6-di-O-methyl 2,3,4-tri-O-m ethyl 2,3,5-tri-O-m ethyl 2,3,6-tri-O-methyl

PI 200 He, 75

P2 180 N2, 18

P3 200 N2, 30

P4 T1 He, 35

P5 T2 He, 30

P6 T3 N2, 40

P6 175 N2, 40

120 0.38 U-tube glass FI 1

300 0.216 na

300 0.216 na

183 0.318 na

183 0.318 na

SS

SS

SS

SS

FI 2

FI 2

FI 3

FI 3,4

180 0.3 na glass FI 5

180 0.3 na glass FI 5

r“

Tb

Tb

Tb

rc

rc

re

1.64 2.07 1.55 1.00 0.74 0.65 1.00 0.65 0.39 0.34



















— — —

— — —

















— — — — —

— — — — —

— — — — —

— — — — — —

— — — — — —

— — — — — — — — — —

— — — — — — — — — — — — — — —

4.79 7.16 6.70 4.62 4.04 3.34 2.67 5.13 3.56 2.93 2.23 2.06 1.44 2.23 1.00

5.26 8.85 6.81 4.63 5.11 3.28 2.77 5.31 4.07 2.89 2.27 2.50 1.40 2.13 1.00

— — — — 2.12 2.60 — 2.06 — — 1.67 1.42 1.33 — 1.00

— — — — 3.50 2.84 — 3.58 — — 2.00 2.00 1.45 1.85 0.94

— — — — — — — — — — — — — — —

— — — — — — — — — — — — — — —

— — — — —

— — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — —

— — — — — 2.55 3.16 — 2.28 3.68 — 2.85 2.45 — 2.03 — 1.65

2.06 2.40 2.34 1.91 1.74 1.82 1.89 1.91 1.61 2.13 1.91 1.82 1.66 1.43 1.53 1.47 1.37

6.53 9.89 9.53 4.99 3.61 4.30 4.95 4.99 2.96 7.16 4.99 4.30 3.16 2.28 2.58 2.38 2.11



— — — — — — — — — — —

Carbohydrates

19

Table GC 6 (continued) ACETYLATED ALDONONITRILES DERIVED FROM METHYLATED SUGARS Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Reference

Parent sugar 2,4,6-tri-O m ethyl 2,5,6-tri-O m ethyl 3,4,6-tri-O m ethyl 3,5,6-tri-O m ethyl 2,3,4,6-tetra-O m ethyl 2,3,5,6-tetra-O m ethyl

PI 200

He, 75 120

0.38 U-tube glass FI

P2 180 N 2, 18

N2, 30

300 0.216 na SS FI

300 0.216 na SS FI

1 r“

P3 200

2 Th

2

r*

P4 T1 He, 35

P5 T2 He, 30

P6 T3 N2, 40

P6 175 N2, 40

183 0.318 na SS FI 3

183 0.318 na SS FI 3,4

180 0.3 na glass FI 5

180 0.3 na glass FI 5

r*

Te

re

Tc

1.31 1.29 1.51 1.54

1.71 1.65 2.47 2.65

__



__

__

1.59



















1.89



















1.00

1.00

1.00











0.95

0 .8 6

“ t r relative to derivatives from 2,3- and 3,4-di-O-methyl-D-xylose (9 min). b tr relative to 5-0-acetyl-2,3,4,6-tetra-0-m ethyl-D -glucononitrile (8.03 min with P2, 6.53 min with P3). c tr relative to 5-0-acetyl-2,3,4,6-tetra-0-m ethyl-D -m annononitrile (6.4 min in Reference 4, about 40 min with T3 in Reference 5). Packing

P1 P2 P3 P4 P5 P6 T em perature T 1 T2 T3

10% LAC-4R-886 on acid-washed Chrom osorb W (100 to 120 mesh). 3% QF-1 on Gas-Chrom Q (100 to 120 mesh). 3% SP-2340 on Supelcoport (100 to 120 mesh). 5% Apiezon L on Gas-Chrom W (60 to 80 mesh). 5% butanediol succinate polyester on Supelcoport (60 to 80 mesh). 3% ECNSS-M on Gas-Chrom Q (100 to 120 mesh), tem perature program m ed, 15 0 -» 2 1 0 °C at l°/m in . tem perature program m ed, 185 -» 210°C at l 0 /m in, then held at 210°C. tem perature program m ed, 1 1 0 -* 180°Cat l°/m in .

REFERENCES 1. 2. 3. 4.

5.

Lance, D. G. and Jones, J. K. N ., Gas chrom atography of derivatives of the methyl ethers of D-xylose, Can. J. C hew ., 45, 1995, 1967. Anderle, D. and Kovac, P ., Separation of O-acetyl-O-methyl-D-glucononitriles by gas chrom atography, Chem. Zvesti., 30, 355, 1976. Seymour, F. R ., Slodki, M. E ., Plattner, R. D ., and Jeanes, A ., Six unusual dextrans: methylation structural analysis by combined g.l.c.-m .s. of per-O-acetyl-aldononitriles, Carbohydr. Res., 53, 153, 1977. Seymour, F. R ., Plattner, R. D ., and Slodki, M. E ., Gas-liquid chromatography-m ass spectrometry of m ethylated and deuteriom ethylated per-O-acetyl-aldononitriles from D-mannose, Carbohydr. Res., 44, 181, 1975. W oolard, G. R ., Rathbone, E. B., and Dercksen, A. W ., Gas-liquid chrom atography of methyl a-D-mannosides and their O m ethyl ethers. II. Acetylated aldononitriles derived from methyl a-D-mannopyranoside and methyl a-D -m annofuranoside, S. A fr. J. C hem .,30, 169, 1977.

20

CRC Handbook o f Chromatography Table GC 7 BUTANEBORONIC ESTERS AND TRIMETHYSILYLATED BUTANEBORONATES OF SOME SUGARS AND ALDITOLS

L-Arabinose D-Xylose D-Ribose L-Rhamnose L-Fucose D-Galactose D-Mannose D-Glucose D-Fructose E rythritol

P2 T1 N2, 45

PI

Packing Tem perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Reference Parent com pound

200

na

213

180 0.5 U-tube glass D1

0 .6

coiled glass FI 2

1



_ — — — —

0 .8 8

0.44; 0.79 0.70; 0.86;d 1.02

1.24 1.39 1.40c 1 . 10 ;' 1 .44 1.07

e e



* * 0.73



2.03'



L-Arabinitol

Tb

rb (TMS) 1.24 1.36 0.60; 0.90d 0.45; 1.43d 1.07 1.28 1.34 1.56 1.21

0.44;d 0.47;d 0.69;d 0.78 0.61;d 0.95;d 0.11

Xylitol D-Glucitol D-Mannitol

0.79 0.70

2.09' 1.97 1.76

Galactitol

0.96

2 .1 0

° b c d e 1



1.06 1.93 1.22;d 1.27;d 1.74 1.18; 2.07d

tr relative to myo- inositol buraneboronate (5.7 min). tr relative to methyl palm itate. Peak with shoulder. M inor peak. No peak. Broad peak.

Packing

PI P2 Tem perature T 1 Detector D1

3% OV-17 on Gas-Chrom Q (100 to 120 mesh). 1 % ECNSS-M on Gas-Chrom Q (80 to 100 mesh), tem perature program m ed, 115 -► 190°C at 5°/m in. argon ionization detector. REFERENCES

1.

2.

Eisenberg, F ., Cyclic butaneboronic acid esters: novel derivatives for the rapid sep­ aration of carbohydrates by gas-liquid chrom atography, Carbohydr. Res., 19, 135, 1971. W ood, P. J., Siddiqui, I. R ., and Weisz, J., The use of butaneboronic esters in the gas-liquid chrom atography of some carbohydrates, Carbohydr. Res., 42, 1 , 1975.

Carbohydrates

21

Table GC 8 CHIRAL GLYCOSIDES: RESOLUTION OF ENANTIOMERS Phase Tem perature Gas; flow rate (m i/m in )

PI T1 N2, 1 m i/m in

PI T2 N2, 1 m i/m in

P2 230°C N2, 0.4 m i/m in

Column Length, m Diameter (I. D.), mm Form M aterial Detector Reference

25 0.31 capillary glass FI

25 0.31 capillary glass FI

25 0.25 WCOT glass FI

1

1

Glycoside

TMS(--)- 2 -butyl

Parent sugar D-Arabinose L-Arabinose D-Xylose L-Xylose D-Ribose L-Ribose D-Rhamnose L-Rhamnose D-Fucose L-Fucose D-Glucose L-Glucose D-Galactose L-Galactose D-Mannose L-Mannose

“ b c d '

2

Acetylated( + )-2-octyl r°

ra 0.55;c 0.56;d 0.58;"0.67d 0.54;d 0.55;c 0.58;"0.70d 0.80;c 1.04c 0.79;c 1.06c 0.62; 0.64c 0.65;" 0.66 0.59;c 0.71" 0.57;c 0.70c 0.59;d 0.69;c 0.72;"0.82d 0.62;d 0.67;c 0.78;"0.80d 1 .6 6 ;" 2 . 10 " 1.58;c 2.10c 1.27;d 1.47;" 1.61" 1.30;d 1.41 ;c 1.70c 1.24;c 1.64c 1.18;c 1.61c

0.70;c0.71;d 0.73;"0.80d 0.69;d0.70;c 0.73;"0.82d 0 . 8 8 ;c 1 .0 2 " 0.87;" 1.03" 0.75; 0.76c 0.77;c 0.78 0.72;c 0.82c 0.71;" 0.81c 0.72;d0.79;c 0.82;"0.89d 0.74;d 0.78;" 0.87;c0.88d 1.36;" 1.53" 1.32;" 1.53" 1.18;d 1.28;" 1.34" 1.21 ;d 1.25;" 1.38" 1.15;" 1.34" 1.12;" 1.32"

r* 2.82; 2.75; 2.41; 2.63; 2.48; 2.97; 2.83; 3.00; 3.02; 1.69; 1.69; 1.96; 2.05; 2.25; 2.15;' 7.24; 7.24; 8.22; 6.56; 7.29; 7.78; 6.22; 7.88;

3.21; 3.31 2.93; 3.41 3.11; 3.28 3.02; 3.28 3.26; 3.47 3.13; 3.36 2.21; 2.39 2.21; 2.44 2.40; 2.96 2.61' 8.03;10.30 8.46; 8.94 8.08; 10.47 8.81; 9.01 8.40; 8.50

6 . 8 6 ; 7.13; 7.91; 8.90

t r relative to TMS methyl a-D-galactopyranoside (~ 26 min at 150°C, ~ 10 min at 175°C, ~ 31 min at program T2). tr relative to l,5-di-0-acetyl-2,3,4,6-tetra-0-m ethyl-D -glucitol (~ 7 min). Pyranose (ring sizes from mass spectrometry). Furanose. Peak shape indicated m ore than one component.

Phase

PI P2 Tem perature T 1 T2

= = = =

SE-30, wall-coated. SP-IOOOWCOT column (Varian). isotherm al; pentoses and 6 -deoxyhexoses at 150°C, hexoses at 175°C. tem perature program m ed, 135 200°C at l° /m in .

REFERENCES 1. Gerwig, G. J ., Kamerling, J. P ., and Vliegenthart, J. F. G ., Determ ination of the D and L configuration of neutral m onosaccharides by high-resolution capillary g.l.c., Carbohydr. Res., 62, 349, 1978. 2. Leontein, K., Lindberg, B., and Lonngren, J ., Assignment of absolute configuration of sugars by g.l.c. of their acetylated glycosides formed from chiral alcohols, Carbohydr. R es.,62, 359, 1978.

22

CRC Handbook o f Chromatography Table GC 9 O-ISOPROPYLIDENE DERIVATIVES OF COMMON ALDOSES Packing Tem perature (°C) Gas; flow rate (m f/m in) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Parent sugar

PI T1 N2, 20

P2 T2 N,, 20

200 0 .2 2

183 0.15 na glass FI

na SS FI M ain acetal

r“

r"

0.24 0.39 0.33 0.097

0.38 0.49 0.45 0.17

L-Rhamnose L-Fucose D-Glucose

1,2:3,4 (p) 1,2:3,5 (a) 2,3 l,5-anhydro-2,3-0isopropylidene-/TDribofuranose 2,3 1,2:3,4 (a) 1,2:5 ,6 (a)

0.72

D-Galactose D-Mannose

1,2:3,4 (a) 2,3:5 ,6

0 .8 6

0.78;h 0.82 0.32 0.60;f 0.87; 0.94 0.90

1.00

1.00

L-Arabinose D-Xylose D-Ribose

0 .2 0

0.59;c 0.82;* 0.90

Note: This table supplements Table GC 13 in Section A, Volume I, Section II.1. a 6

c

tr relative to 2,3:5,6-di-0-isopropylidene-D-m annose. M inor peak. Trace.

Packing

PI P2 Tem perature T 1 T2

3% OV-225 on Gas-Chrom Q (80 to 100 mesh). 3% XE-60 on Chrom osorb G AW DMCS (80 to 100 mesh), isotherm al at 110°C for 12 min, then program m ed at 1.5°/m in for 30 min. 120°C for 5 min, then program m ed at 2°/m in for 30 min.

From Morgenlie, S., Carbohydr. Res., 41,285, 1975. With permission.

Carbohydrates Table GC 10 ACETATES OF SOME PARTIALLY METHYLATED SUGARS Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector

M ethylated sugar D-Glucose 2,3-di-O-methyl 2,3,6-tri-O-m ethyl 2,3,4,6-tetra- O m ethyl D-Galactose 2,3,4,6-tetra-O m ethyl 2,3,5, 6 -tetra- O m ethyl D-Mannose 2,3,6-tri-O m ethyl 2,3,4,6-tetra- O m ethyl L-Rhamnose 2,3-di- O m ethyl “

PI 170 He, 86

PI 185 He, 86

P2 Tl He, 86

244 0.48 na glass FI

244 0.48 na glass FI

0.64 na glass FI

r“

r*

6.69; 7.63 2.85; 3.27 0.92; 1.00

— — 0.92; 1.00

ra

— —

0.82; 1.00 1.16; 1.76 1.31 —

1.56; 2.02 —

122



— —









6 .0



1.22; 1.35

tr relative to l,5-di-O acetyl-2,3,4,6-tetra-O m ethyl-D -glucose, second peak (11.5 min at 170°C, 5.2 min at 185°C with P I, 30.2 min with P2 and program m e T l).

Packing

PI P2

Tem perature T l

= 3°7o ECNSS-M on Gas-Chrom Q (100 to 120 mesh). = 5% butanediol succinate polyester on D iatoport S (80 to 100 mesh). = tem perature program m ed, 110 210°C at 2°C /m in. REFERENCE

1.

Bebault, G. M ., D utton, G. G. S., and W alker, R. H ., Separation by gas-liquid chrom atography of tetra-O m ethylaldohexoses and other sugars as acetates, Carbohydr. Res., 23,430, 1972.

23

PI 175 Ar, 80— 100

Ar, 80— 100

120

120

0.5 na glass D1

0.5 na glass D1

1

Methyl glycoside L-Arabinoside 2 -O-methyl 3-O m ethyl

P2 200

91 0.6 (O .D .) na glass FI 2

1

r*

P3 155 He, 60

r*

r“

2,3-di-O m ethyl

1.56; 1.93

1.10; 1.45 1.26; 1.45; 1.58 0.63; 0.95

2,4-di-O-m ethyl 2,5-di-O-methyl 3,4-di-O m ethyl 3,5-di-O-methyl 2,3,4-tri-O -m ethyl 2,3,5-tri-O -m ethyl D-Xyloside 2 -O m ethyl 3-O m ethyl 4-O-methyl 5-O-methyl 2,3-di-O m ethyl

2.26;* 2.37 1.89; 3.47 2.15 1.08; 2.55 1.04 0.56; 0.72

1.09;* 1.13 0.70; 1.03 0.99; 1.52 0.60; 0.84 0.83 0.47; 0.59

— 4.55; 6.57; 11.1; 14.6 1.96; 2.29; 2.54 2.94;* 3.16 2.33; 4.63 2.82 1.28; 3.21 1.26 0.59; 0.79

4.11; 6.23 3.55; 5.57 4.35 — 1.50; 1.79

1.01; 1.34 0.94; 1.15 1.09 — 0.64; 0.75

5.88; 9.35 5.00; 8.12 — — 1.75; 1.95;

2,4-di-O m ethyl 2,5-di-O m ethyl

1.49; 1.97 —

0.73; 0.93 —

6 .1 0

4.47; 6.95

P4 167 Ar, 62

P5 200

He, 25

P6 190 He, 55

P7 112

Ar, na

P8 160 N2, 39

P9 175 N2, 14.1

P10 195 N2, 20 300 0.3 na glass FI 9

120

200

365

120

183

120

0.5 na glass D1 3

0.4 na glass FI 4

0 .6

0.5 na glass D1

0 . 6 ( 0 .D.)

0.3 (O.D.) na brass FI

rfc

Tc

na glass D2 5

6

r'

r-

na glass FI 7

8

r*

r'

r*

— —

— —

— —

— —

— —

— —

— —















— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — —

— — — — —

— — — — —

— — — — —

3.72;* 8.21 ‘ 3.06;'9.43' — 3.75;* 8.16' 2.31;* 2.64*

— — — — —

— — — — —

— —

— —

— —

— —

— 2.05;* 2.95*

— —

— —

2 .2 0

— —

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

Packing T em perature (°C) Gas; flow rate (m l/m in ) Colum n Length, cm Diam eter (I.D .), cm Form M aterial Detector Reference

24

Table GC 11 METHYL GLYCOSIDES OF METHYLATED SUGARS

3,4-di-O-methyl 3,5-di-O-methyl 2,3,4-tri-O -m ethyl 2,3,5-tri-O-methyl L-Rham noside 3-O-methyl 3,4-di-O m ethyl 2,3,4-tri-O -m ethyl L-Fucoside 2-O-methyl

1.36; 1.63 — 0.46; 0.57 —

0.71; 0.76 — 0.45; 0.54 —

— — 0.44; 0.59 —

— — — —

— — — —

— — — —

— — — —

3.66 0.73; 1.01 0.46

1.01 0.61 0.46

4.86 1.10 0.44

— — —

— — —

— — —

— — —

4.25









2,3,4-tri-O -m ethyl D-Glucoside 3-O-methyl

0.72

0.99; 1.13; 1.15; 1.54 —





— 16.5; 23.6 6.86 — 4.26 4.50 2.38 2.20; 2.92

— 1.53;'4.59' 1.00 1.31;' 1.45'

— — — —

— — — —

— — —

— — —

— —





























— — — — — —

— — — — — 2.36; 3.55; 4.68 — — 1.00; 1.36

— — — — — —

— — — — — —

— — — — — —

— — —

— — —

— — —





2,3-di-O-methyl 2,4-di-O-methyl 3,4-di-O-methyl 4,6-di-O-methyl 2,3,4-tri-O -m ethyl 2,3,6-tri-O-m ethyl

— — — — 2.59; 3.70 3.52; 4.78

2.46; 3.22 2.30; 3.22 — — 1.35; 1.83 1.71; 2.18

— — — — — —

— — — — — —

2,4,6-tri- O-methyl 3,4,6-tri-O -m ethyl 2,3,4,6-tetra- O-methyl

3.31; 4.88 3.12; 3.73 1.00; 1.43

1.64; 2.24 — 1.00; 1.32

— — 1.00; 1.52

— — —

2.15 2.00 0.74; 1.00

— — —

D-Galactoside 2-O-methyl 3-O-methyl 4 -O-methyl 6 -O-methyl 2,3-di-O-methyl

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

2.70 2.70 2.84 2.55 2.24

— —

— — — — 14.6; 18.3; 19.7; 25.3 25.5; 30.0 14.1; 17.3; 20.1; 26.9

— — — — 6.51; 7.72

2,4-di-O-methyl 2,6-di- O-methyl

— — — — 2.46; 3.65; 4.20 3.72; 4.40 2.51; 3.21; 3.77

— 4.89; 5.63

— —

— —

— —

— —

— —

2.30 2.15

25



C a rb o h y d ra te s



PI 175 Ar, 80— 100

2,4, 6 -tri- O-methyl 3,4, 6 -tri-O-methyl 2,3,4,6-tetra- O-methyl 2,3,5,6 -tetra- O-methyl D-M annoside 2 ,3-di-O-methyl 2 ,4-di-O-methyl 2 ,5-di-O-methyl 2 , 6 -di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 3,6-di-O-methyl 4,6-di-O-m ethyl

Ar, 80— 100

P3 155 He, 60

P4 167 Ar, 62

P5 200

He, 25

P6 190 He, 55

P7 112

Ar, na

120

120

91

120

200

365

120

0.5 na glass D1

0.5 na glass D1

0 .6 (0 . D.)

0.5 na glass D1 3

0.4 na glass FI 4

0 .6

0.5 na glass D1

1

na glass FI 2

1

M ethyl glycoside 3,4-di-O-m ethyl 3,6-di-O-methyl 4,6-di-O-m ethyl 2,3,4-tri-O -m ethyl 2,3,5-tri- O-methyl 2,3,6-tri-O -m ethyl

P2 200

r— — — 7.50 — 3.21; 4.30; 4.70 4.17; 4.70 — 1.80 — — — — — — — — —

— — — 2.62; 2.89 — 1.61; 2.23; 2.49 2.08; 2.38 — 1.52*; 1.60 —

— — — 9.3fc; 9.8 — 3.90; 4.77; 5.48; 6.20 5.12; 6.08 —

3.37

— — — — — — — 16.4

— — — 2.26 — — —

r*

r*

r*

2 .0 0



10.9; 11.1 — — 3.16; 3.64 2.17; 2.23 1.70; 1.80; 2.18; 2.30 2.19; 2.60 1.54; 1.62 1.00

0.82 — — — — — — — —

na glass D2 5

r

T *

6

P8 160 N „ 39

P9 175 N2, 14.1

P10 195 N2, 20

183 0.6 (O .D .) na glass FI 7

120

0.3 (O.D.) na brass FI

300 0.3 na glass FI 9

r*

r*

8 Tf

r*

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — —

— — — —

— — — —

— — — —

— — — —

1.51 1.45

— — — — — — — —

2.28 1.60 1.46 1.47; 1.93 1.45 1.74 1.32; 1.82

— — — — — — — —

— — — — — — — —

— — — — — — — —

— — — — — — — —

2 .1 0

2.28 2 .0 2

1.96 1.84 — 1.51

1.00



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

Packing T em perature (°C) Gas; flow rate (m f/m in ) C olum n Length, cm Diam eter (I.D .), cm Form M aterial Detector Reference

26

Table GC 11 (continued) METHYL GLYCOSIDES OF METHYLATED SUGARS

2,3,4-tri-O -m ethyl 2,3,6-tri-O-m ethyl 2,4,6-tri- O-methyl 2,5,6-tri-O-m ethyl 3,4, 6 -tri-O-methyl 3,5, 6 -tri-O-methyl 2,3,4,6-tetra- O-methyl D-Fructoside 3,4-di-O-methyl 1 ,3,4-tri-O-methyl

— — — — 3.74 — 1.54

— — — — — — —

— — — — — — —





1.89; 2.49; 3.94; 4.43

1.72; 2.23; 3.02 1.10; 1.31; 1.71; 2.39



2.74; 4.12 1.04; 1.26

1.52; 1.79 1 .0 1 ; 1.16

— —

3.11 5.08 — — 3.08 — 1.42 —

1.65 2.35 — — 1.71 — 1.29

0.73 0.82 0.67 0.74 0.67 —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

























— —

— —

— —

0.9; 3.9; 5.2 1.70; 2.72; 3.21; 3.53; 4.00 0.9; 2.5; 3.72; 4.6 — —

— —

— —

— —

1.00

1,3,6 -tri- O m ethyl

1,4,6 -tri-O-methyl



































8.4; 9.3 — — 2.53; 3.24

— 2.47; 3.12 — — 1.77; 2.21

— 11.7; 15.8 — — 2.78;3.72

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

7.18

3.90; 4.26















5.72 3.67 4.21 2.25 2.37 1.81 1.00



— —

— — — — —

C a rb o h y d ra te s

3,4, 6 -tri-O-methyl 1,3,4,6-tetra- O-methyl Methyl(methylcr-Dglucopyranosid) uronate 2 - O-methyl 3-O-methyl 4 -O-methyl 2,3-di- O-methyl 2 ,4-di-O-methyl 3,4-di-O-methyl 2 ,3,4-tri-O m ethyl Methyl(methylcr-Dgalactopyranosid) uronate 2 ,3,4-tri-O m ethyl



27

tr relative to methyl 2,3,4,6-tetra-O-methyl-0-D-glucopyranoside. tr relative to methyl 2,3,4,6-tetra-O-methyl-0-D-galactopyranoside (6.2 min). t r relative to methyl 2,3,4,6-tetra-O-methyl-cr-D-glucopyranoside. tr relative to methyl 2,3,6-tri-O-methyl-a-D-mannopyranoside (10.6 min). tr relative to methyl 2,3,4-tri-O-methyl-0-D-xylopyranoside (1.55 min). tr relative to methyl (methyl 2,3,4-tri-O-methyl-a-D-glucopyranosid) uronate (3.07 min). tr relative to methyl 2,3,4,6-tetra-O-methyl-a-D-galactopyranoside. Shoulder (incomplete resolution). Furanoside.

Packing

D etector

PI P2 P3 P4 P5 P6 P7 P8 P9 P10 D1 D2

15% butane 1,4-diol succinate polyester on acid-washed Celite (80 to 100 mesh). 10% polyphenyl ether [m-bis(m-phenoxyphenoxy)benzene] on acid-washed Celite. 14% ethylene glycol succinate polyester on C hrom osorb W (80 to 100 mesh). 5% neopentyl glycol succinate polyester on acid-washed Chrom osorb W (60 to 80 mesh). 5% ethylene glycol succinate polyester on Chrom osorb W. 2% neopentyl glycol succinate polyester on C hrom osorb W. 10% poly(ethylene glycol adipate) on HM DS-treated Celite (80 to 100 mesh). 5% XE-60 on Embacel AW (60 to 70 mesh). 4% GE-XE-60 on Gas-Chrom Z (80 to 100 mesh). 3% Carbow ax 6000 on HM DS-treated Chrom osorb W (100 to 120 mesh). 0 -ionization. therm al conductivity.

REFERENCES 1. 2. 3. 4. 5.

Aspinall, G. O ., Gas-liquid partition chrom atography of methylated and partially methylated methyl glycosides, J. Chem. Soc., 1676, 1963. Stephen, A. M ., Kaplan, M ., Taylor, G. L ., and Leisegang, E. C ., Application of gas-liquid chrom atography to the structural investigation of polysac­ charides. I. The structures of the gums of Virgilia oroboidesand of Agave americana, Tetrahedron, Suppl. 7, 233, 1966. O vodov, Yu. S. and Pavlenko, A. F ., Gas-liquid chrom atography of methylated D-galactose derivatives, J. Chrom atogr., 36, 531, 1968. Heyns, K ., Sperling, K. R ., and G riitzm acher, H . F., Com bination of gas chrom atography and mass spectrom etry for analysis of partially methylated sugar derivatives. The mass spectra of partially methylated methyl glucosides, Carbohydr. Res., 9, 79, 1969. Bhattacharjee, S. S. and G orin, P . A. J ., Identification of di-O-, tri-O , and tetra-O-methylmannoses by gas-liquid chrom atography, Can. J. Chem., 47,1207,1969.

CRC Handbook of Chromatography

" h c d ' 1 * h '

28

Table GC 11 (continued) METHYL GLYCOSIDES OF METHYLATED SUGARS

6.

7. 8.

9.

Percival, E. and Young, M ., C haracterisation of sucrose lactate and other oligosaccharides found in the Cladophorales, Carbohydr. Res., 20, 217, 1971. Anderle, D ., Kovac, P ., and Anderlova, H ., Separation of some methyl O-methyl-D-xylofuranosides by gas chrom atography. II, J. Chrom atogr., 64, 368, 1972. Anderle, D. and Kovac, P ., Separation of methyl(methyl-O-methyl-a-D-glucopyranosid) uronates by gas chrom atography, J. Chrom atogr., 91, 463, 1974. F oum et, B., Dhalluin, J.-M ., Leroy, Y., M ontreuil, J., and Mayer, H ., Analytical and preparative gas-liquid chrom atography of the fifteen methyl ethers of methyl-a-D-galactopyranoside, J. Chromatogr., 153, 91, 1978.

C a rb o h y d ra te s

29

30

CRC Handbook of Chromatography Table GC 12 TRIFLUOROACETATES OF COMMON MONO- AND OLIGOSACCHARIDES Phase Tem perature Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

PI T1 N2, 7.5

P2 T2 N2, 63

P3 T3 N2, 62

P4 T4 N2, 0.79

200 0 .2

U-tube glass FI

183 0.39 na glass FI

183 0.39 na glass FI

1

2

2

5000 0.034 capillary glass D1 3

Parent sugar L-Arabinose

0.47; 0.55; 0.70* 0.39; 0.58; 0.75* 0.67; 0.78

D-Xylose D-Ribose D-Lyxose L-Rhamnose L-Fucose D-Glucose D-Galactose D-Mannose D-Fructose 2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 2-Amino-2-deoxy-D-mannose Sucrose Lactose Maltose M altotriose Raffinose Stachyose ° *

ra

r“

— 0.68; 0.77 —

r" 0.64; 0.87* 0.61; 0.76; 0.81* —







0.33; 0.47; 0.61* 0.43; 0.50 0.92; 1.00 1.00; 1.05;* 1.17* 0.94; 1.11 —







0.95; 1.00 — —

— — —

0 . 8 6 ; 1.61

1.44; 1.61;* 1.67* 1.47; 1.56







1.76; 1.82 —

— — —

2.14 2.48

0.47;* 0.54; 0.69;* 0.70 0.43; 0.61 0.64; 0.70; 0.90;*0.95* 0.37; 0.61;* 0.74;* 1.32* 0.33; 0.66 — 0.77; 1.00 0.84;* 1.15;* 1.17 1.33 0.88;* 0.91



— —















0.82; 1.00 0.76; 1.10;* 1.17* 0.74 0.80;* 0.84;* 0.90

r*

1.77 1.93; 2.00



1.86



2.52 2.27 2.70









tr relative to trifluoroacetate of p-D-glucose (36 min in Reference 1, 8.4 min and 5.6 min for P2 and P3 in Reference 2, 2.37 min in Reference 3). Minor peak.

Phase

PI P2 P3 P4 Tem perature T 1 T2 T3

Detector

T4 D1

= = = = = = =

5% OV-210 on V araport 30. 3% OV-210 on C hrom osorb W H P (80 to 100 mesh). 10% OV-17 on Chrom osorb W H P (80 to 100 mesh). SE-54 W COT capillary column (G. C. Labor, Jaeggi, Switzerland). tem perature program m ed, 90 190°C at 1°/m in. 75°C for 1 min, then tem perature program m ed 75 -* 225°C at 10°/m in. 75°C for 1 min, then tem perature program m ed 75 -* 225°C at 10o/m in; iso­ therm al at 225°C for 5 min. = 74°C for 4 min, then tem perature program m ed 74-► 130°C at l°/m in . = 63Ni electron-capture detector.

Carbohydrates

31

Table GC 12 (continued) TRIFLUOROACETATES OF COMMON MONO- AND OLIGOSACCHARIDES REFERENCES 1.

2.

3.

Z anetta, J. P ., Breckenridge, W. C ., and Vincendon, G ., Analysis of monosaccharides by gasliquid chrom atography of the O-methyl glycosides as trifluoroacetate derivatives. Application to glycoproteins and glycolipids., J. Chromatogr., 69, 291, 1972. Sullivan, J. E. and Schewe, L. R., Preparation and gas chrom atography of highly volatile trifluoroacetylated carbohydrates using N-methylbis (trifluoroacetamide), J. Chromatogr. Sci., 15, 196,1977. Eklund, G ., Josefsson, B., and Roos, C ., Gas-liquid chrom atography of monosaccharides at the picogram level using glass capillary columns, trifluoroacetyl derivatization and electron-capture detection, J. Chrom atogr., 142, 575, 1977.

Table GC 13 TRIFLUOROACETATES OF SOME HEXOPYRANOSIDES AND -FURANOSIDES Packing T em perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Parent com pound D-Glucopyranoside methyl omethyl fiethyl pphenyl aphenyl PD-Glucofuranoside methyl qmethyl P~ ethyl pphenyl P~ D-Galactopyranoside methyl qmethyl P~ ethyl aethyl P~ phenyl aphenyl PD -G alactofuranoside methyl omethyl P~ ethyl pphenyl p~ D-M annopyranoside methyl amethyl P~ D -M annofuranoside methyl a-

PI 115 N2, na

P2 140 N2, na

P3 165 N 2, na

P4 165 N2, na

P5 155 N2, na

P6 160 N2, na

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

rB

rB

rB





r"

1.00

1.05 1.16 1.38 7.34 9.35

1.09

1.11

1.10

1.21

1.46 4.64 6.26

1.99 1.48 2.26* 5.22*

2.14 1.96 2.74c 7.09c

1.03 1.38 1.75 d d

0.93 0.90 0.92 5.70

0.93 0.90 1.04 4.12

1.06 0.97 0.87 2.09b

1.10

0.98 1.30 1.13 1.51 4.37 7.03

1.19 2.50 1.04 2.57 2.83* 7.52*

1.16 2.78

1.29 8.60 10.29

0.97 1.27 1.03 1.38 6.76 10.47

0.79 0.78 0.90 7.33

0.91 0.80 0.84 5.98

0.97 0.89 0.92 4.64

1.23 1.05 0.96 2.70*

1.34 1.13

1.00

1.00

1.00

1.00

1.00

1.00

1.29

1.46

1.42

2.70

3.30

1.97

1.05

1.18

1.09

1.32

1.43

1.30

1.08 1.31 8.55 10.43 0 .8 6

0.79 0.95 7.19 0.92 1.03 1 .00

1.04 0.85 2.65c

1.00

2.83 3.20c 8.91c

1.00

3.03c

0.98 0.80 0.90 d 0.96 1.70 1.08 1.35 d d 1.08 0.80 0.95 d

32

CRC Handbook o f Chromatography

Table GC 13 (continued) TRIFLUOROACETATES OF SOME HEXOPYRANOSIDES AND -FURANOSIDES a b c d

tr relative to trifluoroacetate of methyl a-D-m annopyranoside (1.05, 1.07, 0.82, 1.15, 1.15, and 1.00 min for P I, P2, P3, P4, P5, and P 6 , respectively. Colum n tem perature 190°C. Colum n tem perature 180°C. Not eluted during 20 min at 200°C.

Packing

PI P2 P3 P4 P5 P6

= = = = = =

1.5% SE-30 on C hrom osorb W (60 to 80 mesh). 3% SE-52 on Chrom osorb W (60 to 80 mesh). 2°7o QF-1 on Chrom osorb W (60 to 80 mesh). 3°7o neopentyl glycol succinate polyester on C hrom osorb G (60 to 80 mesh). 3% butanediol succinate polyester on Chrom osorb G (60 to 80 mesh). 5% Ucon oil 50 LB 550X on C hrom osorb W (60 to 80 mesh).

From Yoshida, K., H onda, N., lino, N., and Kato, K., Carbohydr. Res., 10, 333, 1969. With permission.

Table GC 14 (continued) TRIFLUOROACETATES OF O-METHYL GLYCOSIDES PRODUCED ON METHANOLYSIS OF POLYSACCHARIDES AND GLYCOPROTEINS Phase T em perature Gas; flow rate (m i/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

P2 T2 N2, 44

P3 T3 A ra, 1—2

200

183

0 .2

0 .2

U-tube glass FI

na glass D1

1

2

7000 0.05 capillary glass D2 3

PI T1 N 2, 7.5

P arent sugar

Tb

r6

L-Arabinose D-Xylose

0.42; 0.48 0.36; 0.48



D-Ribose

0.45;c 0.48; 0.62



D-Lyxose



L-Rhamnose L-Fucose

0.32; 0.45 0.39; 0.52

D-Glucose D-Galactose

1.00; 1.05 0.88; 0.91; 1.08c

D-Mannose 2-Amino-2-deoxy-D-glucose

0.95; 1.12c 1.44; 1.52

2-Amino-2-deoxy-D-galactose

1.62; 1 . 6 8 c

2-Amino-2-deoxy-D-mannose 2-Acetamido-2-deoxy-Dglucose 2-Acetamido-2-deoxy-Dgalactose

1.52; 1.62 1.74; 2.18 1.77; 1.86c



— — 0.38; 0.40;c 0.64 1.00; 1.23 1.02; 1.42 0.96; 1.40c 1.80;c 1.90; 2.18 1.84; 2.03;c 2.14; 2.49c —

1.79; 1.90;c 2.18;c 2.44c 1.83;c 2.02; 2.14;c 2.49c

T b

0.47; 0.53 0.45; 0.46;c 0.50 0.50; 0.54;c 0.55; 0.67 0.43; 0.47; 0.54c 0.38; 0.50 0.44; 0.45;c 0.57 1.00; 1.07 0.89;c 0.92;c 0.94; 1.10 0.96; 1.14c — — — 1.42;c 1.66; 1.73c 1.51;c 1.57;c 1.62;c 1.72

Carbohydrates Table GC 14 (continued) TRIFLUOROACETATES OF O-METHYL GLYCOSIDES PRODUCED ON METHANOLYSIS OF POLYSACCHARIDES AND GLYCOPROTEINS N-Acetylneuraminic acid

2.42; 2.48

D-Glucuronic acidd D-Galacturonic acidd

1.30; 1.44 0.76; 0.88c

° b c d

3.05;c 3.1 l ; c 3.24

2.32

Containing m ethane (5%). t r relative to trifluoroacetate of methyl a-D-glucopyranoside (33 min in Reference 1, 16.8 min in Reference 2, 37.2 min in Reference 3). M inor peak. Methyl ester.

Phase

PI P2 P3

T em perature T 1 T2 T3 Detector D1 D2

5% OV-210 on Varaport 30. 2°7o X F-1105 on Gas-Chrom P (80 to 100 mesh), capillary packed with Supelcoport (80 to 100 mesh), then 10 m l 1% solution of OV-210 in dichlorom ethane-acetone (4:1) passed through column under N 2 pressure 1.5 kg/cm 2; solvent evaporated by passing N 2 through column before conditioning at 210°C over­ night at N 2 flow-rate 2 m i/m in . tem perature program m ed, 90 190°C at l°/m in . tem perature program m ed, 80 -*■ 200°C at 2°C /m in. tem perature program m ed, 120 -►210°C at 1°/m in. 63Ni linear electron capture detector. Hewlett-Packard 5709 electron capture detector. REFERENCES

1.

2.

3.

Zanetta, J. P ., Breckenridge, W. C ., and Vincendon, G ., Analysis of m onosacchar­ ides by gas-liquid chrom atography of the O-methyl glycosides as trifluoroacetate derivatives. Application to glycoproteins and glycolipids, J. Chromatogr., 69, 291, 1972. Pritchard, D. G. and Niedermeier, W ., Sensitive gas chrom atographic determination of the m onosaccharide com position of glycoproteins using electron capture detec­ tion, J. Chrom atogr., 152, 487, 1978. W rann, M. M. and Todd, C. W ., Sensitive determ ination of sugars utilizing packed capillary columns and electron capture detection, J. Chromatogr., 147, 309, 1978.

33

CRC Handbook of Chromatography

34

Table GC 15 TRIFLUOROACETATES OF ALDITOLS AND MONO-OMETHYL-dGLUCITOLS Phase Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

P2 T1 N2, 7.5

1524 0.051 SCOT glass FI

200 0 .2

U-tube glass FI 2

1

Parent com pound Erythritol Threitol L-Arabinitol Ribitol Xylitol D-Glucitol Galactitol D-Mannitol Allitol L-Iditol D-Talitol Fucitol 2-Amino-2-deoxy-D-glucitol 2-Amino-2-deoxy-D-galactitol D-Glucitol 2 -O-methyl 3-O-methyl 4-O-methyl 6 -O-methyl ° b c

PI 160 He, 4

Tb

rc

0.52 0.62



1.00



0.93 1.06 1.54



1.68

1.43 1.77 1.47 1.47



— 1.25 1.35 1.19 —

P3 T2 N2, 44

P4 T3 Ar°, 1—2

P5 T4 He, 25

183 na glass D1 3

7000 0.05 capillary glass D2 4

305 0.32 (O.D.) na SS FI 5

rc

rc

rc

0 .2





— 0.70 0.65 — 0.96

— 0.99

1.00

1.28 1.18

0.87



— —







— —

1.22

— —

























































0.63 0.79









0 .88









0.29

0.84 1.82 1.94

— — —

Containing m ethane (5%). tr relative to trifluoroacetate of L-arabinitol (3.22 min). tr relative to trifluoroacetate of meso-inositol (24 min in Reference 2 , 26.3 min in Reference 3, 27.5 min in Reference 4, 15.7 min in Reference 5). PI P2 P3 P4

= = =

P5 Tem perature T1 T2 T3 T4 Detector D1 D2

= = = = = = =

Phase

=

FS-1265 SCOT column (Perkin-Elmer). 5°7o OV-210 on V araport 30. 2°/o X F-1105 on Gas-Chrom P (80 to 100 mesh). OV-210 on Supelcoport (80 to 100 mesh) in packed capillary (see P3 in Table GC 14 for method of preparation). 1°7o XE-60 on Gas-Chrom Z (80 to 100 mesh). tem perature program m ed, 90 -* 190°C at 1°/m in. tem perature program m ed, 80 -* 200°C at 2°/m in. tem perature program m ed, 120 -* 210°C at 1°/m in. tem perature program m ed, 130-*- 150°C at l°/m in . 63Ni linear electron capture detector. H ewlett-Packard 5709 electron capture detector.

Carbohydrates Table GC 15 (continued) TRIFLUOROACETATES OF ALDITOLS AND MONO- O-METHYL-dGLUCITOLS REFERENCES 1. 2.

3.

4. 5.

Shapira, J ., Identification of sugars as their trifluoroacetyl polyol derivatives, Nature (Lon­ don), 222,792,1969. Z anetta, J. P ., Breckenridge, W. C ., and Vincendon, G ., Analysis of monosaccharides by gas-liquid chrom atography of the O-methyl glycosides as trifluoroacetate derivatives. Ap­ plication to glycoproteins and glycolipids, J. Chromatogr., 69, 291, 1972. Pritchard, D. G. and Niedermeier, W ., Sensitive gas chrom atographic determination of the m onosaccharide composition of glycoproteins using electron capture detection, J. Chro­ m atogr., 152,487,1978. W rann, M. M. and Todd, C. W ., Sensitive determ ination of sugars utilizing packed capil­ lary columns and electron capture detection, J. Chromatogr., 147, 309, 1978. Anderle, D. and Kovac, P ., Gas chrom atography of partially methylated alditols as trifluo­ roacetyl derivatives. Separation of mono-O-methyl-per-O-TFA-D-glucitols, J. Chroma­ togr., 4 9,419,1970.

35

Parent sugar Erythrose L-Arabinose D-Xylose D-Ribose D-Lyxose D-Glucose D-Galactose D-Mannose D-Allose D-Altrose D-Gulose D-Talose D-glycero-D-galacto-Heptose D-glycero-D-gu/o-Heptose 2-Deoxy-D-ribose

P2 PI PI T1 250 140 Ar, 75 (monosaccharides), 150(oligo-) 183 0 . 6 ( 0 . D.) coiled SS FI 1 r“ 0.10; 0.12; 0.14 0.28; 0.33; 0.38* 0.31;* 0.43; 0.54 0.27;* 0.32; 0.35 0.26; 0.33 1.00; 1.57 0.76;* 0.88; 1.08 0.70; 1.08 0.76; 0.81; 0.91 0.65; 0.68; 0.94* 0.66; 0.74; 0.95* 0.86; 1.00;* 1.13 4.27 2.29 0.16

244 0.6 U—tube glass D1

183 0.6 (O.D.) coiled SS FI 1

1

P2 150

P3 170 H2, 75

P4 144 N2, 15.5

P5 T2 N 2, 50

P6 T3 N 2, 40

P7 T4 N 2, 20

244 0.6 U—tube glass D1 1

200 0.47 coiled copper D2 2

300 0.32 na SS FI 3

250 0.32 na glass FI 4

152 0.6 coiled glass FI 5

183 0.2 na glass FI 6

r'

r/

r*

Tc

Tb

rd

-

0.29; 0.35

-



0.97; 1.10;* 1.31 1.64; 2.11



0.32(0)



0.50(a)



0.38; 0.40



0.30; 0.39

1.00; 1.94 0.91;* 1.03; 1.38 0.62; 1.31 0.78; 0.91; 1.20* 0.63; 0.75; 1.16* 0.84; 0.95; 2.11* 1.06;* 1.22; 1.51 — — —

1.14; 1.18 1.00(a)

— — — —

1.22; 1.33; 1.48* 0.94; 1.26; 1.42 5.48 —

— —



















— — —

— — 0.49

-

-

0.78; 1.18

0.31

0.23; 0.24; 0.25; 0.28 0.21; 0.26*

0.36; 0.37 0.31; 0.38



0.58; 1.00 0.46; 0.55; 0.67;* 0.71 0.42; 0.69

0.86; 1.13 0.72;* 0.80; 0.90 0.66; 0.89 0.72; 0.76; 0.82* 0.64; 0.67; 0.82 0.65; 0.71; 1.00* 0.74; 0.78; 0.91 — — —

1.00; 1.20 0.87;* 0.94; 1.03 0.86; 1.03



















— — 0.20

0.45; 0.57

r"

0.17; 0.18

0.19; 0.22; 0.26; 0.27* 0.30; 0.37

— 0.18



r*

0.46; 0.51; 0.56* 0.49;* 0.61; 0.72 0.50; 0.53; 0.54

0.66; 0.76 0.65; 0.73 0.70 — 1.00; 1.05 — —

















— — 0.24;* 0.27

— — —

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

Packing Temperature (°C) Gas; flow rate (m l/h r) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

36

TABLE GC 16 TRIMETHYLSILYL ETHERS OF NEUTRAL MONOSACCHARIDES AND COMMON OLIGOSACCHARIDES

2-Deoxy-D-glucose 2-Deoxy-D-galactose L-Rhamnose L-Fucose 6-Deoxy-D-glucose D-eryt/jro-Pentulose

0.16;* 0.25; 0.46; 0.64 0.42; 0.45; 0.53 0.30 0.33; 0.38; 0.45 — 0.23;* 0.25; 0.33; 0.35









0.59









0.20 0.25

























0.56



0.69

D- t/ireo-Pentulose D-Fructose

0.69

L-Sorbose

0.85

D-Tagatose D-a/fro-Heptose Sucrose Trehalose Maltose Cellobiose

Gentiobiose Lactose Melibiose











0.21; 0.28* 0.20; 0.26; 0.30;* 0.32 —

0.33; 0.42 0.35;* 0.39; 0.45 —















0.41; 0.42; 0.44; 0.51* 0.34;* 0.44; 0.51;* 0.67



0.48; 0.59 0.50;* 0.55; 0.60 — 0.47; 0.54; 0.59 0.42; 0.46; 0.54; 0.61* 0.85; 0.91*





— —

— — —





0.34 0.42 0.52; 0.72

0.80 — —



— — —

— — —

— — —









1.12



0.81;* 0.94; 1.02* 0.88;* 0.94;* 0.99 — 2.40 2.55 2.52; 2.63



— — — — 0.92 — — 1.02 1.59 1.66 —

2.50; 2.71

7.98 6.40 52.4

_

_

_

_

_

_

























— — —

— — —

— — —

— — —

— — —

_

2.98 2.41; 2.69 2.85; 2.90 3.86 3.72

— — —



1.99 2.88

tr relative to trimethylsilylated a-D-glucose (20 min at 140°C, 1.3 min at 210°C on P I; 25.2 min on P2 at 150°C; 11.0 min in Reference 5; 7.25 min in Reference 6). tr relative to trimethylsilylated sucrose (2.3 min). tr relative to trimethylsilylated methyl a-L-arabinopyranoside (10.85 min). tr relative to trimethylsilylated a-D-galactose. tr relative to trimethylsilylated /?-D-glucose (about 26 min). tr relative to trimethylsilylated D-mannitol (about 58 min). Minor peak. Trace.

37

" fc c d ' ' * *



Carbohydrates

Melezitose Raffinose Stachyose

22.6 10.5 15.1;* 19.0; 20.0 120.5 99.0 —





1.00

0.65; 0.78





— 1.12 10.4 9.04;* 13.5 11.7;* 13.1; 1.16 11.9;* 16.6; 1.14;* 1.37



Table GC 16 (continued) TRIMETHYLSILYL ETHERS OF NEUTRAL MONOSACCHARIDES AND COMMON OLIGOSACCHARIDES

Temperature

Detector

PI P2 P3 P4 P5 P6 P7 T1 T2 T3 T4 D1 D2

3% SE-52 on acid-washed, silanized Chromosorb W (80 to 100 mesh). 15% poIy(ethyIene glycol succinate) on Chromosorb W (80 to 100 mesh). 10% SE-52 on acid-washed, silanized Chromosorb W (80 to 100 mesh). 1.2% XE-60 on acid-washed DMCS-treated Chromosorb W (60 to 80 mesh). 3% SE-30 on Diatoport S (80 to 100 mesh). 2% SE-52 on acid-washed Diatomite C (85 to 100 mesh, BSS). 5% Dexsil 300 GC on Chromosorb W AW DMCS (80 to 100 mesh). 140°C for monosaccharides, 210°C for oligosaccharides, temperature programmed, 140 -* 200°C at 0.5°/m in. temperature programmed, 140 -*■ 290°C at 4°/m in. temperature programmed, 160 -» 240°C at 10o/m in, then 240 -*• 350°C at 30°/min; isothermal at 350°C for 11 min. argon ionization detector. thermal conductivity (katharometer). REFERENCES

1. 2. 3. 4. 5. 6.

Sweeley, C. C ., Bentley, R., Makita, M., and Wells, W. W ., Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances, ./. Am. Chem. Soc., 85, 2497, 1963. Cheminat, A. and Brini, M ., Separation of various monosaccharides by gas chromatography, Bull. Soc. Chim. Fr., 80, 1966. Ellis, W. C ., Liquid phases and solid supports for gas-liquid chromatography of trimethylsilyl derivatives of monosaccharides, J. C h rom atogr.,4\, 335, 1969. Bhatti, T ., Chambers, R. E., and Clamp, J. R., The gas chromatographic properties of biologically important N-acetylglucosamine derivatives, monosaccharides, disac­ charides, trisaccharides, tetrasaccharides, and pentasaccharides, Biochim. Biophys. Acta, 222, 339, 1970. Holligan, P. M. and Drew, E. A ., Routine analysis by gas-liquid chromatography of soluble carbohydrates in extracts of plant tissues. II. Quantitative analysis of standard carbohydrates, and the separation and estimation of soluble sugars and polyols from a variety of plant tissues, New Phytol., 70, 271, 1971. Janauer, G. A. and Englmaier, P ., Multi-step time program for the rapid gas-liquid chromatography of carbohydrates, J. Chromatogr., 153, 539, 1978.

00

CRC Handbook of Chromatography

Packing

U>

Carbohydrates Table GC 17 TRIMETHYLSILYL ETHERS OF NEUTRAL OLIGOSACCHARIDES Packing T em perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

PI T1 N2, 50

P2 228 N2, 18

P3 T2 He, 50

122

100

0.4 na glass D1

0.32 na glass FI

270 0.32 coiled SS FI 3

183 0.32 na SS FI 4

PI 210

Ar, 70—80

Parent sugar Disaccharides Sucrose a, a-Trehalose /T/?-Trehalose Kojibiose Sophorose Lam inaribiose Maltose Cellobiose Isomaltose Gentiobiose Lactose Neolactose Melibiose Lactulose Turanose Palatinose

2

ra

r-

r*

1.00





















1.38 1.90 1.40; 1.82 1.66; 1.99 1.64; 1.80 1.19; 1.33 1.22; 1.70 2.37; 2.77 2.36; 2.76 1.09; 1.54 0.81; 0.87 2.16; 2.33 0.94 1.28; 1.56 1.39;c 1.48; 1.79c 2.40; 2.80 — 2.38 0.95 0.73; 0.90; 1.03 —





— — — 1.59; 1.86 1.57; 1.77 1.17; 1.36 1.18; 1.77 1.96; 2.36 2.56; 2.68 0.99; 1.58







— — 1.22; 1.64 — — — — —

— 1.92;1.96 1.18; 1.52 1.70; 2.08 — — — —

— — — — — — — —



— — — 1.62 1.40 1.19;* 1.73 — —



3)-L-Ara

ra

1.00

1.00

a-D-Glcp-(l -5 )-D -G lc f /TD-Galp-(1 3)-D-Galp 0-D-Galp-(l — 6 )-D-Galp /TD-Glcp-(1 2)-L-Ara /TD-Galp-(1 3)-L-Ara 0-L-Arap-(l

1



1.00; 1.97 2.5 — 0.80; 0.96; 1 .19C 0.67; 0.74;c

1.37;* 1.49 — — — — —



— — — 0.92; 1.00 — — — — — — —

— — —

— — — — —

1.10

0-D-Xylp-(l

3)-D-Xyl

P-D-Xylp-(1 - 4)-D-Xyl ft-D-Xy\p-(\ 6 )-D-Glc /TD-Manp-(1 -►4)-D-M anp a-D-M anp-(l -►6 )-D-Glc 0-D-M anp-(l — 4)-D-Gul a-L-Fucp-(l 2)-L-Fucp a-L-Fucp-(l 3)-L-Fucp a-L-Fucp-(l 4)-L-Fucp Higher oligosaccharides Raffinose M altotriose Isom altotriose M annotriose Stachyose M altotetraose Isom altotetraose

0.42;c 0.56; 0.92c 0.69;c 0.89 — 1.03; 1.40 — 1.31 0.51 0.46; 0.95c 0.51 ;d 0.57 — — — — — — —

1 .0 0 '

— 1.87' 1.2 2 * 1 .0 0 ' — 1.67'

— — — — — — —

Note: This table supplements Table GC 124 in Section A, Volume I, Section I.II.

— 1.40 — — — 1.92 —

39

40

CRC Handbook o f Chromatography Table GC 17 (continued) TRIMETHYLSILYL ETHERS OF NEUTRAL OLIGOSACCHARIDES a h c d ' 1

t r relative to trimethylsilylated tr relative to trimethylsilylated Minor peak. Shoulder. tr relative to trimethylsilylated tr relative to trimethylsilylated

Packing

PI P2 P3 Tem perature T1 T2 Detector

D1

sucrose (12.1 min in Reference 3). /3-maltose (about 13 min,).

raffinose. stachyose.

3% SE-30 on Gas-Chrom P (Reference 1) or D iatoport S (Reference 2). 3% OV-17 on C hrom osorb W — HP (80 to 100 mesh). 3% OV-17 on silanized C hrom osorb W (60 to 80 mesh). 220°C for disaccharides, 300°C for tri- and tetrasaccharides. tem perature program m ed, 150 325°C at 10°/m in, then isothermal at 325°C for 12 min. /3-ionization. REFERENCES

1. 2.

3. 4.

Percival, E ., Gas chrom atography of the trimethylsilyl ethers of some less-common disacchar­ ides, Carbohydr. Res., 4,441, 1967. Bhatti, T ., Cham bers, R. E ., and Clam p, J. R., The gas chrom atographic properties of biologi­ cally im portant N-acetylglucosamine derivatives, m onosaccharides, disaccharides, trisaccharides, tetrasaccharides and pentasaccharides, Biochim. Biophys. Acta, 222, 339, 1970. H averkam p, J., Kamerling, J. P ., and Vliegenthart, J. F. G ., Gas-liquid chrom atography of trimethylsilyl disaccharides, J. Chrom atogr., 59, 281, 1971. Brobst, K. M ., Gas-liquid chrom atography of trimethylsilyl derivatives. Analysis of corn syrup, in M ethods in Carbohydrate Chemistry, Vol. 6 , W histler, R. L. and BeMiller, J. N., Eds., Aca­ demic Press, New York, 1972, 3.

Table GC 18 TRIMETHYLSILYL ETHERS OF VARIOUS GLYCOSIDES OF COMMON SUGARS Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diam eter (I.D .), cm Form M aterial Detector Reference P arent com pound

q

q

183 0.63 coiled SS FI

PI N2, na

P2 170 N2, na

P3 160 N2, na

P4 180 N 2, na

P5 160 N2, na

N2, na

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

200

2

1

2

2

2

P6 200

2

2



r*

r*

r*

r*

r*

r*

0.21

— —

— —



— —

— —



0 .2 0





0.31 0.34

























0.92 1.07 — — 7.16 4.67

1.47 1.54 1.70 5.96 7.00 —

1.45 1.56 1.95 6.76 8.57 —

1.52 1.65 1.74 6.52 8.60 —

1.71 1.81

1.85 2 .0 2

1.67 1.85

1.06 1.13 4.87

1.06 1.04 1.19 5.81

1.00

1.00

1.14

1.13

— — — —

2.13 11.73 19.47 —

10.38 —

1.25 1.23 1.30

1.16 1.14

1.15

1.15

1.01

1.11

1.20

1.13

1.13

6 .1 0

9.50

10.00

6 .6 6

1.00

1.00

1.00

1.00

1.39

1.26

1.29

1.22

2 .2 0

1.88 8 .0 0

41

0.59 0.67

1.02

11.40 16.80 —

Carbohydrates

L -A rabinopyranoside methyl methyl ftD-Xylopyranoside methyl amethyl f t D-Glucopyranoside methyl crmethyl ftethyl ftphenyl phenyl ftmethyl 4,6-benzylidene-aD-G lucofuranoside methyl amethyl ftethyl ftphenyl ftD-M annopyranoside methyl amethyl ft-

PI 140 na, 75

42

Table GC 18 (continued) TRIMETHYLSILYL ETHERS OF VARIOUS GLYCOSIDES OF COMMON SUGARS

P arent com pound D -M annofuranoside methyl aD-Galactopyranoside methyl a methyl pethyl aethyl pphenyl aphenyl pmethyl 4,6-benzylidene-amethyl 4,6-benzylidene-/?D-G alactofuranoside methyl a methyl pD -G alactofuranoside ethyl pphenyl pL-Idopyranoside methyl a methyl pmethyl 4,6-benzylidene-amethyl 4,6-benzylidene-/T

PI 140 na, 75 183 0.63 coiled SS FI

PI N2, na

P2 170 N2, na

P3 160 N2, na

P4 180 N2, na

P5 160 N2, na

N2, na

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

150 0.3 straight SS FI

200

2

1

ra

2

1.31

1.39

1.41

1.51

1.37

1.16 1.30 1.17 1.43 4.74 6.70 — —

1.15 1.32 1.19 1.43 5.24 7.62 — —

1.19 1.57 1.14 1.53 5.55 10.70 — —

1.33 1.61

9.50 20.60 — —

1.40 1.90 1.13 1.87 9.67 25.73 — —

1.19 1.50 1.09 1.56 6.28 10.50 — —

1.43 1.13

1.35 1.08

1.50 1.23

1.32 1.09

1.07 10.69

1.06 6.50



1.28

0.72 0.82

1.20 2 .1 0



1.20



1.00

1.17 1.03



1.09 4.61

1.10

1.11

1.10

5.14

6.65

9.30

— — — —

— — — —

— — — —

— — — —



0.75 0.64 2.96‘ 2.52;c 3.1 l c

2

r*

Tb



2

2

r*

Tb

— — — 2.77c 3.00c

2

P6 200

Tb

— — — —

r*

— — — —

CRC Handbook of Chromatography

Packing T em perature (°C) Gas; flow rate (m f /m in) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

" b r

tr relative to trimethylsilylether of a-D-glucose (20 min at 140°C, 1.3 min at 210°C). tr relative to trimethylsilylether of methyl a-D -m annopyranoside (1.15, 1.05, 0.90, 0.50, 0.75, and 1.60 min for P I, P2, P3, P4, P5, and P 6 , respectively). Colum n tem perature 210°C.

Packing

P1 P2 P3 P4 P5 P6

3% SE-52 on C hrom osorb W (80 to 100 mesh in Reference 1,60 to 80 mesh in Reference 2). 1.5% SE-30 on C hrom osorb W (60 to 80 mesh). 2°7o QF-1 on C hrom osorb W (60 to 80 mesh). 3% neopentyl glycol succinate polyester on Chrom osorb G (60 to 80 mesh). 3% butanediol succinate polyester on C hrom osorb G (60 to 80 mesh). 5% Ucon oil 50 LB 550X on Chrom osorb W (60 to 80 mesh). REFERENCES

1. 2.

Sweeley, C. C ., Bentley, R ., M akita, M ., and Wells, W. W ., Gas-liquid chrom atography of trimethylsilyl derivatives of sugars and related substances, J. A m . Chem. Soc., 85, 2497, 1963. Yoshida, K., H onda, N ., lino, N ., and Kato, K., Gas-liquid chrom atography of hexopyranosides and hexofuranosides, Carbohydr. Res., 10, 333, 1969.

Carbohydrates 43

P arent sugar L-Arabinose D-Xylose D-Ribose D-Lyxose L-Rhamnose L-Fucose D-Glucose D-Galactose D-Mannose D-Allose D-Altrose D-Gulose D-Talose 2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose

PI T1 N2, 50

P2 T2 He, 50

P3 T3 N2, 60

P4 T4 N2, 20

P5 130 N2, 13

250 0.32 U—tube glass FI 1,2

152 0.3 na SS FI 3

200 0.3 spiral glass FI 4

200 0.3 spiral glass FI 4

200 0.2 spiral SS FI 5

r° 0.22; 0.23 0.34; 0.37 0.24; 0.25 0.24; 0.28* 0.26 0.25;* 0.28; 0.31 0.79; 0.86 0.61;* 0.68; 0.74 0.59; 0.65* 0.53; 0.55; 0.60;' 0.63 0.42; 0.49; 0.57; 0.66 0.37; 0.51; 0.55; 0.66* 0.65; 0.70* — —

r6

rc

rc

Td

0.45; 0.49 0.59 — — — 0.50;* 0.52; 0.55 0.87 0.77;* 0.79; 0.83 0.72; 0.74* —

_

_

0.66; 0.69 — — — 0.56;* 0.59; 0.62 0.85; 0.87 0.73; 0.77; 0.81 0.74 —

0.36; 0.39 — — — 0.29;* 0.30; 0.34 0.67; 0.71 0.57;* 0.60; 0.65 0.54 —

— — — 0.19;* 0.23; 0.26 1.00(a) 0.62;* 0.74; 0.84 0.51; 0.57* —

















— 0.87; 0.93 0.76;* 0.80; 0.95

— 0.67; 0.75 0.52;* 0.64; 0.88

— — —

— 0.87(a) —

_ 0.31

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

Packing T em perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Reference

44

Table GC 19 TRIMETHYLSILYL ETHERS OF O-METHYL GLYCOSIDES OF NEUTRAL SUGARS AND OTHER SUGAR CONSTITUENTS OF GLYCOPROTEINS AND GLYCOSAMINOGLYCANS

2-Acetamido-2-deoxy-D-glucose 2-Acetamido-2-deoxy-D-galactose N-Acetylneuraminic acid D-Glucuronic acid' L-Iduronic acid'

1.05;' 1.17; 1.31; 1.38' 1.08; 1.21 2 .2 2

0.45; 0.82 0.37

1.20; 1.31

0.84; 1.04'

0.58; 0.96'

1.16; 1.26 — — —

0.78; 0.99' 1.09 0.88 0.90

0. 55;0.96' 1.14 0. 73 0. 74

“ tr relative to trimethylsilylated D-mannitol (about 57 min in Reference 2). b tr relative to trimethylsilylated m e s o inositol (about 9 min). c tr relative to trimethylsilylated myo-inositol (about 17 min for P3, 33 min for P4). d tr relative to trimethylsilylated methyl a-D-glucopyranoside (50 min). ' M inor peak. ' Methyl ester. Packing

Tem perature

PI P2 P3 P4 P5 T1 T2 T3 T4

3.8% SE-30 on D iatoport S (80 to 100 mesh). 0.05% OV-17 on glass beads (Corning G L C -110; 120 to 140 mesh). 3% Apiezon M on C hrom osorb W AW DMCS (100 to 120 mesh). 3% SE-30 on C hrom osorb W AW DMCS (100 to 120 mesh). 3% OV-17 on Gas-Chrom Q (100 to 120 mesh), tem perature program m ed, 140 200°C at 0.5°/m in. tem perature program m ed, 80 -*• 250°C at 10o/m in. tem perature program m ed, 110 280°C at 8 °/m in. tem perature program m ed, 110 200°C at 2°/m in. REFERENCES

1.

3. 4. 5.

Carbohydrates

2.

Clam p, J. R ., D awson, G ., and Hough, L ., The sim ultaneous estim ation of 6 -deoxy-L-galactose (L-fucose), D-mannose, D-galactose, 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) and N-acetylneuraminic acid (sialic acid) in glycopeptides and glycoproteins, Biochim. Biophys. Acta, 148, 342, 1967. Bhatti, T ., Cham bers, R. E ., and Clam p, J. R ., The gas chrom atographic properties of biologically im portant Nacetylglucosamine derivatives, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides, Biochim. Biophys. A cta, 222, 339, 1970. Reinhold, V. N ., Gas-liquid chrom atographic analysis of constituent carbohydrates in glycoproteins, in M ethods in E nzym ology, Vol. 25 (P art B), Hirs, C. H. W. and Tim asheff, S. N., Eds., Academic Press, New York, 1972, 244. Y okota, M . and M ori, T ., Gas-liquid chrom atographic analysis of the m onosaccharide composition of acid glycosaminoglycans (mucopolysaccharides) derived from animal tissues, Carbohydr. Res., 59, 289, 1977. C ahour, A . and H artm ann, L ., Study of neutral and amino-m onosaccharides by gas-liquid differential chrom atography: application to three reference glycoproteins, J. Chrom atogr., 152, 475, 1978.

45

Table GC 20 TRIMETHYLSILYLATED OXIME AND O-METHYL OXIME DERIVATIVES OF MONO- AND DISACCHARIDES P3 170 Nj, 35

P4 160 N j,3 0 — 35

244 0.6 (O .D .) U — tube glass D1

200 0.3 na na FI 2

200 0.3 na na FI 2

200 0.2 na SS FI 3

1

P4 160

200 0.2 na SS FI 3

P5 160 Nj, 30— 35

P5 160 N,, 30— 35

P6 120 Nj, 30—35

P6 120 N j,3 0 — 35

P7 215 Nj, 40

P8 215 Nj, 40

P9 272 He, 0.9

P10 140 Nj, na

200 0.2 na SS FI 3

200 0.2 na SS FI 3

200 0.2 na SS FI 3

200 0.2 na SS FI 3

200 0.3 coiled SS FI 4

200 0.3 coiled SS FI 4

4500 0.025 capillary glass FI 5

4500 0.02 capillary SS FI 6

Oxime

MO

MO

Oxime

MO

Oxime

MO

Oxime

MO

Oxime

Oxime

MO

Oxime

r*

r*

r*

r‘

rc

r'

r*

rc

rc

X*

x*

x*

x’

— — 0.32 — — — 0.97 1.00 3.05 2.96 — —

— — 0.35 — — — 0.99 1.00 2.60 2.46 — —

0.06 0.05 0.16 — 0.14 0.43 0.42 0.48 1.34 1.27; 1.41 1.30 —

— — — — — — 0.39 — 1.04 — 0.96

0.06 0.05 G. 15 —

— — — — — 0.25 — 0.84 — 0.83; 1.02

— — — — — — — — — — —

— — — — — — — — 2.16 1.98 2.00 2.08 1.98 2.10 — — — — — —













0.60 — — — 1.36 —

0.68 — — — 1.22 —

0.08; 0.09 0.09 0.20; 0.23 _ 0.20 0.53 0.55 0.60 1.55 1.43; 1.57 1.45

0.13 0.40 0.40 0.46 1.32 1.26; 1.54 1.31; 1.51











— — — — — — — —

— —



— —





— — — — — —

— — — — — — —

















8.29

5.90















-

0.69

0.82

-

-

-

-

-

-

-







2.79

2.30



0.73 —

0.41; 0.44 1.01; 1.08 1.02; 1.10 0.68 0.69 0.50 0.51 1.13 1.19

— — — — — — —

0.31 0.89 0.90 0.58 0.57 0.37 0.36 1.03 1.02



0.28 0.78 0.78 0.58 0.58 0.41 0.40 1.07 1.07

— — — — — 0.25 — 0.80 — 0.77



0.94; 1.02





0.72



0.78; 1.00 1.00

2.28; 2.78 2.95; 3.00 2.36; 2.47

CRC Handbook of Chromatography

M onosaccharides G lyceraldehyde Dihydroxyacetone D-Erythrose D-Threose L- g /y ce ro Te t r u lose L-Arabinose D-Xylose D-Ribose D-Glucose D-Galactose D-M annose D-AIlose D-Altrose D-Gulose 2-Deoxy-D-ribose 2-Deoxy-D-glucose 2-Deoxy-D-galactose L-Rham nose L-Fucose D- eryf/jro-Pentulose D- t h r e o - Pentulose D-Fructose L-Sorbose D - g l y c e r o - D - g u l o - H eptose M ethyl ethers D-Ribose 2-Q m eth y l

P2 170 N*. 35



P a ren t com pound

PI 170 Ar, 75

z

Phase T em perature (°C) G as; flow rate (m l/m in ) Colum n Length, cm D iam eter (I.D .), cm Form M aterial D etector Reference

On

3 -O m eth y l 2 ,3-di-O m ethyl D-Glucose, 2 ,3 ,6 -tri-O m e th y l D-G alactose, 2 ,3 ,4 ,6 -te tra -O m ethyl D isaccharides Kojibiose Nigerose M altose Cellobiose G entiobiose Lactose N eolactose M elibiose Lactulose Turanose Palatinose N ote:

“ b c d * 1 *

— — — —

0.71 0.47 3.15' 2.56'

0.84 0.72 —

— — —

— — —























— —

— —

— —

— —









— —

— —

— —

— —

— —

— —

— —

— — —

— — —

— — —

— — —

— — —

— — —

— — —

— — —

— — —













































































































— —

— —

— —

















— —

— —

— —

1.56; 1.75* 1.21;* 1.48; 1.57* 1.45 1.24; 1.35* 2.01; 2.16* 1.16 1.22; 1.35* 2.20; 2.46 1.15 1.47 —

1.36; 1.50* 1.05;* 1.23; 1.35* 1.26 1.09; 1.16* 1.82 0.97 0.99; 1.08* 1.95;2 .12* 0.94 1.23 —

1.02; 1.10* 0.94;1.00* 1.24; 1.31* 0.93; 0.95* —

1.39;1.49*

— — — — — —









1.31



M O = O m e th y l oxime.

tr relative to trimethylsilylated a-D-glucose (6.0 min). tr relative to trimethylsilylated O-methyl oxime of D-ribose (about 2 min). tr relative to trimethylsilylated D-glucitol (13.2 min, 6 .6 min, 14.4 min for P4, P5, P 6 , respectively). tr relative to trim ethylsilylated trehalose (14.5 min for P7, 17.05 min for P 8in Reference 4; 30 min for P9 in Reference 5) tr relative to trimethylsilylated oxime of dihydroxyacetone (about 12 min). Estim ated from published chrom atogram . M inor peak. 15% poly(ethylene glycol succinate) on C hrom osorb W (80 to 100 mesh). 3% SE-30 on Supelcoport (100 to 120 mesh). 3% OV-17 on Supelcoport (100 to 120 mesh). 0.5% OV-1 on C hrom osorb G (100 to 120 mesh). 0.5% OV-17 on Chrom osorb G (100 to 120 mesh). 3% DC QF-1 on Gas-Chrom Q (100 to 120 mesh). 1.5% SE-52 on C hrom osorb W AW DMCS (60 to 80 mesh). 1.5% OV-17 on Shimalite W (80 to 100 mesh). OV-101 coating in open tubular capillary column. OV-17 coating in capillary column, argon ionization detector.

Carbohydrates

Packing PI P2 P3 P4 P5 P6 P7 P8 P9 P10 D etector D1

^4

1. Sweeley, C. C ., Bentley, R ., M akita, M „ and Wells, W. W ., Gas-liquid chrom atography of trimethylsilyl derivatives of sugars and related substances, J. A m . Chem. Soc., 85, 2497, 1963. 2. Laine, R. A. and Sweeley, C. C., O-Methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chrom atography and mass spec­ trom etry, Carbohydr. Res., 27, 199, 1973. 3. Petersson, G ., G as-chrom atographic analysis of sugars and related hydroxy acids as acyclic oxime and ester trimethylsilyl derivatives, Carbohydr. Res., 33, 47, 1974. 4. T oba, T. and A dachi, S., Gas-liquid chrom atography of trimethylsilylated disaccharide oximes, J. Chromatogr., 135,411, 1977. 5. A dam , S. and Jennings, W. G., Gas chrom atographic separation of silylated derivatives of disaccharide mixtures on open tubular glass capillary colum ns, J. Chrom atogr., 115, 218, 1975. 6 . Anderle, D., Konigstein, J ., and Kovacik, V., Separation of trioses and tetroses as trimethylsilyl oximes by gas chrom atography, A nal. Chem., 49, 137,1977.

Table GC 21 TRIMETHYSILYL ETHERS OF POLYOLS, CYCLITOLS, AND REDUCED OLIGOSACCHARIDES Packing Tem perature (°C) Gas; flow rate (m l/m in ) Colum n Length, cm Diameter (I.D .), cm Form M aterial D etector Reference Parent com pound Acyclic polyols Ethylene glycol

P2 140 Ar, 75

P3 T1 N2, 40

P2 T2 He, na

P2 T3 He, na

P4 T2 He, na

P4 T3 He, na

P5 T4 He, 88

P6 175 N2, 42

P7

P8

200

210

N2, 45

Ar, 70—80

244 183 0.63 0.63 U —tube coiled glass SS D1 FI

152 0.63 coiled glass FI

183 0.32 na SS FI 3

183 0.32 na SS FI 3

183 0.32 na SS FI 3

183 0.32 na SS FI 3

244 0.63 coiled copper D2 4

183 0.4 na glass FI 5

183 0.4 na glass FI 5

122

200

200

0.4 na glass D3

0.35 na glass FI 7

0.35 na glass FI 7

PI 140 Ar, 75

6

P9 263 He, na

P10 218 He, na

1

2

r*

rfc

rfc

rc

rc

rc

rc

rd

r*

V

r'

r*

r*









0.14



0.09

0.43











1

CRC Handbook of Chromatography

REFERENCES

48

Table GC 20 (continued) TRIMETHYLSILYLATED OXIME AND O-METHYL OXIME DERIVATIVES OF MONO- AND DISACCHARIDES

0.11 0.49 2.13 2.06 2.08

0.16 0.46 0.46 0.42 1.24 1.28 1.21 1.30

0.32 0.68 0.67 0.64 1.11 1.13 1.09 i 11 1.11 1.12































1.37

0.09 0.29 0.61

0.51 0.71 0.86

0.07 0.24 0.58













1.00 —

1.00

1.00 —

1.00

1.00

0.45

1.57



2.6 6



0.68 0.85

2.70 —

1.00



1.00



— —



1.00

_

_

_

0.39 0.79 0.79 0.79 0.67

0.36 0.77 0.79 0.77 0.60 0.35 0.42

0.76 0.92 0.93 0.92

— — — —

0.86



0.46

0.77 0.93 0.93 0.93 0.89 0.76 0.81

0.75 0.79

— —

1.26

1.08

1.27

1.09 — — — — — — — —

— — — — — — — — —

1.00 1.17 0.73 0.88 0.56 0.50 0.46 0.45 0.81

1.00 0.86 0.79 0.78 0.63 0.55 0.51 0.49 0.71

— — — — — —

0.66 0.68 0.74 1.26 1.25 0.45

0.62 0.64 0.54 0.82 1.16 0.45



0.39

0.40



Carbohydrates

Glycerol Threitol E rythritol A rabinitol Ribitol Xylitol Glucitol Galactitol M annitol Iditol Talitol 2-Deoxy-D-ribitol 2-Deoxy-D-glucitol 3-Deoxy-D-glucitol 3-Deoxy-D-mannitol L-Rhamnitol 1, 6 -Dideoxyhexitols 2,6-Dideoxy-D -ribohexitol Cyclitols myo- Inositol c/s-Inositol scyllo- Inositol epj-lnositol chiro- Inositol muco- Inositol n eo in o sito l a //o ln o sito l m y o in o sito l, l-Omethyl 4-O-methyl 5-O-methyl 1,3-di-O-methyl 2 -C-methyl 2 - C-hydroxymethyl chiro-1 nositol, 2- Omethyl 4-O-methyl

VO

Table GC 21 (continued) TRIMETHYSILYL ETHERS OF POLYOLS, CYCLITOLS, AND REDUCED OLIGOSACCHARIDES

P arent com pound Reduced oligosaccharides Lam inaribitol M altitol Cellobiitol G entiobiitol Lacitiol Melibiitol D-M annitol,4- O-p-Dm annopyranosyl L-Gulitol,4- O-ft-Dm annopyranosyl L-Arabinitol,3- O-p-Larabinopyranosyl D-Xylitol,4- O-p-Dxylopyranosyl L-Fucitol,2- O-q-Lfucopyranosyl 3-O-a-Lfucopyranosyl 4-O-a-Lfucopyranosyl

P2 140 Ar, 75

P3 T1 N2, 40

P2 T2 He, na

P2 T3 He, na

P4 T2 He, na

P4 T3 He, na

P5 T4 He, 88

P6 175 N2, 42

P7 200 N2, 45

P8 210 Ar, 70—80

P9 263 He, na

P10 218 He, na

244 183 0.63 0.63 U —tube coiled glass SS D1 FI

152 0.63 coiled glass FI

183 0.32 na SS FI 3

183 0.32 na SS FI 3

183 0.32 na SS FI 3

183 0.32 na SS FI 3

244 0.63 coiled copper D2 4

183 0.4 na glass FI 5

183 0.4 na glass FI 5

122 0.4 na glass D3 6

200 0.35 na glass FI 7

200 0.35 na glass FI 7

rc

rc

rc

rc

Td

r*

r*

r'

r*

r*

— —































-











0.67 0.76 0.64 0.85 0.62 1.00

0.63 0.68 0.59 0.89 0.54 1.00

PI 140 Ar, 75

1

r

2

1

Tb

r6

















1.90 1.90 1.70





















































2.7 2.0

















— —



















































0.75











0.69

2.0 —



-





0.37



-





0.46 —









0.77

CRC Handbook of Chromatography

Packing Tem perature (°C) Gas; flow rate (m f/m in ) Column Length, cm Diameter (I.D .), cm Form M aterial Detector Reference

O

“ tr relative to trimethylsilylated methyl a-L-arabinopyranoside (10.85 min). b tr relative to trimethylsilylated a-D-glucose (20 min in Reference 1,11 min in Reference 2). c tr relative to trimethylsilylated D-glucitol (22.8 min for T 1, 38.6 min for T2, with P2; 20.8 min for T2, 36.3 min for T3, with P4). d tr relative to trimethylsilylated butane-1,4-diol (about 10.5 min). e tr relative to hexak/s trimethylsilyl-myo-inositol (3.7 min with P 6 , 10.8 min with P7). 1 tr relative to trimethylsilylated sucrose. * tr relative to trimethylsilylated melibiitol. Packing

PI P2 P3 P4 P5 P6 P7 P8 P9 P10 T em perature T1 T2 T3 T4 Detector D1 D2 D3

15% polyethylene glycol succinate) on Chrom osorb W (80 to 100 mesh). 3% SE-52 on acid-washed, silanized Chrom osorb W (80 to 100 mesh). 2% SE-52 on acid-washed Diatom ite C (85 to 100 mesh BSS). 3% JX R silicone gum on Gas-Chrom Q (100 to 120 mesh). 20% SF-96 on D iatoport S (60 to 80 mesh). 3% XE-60 on Supelcoport (80 to 100 mesh). 3% OV-1 on Gas-Chrom Q (100 to 120 mesh). 3% SE-30 on Gas-Chrom P. 2.2% SE-30 on Gas-Chrom S (100 to 120 mesh). 1% OV-22 on Supelcoport (80 to 100 mesh), tem perature program m ed, 140 290°C at 4°/m in. tem perature program m ed, 120-** 180°C at 2°/m in. tem perature program m ed, 60 70°C at 1°/m in, then 70 210°C at 4°/m in. isothermal at 130°C for 6 min, then tem perature program m ed, 130 220°C at 3°/m in. argon ionization detector, therm al conductivity detector. /5-ionization detector. REFERENCES

Carbohydrates 51

1. Sweeley, C. C ., Bentley, R., M akita, M ., and Wells, W. W ., Gas-liquid chrom atography of trimethylsilyl derivatives of sugars and related substances, J. A m . Chem. Soc., 85, 2497, 1963. 2. H olligan, P. M. and Drew, E. A ., Routine analysis by gas-liquid chrom atography of soluble carbohydrates in extracts of plant tissues. II. Q uantitative analysis of standard carbohydrates, and the separation and estim ation of soluble sugars and polyols from a variety of plant tissues, N ew P hytol.,10, 271, 1971. 3. El-D ash, A. A. and H odge, J. E ., Determ ination of 3-deoxy-D-eryfhro-hexosulose (3-deoxy-D-glucosone) and other degradation products of sugars by borohydride reduction and gas-liquid chrom atography, Carbohydr. Res., 18, 259, 1971. 4. D utton, G. G. S., Gibney, K. B., Jensen, G. D ., and Reid, P. E., The simultaneous estimation of polyhydric alcohols and sugars by gasliquid chrom atography. Applications to periodate-oxidixed polysaccharides, J. Chromatogr., 36, 152, 1968. 5. Loewus, F. and Shah, R. H ., Gas-liquid chrom atography of trimethylsilyl ethers of cyclitols, in Methods in Carbohydrate Chemistry, Vol. 6 , W histler, R. L. and BeMiller, J. N., Eds., Academic Press, New York, 1972, 14. 6 . Percival, E ., Gas chrom atography of the trimethylsilyl ethers of some less-common disaccharides, Carbohydr. Res., 4, 441, 1967. 7. K arkkainen, J ., Determ ination of the structure of disaccharides as trimethylsilyl derivatives of disaccharide alditols by gas-liquid chrom atog­ ra p h y — mass spectrom etry, Carbohydr. Res., 11,247, 1969.

52

CRC Handbook o f Chromatography

Table GC 22 TRIMETHYLSILYL ETHERS OF ALDITOLS DERIVED FROM PARTIALLY METHYLATED SUGARS Packing T em perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (O .D .), cm Form M aterial Detector Reference Parent sugar L-Arabinose 2,3-di-O-methyl 2,5-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 2,3,5-tri-O-m ethyl D-Xylose 2,3,4-tri-O-m ethyl L-Rhamnose 3-O-methyl 4- O m ethyl 2,4-di-O-methyl 3,4-di-O-methyl 2,3,4-tri-O-m ethyl D-Glucose 2 - O-methyl 3-O-methyl 6 - O-methyl 2,3-di-O-methyl 2 ,6 -di-O-methyl 3,4-di-O-methyl 3, 6 -di-O-methyl 2,3,4-tri-O-m ethyl 2 ,3, 6 -tri-O-methyl 2,4, 6 -tri- O-methyl 2,3,4-6-tetra- O-methyl D-Galactose 2 - O-methyl 3-O-methyl 4 -O-methyl 6 - O-methyl 2,3-di-O-methyl 2,4-di-O-methyl 2 , 6 -di-O-methyl 3,4-di-O-methyl 2,3,4-tri-O-m ethyl 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-Mannose 6 - O-methyl 2,3-di-O-methyl 3, 6 -di-O-methyl

PI 175 He, 20

P2 140 He, 60

P3 140 He, 60

P4 140 He, 60

P5 140 He, 60

P5 140 He, 60

400 0.2(1. D.) na copper FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI 3

2

2

1

2

2

r"

r*

r*

r*

r*

Tb

_

0 .6 8

1.55 0.73 0.62 0.73 0.62 0.42

1.54 0.73 0.62 0.73 0.62 0.42

1.38 0.69 0.58 0.69 0.58 0.41

— — — — — —

— 1.93 1.26

— 1.92 1.25

— 1.68

0.52 — — 0.97 0.71 0.79 — 4.03 2.93 — — 2.13 1.78

— —

— — —

— —

— — — —

0.50 0.41 0.50 0.41 0.37 — 0.77 0.61 — —

— 0.49















— — — — — — 1.83 1.35 1.35 1.35 1.39 1.27 1.14



1.00



1.26



1.20



1.00



0.92



1.22

0.58 — — — — — — — — — — — — 4.63 2.89 2.94 2.94 2.99 2.08 1.99 1.79 2.18 1.57 1.34 1.25 1.39



1.00

1.00



2.87 2.19 2.18 1.97 1.75 1.58 — 1.52 — 1.29 — 1.00

— — — — — —



— — — —

_

1.14



— 0.58 — — — — — — — — — — — — 4.61 2.88

2.92 2.92 2.98 2.09 1.99 1.79 2.19 1.57 1.35 1.25 1.38 1.00

— 0.57 — — — — — — — — — — — — 4.13 2.62 2.64 2.64

2 .1 0

— 1.67 1.32 1.35 1.01

— — — —

2 .6 6



1.95 1.84 1.64





2 .0 0



1.50 1.28 1.19 1.32



— — — —

1.00

1.00





















3.90 2.33











1.88











1.63

53

C a rb o h yd ra tes

Table GC 22 (continued) TRIMETHYLSILYL ETHERS OF ALDITOLS DERIVED FROM PARTIALLY METHYLATED SUGARS Packing T em perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diameter (O .D .), cm Form Material Detector Reference

“ 6

PI 175 He, 20

P2 140 He, 60

P3 140 He, 60

P4 140 He, 60

P5 140 He, 60

P5 140 He, 60

400

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI

180 0.63 coiled glass FI 3

0 .2 (I.D .)

na copper FI

2

1

Parent sugar

r“

4,6-di-O-methyl 2,3,6-tri-O-m ethyl 2,4,6-tri-O-m ethyl 3,4,6-tri-O-m ethyl 2,3,4,6-tetra- O-methyl

_ — — — —

r* — —

— — —

2

2

r*

r*

_ — — — —

2

r*

Tb

_

_

— — — —

— — — —

1.64 1.35 1.19 1.35 1.00

tr relative to trimethylsilylated 2,3,4,6-tetra-O-methyl-D-glucitol (7.8 min). tr relative to trimethylsilylated 2,3,4,6-tetra-O-methyl-D-galactitol ( 8 , 9, 11, and 5 min for P2, P3, P4, and P5, respectively).

Packing

PI P2 P3 P4 P5

= = = = =

2% SE-52 on acid-washed Chrom osorb G. 3% OV-17 on C hrom osorb W (60 to 80 mesh). 3% OV-101 on C hrom osorb W (60 to 80 mesh). 3.8% SE-30 on Supelcoport (60 to 80 mesh). 3% SE-52 on Chrom osorb W AW DMCS (80 to 100 mesh).

REFERENCES 1. 2. 3.

Patel, S., Rivlin, J ., Sam uelson, T ., Stamm , O. A ., and Zollinger, H ., A method for elucidation of the structure of products of the reaction of cellulose with formaldehyde, Helv. Chim. Acta, 51, 169, 1968. Freem an, B. H ., Stephen, A. M ., and Van der Bijl, P ., Gas-liquid chrom atography of some partially m ethylated alditols as their trimethylsilyl ethers., J. Chromatogr., 73, 29, 1972. M errifield, E. H ., unpublished laboratory data, 1975.

Parent com pound D-Xylose 2-O-methyl 3-O-methyl 4- O-methyl 2,3-di-O-methyl 2,3,4-tri-O-methyl D-Glucose 2-O-methyl 3- O-methyl 4- O-methyl 6- O-methyl 2,3-di-O-methyl 2,4-di-O-methyl 2 , 6 - O-methyl 3 , 4 - O-methyl 3,6-di-O-methyl 4,6-di- O-methyl 2,3,6-tri-O-methyl 2,3,4,6-tetra-O-methyl D-Mannose 2,4-di-O-methyl

PI T1 na

P2 150 He, na

P3 100 na

P4 125 Ar, 20— 35

P5 136 He, 60

183 0.63 (O.D.) straight copper FI 1

366 0.63 (O.D.) straight copper FI 2

244 na na SS FI 3

120 0.4 straight glass D1 4

366 0.63 na glass D2 5,6

r“

rfc

rc

vd

r

1.73 1.57; 1.67 1.38 1.28 1.00

— — — — —

— — — — —

— — — — —

— — — — —

— — — — — — — — — — — —

1.08; 1.50 0.76; 1.25 0.90; 1.67 1.52; 1.85 — — — — — — — —

— — — — 0.91; 1.00 0.86; 1.15 — 0.78; 1.15 — — — —

5.98; 9.26 4.80; 9.50 — 7.81; 9.67 2.63; 3.44 — 4.33 — 3.40; 3.99 3.91; 4.86 1.82; 1.59 1.00; 0.91

— — — — — — — — — — — 0.69; 0.76









0.92

CRC Handbook of Chromatography

Packing Temperature (°C) Gas; flow rate ( m l / m i n ) Column Length, cm Diameter (I.D.), cm Form Material Detector Reference

54

Table GC 23 TRIMETHYLSILYL ETHERS OF PARTIALLY METHYLATED SUGARS AND METHYL GLYCOSIDES

tr relative to trimethylsilylated tr relative to trimethylsilylated tr relative to trimethylsilylated tr relative to trimethylsilylated tr relative to trimethylsilylated M inor peak. Furanose. Pyranose.















































































































































































































2,3,4-tri-O-methyl-a-D-xylose (about 16 min). a-D-glucose. 2,3-di- 0-methyl-/?-D-glucose (about 56 min). 2,3,4,6-tetra-O-methyl-a-D-glucose. 2,3,4,6-tetra-O-m ethyl-a-D-m annoside (29.4 min).

1.27; 1.67' 0.67 1.41; 1.70' 1.45; 1.92' 0.66 0.97 0.42 1.05;' 1.23 0.95; 1.04' 1.12

0.87 0.84 1.11 1.02;* 1.30'’ 0.62 1.20 0.78;* 1.09* 0.74 1.30 0.82 1.20 1.14 0.58 0.99 0.58 0.99 1.00

55

“ b c d e 1 * h



Carbohydrates

2,6-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 5,6-di-O-methyl 2,3,4-tri-O-m ethyl 2,5,6-tri-O-m ethyl 3,4,6-tri- O-methyl 3,5,6-tri-O-m ethyl 2,3,4,6-tetra-O-m ethyl 2,3,5,6-tetra-O-m ethyl Methyl glycosides Methyl D-mannoside 2 ,3-di-O-methyl 2,4-di-O-methyl 2 ,5-di-O-methyl 2,6-di-O-methyl 3,4-di-O-methyl 3,5-di-O-methyl 3,6-di-O-methyl 4 ,6-di- O-methyl 5,6-di-O-methyl 2,3,4-tri-O-m ethyl 2 ,3 ,5-tri- O-methyl 2,3,6-tri-O-methyl 2,4,6-tri- O-methyl 2,5,6-tri-O-m ethyl 3,4,6-tri-O-m ethyl 3,5,6-tri-O-m ethyl 2 ,3 ,4-6-tetra- O-methyl

PI P2 P3 P4 P5 T em perature T 1 D etector D1 D2

3% SE-52 on Gas-Chrorrl A. 19.5% Carbowax 20M on Chrom osorb W (80 to 100 mesh). 5% butanediol succinate polyester on Chrom osorb W. 8.7% poly(ethylene glycol succinate) on kieselguhr (60 to 100 mesh). 2% neopentyl glycol succinate polyester on Chrom osorb W (80 to 100 mesh). tem perature program m ed, 50 -► 125°C at 5.6°/m in, then isothermal at 125°C for about 20 min. /3-ionization. thermal conductivity. REFERENCES

1. 2. 3. 4. 5. 6.

Sephton, H . H ., Separation and determ ination of the hydrolysis products of methylated xylan as their trimethylsilyl derivatives by vapor phase chrom atography, J. Org. Chem .,29, 3415, 1964. Dick, W. E ., Baker, B. G ., and H odge, J. E ., Preparation and characterization of 1,2,6 ,2', 3', 4', 6'-hepta-0-acetyl-/3m altose, Carbohydr. Res., 6 , 52, 1968. N orrm an, B., Partial m ethylation of dextran, Acta Chem. Scand.,22 , 1381, 1968. H aw orth, S., Roberts, J. G ., and Sagar, B. F., Q uantitative determ ination of mixtures of alkyl ethers of Dglucose, Carbohydr. R es.,9 , 491, 1969. B hattacharjee, S. S. and G orin, P. A. J ., Identification of di-O-, tri-O , and tetra-O-methylmannoses by gas-liquid chrom atography, Can. J. Chem ., 47, 1207, 1969. Jones, H . G ., Gas-liquid chrom atography of methylated sugars, in M ethods in Carbohydrate Chemistry, Vol. 6 , W histler, R. L. and BeMiller, J. N., Eds., Academic Press, New York, 1972, 25.

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

Packing

56

Table GC 23 (continued) TRIMETHYLSILYL ETHERS OF PARTIALLY METHYLATED SUGARS AND METHYL GLYCOSIDES

Table GC 24 TRIMETHYLSILYL ETHERS AND ESTERS OF URONIC, ALDONIC, AND ALDARIC ACIDS, AND THEIR DERIVATIVES Packing Tem perature (°C) Gas; flow rate (m l/m in ) Colum n Length, cm Diameter (I.D .), cm Form M aterial Detector Reference

P2 170 N2, 29—32

P3 T1 N2, 50

200

200

0.3 na na FI

0.3 na na FI

250 0.32 U-tube glass FI

PI 190 N2, 29—32

1





Parent com pound Hexuronic acids D-Glucuronic acid

1

D-M annuronic acid

4.9; 6.3 7.8 4.1; 5.5; 6.0; 7.7 4.1; 6.1

5.3; 6 . 8 ; 9.2 4.5; 5.9; 6.4; 8.3 4.4; 6.7

L-Guluronic acid

4.4

4.8

D-G alacturonic acid

L-Iduronic acid



2

1.07; 1.27 0.82;* 1.18 —



P8 160 N2, 30

P9 160 N2, 30

P10 T3 N2, 30

122

120

0.32 na SS FI 4

0.5 straight glass D1 5 ,6

200 0 .2

200 0 .2

200 0 .2

na na FI 7,8

na na FI 7, 8

na na FI 7, 8

r'

r'

r'

r'











1.00







































N2, 40

P6 T2 N2, 25

152 0.4 coiled glass FI 3

152 0.4 coiled glass FI 3

rc

rc

1.73; 2.10; 2.53 1.48; 1.89; 2 .0 2 ; 2.60 1.52; 2.11; 2.21; 2.54 1.60; 1.91; 2.42; 2.67 1.48; 1.80;

2.52; 3.26; 4.17 2.04; 2.73; 4.06; 4.69 2.46; 3.51; 3.96 2.75; 2.97; 4.06 2.09; 2.51; 2.94

TJ

0.45; 0.82















3.0;* 2.5;* 3.9;'4.0' 3.0;* 3.5;* 2 .6 ;'3 .0 ;' —

0.49; 0.56; 0.70 0.52









































Methyl L-iduronate



















Methyl D-galacturonate Methyl D-m annuronate

0.30; 0.42; 0.55 0.37

57

3.0;* 4.8'

Methyl L-guluronate

3.1;* 2.9;* 4.9;' 4.7' 3.1;* 2.6;* 4.1;' 4.3‘ 2.9;* 3.5;* 2.7;' 3.2;' —

Carbohydrates

2 .0 0

Methyl ester methyl glycosides Methyl D-glucuronate

P7 170 Ar, 140

P5 175 N2, 40

r*

— —

P4 200

P2 170 N2, 29—32

P3 T1 N2, 50

200

200

0.3 na na FI

0.3 na na FI

250 0.32 U-tube glass FI

PI 190 N 2, 29—32

1

L-Gulurono- 6 ,3-lactone L -Idurono- 6 ,3-lactone O ligogalacturonic acids Digalacturonic acid methyl ester T rigalacturonic acid methyl ester Aldonic acids Glyceronic (glyceric) acid 2-C-Methylglyceronic acid L-Threonic acid D-Erythronic acid 2-C-Methyl-D-erythronic acid D-Arabinonic acid

— —

1.20; 1.27 1.22; 1.48

2.4; 3.2 —

2.2; 3.8 —

— —

0.86; 1.15

— — — —

— — — —

— — — —

— — — —



— — — — —





— — — —

122

120

0.32 na SS FI 4

0.5 straight glass D1 5, 6

200 0 .2

200 0 .2

200 0.2

na na FI 7, 8

na na FI 7, 8

na na FI 7, 8

152 0.4 coiled glass FI 3

3.0; 3.2 3.1; 4.1



P10 T3 N2, 30

152 0.4 coiled glass FI 3

3.6 3.6; 4.7



P9 160 N2, 30

N2, 40

ra

— —

P8 160 N2, 30

P6 T2 N2, 25

r“

P arent com pound Hexuronolactones D-Glucurono- 6 ,3-lactone D -M annurono- 6 ,3-lactone

2

1

P7 170 Ar, 140

P5 175 N2, 40

P4 200

r*

rc

rc

r'

r'

r'

— —

— —

— —

— —

— —

— —

— —

— —

— —

— —

— — — —

4.32 4.04 6.51 6.05

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —





















0.052 0.054 0.156 0.151 0.184 0.463

0.092 0.082 0.251 0.218 0.246 0.646

1.00

7.18; 9.13

r'

Td

10 . 10 ;

16.73 — 4.14; 4.32

— —

— — 0.086 0.072 0.237 0 .212

0.244 0.632

CRC Handbook of Chromatography

Packing Tem perature (°C) Gas; flow rate (m jf/min) Colum n Length, cm Diameter (I.D .), cm Form M aterial Detector Reference

58

Table GC 24 (continued) TRIMETHYLSILYL ETHERS AND ESTERS OF URONIC, ALDONIC, AND ALDARIC ACIDS, AND THEIR DERIVATIVES

— — — — — — — — — 6.9

— — — — — — — — — 8.1

6 .8

8 .2

6.1

7.2

— — — — — — — — — — — —













6.1

6.9

7.4

8 .6

— —

— — — — — — — — — — — — —

— — — — — — — — — — — — — — — —



— — — —



























— — — — — — — — — — — — — — —



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

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

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

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

0.428 0.438 0.457 0.252 0.251 0.236 0.252 0.554 0.564 1.286 1.256 1.113 1.115 1.247 1.087 1.368 1.235 0.615 0.528 0.659 0.673 0.676 0.644 0.673 0.659 0.612 0.477 0.590 0.728

0.617 0.569 0.599 0.379 0.373 0.380 0.419 0.674 0.689 1.569 1.561 1.254 1.276 1.531 1.256 1.743 1.507 0.809 0.629 0.912 0.935 0.971 0.894 0.946 0.995 1.271 0.986 1.241 1.038

0.570 0.565 0.621 0.395 0.392 0.375 0.418 0.710 0.742 1.639 1.570 1.385 1.410 1.568 1.309 1.812 1.656 0.801 0.687 1.007 1.035 1.004 0.973 1.007 1.026 1.928 1.416 1.776 0.990

0.06 0.08 0.08

0.17 0.27

0.56 0.92

0 .2 0

0 .6 8

0 .2 0 0 .2 2

0.49 0.54 0.67 0.64 0.83

1.13 1.58 1.79 2.06 2.47

_

_

_

_

— — — — — — —

— — — — — — —

— — — — — — —

— — 0 .6 8

0.79 —

1.00

1.58

0.26 0.25 0.32

Carbohydrates

D-Xylonic acid D-Ribonic acid D-Lyxonic acid 2-Deoxy-D-eryfbro-pentonic acid 2-Deoxy-D- fbreo-pentonic acid 3-Deoxy-D-eryfbro-pentonic acid 3-Deoxy-D- fbreo-pentonic acid 2-C-M ethyl-D-arabinonic acid 2-C-Methyl-D-ribonic acid D-Gluconic acid D-Galactonic acid D-M annonic acid D-Allonic acid D-Altronic acid L-Gulonic acid L-Idonic acid D-Talonic acid 6 -Deoxy-L-galactonic acid 6 -Deoxy-L-mannonic acid 2-Deoxy-D-arab/no-hexonic acid 2-Deoxy-D-/yxo-hexonic acid 3-Deoxy-D-arab/no-hexonic acid 3-Deoxy-D-/yxo-hexonic acid 3-Deoxy-D-r/bo-hexonic acid 3-Deoxy-D-xy/o-hexonic acid 2,5-Anhydro-D-gluconic acid 2,5-A nhydro-D-m annonic acid 2,5-Anhydro-D-talonic acid 3-O-Methyl-D-gulonic acid A ldonolactones L-Threono-1,4-lactone D -Erythrono-1,4-lactone 2- C-M ethyl-D -erythrono-1,4-lactone L-A rabinono-1,4-lactone D-Xylono-1,4-lactone 1,5-lactone D-Ribono-1,4-lactone D-Lyxono-1,4-lactone

VO

P2 170 N2,2 9 —32

P3 T1 N2, 50

200

200

0.3 na na FI

0.3 na na FI I

250 0.32 U-tube glass FI

PI 190 N 2, 2 9 - -32

1

2-Deoxy-D-eryt/iro-pentono-l ,4lactone 2-Deoxy-D- threo- p entono-1,4-lactone 3-D eoxy-D -eryt/iropentono-l ,4lactone 3-Deoxy-D- threo- p entono-1,4-lactone 2 -C -M ethyl-D -arab/nono 1,4-lactone 2- C-Methyl-D- ribono-1,4-lactone D-G lucono-1,4-lactone 1,5-lactone L -G alactono-1,4-lactone D -M annono-1,4-lactone 1,5-lactone D -A llono-1,4-lactone D -A ltrono-1,4-lactone L-Gulono-1,4-lactone L-Idono-1,4-lactone D-T alo n o -1,4-lactone 6-D eoxy-L-galactono-l,4 -lactone 6 -Deoxy-L-m annono-l ,4-lactone

r"

ra

P arent com pound

2

P7 170 Ar, 140

P8 160 N 2, 30

P9 160 N2, 30

P10 T3 N2, 30

122

120

0.32 na SS FI 4

0.5 straight glass D1 5 ,6

200 0 .2

200 0 .2

200 0 .2

na na FI 7 ,8

na na FI 7, 8

na na FI 7, 8

N2, 40

P5 175 N2, 40

P6 T2 N 2, 25

152 0.4 coiled glass FI 3

152 0.4 coiled glass FI 3

P4 200

r*

rc

rc

r'

Td

r'

r/

r'













0.69

0.11

0.33

1.48

— —

— —

— —

— —

— —

— —

— —

0 .1 2 0 .1 0

0.44 0.34

1.81 0.98

— — —

— — —

— — — — — — — — — — — — — — —

— — — — — — — — — — — — — — —

— — — — — — — — — — — — — — —

— — — — — — — — — — — — — — —

— — —

0.11 0.21 0.21

1.02 1.01

1.64

0.71 0.67 0.69

0.38 0.42 0.45 1.48 1.49 1.37

1.01

2.21

0.65 0.81 0.62 0.78 0.79 0.80 0.25 0.38

1.35 1.70 1.19 1.72 1.63 1.70 0.56 0.94

4.6 4.4 4.5 6.5

4.5 4.4 4.3 6.3

— — —

— — — 5.0 5.2

5.0 4.9

— — —

— — —

— 1.53 3.07

— —

2.03 2.36 1.88 2.01

0.76 1.65

1.24 3.19 2.99 2.82 4.64 3.27 4.80 2.28 3.58 4.31 4.87 1.31 2.57

CRC Handbook of Chromatography

Packing T em perature (°C) Gas; flow rate (m f/m in ) Colum n Length, cm Diameter (I.D .), cm Form M aterial D etector Reference

60

Table GC 24 (continued) TRIMETHYLSILYL ETHERS AND ESTERS OF URONIC, ALDONIC, AND ALDARIC ACIDS, AND THEIR DERIVATIVES















0.42

1.28

3.64

— —

— —

— —

— —

— —









0.39 0.37

1.00 1.01

4.17 2.24















— — —

— — —

— — —

— — —

— — —

— — —

— — 2.63

0.41 0.38 0.40 —

1.09 0.92 1.13 —

2.99 2.40













2.81



















4.44



















4.56

















— —





2.92









0 .8 8

2.01

— 4.72 0.190 0.471 0.354 0.945 1.034 1.037 0.655 0.736 0.673 0.774 1.920





2 .6 6

















0.064















0 .2 1 2

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —







— — — — — — — —

— — — — — —



— — — — — — — —

7.4 5.5

7.4 5.6 6.3











0.174 0.522 0.545 0.532 0.278 0.291 0.280 0.295 1.295

0.152 0.432 0.313 0.864 0.978 0.873 0.517 0.573 0.538 0.593 1.814



















































1.500 1.074

2.272 1.346

2.347 1.401

6.1 2.8

8.4

2 .2

— —



















5.3

9.5 6.5 4.0



























































1.242 1.419 1.602

1.669 2.098 2.518

1.904 2.195 2.589

6 .2

Carbohydrates

2-Deoxy-D-arab/no-hexono-l ,4lactone 2-Deoxy-D- lyxo- hexono-1,4-lactone 3-Deoxy-D-arab/no-hexono-1,4lactone 3-Deoxy-D- lyxo- hexono-1,4-lactone 3-Deoxy-D- ribo- hexono-1,4-lactone 3-Deoxy-D-xy/o-hexono-1,4-lactone D -giycero-L-ga/acfoH eptono-1,4lactone D-glycero-D-galacto-Heptono-1,4lactone D-glycero-L-mann o-H eptono-1,4lactone D-g/ycero-D-gu/o-Hep to no-1,4lactone D-g/ycero-D- fa/o-Heptono-1,4-lactone L- f/jreo-Hex-2-enono-l ,4-lactone; Aldaric acids and lactones Glyceraric acid T hrearic acid E rythraric acid A rabinaric acid Xylaric acid Ribaric acid 2-Deoxy-eryt/iro-pentaric acid 2-Deoxy- fbreo-pentaric acid 3-Deoxy-eryf/jro-pentaric acid 3-Deoxy- t/ireo-pentaric acid Glucaric acid 1,4-lactone 6,3-lactone 1,4:6,3-dilactone G alactaric acid M annaric acid 1,4:6,3-dilactone Allaric acid A ltraric acid Idaric acid

61

62

Table GC 24 (continued) TRIMETHYLSILYL ETHERS AND ESTERS OF URONIC, ALDONIC, AND ALDARIC ACIDS, AND THEIR DERIVATIVES

P arent com pound 2-D eoxy-arab/nohexaric acid 2-Deoxy-/yxo-hexaric acid 3-Deoxy-arab/no-hexaric acid 3-Deoxy-/yxo-hexaric acid 3-Deoxy-ribo-hexaric acid 3-D eoxy-xy/ohexaric acid 3-O-M ethylgularic acid " b c d * 1 * h * '

PI 190 N2,2 9 —32

P2 170 N2, 29—32

P3 T1 N2, 50

P4 200 N2, 40

P5 175 N2, 40

P6 T2 N2, 25

P7 170 Ar, 140

P8 160 N2, 30

P9 160 N2, 30

P10 T3 N2, 30

200 0.3 na na FI 1

200 0.3 na na FI 1

250 0.32 U-tube glass FI 2

152 0.4 coiled glass FI 3

152 0.4 coiled glass FI 3

122 0.32 na SS FI 4

120 0.5 straight glass D1 5 ,6

200 0.2 na na FI 7, 8

200 0.2 na na FI 7, 8

200 0.2 na na FI 7, 8

rc



















— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

T b

Tc

Td

tr relative to trim ethylsilylated meso-erythritol. tr relative to trim ethylsilylated D-mannitol (about 58 min.) t Trelative to trim ethylsilylated ribitol. tr relative to trim ethylsilylated D-galacturonic acid (m ajor peak, 2.2 min). tr relative to 2,3,5-tri-0-(trim ethylsilyl)-D -ribono-l ,4-lactone (about 10 min). tr relative to trim ethylsilylated D-glucitol (about 12 min for P8, 7 min for P9; for P10 15 min at 120°, 3 min at 160°). M inor peak. Furanose. Pyranose. L-Ascorbic acid (vitamin C).

r*

Tf

Tf

T1

0.777 0.847 0.767 0.789 0.732 0.761 0.909

1.277 1.467 1.425 1.470 1.290 1.317 1.610

1.565 1.751 1.675 1.713 1.550 1.639 1.770

CRC Handbook of Chromatography

Packing T em perature (°C) Gas; flow rate (m i/m in ) Column Length, cm Diam eter (I.D .), cm Form M aterial Detector Reference

Packing

PI P2 P3 P4 P5 P6 P7 P8 P9 P10 Tem perature T1 T2 T2 Detector D1

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

2.5% SE-52 on Chrom osorb G AW DMCS (80 to 100 mesh). 1% SE-30 on Chrom osorb G AW DMCS (80 to 100 mesh). 3.8% SE-30 on D iatoport S (80 to 100 mesh). 10% SE-30 on Celite( 100 to 120 mesh). 4% XE-60 on Celite (100 to 120 mesh). 0.5% SE-30 on acid-washed, silanized Chrom osorb W (80 to 100 mesh). 10% neopentyl glycol sebacate polyester on acid-washed Chrom osorb W (100 to 120 mesh). 0.5% OV-1 on Chrom osorb G (100 to 120 mesh). 0.5% OV-17 on Chrom osorb G (100 to 120 mesh). 3% DC QF-1 on Gas-Chrom Q (100 to 120 mesh). tem perature program m ed, 140 -►200°C at 0.5°/m in. tem perature program m ed, 130 300°C at 12°/min; isothermal at 300°C for 6 min. 120°C for aldonic and aldaric acids, 160°C for lactones ionization detector ( 90Sr). REFERENCES

1. 2. 3. 4. 5. 6.

7.

Carbohydrates

8.

R aunhardt, O ., Schmidt, H. W. H. and Neukom , H ., Gas-chromatographic investigations of uronic acids and uronic acid derivatives, Helv. Chim. Acta, 50, 1267, 1967. Bhatti, T ., Cham bers, R. E ., and Clam p, J. R., The gas chromatographic properties of biologically im portant N-acetylglucosamine derivatives, m on­ osaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides, Biochim. Biophys. Acta, 222, 339, 1970 Kennedy, J. F ., Robertson, S. M ., and Stacey, M ., G .l.c. of the O-trimethylsilyl derivatives of hexuronic acids, Carbohydr. Res., 49,243, 1976. Raym ond, W . R. and Nagel, C. W ., Gas-liquid chrom atographic determination of oligogalacturonic acids, Anal. C h e m .,4 \, 1700, 1969. Perry, M. B. and Hulyalkar, R. K., The analysis of hexuronic acids in biological materials by gas-liquid partition chrom atography, Can. J. Biochem., 43, 573,1965. M orrison, I. M. and Perry, M. B., The analysis of neutral glycoses in biological materials by gas-liquid partition chrom atography, Can. J. Biochem., 44, 1115, 1966. Petersson, G ., G as-chrom atographic analysis of sugars and related hydroxy acids as acyclic oxime and ester trimethylsilyl derivatives, Carbohydr. Res., 33,47,1974. Petersson, G ., Retention data in GLC analysis; carbohydrate-related hydroxy carboxylic and dicarboxylic acids as trimethylsilyl derivatives, J. Chromatogr. Sci., 15, 245, 1977.

63

Parent com pound 2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 2-Amino-2-deoxy-D-mannose 2-Acetamido-2-deoxy-D-arabinose

PI T1 na, 75

P2 187 Ar, 15

183 0.63 coiled SS FI 1

2

2

ra



ra

P3 140 Ar, 15

P4 T2 N2, 50

200

200

0.35 coiled glass FI

0.35 coiled glass FI

250 0.32 U—tube glass FI 3

0.91; 1.15 0.79 — —

0 . 8 8 ; 1.12 0.77 — —





1.20; 1.38

P5 T3 N2, 30

P6 T4 N2, 30

P7 140 He, na

183 0 .2

200 0 .2

coiled glass FI 4

spiral SS D1 5

91 0.32 na glass FI 6 , 7, 8

0.4 na glass FI 9

r'

r'

_

_





rc

Tb

Td

0.92 0.83* 0.81 0 .6 6 ;*





— —

0.79; 0.73; 0.63; 0.60;

— — —

— — —



0.55;* 0.60;



1 . 11 ; 1.22

P8 220

N2, 40 200















0 .6 8

2-Acetamido-2-deoxy-D-ribose

0 .6 8

2-Acetamido-2-deoxy-D-glucose 2-Acetamido-2-deoxy-D-galactose 2-Acetamido-2-deoxy-D-mannose 2-Acetamido-2-deoxy-D-allose

2.27 2.08 1.71 —

2.25 2.04

8.3; 10.5 —













2-Acetamido-2-deoxy-D-altrose



2-Acetamido-2-deoxy-D-gulose 2-Acetamido-2-deoxy-D-idose













N-Acetylneuraminic acid N-Glycolylneuraminic acid

— —

— —

— —

1.38 1.30 0.97; 1.15 1.05;* 1.19; 1.24 0.96;* 1.09;* 1 . 11 ; 1.21 1.05; 1.07 0.85; 1.00; 1.04; 1.11 2.41 2.80

1.00

0.95 0.68; 0.78 — — — — — —











































— —









CRC Handbook of Chromatography

Packing Tem perature (°C) Gas; flow rate (m l/m in ) Column Length, cm Diameter (I.D .), cm Form Material Detector Reference

64

Table GC 25 TRIMETHYLSILYL ETHERS OF AMINODEOXY AND ACETAMIDODEOXY SUGARS, NEURAMINIC ACIDS, AND THEIR DERIVATIVES

Methyl glycosides Methyl 2-amino-2-deoxy-D-glucoside Methyl 2-amino-2-deoxy-D-galactoside Methyl 2-acetam ido-2-deoxy-D-arabinoside Methyl 2-acetamido-2-deoxy-D-riboside Methyl 2-acetamido-2-deoxy-D-glucoside

— — — — —

0.70 0.59 — — 2.05

1.02 1.06 — — —

3-O m ethyl Methyl 2-acetamido-2-deoxy-D-galactoside

— —

— 1.77

— —

Methyl 2-acetamido-2-deoxy-D-mannoside







Methyl 2-acetamido-2-deoxy-D-alloside Methyl 2-acetamido-2-deoxy-D-altroside

— —

— —

— —

Methyl 2-acetamido-2-deoxy-D-guloside







Methyl 2-acetamido-2-deoxy-D-idoside







7.25 —

— —

— —

— — —



1.15 1.15 2.12 2.23

— — —

— — — — —

— — — — — —



— — 1.33 1.37

— — — — —

— — — — —

— —

— —







1.12;* 1.15 —

— —

— —

— —

















— —

— —

0.73* 0.72;' 0.73;' 0.87;* 0.92* —

— —

— — — — 2.76;* 3.38;* 4.00; 5.28* — 2.92; 3.55

9.15; 10.10* 9.15; 10.10

— — — —

— — — —

— — — —

— — — —

— 1.00 1.66

— — —

— — —

— — —

— — —

2.04; 2.32 3.20 —

— — — —

— — — —

— — — —

— — — —

— —

— —

— —

— —

1.70; 1.29; 1.00; 1.89; 0.89; 1.42

2.79 1.75* 1.68 2.27* 1.29

— — — —

— —

65

— — —

1.44 1.67 — —

0.94; 1.04;* 1.18;* 1.22* 0.99; 1.04;* 0.84;* 0.88; 1.00;* 1.08 0.88; 1.04; 1.18;* 1.20 0.86; 0.92;* 0.95; 1.00 2.22 —

— — — — 0.86;* 0.93*

C a rb o h y d ra te s

Methyl N-acetylneuram inate Methyl N-glycolylneuraminate Alditols 2-Amino-2-deoxy-D-glucitol 2-Amino-2-deoxy-D-galactitol 2-Acetamido-2-deoxy-D-glucitol 2-Acetamido-2-deoxy-D-galactitol Disaccharides p-D-Gal p-( 1-M)-D-Glc p N A c P~D-Glc p N Ac-( 1-^4)-D-G1cp N A c 0-D-Glc p N Ac-(1 ->6)-D-Glc p N Ac M ethylated derivatives 2-Deoxy-2-methylamino-D-glucose 3-O-methyl 4-O-m ethyl 6-O-methyl 3,4-di-O m ethyl 3,6-di-O-m ethyl

— — 0.53; 0.55 0.45; 0.50 1.05;* 1.17;* 1.31; 1.38* — 1.08; 1.21

P arent com pound 4,6-di-O-m ethyl 3,4,6-tri- O-methyl 2-Deoxy-2-methylamino-D-galactose 3 -O-methyl 4 -O-methyl 6 - O-methyl 3,4-di-O-methyl 3, 6 -di- O-methyl 4,6-di-O-m ethyl 3,4, 6 -tri-O-methyl 2-Deoxy-2-methylamino-D-glucitol 3 -O-methyl 4 -O-methyl 6 - O-methyl 3,4-di-O-m ethyl 3, 6 -di-O-methyl 4,6-di-O-m ethyl 3,4,6-tri-O-m ethyl 2-Deoxy-2-methylamino-D-galactitol 3 -O-methyl

PI T1 na, 75

P2 187 Ar, 15

183 0.63 coiled SS FI 1

2

2

r* — — — — — — — — — — — — — — — — — — — —

P3 140 Ar, 15

P4 T2 N 2, 50

200

200

0.35 coiled glass FI

0.35 coiled glass FI

250 0.32 U—tube glass FI 3

r“ — — — — — — — — — — — — — — — — — — — —

r° — — — — — — — — — — — — — — — — — — — —

P5 T3 N2, 30

P7 140 He, na 91 0.32 na glass FI 6 , 7, 8

183

200

0 .2

0 .2

coiled glass FI 4

spiral SS D1 5 rc

T b

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

P6 T4 N2, 30

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

r'

Td

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

1.11; 1.45* 1.00

1.26; 1.36; 1.30; 1.16; 1.70; 1.19; 1.05; 1.40; 1.29 1.14 1.07 0.93 1.12

0.90 0.79 0.93 1.20

1.26

1.77 1.59 1.99 1.57* 2.16 1.40 1.67 1.75

P8 220

N2, 40 200

0.4 na glass FI 9 Tf

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

CRC Handbook of Chromatography

Packing T em perature (°C) Gas; flow rate (m l/m in ) C olum n Length, cm Diam eter (I.D .), cm Form M aterial Detector Reference

66

Table GC 25 (continued) TRIMETHYLSILYL ETHERS OF AMINODEOXY AND ACETAMIDODEOXY SUGARS, NEURAMINIC ACIDS, AND THEIR DERIVATIVES

4-O-methyl 6 -O-methyl 3,4-di-O-methyl 3,6-di-O-methyl 4,6-di-O-m ethyl 3,4,6-tri- O-methyl 1,2- 0-M e-NeuN(Ac,M e)' 4-O-methyl 9 -O-methyl 4,9-di-O-methyl 4 ,7 , 8 -tri-O-m ethyl 4,7,9-tri-O-methyl 4 , 8 ,9-tri-O-methyl 4,7,8,9-tetra- O-methyl a b c

d * f * h ' *

— — — — — — — — — — — — — —

— — — — — — — — — — — — — —

— — — — — — — — — — — — — —

— — — — — — — — — — — — — —

— — — — — — — — — — — — — —

— — — — — — — — — — — — — —

1.04 0.99 1.26 1.04 0.92 1.32 — — — — — — — —

— — — — — — 1.89 1.70 1.43 1.27 1.30 1.14 1.07 1.00

tr relative to trimethylsilyl ether of cr-D-glucose (20 min at 140°C, 6.2 min at 160°C, in Reference 1). tr relative to trimethylsilylated D-mannitol (about 57 min). tr of acetam idodeoxyhexoses and glycosides of 2-acetamido-2-deoxy-D-glucose relative to TMS ether of 2-acetamido-2-deoxy-D-glucose (37.6 min); tr of 2-acetamido-2-deoxy-D-galactose glycosides relative to TMS ether of 2-acetamido-2-deoxy-D-galactose (7.5 min at 180°C); tr of oligosaccharides relative to TMS ether of /J-D-GlcpNAc-(l-*'4)-D-GlcpNAc (13.1 min at 243°C). tr relative to TMS methyl glycoside of racephedrine (6 min). tr relative to TMS ether olf 3,4,6-tri-0-methyl-2-deoxy-2-methylamino-D-glucose (7.3 min). tr relative to 1,2,4,7,8,9-O M e-N euN (A c,M e) (about 12 min). M inor peak. Pyranoside. Furanoside. N ,N -Acetyl, m ethyl-neuraminic acid methyl ester /J-D-methyl glycoside. P1 P2 P3 P4 P5 P6 P7 P8

3% SE-52 on acid-washed, silanized Chrom osorb W (80 to 100 mesh). 2.2% SE-30 on Gas-Chrom S (100 to 120 mesh). 3% QF-1 on Gas-Chrom Q (80 to 100 mesh). 3.8% SE-30 on D iatoport S (80 to 100 mesh). 3% SE-30 on C hrom osorb W H P (80 to 100 mesh). 4% SE-30 on C hrom osorb W H P (80 to 100 mesh). 10% neopentyl glycol sebacate polyester on acid-washed, silanized Chrom osorb W (80 to 100 mesh). 3.8% SE-30 on C hrom osorb W AW DMCS H P (80 to 100 mesh).

Carbohydrates

Packing

67

Table GC 25 (continued) TRIMETHYLSILYL ETHERS OF AMINODEOXY AND ACETAMIDODEOXY SUGARS, NEURAMINIC ACIDS, AND THEIR DERIVATIVES

Detector

T1 T2 T3 T4 D1

140°C for aminodeoxyhexoses, 160°C for others. tem perature program m ed, 140 -*■ 200°C at 0.5°/m in for monosaccharides; isothermal at 220°C for disaccharides, tem perature program m ed, 140 -*■ 160°C at 0.5°/m in for acetamidodeoxyhexoses and G lcpN A c glycosides; isothermal at 180°C for GalNAc glycosides; isothermal at 243°C for disaccharides. isothermal at 175°C for 30 min, then tem perature programmed, 175 -*■ 200°C at 1°/m in; held at 200°C for 10 min. thermionic nitrogen-phosphorus selective detector.

REFERENCES 1. 2. 3. 4. 5. 6.

7. 8.

9.

Sweeley, C. C ., Bentley, R., M akita, M ., and Wells, W. W ., Gas liquid chrom atography of trimethylsilyl derivatives of sugars and related sub­ stances, J. A m . Chem. Soc. 85, 2497, 1963. K&rkkainen, J. and Vihko, R., Characterisation of 2-amino-2-deoxy-D-glucose, 2-amino-2-deoxy-D-galactose and related com pounds, as their tri­ methylsilyl derivatives by gas-liquid chrom atography-m ass spectrometry, Carbohydr. Res., 10, 113, 1969. Bhatti, T ., Cham bers, R. E ., and Clamp, J. R., The gas chromatographic properties of biologically im portant N-acetylglucosamine derivatives, m onosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides, Biochim. Biophys. Acta, 222, 339, 1970. C oduti, P. L. and Bush, C. A ., Structure determ ination of N-acetyl amino sugar derivatives and disaccharides by gas chrom atography and mass spectroscopy, Anal. Biochem .,78, 21, 1977. C ahour, A. and H artm ann, L., Study of neutral and aminomonosaccharides by gas-liquid differential chrom atography: application to three refer­ ence glycoproteins, J. Chromatogr., 152, 475, 1978. G orin, P . A. J. and Finlayson, A. J., Synthesis and chrom atographic properties of partially O-methylated 2-deoxy-2-methylamino-D-glucoses; standards for m ethylation studies on polysaccharides, Carbohydr. Res., 18, 269, 1971. G orin, P. A. J ., Syntheses and chrom atographic properties of 2-deoxy-2-methylamino-D-galactose and its methyl ethers, Carbohydr. Res., 18, 281, 1971. G orin, P. A. J. and M agus, R. J ., A method for gas-liquid chrom atographic identification of O-methyl ethers of 2-deoxy-2-methylamino-D-glucose and 2-deoxy-2-methylamino-D-galactose, Can. J. Chem., 49, 2583, 1971. H averkam p, J ., Kamerling, J. P ., Vliegenthart, J. F. G ., Veh, R. W ., and Schaur, R., M ethylation analysis determination of acylneuraminic acid residue type 2-**8 glycosidic linkage. Application to G T ,ft ganglioside and colominic acid, F E B SL ett., 73, 215, 1977.

00

CRC Handbook of Chromatography

Tem perature

as

Carbohydrates

69

Section I.II LIQUID CHROM ATOGRAPHY TABLES The term “ liquid chromatography” covers many different types of system (see Sec­ tion A, Volume II, Section I.III). To facilitate reference, the data on liquid chroma­ tography of carbohydrates presented here are grouped according to the separation mechanism and type of packing involved as follows: Chrom atographic system H igh-perform ance liquid chrom atography On bonded-phase packings On m icroparticulate silica On cation-exchange resins Partition chrom atography on ion-exchange resins in aqueous ethanol Ion-exchange chrom atography Gel-perm eation chrom atography

Table numbers

LC 1—4 L C 5 — 10 LC 11, 12 LC 13— 16 LC 17—27 LC 28—45

The application of affinity chromatography to carbohydrates is reviewed at the end of this section. The assistance of Dr. Kirsti Granath (Pharmacia AB, Uppsala, Sweden), Professor L. Hough and Dr. R. Sidebotham (Queen Elizabeth College, University of London), and Dr. F. M. Rabel (Whatman Inc., Clifton, New Jersey) in the compilation of this section is gratefully acknowledged. H IGH-PERFORM ANCE LIQUID CHROM ATOGRAPHY The term high-performance liquid chromatography (HPLC) is used here for sepa­ rations complete in 1 hr or less. Under the conditions stated, monosaccharides can be separated in approximately 20 min and oligosaccharides in approximately 40 min, in the majority of cases. To facilitate comparisons between systems, values of the capac­ ity factor k' are tabulated, where possible, in preference to actual retention times. Separations on bonded-phase packings depend upon partition chromatography, those on microparticulate silica on partition and/or adsorption, and the chromatog­ raphy of sugars on a cation-exchange resin in a salt form (usually Ca2+form) is believed to involve ligand exchange. Tables LC 1, LC 2, LC 4, LC 7, and LC 11 are largely based on data compiled by Dr. F. M. Rabel (Whatman Inc., Clifton, New Jersey).

PI

P2

P2

P2

P2

25 cm 4.6 mm SS SI 1 m l/m in 25°C RI

30 cm 3.9 mm SS S2 2.5 m l/m in 25°C RI

30 cm 3.9 mm SS S3 1.5 m l/m in 25 °C RI

25 cm

25 cm 2.1 mm

2

30 cm 3.9 mm SS S4 1 m l/m in 22°C RI 3

2.1 mm

1

30 cm 3.9 mm SS S2 1 m l/m in 22°C RI 3

SS S5 1 m l/m in 25 °C UV" 4

SS S3 1 m l/m in 25°C UV“ 4

P3

Capacity factor (k )

Com pound L-Arabinose D-Xylose D-Ribose D-Lyxose D-Fructose D-Glucose D-Galactose D-Mannose L-Rhamnose L-Fucose 2-Deoxy-D-glucose 6 -Deoxy-D-glucose Sophorose Kojibiose M altose Cellobiose Isomaltose Lactose Melibiose Sucrose

2

P3

1.42 1.16 0.89

2.9 2.4 —

1.12



1.53 1.84 1.93/2.26* 1.58





1 .0 2 / 1 . 2 2 *

5.2 5.9 4.6 1.7

2.2





3.7 3.0



1.6 2.1





2 .0



3.0

3.5 5.1 5.9 4.4 1.7

















1.25 —

0.9 —

5.0 7.9

— —



















2 .0













1.7













2 .0

















2.1











2.1









2.1



8 .6







2.1







2.5 2.4 2.7 1.5





3.47 3.47 —

3.86 4.05 2.92

2.75



















2 .1 2

3.25



9.6







6.8

CRC Handbook of Chromatography

Packing Colum n Length Diameter M aterial Solvent Flow rate T em perature Detection Reference

70

Table LC 1 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF SUGARS ON BONDED-PHASE PACKINGS

10.6

Trehalose Raffinose “ b

3.4

7.26

20.0

192 nm. A nom er separation.

Packing

Solvent

PI P2 P3 SI S2 S3 S4 S5

= Partisil-10 PA C (W hatm an® ); m icroparticulate (10 ^m) silica with bonded polar amino-cyano phase. W aters® \ Bondapak Carbohydrate Analysis Column (10 jum silica with bonded polar phase). = M icroPak-N H 2 (V arian® ); m icroparticulate silica with bonded amino phase. = acetonitrile-H 20 (75:25), pH adjusted to 5.0 by addition of H 3P 0 4. = acetonitrile-H 20 (85:15). = acetonitrile-H 20 (80:20). = acetonitrile-H 20 (75:25). = acetonitrile-H 2Q (88:12). =

a

REFERENCES 1.

2. 3. 4.

Rabel, F. M ., C aputo, A. G ., and Butts, E. T ., Separation of carbohydrates on a new polar bonded phase m aterial, J. Chrom atogr., 126,731,1976. Palm er, J. K., A versatile system for sugar analysis via liquid chrom atography, Anal. Lett., 8 , 215, 1975. C hurm s, S. C. and Seeman, U. A ., unpublished laboratory data, 1977. H ettinger, J. and M ajors, R. E ., Separation of carbohydrates by high perform ance liquid chrom atography, Varian Instrum . A ppl., 10, 6,1976.

Carbohydrates 71

72

CRC Handbook of Chromatography Table LC 2 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF MALTODEXTRINS ON BONDED-PHASE PACKINGS Packing Column Length Diameter M aterial Solvent Flow rate Tem perature Detection Reference

25 cm 4.6 mm SS SI 1 m f/m in 25°C RI

30 cm 3.9 mm SS S2 1 m f/m in 25°C RI

1

2

Degree of polymerization 1 2

3 4 5 6

7 8

9 10

"

P2

P2

PI

30 cm 3.9 mm SS S3 1.8 m f/m in 25°C RI 3

k' 0.30 0.55 0.95 1.45 1.95 2.60 3.30 4.15 5.05 6 .2 0 “



0.9 1.4 2.5 5.2 6.5 — — — —





0.72 0.94 1.25 1.69 2 .2 2 — — —

Eluted after about 40 min.

Packing

Solvent

PI = Partisil-10 PAC (W hatm an® ). P2 = jiBondapak Carbohydrate Analysis Col­ umn (W aters® ). SI = acetonitrile-0.0025 M sodium acetate (65:35), pH adjusted to 5.0 with acetic acid. 52 = acetonitrile-H 20 (65:35). 53 = acetonitrile-H 20 (75:25). REFEREN CES

1. Rabel, F. M ., C aputo, A. G ., and Butts, E. T ., Separation of carbohydrates on a new polar bonded phase m aterial, J. Chrom atogr., 126, 731, 1976. 2. Palm er, J. K., A versatile system for sugar analysis via liquid chrom atography, A n a l. Lett., 8 , 215, 1975. 3. Linden, J. C. and Lawhead, C. L ., Liquid chro­ m atography of saccharides, J. Chromatogr., 105, 125,1975.

Carbohydrates Table LC 3 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF CELLODEXTRINS AND REDUCED DERIVATIVES ON BONDED-PHASE PACKINGS Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection

30 cm 3.9 mm SS SI Program m ed 0 25°C RI

C om pound D-Glucose Cellobiose -triose -tetraose -pentaose -hexaose D-Glucitol Cellobiitol -triitol -tetraitol -pentaitol -hexaitol “

6

P2

Pi

25 cm 4.6 mm SS S2 1.5 m l/m in 25°C RI

k' 1.2

0.5

2.3 4.4

1.0

8.1

2.5 3.8 5.4

— —

1.2

1.6

0 .6

2 .6

1.3

5.2 9.2 14.6

2.1



3.2 4.5 6.5b

W aters Model 660 Solvent Program m er: 2 m l/m in for 10 min, then flow rate increased to 4.6 m l/m in over 45-min period. U nder these conditions, cellopentaitol was eluted after 40 min. Com plete separation of oligomers to cellohexaitol in 25 min.

Packing

PI = ^ Bondapak Carbohydrate Analysis Column (W aters® ). P2 = P artisil-10 PA C (W hatm an® ).

Solvent

SI = acetonitrile-H 20 (75:25). S2 = acetonitriIe-H20 (71:29). REFEREN CE

1. Gum , E. K. and Brown, R. D ., Two alternative H PLC separation m ethods for reduced and nor­ mal cellooligosaccharides, Anal. Biochem ., 82, 372,1977.

73

74

CRC Handbook of Chromatography Table LC 4 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF CHITIN OLIGOSACCHARIDES0ON BONDEDPHASE PACKING Packing

PI

Column Length Diameter M aterial Solvent Flow rate

30 cm 3.9 mm SS SI 1.5 m 1 / min 25°C RI

Tem perature Detection

k'

Com pound 2-Acetamido-2-deoxy-D-glucose Chitobiose -triose -tetraose -pentaose “ b

0.7 1.3 2.1

3.4 5.3b

/3-(l 4)-linked oligomers of 2-acetamido-2deoxy-D-glucose. Eluted after about 15 min.

Packing Solvent

PI = ^ Bondapak C arbohydrate Analysis Column (W aters® ). SI = acetonitrile-H 20 (70:30). REFEREN CE

1. Van Eikeren, P. and M cLaughlin, H ., Analysis of the lysozyme-catalysed hydrolysis and transglycosylation of N-acetyl-D-glucosamine oligo­ mers by high-pressure liquid chrom atography, A nal. Biochem ., 77, 513, 1977.

Carbohydrates Table LC 5 CAPACITY FACTORS OF SUGARS AND ALDITOLS ON MICROPARTICULATE SILICA PACKINGS Packing Colum n Length Diameter M aterial Solvent Flow rate T em perature Detection Reference

PI

PI

PI

P2

P2

20 cm 4.6 mm SS SI 1.7 m l/m in rt R1

20 cm 4.6 mm SS S2 1.5 m i/m in rt RI

20 cm 4.6 mm SS S3 0.5 m i/m in rt Rl

25 cm 4.6 mm SS S3 0.5 m i/m in 22°C Rl

25 cm 4.6 mm SS S4 0.5 m i/m in 22°C RI

1

1

1

2

2

1.06/1.30“ 1.00/1.13° 0.80/0.96“

0.79/0.97“ 0.93 0.87









1.23 1.58fc 1.00/1.25“ —

Com pound

k'

L-Arabinose D-Xylose D-Ribose L-Sorbose D-Fructose D-Glucose D-Galactose D-Mannose D-GIucitoI Galactitol D-M annitol L-Rhamnose L-Fucose Sucrose M altose Cellobiose Lactose Trehalose Raffinose “ b









1.25 1.20/1.30° 1.06 1.27 1.50 1.62 1.70/1.94°





1.71 2.13fc 1.46

1.88





1.0

1.4 1.5



1.7



0.54 0.70 0.77

1.00









1.92 1.92













2 .2

1.2

2.3 2.3

1.4 1.4

2 .8

1.8









2.42 2.60 2.65 3.40 3.15 4.60









1 .0 0 / 1 . 2 2 “ 1.12/1.33“

0.75 0.80





2 .8

2 .0





3.6

2.8





4.8

3.3

Anom er separation. Broad peak.

Packing

PI = LiChrosorb Si 60 (M erck® ; particle size 5 /urn). P2 = Partisil 10 (W h atm an ® ; particle size 10/um).

Solvent

SI 52 53 54

= = = =

ethyl ethyl ethyl ethyl

form ate-m ethanol-w ater (55:20:15). form ate-m ethanol-w ater (55:25:10). form ate-m ethanol-w ater (60:20:10). acetate-m ethanol-w ater (60:20:10). REFERENCES

1. Rocca, J. L. and Rouchouse, A ., Separation of sugars on m icroparticulate silica by high perform ance liquid chrom atography, J. Chromatogr., 117, 216, 1976. 2 . Churm s, S. C. and Seeman, U. A ., unpublished laboratory data, 1977.

75

76

CRC Handbook of Chromatography Table LC 6 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF GLYCOSIDES AND OTHER DERIVATIVES OF SUGARS ON MICROPARTICULATE SILICA Packing

PI

Column Length Diameter M aterial Solvent Flow rate T em perature Detection

25 cm 4.6 mm SS SI 1 .2 m l/m in rt RI

PI

PI

25 cm 4.6 mm SS S3 1 . 2 m i/m in rt RI

25 cm 4.6 mm SS S2 1 .2 m jf/m in rt RI

tr(min)

Com pound L-Arabinopyranoside, methyl aD-Xylopyranoside methyl omethyl (3D-Ribopyranoside, methyl (3D-Glucopyranoside methyl amethyl (3phenyl aphenyl (3D-Galactopyranoside methyl amethyl (3D-M annopyranoside methyl amethyl (3D-Altropyranoside, methyl aD -G ulopyranoside,a methyl aD-Glucose 3-O-methyl2,3,6-tri-O-m ethyl2,4,6-tri-O-m ethylD-Glucopyranose 1 , 2 - O-isopropylidene4,6-O-benzylideneD-Glucofuranose, 1,2:5,6-di-O isopropylideneD-M annofuranose, 2,3:5,6-di-0-isopropylidene“

14.2



12.4

— — —

— — —

— — — —

— — — —

19.5

— —

— —

15.3 18.3 15.3 18.3

— — — —

— — — —

15.9

— 10.4 10.9

— — —

10.4

— — io.: 8 .(

11.8

11.5 15.9 17.7 10.0 8.8 20.1

8.1

— —

H ydrate.

Packing

PI = Partisil 10 (W h atm an ® ; particle size 10 ^m).

Solvent

SI = acetonitrile-water (9:1). 52 = acetonitrile-water (18:1). 53 = n-hexane-ethyl acetate (1:3).

From McGinnis, G. D. and Fang, P ., J. Chrom atogr., 153, 107, 1978. W ith permission.

Carbohydrates Table LC 7 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF 4-NITROBENZOATE DERIVATIVES OF CARBOHYDRATES ON MICROPARTICULATE SILICA PI

PI

PI

15 cm 3 mm SS SI 2.1 m i/m in rt° UVb

15 cm 3 mm SS S2 1.5 m i/m in rt" UVb

25 cm 3 mm SS S3 0 .8 m i/m in rt RI

1

1

2

Packing Column Length Diameter M aterial Solvent Flow rate Tem perature Detection Reference

k'

Parent com pound cr-D-Glucopyranose 0-D-Glucopyranose D-Glucopyranoside methyl omethyl 0 Sucrose o-Lactose 0-Lactose A rabinitol Xylitol D-M annitol D-Glucitol M altitol “ b

3.0 5.0

1.9 2.3



4.2





8.1







5.9





8.2





9.8







2.1





2 .6







3.3





3.3 3.6 9.0

20—22 °C 260 nm.

Packing

PI

LiChrosorb Si 60 (Merck; particle size 5 fim).

Solvent

SI S2

n-hexane-ethyl acetate (3:1), with 5% dioxane. /3-h e x a n e -ch lo ro fo rm -a c eto n itrile -tetrah y d ro furan (10:5:1:0.5). n-hexane-chloroform-acetonitrile (5:2:1).

S3

REFEREN CES 1. N achtm ann, F. and Budna, K. W ., Sensitive determ ination of derivatized carbohydrates by high-perform ance liquid chrom atography, J. Chrom atogr., 136, 279, 1977. 2. Schwarzenbach, R ., Separation of some polyhydric alco­ hols by high-perform ance liquid chrom atography, J. Chro­ m atogr., 140, 304,1977.

77

78

CRC Handbook o f Chromatography Table LC 8 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PERACETYLATED CARBOHYDRATES ON MICROPARTICULATE SILICA Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection Reference

PI

P2

25 cm 3 mm SS SI 0.46 m l/m in rt RI, UV“

25 cm 4.6 mm SS S2 1.2 m l/m in rt RI

1

2

k'

tr (min)

11.49 7.74 8.33 7.02 10.51 5.97

— —

15.13* 17.7

17.2 15.9

13.04 18.3

15.9 16.0

Parent com pound D-A rabinopyranose Q-

PD-Xylopyranose, aD-Lyxopyranose, aD-Ribopyranose, (1L-Rham nopyranose, aD-Glucopyranose a-

P~ D-Galactopyranose a-

PD-M annopyranose a-

16.37 24.51 19.63 21.38 21.92

PD-Allopyranose, pD-Idopyranose, aD-Talopyranose, aErythritol L-Arabinitol D-Glucopyranoside methyl amethyl /3phenyl aphenyl 0 a b

— — —





— — — —



12.6



14.0

_

16.3 17.3 11.7 13.0

— — —

220 nm.

tr = about 70 min.

Packing Solvent

PI P2 SI S2

= = = =

LiChrosorb Si60 (M erck® ; particle size 5 pim). Partisil 10 (W hatm an® ; particle size 10 pirn). n-hexane-acetone ( 10 : 1). /j-hexane-ethyl acetate (1:1). REFERENCES

1.

2.

Thiem, J., Schwentner, J., Karl, H ., Sievers, A ., and Reimer, J., Separation of peracetylated mono- and disaccharides and quanti­ tative analysis of guaran by high-perform ance liquid chrom atog­ raphy on silica gel, J. Chrom atogr., 155, 107, 1978. McGinnis, G. D. and Fang, P ., Separation of substituted carbo­ hydrates by high-perform ance liquid chrom atography 11, J. Chro­ matogr., 153,107, 1978.

Carbohydrates Table LC 9 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PERACETYLATED OLIGOSACCHARIDES ON MICROPARTICULATE SILICA Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection Reference

PI

P2

25 cm 3 mm SS SI 0.39 m i/m in rt RI

25 cm 4.6 mm SS S2 1.2 m i/m in rt RI

1

2

k'

tr (min)

Parent oligosaccharide a-D-Glucopyranose 3- O-a-D-glucopyranosyl3- 0-/3-D-glucopyranosyl4- O/J-D-glucopyranosyl4- 0-/3-D-galactopyranosyl/J-D-Glucopyranose 4- O-a-D-glucopyranosyl4- 0-/3-D-glucopyranosyl6 - 0-/3-D-glucopyranosyla-D-M annopyranose 4- O/J-D-mannopyranosyl/3-D-Mannopyranose 4- 0-/3-D-mannopyranosyla-D-Glucopyranoside a-D-glucopyranosyl/J-D-Glucopyranoside /3-D- glucopyranosyl/J-D-Fructofuranoside 2- O-a-D-glucopyranosyl“



7.29 10.33 8.96“ 10.63

38.0 —

6.73 9.47 10.82

36.1 —



_

9.53 11.53 7.14

-

10.73

-

6.14

_

tr = about 50 min.

Packing Solvent

PI P2 SI S2

LiChrosorb Si60 (M erck® ; particle size 5 ^m). Partisil 10 (W hatm an® ; particle size 10 ^m). n-pentane-acetone (7:2). n-hexane-ethyl acetate ( 1 : 1). REFERENCES

1.

2.

Thiem , J ., Schwentner, J ., Karl, H ., Sievers, A ., and Reimer, J., Separation of peracetylated mono- and disaccharides and quanti­ tative analysis of guaran by high-perform ance liquid chrom atog­ raphy on silica gel, J. Chrom atogr., 155, 107, 1978. M cGinnis, G. D. and Fang, P ., Separation of substituted carbo­ hydrates by high-perform ance liquid chrom atography. II, J. Chrom atogr., 153, 107, 1978.

79

80

CRC Handbook o f Chromatography Table LC 10 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF GLYCOLIPIDS ON MICROPARTICULATE SILICA Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection

25 cm

25 cm

2 .8 mm

2 .8 mm

SS SI naa rt D1

SS S2 na* rt D1

k'

C om pound Brain gangliosides G m, G m2 GV fi g d1o g d16

Gri Neutral glycosphingolipids G L 1o G L 2a gl3 gl4

“ b c

PI

PI

0.38 0.98 2.16 2.98c 4.31c 6 .0 2 c

— — — — — —

— — — —

0.63 1.38 2.21

4.06

Analysis time 40 min. Analysis time 30 min. Doublet peak.

Packing

PI

Solvent

SI

Detection

S2 D1

Silica Si60 (M erck® ), with particle size range 63—200 /^m, ground in agate m ortar and fraction­ ated by air classifier; fraction having particle size 9 ± 1.5 nm used to pack column, chloroform -m ethanol-aqueous HC1 (60:35:4); fi­ nal HC1 concentration 0.01 M. chloroform -m ethanol (3:1). moving-wire system equipped with flame-ioniza­ tion detector (Pye Unicam LCM2). REFEREN CE

1.

Tjaden, U. R., Krol, J. H ., Van Hoeven, R. P ., Oomen-Meulemans, E. P. M ., and Em m elot, P ., High-pressure liquid chrom a­ tography of glycosphingolipids (with special reference to ganglio­ sides), J. Chrom atogr., 136, 233, 1977.

Cation Colum n Length Diam eter M aterial Solvent Flow rate Tem perature Detection

T risb

Li+

K+

Na*

73 or

Table LC11 CAPACITY FACTORS OF SUGARS AND POLYOLS ON HIGH-PERFORMANCE LIGAND-EXCHANGE CHROMATOGRAPHY: VARIATION WITH CATION ASSOCIATED WITH RESIN" Tr

Mg2+

C a2+

Sr2+

Ba2+

C d2+

La3+

— 0.50 0.25 0.35

— 0.35 0.15 0.25 0.30 ~ 4 .0

50 cm Conditions same in all cases

2 .8 mm

Glass h 2o 0.1 m l/m in rt RI Jk _

C om pound D-Xylose D-Fructose D-Glucose D-Galactose D-Mannose D-Talose D-Gulose Sucrose Glycerol D-M annitol Galactitol

Ag+

_

_

_

_

_

0.35 0.35 0.35

0.45 0.35 0.50 0.45

0.65 0.40 0.55 0.70 0.50

1.05 0.70 0.80/1.10*

0.90 0.75* 0.75/1.00* 0.75/1.25* 0.95* 1.25 0.40 0.75 0.55 0.60

— —







0 .2 0

0 .2 0

0.45

0.90 1.05

0.65 0.75









0 .1 0 —





0.40 0.80 —





1.10



1.10

0.70 0.85 —



0.35 0.30 0.40 0.40









0.45 1.00

0.80







0.15 0.70 0.40 —

0.45/0.60* 1.15" 0.30/0.45* 0.45/0.65* 0.45/0.65* ~ 3.8 1.3 0.15 1.05 1.40 2.1





1 . 10 "

1 .0 0 *

0.30/0.45* 0.40/0.55* —

0.40* 0.45* 0.70*









0 .2 0 —

0.25 0.60



1.00









— — —

0.15 0.80

1.0 0 .1 0

0.80 1.70 —

Carbohydrates

■ Aminex A-5 (Bio-Rad® ; particle size 11 ± 2 ^m). b Tris = N H 3C (C H 2O H )3. c A nom er separation. d Skewed peak. * Asymm etric peak; partial separation of anomers.



1.15 0.75 0.85 0.95' 1.7 1.15 0.45

From G oulding, R. W ., J. Chrom atogr., 103, 229, 1975. With permission.

81

82

CRC Handbook o f Chromatography T a b l e L C 12 H IG H -P E R F O R M A N C E L IQ U ID C H R O M A T O G R A P H Y O F S U G A R S , A L D IT O L S , A N D O L IG O S A C C H A R ID E S O N IO N -E X C H A N G E R E S IN S (C A L C IU M F O R M )

Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection Reference C om pound D-Glucitol D-Mannitol L-Arabinose D-Xylose D-Galactose D-Mannose D-Glucose D-Fructose Sucrose Maltose Cellobiose Lactose M altotriose Cellotriose Cellotetraose Cellopentaose Cellohexaose Celloheptaose a b c d * /

PI P2

PI

P2

P2

60 cm 7 mm na h 2o 1 m l/m in 80°C RI 1

50 cm 7 mm SS h 2o 0.6 m l/m in 85°C RP 2

61 cm 8 mm SS h 2o 0.6 m l/m in 85°C RP 3

61 cm 8 mm SS h 2o 0.55 m l/m in 85°C RP 3

rc

rd

r*

k'

_

_

_





1.63 1.35 1.19 — 1.10 1.12 1.00 1.18 0.84 — — — —

R efractom eter Refractom eter t r of D-glucose tr of D-glucose t r of D-glucose tr of D-glucose

Packing

PI

— — — — 1.00 — — 0.83 — — 0.74

1.26 1.11 1.13 1.16 1.00 1.18 0.86 —

— 0.89 —































— — — — — 1.43' — — — 1.02 — — 0.70 0.49 0.35 0.25 0.18

tem perature 45°C. tem perature 30°C. = about 15 min. = 19.6 min. = about 28 min. = about 35 min.

= Aminex Q15-S (B io-R ad® ; particle size 22 ± 3 p2)a-D-Glc pNAc( 1-*4)a-D-GIcpN Ac( 1-*,4)a-L -F ucp(l-*2)-

0 0

a-D-Glc pN Ac( 1-*,4)0

a-L-Fucp(l-*2)0

a-D-GlcpN Ac( 1-*4)a-L -Fucp(l-*2)a-L -Fucp(l-*2)-

Carbohydrates

C E 13.1

R2

97

APPENDIX TO TABLE LC 24: STRUCTURES OF OLIGOSACCHARIDES (continued)

VO 00

K M 14.2

0 0 0

a-D-GlcpNAc( 1-*,4)14.4 15.1 15.3 15.5

Z G 15.2 15.4 16.1 16.2 16.4

0

R2 0

o-D-GlcpN Ac( 1-*•4)a-D-GlcpNAc(l-M )0

a-L-Fucp(l-*2)-

a-L -F ucp(l-*2)a -D -G lcpN A c(l-4) a-L -F ucp(l-*2)a-L -F ucp(l-"2)-

a-D-GlcpNAc(l —4)a-D-GlcpN Ac( 1-*4)a-L-Fucp(l-*,2)-

R* 0 a-D-GIcpNAcO-M )0 a -D -G lcpN A c(l-4)-

R2 0 0 a-D-GlcpN Ac( 1-*4)0

0 a-L -F ucp(l-*2)a-D-GlcpNAcO-M )a-D-Glc pNAc( 1- 4 ) a-L -F ucp(l-*2)-

0

a-L -Fucp(l -*2)0 a-D-GlcpNAcO-M )a-L-Fuc p (1—2)a-L -Fucp(l~>2)-

CRC Handbook of Chromatography

R*

Carbohydrates

99

100

CRC Handbook of Chromatography Table LC 25 SEPARATION OF AMINODEOXY SUGARS, AMINODEOXYALDITOLS, AND REDUCED OLIGOSACCHARIDES ON AN AMINO ACID ANALYSER Packing Column Length Diameter M aterial Solvent F low rate Tem perature Detection

PI

PI

40 cm 1cm glass SI 45 m i/h r 50°C Ninhydrin

40 cm 1cm glass S2 45 m i/h r 50°C Ninhydrin

Com pound

tr (min)

2-Amino-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 2-Amino-2-deoxy-D-mannose 2-Amino-2-deoxy-D-glucitol 2-Amino-2-deoxy-D-galactitol 2-Amino-2-deoxy-D-mannitol GlcN0(l-*3)galactitol G lcN p(l-* 6 )galactitol G a lN a (l-3 )g a la c tito l Gal^(l-*3)2-am ino-2-deoxy-D-galactitol Gal^(l-*3)2-amino-2-deoxy-D-glucitol Gal/l(1^6)2-amino-2-deoxy-D-glucitol Gal/?(l-*4)2-amino-2-deoxy-D-glucitol Gal/?(l-*4)GlcN/?(l-*6)2-amino-2-deoxy-D-galactitol GlcN/J(l-*4)2-amino-2-deoxy-D-glucitol GalNa(l-*3)Gal/?(l-*3)2-amino-2-deoxy-D-glucitol GalNa(l-*3)Gal/?(l-*4)2-amino-2-deoxy-D-glucitol

170 318 194 363 387 285 — — — — — — — — — — —

°

tr (min) — — — 366 387 — 83° 83° 100

146 158 179 192 416 533 552 608

Separate if pH of eluting buffer adjusted to 7.0.

Packing

PI

Solvent

SI

S2

= Locarte No. 12 resin (8 % cross-linked sulfonated polystyrene; Locarte, London), Na+ form. = buffer, 0.1 M in N a+, containing trisodium citrate dihydrate (49.0 g), boric acid (1.55 g), 33% (w/v) Brij-35 solution (15 m i), water to 5 i , and HC1 to bring pH to 7.5. = SI for 380 min, then buffer changed to solution 0.2 M in N a+, containing trisodium citrate dihydrate (91.88 g), borax (11.9 g), 33% (w/v) Brij-35 solution (15 m i), water to 5 i , and HC1 to bring pH to 8.0. REFERENCE

1.

Donald, A. S. R., Separation of hexosamines, hexosaminitols and hexosam ine-containing di- and trisaccharides on an amino acid analyser, J. Chromatogr., 134, 199, 1977.

Carbohydrates

101

CHROM ATOGRAPHY OF CARBOHYDRATES ON DIETHYLAM INOETHYL-DEXTRAN ANION-EXCHANGERS DEAE-Sephadex ion-exchangers (Pharmacia®) are useful in the separation and isolation of acidic carbohydrates, especially oligosaccharides. The isolation of the in­ dividual oligomers from a mixture of oligogalacturonic acids produced on enzymic hydrolysis of pectic acid serves as a typical example of the application of this type of chromatography; data are presented in Table LC 26.

Table LC 26 CHROMATOGRAPHY OF OLIGOGALACTURONIC ACIDS ON DEAESEPHADEX A-50 Packing Column Length Diameter Material Solvent Flow rate Tem perature Detection Reference

PI 80 cm 10 cm glass SI na rt D1 1

Degree of polymerisation

Relative retention volume"

1

100

2 3 4 5

115 155 191 236 276 317 379 434

6

7 8

9 “

Relative to D-galacturonic acid = 100.

Packing Solvent

PI SI

Detection D1

DEAE-Sephadex® A-50, chloride form , equilibrated with water at pH 6.0. 4 I water at pH 6.0, then stepwise elution with 4-1 volumes of sodium chloride solutions, 0.05, 0.10, 0.125, 0.150, 0.175, 0.200, and 0.225 M, respectively, followed by 6 l each of 0.250 and 0.275 M N aC l. carbazole method. REFERENCE

1.

Liu, Y. K. and Luh, B. S., Preparation and thin-layer chrom atography of oligogalacturonic acids, J. Chrom atogr., 151, 39, 1978.

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CRC Handbook o f Chromatography

DEAE-Sephadex A-50 may be applied to the fractionation of charged polysacchar­ ides, e.g., agar components differing in sulfate and pyruvate content have been frac­ tionated on the chloride form of this exchanger,2by stepwise elution with water and sodium chloride solutions of increasing concentration (0.5—3 M ) . For polysaccharides containing components having chains terminated by D-mannitol, the use of DEAESephadex A-50 in the molybdate form has been recommended3as a means of isolating such components: on chromatography of a laminarin on this packing the components having chains terminated by D-glucose residues are eluted with water, the D-mannitolterminated chains with 0.25 Msodium chloride. DEAE-Sephadex A-25 is useful in the examination of hydrolysates of acidic polysac­ charides, for the separation of acidic from neutral components, and the fractionation of mixtures of acidic oligosaccharides. For example, Aspinall and co-workers,4'6 in studying the products of partial acid hydrolysis of plant gums, have used this ex­ changer in the formate form; neutral sugars present in the hydrolysate are eluted with water, whereas the acidic oligosaccharides, such as the aldobiouronic acid 6-0-(/3-dglucopyranosyluronic acid)-D-galactose, produced from such gums are retained by the exchanger in water but can subsequently be eluted with formic acid solutions (0.50—0.5 M ) . REFERENCES 2. 3. 4. 5. 6.

Duckworth, M ., Hong, K. C ., and Yaphe, W ., The agar polysaccharides of Gracilaria species, Carbo­ hydr. Res., 18, 1, 1971. Stark, J. R ., A new method for the analysis of laminarins and for preparative-scale fractionation of their com ponents, Carbohydr. R es.,47, 176, 1976. Aspinall, G. O ., Molloy, J. A ., and W hitehead, C. C., Araucaria bidwillii gum. III. Partial acid hy­ drolysis of the gum, Carbohydr. Res., 12, 143, 1970. Aspinall, G. O. and B hattacharjee, A. K., Plant gums of the genus Khaya. IV. M ajor component of Khaya ivorensisgum, J. Chem. Soc. C, 361, 1970. Aspinall, G. O. and Sanderson, G. R., Plant gums of the genus Sterculia. IV. Acidic oligosaccharides from Sterculia urensgum, J. Chem. Soc. C, 2256, 1970.

Carbohydrates

103

CHROM ATOGRAPHY OF CARBOHYDRATES ON CELLULOSIC IONEXCHANGERS Diethylaminoethyl-cellulose affords an efficient means of fractionating charged polysaccharides, and in recent years microgranular DEAE-cellulose has been preferred to ECTEOLA-cellulose in fractionation of glycosaminoglycans, since sharper peaks and virtually quantitative recoveries of these polysaccharides have been obtained with the former exchanger. The data presented in Table LC 27 exemplify this application of DEAE-cellulose. Table LC 27 CHROMATOGRAPHY OF GLYCOSAMINOGLYCANS OF DEAE-CELLULOSE PI + P2 (in series)

Packing Column Length Diameter M aterial Solvent Flow rate Tem perature Detection Reference

40 cm 1.5 cm glass SI 8 m l/h r rt

5 cm 1 cm glass S2 5 m l/h r 60°C D1 1

Com pound

V. (m l)

H yaluronic acid" H eparan sulfate 6 Chondroitin 4-sulfatec Derm atan sulfate* Keratan sulfate* H eparin 7

23 44 55 58 58 80

“ 6

c d e 1

M* about 1 x io 6. Mw = 58,000; 0.5 sulfate groups per disaccharide unit. Mw = 19,500; 0.95 sulfate groups per disaccharide unit, Mw = 41,000; about 1 sulfate per disaccharide unit. Mw = 17,500; sulfate as in d. Mw = 13,000; 2.4 sulfate groups per disaccharide unit.

Packing

PI P2

Solvent

SI S2

Detection D1 Technique

Sephadex G-50 (Pharm acia® ), equilibrated with SI before use. DE 52 (W hatm an® ), m icrogranular DEAE-cellulose, chloride form, equilibrated with SI before use. 0.15 M sodium chloride, containing 0.02% sodium azide. lithium chloride gradient (0.2— 1.2 M), buffered at pH 4.0 with 0.05 M sodium acetate buffer. carbazole method. The Sephadex column was connected to the DE 52 column, and the glycosaminoglycans (usually in 2 m l NaCl solution) were applied to the former column and transferred to the DE 52 column by elution with S I. REFERENCE

1.

Hallen, A ., Chrom atography of acidic glycosaminoglycans on DEAE-cellulose, J. Chromatogr., 71, 83,1972.

104

CRC Handbook o f Chromatography

A further example of the value of DE 52 in fractionation of acidic carbohydrates is the good resolution obtained on chromatography of the mixture of sialyl oligosaccha­ rides occurring in human milk on a column (45 x 1.5 cm) packed with this exchanger; stepwise elution with acetic acid-pyridine buffers, pH 5.4, of increasing concentration (0.002, 0.012, and 0.060 M ) yielded fractions from which several of these oligosaccha­ rides, (e.g., 3 - and 6 -sialyllactose) were subsequently isolated.2 In studies of acidic plant-gum polysaccharides, DEAE-cellulose in the phosphate form, eluted with phosphate buffers (0.1—0.5 M ) at pH 6, has been used to fractionate polydisperse gums.3 The use of this exchanger in the carbonate form, eluted with a 0—0.5 M ammonium carbonate gradient, has also been recommended.4 Further examples of applications of DEAE-cellulose in the fractionation of acidic polysaccharides will be found in the two review articles56cited below. REFERENCES 2. 3. 4. 5.

6.

Smith, D. F., Zopf, D. A ., and Ginsburg, V., Fractionation of sialyl oligosaccharides of human milk by ion-exchange chrom atography, Anal. Biochem., 85, 602, 1978. Aspinall, G. O. and B hattacharjee, A. K., Plant gums of the genus Khaya. IV. M ajor com ponent of Khaya ivorensisgum, J . Chem. Soc. C, 361, 1970. Siddiqui, I. R. and W ood, P. J., DEAE-cellulose carbonate form. A useful medium for fractionating polysaccharides, Carbohydr. Res., 16, 452, 1971. Neukom , H. and Kuendig, W ., Fractionation of neutral and acidic polysaccharides by ion-exchange column chrom atography on diethylaminoethyl (DEAE)-cellulose, in M ethods in Carbohydrate Chem­ istry, Vol. 5, W histler, R. L., BeMiller, J. N., and W olfrom , M. L., Eds., Academic Press, New York, 1965, 14. Jandera, P. and Churacek, J., Ion-exchange chrom atography of aldehydes, ketones, ethers, alcohols, polyols and saccharides, J. Chromatogr., 98, 55, 1974.

Carbohydrates

105

GEL-PERM EATION CHROM ATOGRAPHY Gel-permeation chromatography is important in the carbohydrate field, as it is much used in the separation of commercially important oligosaccharides, such as the maltodextrins, and the fractionation and molecular-weight distribution analysis of polysac­ charides. This type of chromatography is, therefore, given extensive coverage here. For some earlier data on polyacrylamide gel chromatography of sugars, see Section A, Volume I, Section II.II, Table LC 71. Tables LC 28-37, LC 40, LC 42, and LC 44 were contributed by Dr. Kirsti Granath (Pharmacia AB, Uppsala, Sweden).

Table LC 28 COLUMN CHROMATOGRAPHY ON TIGHTLY CROSS-LINKED GELS: CALIBRATION WITH SELECTED SUGARS AND ALCOHOLS Packing Column Length Diameter Solvent Flow rate Tem perature Detection Sample C om pound Stachyose Raffinose Maltose Glucose Glycerol Propylene glycol Ethylene glycol M ethanol “

PI

P2

P3

P4

P5

P6

90 cm 1.5 cm SI lO m l/h r rt R1 na

90 cm 1.5 cm SI

90 cm 1.5 cm SI

90 cm 1.5 cm SI

90 cm 1.5 cm SI

90 cm 1.5 cm SI

Conditions same throughout

M

r(A )°

K,

K,

IG

Kd

K,

K„

667 505 342 180 92 76 62 32

5.78 5.28 4.64 3.56 2.89 2.85 2.50 2.13

0.17 0.24 0.35 0.48 0.60 0.67 0.67 0.80

0.13

0.31 0.43 0.52 0.63 0.76 0.78 0.77 0.96

0.31 0.39 0.52 0.63 0.72 0.76 0.75 0.87

0.41 0.49 0.60 0.70 0.77 0.79 0.80 0.87

0.48 0.63 0.65 0.82 0.89 0.85

0.21

0.32 0.53 0.58 0.67 0.67 0.80

0 .8 8 0 .8 6

Unhydrated radius, derived from Corey-Pauling-Koltun molecular models by method of Goldstein and Solomon (see J. Gen. P hysiol.,44, 1, 1960).

Packing

Solvent

PI P2

P3 P4 P5 P6 SI

Sephadex G-10 (Pharm acia® ), allowed to swell in distilled water for at least 24 hr. Sephadex G-10 (1 MHC1). Swollen gel, mixed with equal volume of 1 MHC1, placed in boiling-water bath for 2 hr, with stirring. After supernatant decanted, HC1 removed by repeated washes with distilled water, followed by vacuum filtration, gel extracted twice with 1 volume chloroform -m ethanol ( 1 : 1), followed by 1 volume diethyl ether, vacuum filtration between each extraction. Gel dried in hood for 24 hr then heated in oven at 100°C for 2 hr. Before use, swollen in distilled water, as P I . Sephadex G-10 (6 MHC1), treated with 6 MHC1 as described for P2. Sephadex G-15, prepared as P I . Sephadex G-15 (1 MHC1), treated with 1 MHC1 as P2. Bio-Gel® P-2, polyacrylamide gel (Bio-rad® ), prepared as P I . 0.15 M N aC linO .O l M acetic acid (pH 3.3). REFEREN CE

1.

G oodson, J. M ., Stefano, V., and Smith, J. C ., C alibration of tightly cross-linked gel filtration media for determ ination of the size of low molecular weight, non-interacting solutes, J. Chromatogr., 54, 43,1971.

106

CRC Handbook of Chromatography Table LC 29 COLUMN CHROMATOGRAPHY OF SUGARS ON SEPHADEX G-15; EFFECT OF STRUCTURAL MODIFICATIONS AND MOLECULAR WEIGHT ON RELATIVE ELUTION VOLUME Sephadex G-15

Packing Column L ength(bed) Diameter Solvent Flow rate, linear Tem perature Detection Sample

45 cm 2.54 cm h 2o 8.5 cm /hr na R1 4°7o/0.1 mi

1. Effect of Structural Modifications on Relative Elution (Rg) on Sephadex G-151 Substituent at indicated C-atom M onosaccharide

C orresponding aldopentapyranose"

H*

1.086 1.042 1.029

D-Ribose D-Glucose D-Galactose

OH

OMe

Carbon-2 1.063 1.000

0.974

0.990 Carbon-3

D-Glucose

M annose D-Glucose Galactose

1.000

1.062 1.047 1.024

0.977

C arbon -6 1.040c 1.029 1.000

0.997c

0.957

0.990

Note: Data expressed as Rs values (R* here is V, for solute relative to V„ for D-glucose). “ b c

Hydroxym ethyl substituent on C-5 of the aldohexose replaced by H. Deoxy sugars. M easurements m ade with L-monosaccharide.

II. Effect of Site of Linkage (Disaccharides of D-Glucose) on R g on Sephadex G-151 Disaccharide

Linkage

Maltose Isomaltose Cellobiose Gentiobiose

a-D-(l - 4 ) a-D-(l -*• 6 ) P~ D - ( l - 4) P -D - ( l - 6)

R. 0.922 0.889 0.907 0.870

Carbohydrates

Table LC 29 (continued) COLUMN CHROMATOGRAPHY OF SUGARS ON SEPHADEX G-15; EFFECT OF STRUCTURAL MODIFICATIONS AND MOLECULAR WEIGHT ON RELATIVE ELUTION VOLUME Packing Column Length(bed) Diameter Solvent Flow rate, linear Tem perature Detection Sample

Sephadex G-15 45 cm 2.54 cm h 2o 8.5 cm /hr na R1 4% /OA m l

III. Effect of Configuration at Substituted Anomeric Carbon Atom on R g of Methyl Glycosides and Disaccharides on Sephadex G-151 C om pound

Rf

Methyl a-D-xylopyranoside Methyl /3-D-xylopyranoside Methyl a-D-glucopyranoside Methyl /3-D-glucopyranoside 4-0-a-D-Glucopyranosyl-D-glucopyranose 4-0-/?-D-Glucopyranosyl-D-glucopyranose 6-0-a-D-Glucopyranosyl-D-glucopyranose 6-0-/?-D-Glucopyranosyl-D-glucopyranose

1.021 1.005 0.971 0.954 0.922 0.907 0.889 0.870

IV. Relative Elution Volumes of Groups of Isomeric Saccharides on Sephadex G-152 Pentoses M 150 D-Ribose D-Lyxose D-Xylose L-Arabinose

R. 1.065 1.062 1.047 1.024

Hexoses M 180 D-Psicose D-Mannose L-Sorbose D-Fructose D-Glucose D-Galactose

R, 1.040 1.029 1.026 1.018 1.000 0.990

Disaccharides M 342 Sucrose Maltose Turanose Cellobiose Trehalose Lactose Isomaltose Melibiose Gentiobiose

R. 0.926 0.922 0.920 0.907 0.902 0.895 0.889 0.877 0.870

REFERENCES 1. Bertoniere, N. R., M artin, L. F., and Rowland, S. P ., Stereoselectivity in the elution of sugars from columns of Sephadex G-15, Carbohydr. Res., 19,189,1971. 2. M artin, L. F ., Bertoniere, N. R ., and Rowland, S. P ., The effects of sorp­ tion and molecular size of solutes upon elution from polyhydroxylic gels, J. Chrom atogr., 64, 263, 1972.

107

108

CRC Handbook of Chromatography T a b le L C 30 S E P A R A T IO N O F S U B S T IT U T E D C A R B O H Y D R A T E S BY H IG H P E R F O R M A N C E G E L P E R M E A T IO N CHRO M ATO GRAPH Y Packing Column Length I.D. Solvent Flow rate Tem perature Detection Sample

C om pound a-Cellobiose octaacetate Phenyl tetra-O-acetyl /3-D-glucopyranoside a-D-Glucopyranose pentaacetate /1-D-Glucopyranose pentaacetate a-D-Galactopyranose pentaacetate /1-D-Galactopyranose pentaacetate Methyl tetra-O-acetyl /3-D-glucopyranoside L-Arabinitol pentaacetate E rythritol tetraacetate 4,6-O-Benzylidene D-glucopyranose 1,2:5,6-D i-0-isopropylidene a-D-glucofuranose 2,3,6-Tri-O-methyl-D-glucose 2,4,6-Tri-O-methyl-D-Glucose 1,2-O-Isopropylidene a-D-glucofuranose 3-O-Methyl D-glucose D-mannose D-Fructose D-Glucose D-Xylose D-Glucitol D-Arabinitol Methyl a-D-glucopyranoside Methyl /3-D-glucopyranoside Phenyl /3-D-glucopyranoside Polypropylene glycol standard (M„ = 1220)

PI

P2

61 cm 2 mm SI 0.27 m i/m in rt RI, UV 10 mg /1 m i

122 cm

ra 1.22

2 mm

S2 0.51 m i/m in rt RI, UV 10 mg /1 m i

Tb

Mol wt

0.92 1.03

2 .0 0

1.01

2.16 2.03 1.84 1.84 1.61 1.59

1.01

1.11 1.10

1.15 1.15

678.6 424.4 390.3 390.3 390.3 390.3 362.3 362.3 290.2 268.3 260.3 225.2 225.2

1.23

1.20

2 2 0 .2

2.19 2.16 2.32

1.00

0.99

1.03 1.18 1.07 1.21 1.12

1.00

0.97 1.03 1.00 1.10 1.00 1.00 1.00 1.00 1.22

0.69

Note: Commercial liquid chrom atograph (W aters Assoc. Model 202) was used. “ Relative to D-glucose. b Relative to a-D-glucopyranose pentaacetate. Packing PI

P2 Solvent

SI S2

EM Gel OR-PVA 500 (M erck® ), 50 ptm fractionation range, mol wt 0—300. Swollen in m ethanol for 24 hr. Slurry introduced at top of vertical column under vacuum. High-pressure pump for final packing. Poragel 60 A (W aters® ), 37—75 ptm fractionation range, mol wt 100—2400. Packed by m anufacturer. m ethanol. chloroform .

From McGinnis, G. D. and Fang, P ., J. Chromatogr., 130, 181, 1977. With permission.

Carbohydrates

109

T a b le L C 3 1 P A R T I T I O N C O E F F I C I E N T S ( Kd) F O R M A L T O D E X T R I N S , A T D I F F E R E N T T E M P E R A T U R E S , O N P O L Y A C R Y L A M ID E G E L Packing Tem perature C om pound Glucose Maltose M altotriose M altotetraose M altopentaose M altohexaose M altoheptaose Packing

Column

Solvent Flow rate Sample Detection

P 1=

PI 20°C

PI 30°C

PI 40°C

PI 50°C

PI 60°C

PI 70°C

K„

Kd

Kd

Kd

Kd

Kd

M

d(logK)/dT

0.892 0.800 0.723 0.647 0.575 0.511 0.455

0.887 0.789 0.703 0.625 0.550 0.486 0.430

0.893 0.790 0.700 0.616 0.539 0.473 —

0.883 0.775 0.682 0.599 0.522 0.459 0.398

0.894 0.784 0.683 0.596 0.516 0.448 0.391

0.877 0.766

180 342 504

- 0.86 xlO ' 4 - 3.32 xlO -4 - 6.61 x l 0 ‘4 - 9.09 xlO "4 - 11.51 x 10-4 - 13.52 x lO"4 - 15.78 x lO"4

0 .6 6 6

0.579 0.500 0.435 0.378

666

828 990 1152

Polyacrylam ide (Bio-Gel® P-2). From -400 mesh product, 28-—60 jim gel fraction was sieved out and further fractionated by repeated settling and decanting. Degassed before packing, length: 20 0 cm. diameter: 1.5 cm. material: glass, coated with 1% dichlorodimethylsilane in benzene. h 2o . 25 m l/h r . 20 /Lil, 10 % solution. autom atic analysis of effluent by orcinol m ethod. REFERENCE

1.

Dellweg, H ., John, M ., and Trenel, G ., Gel permeation chrom atography of maltooligosaccharides at different tem peratures, J. Chromatogr., 57, 89, 1971; John, M. and Dellweg, H ., Gel chrom at­ ographic separation of oligosaccharides, Sep. Purif. Methods, 2, 231, 1973.

110

CRC Handbook of Chromatography T a b le L C 32 C O L U M N C H R O M A T O G R A P H Y O F M A L T O D E X T R IN S O N P O L Y A C R Y L A M ID E G E L S ; IN F L U E N C E O F F L O W R A T E A N D T E M P E R A T U R E O N P A R T I T I O N C O E F F I C I E N T K„

Packing Column Length, cm Diameter, cm Solvent Flow rate, m l/h r Tem perature Detection Sample, \xl cone Reference



PI

P2

PI

PI

PI

PI

PI

PI

100

100

100

100

100

82.0

82.0

82.0

1.6

1.6

1.6

1.6

1.6

1.6

1.6

1.6

h 2o

h 2o

h 2o

h 2o

h 2o

h 2o

h 2o

2 0 .0

45 °C RI

18.0 45°C Rl

19.0 45°C Rl

24.6 45°C Rl

30.0 45°C RI

28.8 35°C RI

28.8 45°C RI

H 20 28.8 55°C Rl

200

200

200

200

200

8

L 1 L2

T3 T4 T4 T5 T5 T6 T7 3 4 4 5 5 5 6

Compound

T7 6

T7 T8 T8 T9 7 7 8 6

T10 9

RFx 100 *

*

*

1* 2* * * * * *

L 13 L 14 S16 S3 T10 T10 T T12 9 10 11 12

RGtca x 100 *

*

*

1* 2* **

***

*** *** ** ***

L 15 S 17 T13 13

RSucb x 100 C

L 16 S18 T14 14 RF

x 100

VP

25 6 Isomal totriose to 2 411 Isokes se 75 neo -Ke stose K t 51 1- es ose 41 6-K e stose 29 N y sto se 0 2 3 29 Stac hyose M al to te tr aose 34 17 Is om a lt ot tr ose M alto penta 27 e a se o 23 Maltohexaose N ote: * = single development. ** = double development (monodimensional). *** = threefold development (monodimensional). 1* = twodimensional development, first run. 2* = two-dimensional development, second run orthogonal to first. C = continuous run. VP = Vapor-programmed TLC. b

aR Gtc = mobility relative to glucose. R,uc = mobility relative to sucrose.

Layer

LI L2 L3 L4

= precoated Silica Gel 60 (M erck®), conditioned at 105°C for 1 hr, cooled in desiccator. = precoated Silica Gel 60 (M erck®), impregnated with 0.5 M N aH 2P 0 4. = 0.25 mm Silica Gel G (M erck®), impregnated with 0.03 M H 3BO3; heated at 110°C for 1 hr before use. = Silica Gel H (M erck®).

CRC Handbook of Chromatography

S1 S2 T1 T2 1 2

140

Layer Solvent Technique Reference

Table TLC 1 (continued) SUGARS, MONO- AND OLIGOSACCHARIDES; ALDITOLS L3 L4 L4 L5 L5 L5 L6 L7 L8 L9 L9 L10 L 11 L 12 L 11 S3 S4 S5 S6 S6 S7 S8 S9 S10 S11 S5 S12 S14 S15 S13

L5 = 0.5 mm Kieselguhr G (M erck® ), impregnated with 0.15 M N aH 2P 0 4; plates allowed to dry for at least 24 hr at 18—20°C and stored in presence of air at tem perature not exceeding 20°C. L6 = 0.25 mm Silica Gel G 60 (M erck® ), dried at 110°C for 2 hr. L7 = 0.25 mm layer, mixture of Silica Gel G 60 (2 parts), Kieselguhr G (1 part), prepared as for L6. L8 = 0.25 mm Cellulose MN 300 (Machery-Nagel), allowed to dry overnight. L9 = 0.30 mm layer, 2:1 mixture of Silica Gel G 60 (M erck® ) and Syloid 63 (W. R. Grace), impregnated with 0.03 M sodium tetraborate and 0.05 M sodium tungstate (3:1); dried at 90°C for 30 min. L10 = precoated 0.25 mm Silica Gel G. LI 1 = 0.25 mm Cellulose MN 300 (Machery-Nagel). L12 = 0.30 mm Cellulose MN 300. L13 = precoated 0.10 mm Cellulose F (M erck® ). L14 = 0.20 mm cellulose layer (Chrom edia CC 41, W. R. Balston), air-dried for 15—20 min; heated at 100°C for 30 min. L I 5 = precoated 0.25 mm Silica F 254 (M erck® ). L16 = 0.30 mm Silica Gel G, impregnated with 0.2 M N aH 2P 0 4; plates air-dried for 15 min, heated at 100°C for 30 min.

Solvent

= 2-propanol-acetone-M lactic acid (2:2:1). = 2-propanol-acetone-0.1 M lactic acid (2:2:1). = ethyl acetate-2-propanol-acetic acid-water (4:2:1:1). = chloroform -m ethanol-w ater (16:9:2). = ethyl acetate-m ethanol-acetic acid-water (12:3:3:2). = ethyl acetate-m ethanol-1-butanol-water (16:3:3:2). = ethyl acetate-m ethanol-1-butanol-water (16:3:3:1). = benzene-ethanol (2:1). = benzene-acetic acid-ethanol (2:2:1). = 1-butanol-acetic acid-water (6:1:2). 1 = ethyl acetate-2-propanol-w ater (2:2: 1). = pyridine-ethyl acetate-water (13:33:4). = formic acid-2-butanone-ferf-butanol-water (3:6:8:3). = formic acid-2-butanone-ferf-butanol-water (3:5:7:5). = ethyl acetate-pyridine-water (20:7:5). = ethyl acetate-pyridine-water-acetic acid-propionic acid (10:10:2:1:1). = w ater-saturated 1-butanol-ethanol (5:2). = acetone-m ethanol-water (16:3:1); vapor program in troughs (A = acetone; M = methanol; W = water): trough 3 = A-MW (7:2:1); trough 6 = A-M-W (3:1:1); trough 8 = A-M-W (4:3:3); trough 10 = A-M-W (1:2:2); troughs 12, 14, 16, and 18 = A-M-W (4:5:11); troughs 1,2, 4, 5 ,7 ,9 , 11, 13, 15, 17, 19, 20, and 21 = A only.

Carbohydrates

51 52 53 54 55 56 57 58 59 S10 SI 512 513 514 515 516 517 518

141

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14

= ascending development (13 cm; 3 hr); plates dried in stream of warm air (60°C) before spraying. = ascending development (16 cm; 5 hr); plates dried as above. = ascending development (10 cm) in pre-equilibrated (for 30 min) chamber. = two-dimensional development (18 x 18 cm; about 75 min each development). = samples applied to plates in streaks of about 1 cm, under current of warm air (75—80°C); ascending development (20 cm; 65 min). = double developm ent (35 cm; 325 min each); after each run plates dried at room tem perature for 15 min, then heated at 100°C for 5 min. = ascending development in filter paper-lined glass chamber. = two-dimensional development (12 x 12 cm; first run 120 min, second 50 min); plates dried between two runs at 70°C for 4—5 min. = layer sprayed lightly with solvent before use and dried at 105°C for 2 hr; ascending development (double, 14 cm each) in filter paper-lined cham ber, drying between runs at 50°C for 100 min. = threefold ascending development (about 18.5 cm and 180 min each run), without previous saturation of the chamber. = ascending development (double about 105 min each run) in presaturated paper-lined chamber; plates air-dried between runs. = threefold developm ent, 3 hr total time required. = continuous development for 47—60 hr. = vapor-program m ed (VP) development in 20 cm VP-chamber (Desaga); ambient tem perature 24—25°C, saturation time 10 min, developm ent time 3 hr. REFERENCES

1. H ansen, S. A ., Thin-layer chrom atographic method for identification of oligosaccharides in starch hydrolysates, J. Chrom atogr., 105, 388,1975. 2. H ansen, S. A ., Thin-layer chrom atographic method for the identification of mono-, di-, and trisaccharides, J. Chrom atogr., 107, 224, 1975. 3. N akai, T ., D em ura, H ., and Koyama, M ., Thin-layer chrom atographic detection of glycoladehyde using a fluorescence reaction with o-am inobiphenyl, J. C hrom atogr.,66, 87, 1972. 4. Kartnig, T. and W egschaider, O ., A method for identification of sugars from small amounts of glycosides or from sugar mixtures, J. C hrom atogr., 61,375, 1971. 5. T alukder, M. Q .-K ., A rapid quantitative thin-layer chrom atographic separation of fucose from other neutral m onosaccharides, J. Chro­ m atogr., 57,391, 1971.

CRC Handbook of Chromatography

Technique

142

Table TLC 1(continued) SUGARS, MONO- AND OLIGOSACCHARIDES; ALDITOLS

6 . H aldorsen, K. M ., Vanadium pentoxide in sulfuric acid, a general chromogenic spray reagent for carbohydrates, J. Chromatogr., 134,

467, 1977. 7. G hebregzhaber, M ., R ufini, S., Ciuffini, G., and Lato, M ., A two-dimensional thin-layer chrom atographic method for screening carbo­ hydrate anomalies, J. C hrom atogr.,95, 51, 1974. 8 . Szustkiewicz, C. and D em etriou, J., Detection of some clinically im portant carbohydrates in plasma and urine by means of thin-layer chrom atography, Clin. Chim. Acta, 32, 355, 1971. 9. D am onte, A ., L om bard, A ., Tourn, M. L., and Cassone, M. C ., A modified solvent system and multiple detection technique for the separation and identification of mono- and oligosaccharides on cellulose thin layers, J. Chromatogr., 60, 203, 1971. 10. Raadsveld, C. W. and Klomp, H ., Thin-layer chrom atographic analysis of sugar mixtures, J. Chromatogr., 57, 99, 1971. 11. W alkley, J. W. and Tillm an, J., A simple thin-layer chrom atographic technique for the separation of mono- and oligosaccharides, J. Chrom atogr., 132, 172, 1977. 12. Petre, R ., Dennis, R ., Jackson, B. P ., and Jethwa, K. R., Thin-layer chrom atography of sugars and uronic acids in plant extracts on cellulose, Planta M ed., 21, 81, 1972. 13. Schaffler, K. J. and Morel du Boil, P. G., Thin-layer chrom atographic separation of oligosaccharides isolated from sucrose-enzyme mixtures, J. C hrom atogr.,72, 212, 1972. 14. De Zeeuw, R. A. and Dull, G. G., Rapid analysis of simple carbohydrates by means of vapour-program m ed thin-layer chrom atography and densitom etry and using a new spotting device, J. Chromatogr., 110, 279, 1975.

Carbohydrates 143

144

T ab le T L C 2 M A IN K E T O S E S LI SI T1

LI SI T1

LI S2 T1

LI S2 T1

LI S3 T1

LI S3 T1

Com pound

-

b



b

a

b

186 177 173 145 136 125 116

80 74 72 60 54 50 46 38 47 39 36 32 29

210

Dihydroxyacetone L-g/ycero-Tetrulose D- f/ireo-Pentulose D -eryt/iroPentulose D-Tagatose D-Psicose L-Sorbose D-Fructose D-a/tro-Heptulose D-m anno-H eptulose D -/doH eptulose D -a//oH eptulose D-g/uco-Heptulose L-ga/acTo-Heptulose -epAInosose -2 scy llo-1 nosose

81 231 82 75 214 78 70 200 76 60 171 64 50 143 60 49 140 55 42 120 51 35 100 44 42 120 54 32 91 43 33 94 41 24 69 37 22 63 33 17 49 25 10 29 9 4 11 4

100

123 98 93 84 75 57 20

9

195 189 136 123 114 121 100

124 103 95 84 76 22 58 9 24 4 11 "

Layer Solvent

LI SI 52 53 54 55 56 57

= = = = = = = =

LI S4 T1

LI S4 T1

LI S5 T1

LI S5 T1

LI S6 T1

LI S6 T1

LI S7 T1

LI S7 T1

LI S8 T1

LI S8 T1

LI S9 T1

G

b

-

b

a

b

a

b

a

b

°

71 67 63 50 43 39 34 28 35 27 28

254 239 225 179 154 139

75 71 54

237 210 203 163 137 137 117 100 113 97 100 83 77 63

71 65 63 51 45 43 38 33 37 32 31 28 26 19

21

6

20

7

3

10

74 70 69 55 51 48 44 38 43 38 35 33 31 23 7 4

195 184 182 145 134 126 116

21

71 63 61 49 42 41 35 30 34 29 30 25 23 19

121 100

125 96 100

20

15 6 2

215 197 191 155 136 130 115 100 112 97 94 85 79 58 6 18 3 9

100

113 100

92 87 82 61 18 11

= Rf x 100.

0.25 mm Silica Gel 60 (M erck® ). acetone-water (9 : 1). 2-propanol-acetone-water (4:5:1). 1-propanol-acetone-water (4:5:1). 1-butanol-acetone water (4:5:1). 2-propanol-ethyl acetate-water (4:5:1). 2-propanol-ethyl acetate-water (5:4: 1). 2-propanol-ethyl acetate-water (6:3:1).

Technique

From Papin, J.-P . and Udim an, M ., J. Chromatogr., 132, 339, 1977. With permission.

= = = = = =

b

68 189

70 233

178 178 144 133 125 117

66 220 66 220

64 64 52 48 45 42 36 42 38 35 33 32 23

100

117 106 97 92 89 64 17 6 8 3 6

58 59 510 511 512 T1

LI LI LI LI LI LI LI S9 S10 S10 S ll S ll S12 S12 T1 T1 T1 T1 T1 T1 T1

50 47 41 37 30 40 33 27 27 24 16 4 2

167 157 137 123 100

133 110

90 90 80 53 13 7

a

b

a

b

76 71 69 59 54 49 48 40 48 41 35 33 32 23

190 178 173 148 135 123 120

64 54 46 38 28 29 24

305 257 219 181 133 138 114

100

21

100

120

21

100

103

18 19 14 14

10

5

88

83 80 58 25 13

12 6

3

86

90 67 67 57 29 14

a

b

69 157 66 150 66 150 56 127 56 127 50 114 50 114 44 100 50 114 47 107 41 93 39 89 39 89 29 66 9 20 5 11

= R f>uc x 100 .

2-propanol-ethyl acetate-water (7:2:1). 2-propanol-ethyl acetate-water (83:11 ;6 ). 1-butanol-acetone-m ethanol-w ater (33:36:18:9) ethyl acetate-l-butanol-m ethanol-w ater (16:3:3:2). ethanol-2-butanol-w ater (6:3:1). ascending development (15 cm; 60 min); developing chamber equilibrated with solvent system for 1 hr before use.

CRC Handbook of Chromatography

Layer Solvent Technique

Carbohydrates

145

T ab le T L C 3 M A IN S U G A R C O N S T IT U E N T S O F G L Y C O P R O T E IN S A N D G L Y C O L IP ID S Layer Solvent Technique Reference

LI SI T1

LI S2 T1

LI S3 T1

LI S4 T2

LI S5 T3

L2 S6 T4

L2 S7 T4

1

1

1

1

1

2

2

L3 S8 T5 3

K f x 100

RADCa X 100

*

*

***

1 **

2 **

_

_

_

_

_

_

55 61

100

72 75

30 42













66

50

10





33 7 26

64

53

69 58 65 75 34 79







C om pound

*

*

D-Mannose D-Galactose D-Glucose L-Fucose 2-Amino-2-deoxy-D-glucose 2-Acetamido-2-deoxy-D-glucose 2-Amino-2-deoxy-D-galactose 2-Acetamido-2-deoxy-D-galactose N-Acetylneuraminic acid N-Glycolylneuraminic acid

_ 54 62 —



21

54 13 45 3

26 59 19 51 5





100 —

100

54

31 74

43

22

100

66 2

44 41



1 —

L4 S9 T6 4

*

*

_ — — —

100

116 85 107

52

57



















Note: * = single development. ** = double developments. *** = threefold development. 1 refers to first run in 2 -dimensional development. 2 refers to second run, orthogonal to first, in 2 -dimensional devel­ opm ent. “

R ^ c = mobility relative to 2-amino-2-deoxy-D-glucose.

Layer

Solvent

Technique

LI = 0.25 mm Silica Gel G (M erck® ); plates air-dried for 1 hr, heated at 110°C for 30 min before use. L2 = 0.25 mm Silica Gel F 2s4 (M erck® ), impregnated with 0.2 M N aH 2P 0 4; heated at 110— 120°C for 30 min before use. L3 = precoated Silica Gel 60 (M erck® ), conditioned at 105°C for 1 hr. L4 = Chrom AR 500 (M allinckrodt) sheets (about 70% neutral silicic acid and 30% fiber glass) cut into 83° wedge, base 2.5 cm wide, sides about 12 cm long. SI = 1-propanol-w ater (7: 1). 52 = 1-propanol-w ater (14:3). 53 = m ethanol-water (5:2). 54 = 1-propanol-ethyl acetate-water (5:1:4). 55 = methyl acetate-2-propanol-w ater (18:1:1). 5 6 = 1-butanol-acetone-water (4:5:1). 57 = phenol-water (3:1 w/v). 5 8 = 2-propanol-acetone-M lactic acid (2:2:1). 59 = 1-propanol-concentrated amm onia (4:1). T1 = ascending development, 4 hr. T2 = ascending development, 5 hr. T3 = threefold development, 50 min each run; plates dried after each development. T4 = two-dimensional development, twice to 15 cm in each direction; plate air-dried between runs. T5 = ascending development (13 cm; 3 hr). T 6 = sheets uniform ly sprayed with methanolic solution of C u S 0 4 (0.05%), dried in stream of warm air for 30 min before sample application; ascending development (15 cm) in Gelman cham ber, without presaturation. REFERENCES

1. Gal, A. E ., Separation and identification of m onosaccharides from biological materials by thin-layer chrom atography, A nal. Biochem ., 24,452, 1968. 2 . H otta, K. and K urokawa, M ., Separation of fucose and acetylhexosamine by two-dimensional thinlayer chrom atography, A nal. Biochem .,26, 472, 1968. 3. H ansen, S. A ., Thin-layer chrom atographic method for identification of oligosaccharides in starch hydrolysates, J. Chrom atogr., 105, 388, 1975. 4. M artz, M. D. and Krivis, A. F., Thin-layer chrom atography of hexosamines on copper-impregnated sheets, A nal. Chem., 43, 790, 1971.

LI SI T1 1

L2 S2 T1 1

R , x 100 Com pound D-Glucopyranoside methyl ftmethyl 4,6-O-benzylidene-amethyl 2,3,4-tri-O-m ethyl-amethyl 2,3,4-tri-O-methyl-0methyl 2,3,4,6-tetra-O-m ethyl-amethyl 2,3,4,6-tetra-O-methyl-/?phenyl 2,3,4,6-tetra-O-acetyl-aphenyl 2,3,4,6-tetra-O-acetyl-/?2-chlorophenyl 2,3,4,6-tetra-O -acetyl-a2-chlorophenyl 2,3,4,6-tetra- O-acetyl-04-chlorophenyl 2,3,4,6-tetra-O-acetyl-a4-chlorophenyl 2,3,4,6-tetra- O-acetyl-02-methylphenyl 2,3,4,6-tetra-O-acetyl-a2-methylphenyl 2,3,4,6-tetra- O-acetyl-/?4-methylphenyl 2,3,4,6-tetra- O-acetyl-a4-methylphenyl 2,3,4,6-tetra- O-acetyl-04-nitrophenyl 2,3,4,6-tetra-O-acetyl-a4-nitrophenyl 2,3,4,6-tetra-0-acetyl-/34-phenylphenyl 2,3,4,6-tetra-O-acetyl-a4-phenylphenyl 2,3,4,6-tetra-O-acetyl-0D-Xylopyranoside, methyl fiD-Glucopyranose 4,6- O-ethylidene-a1,2,3,4,6-penta-O-acetyl-a1,2,3,4,6-penta-O-acetyl-/?-

L3 S3 T2 2

L4 S4 T3 3

L4 S5 T3 3

Rc“ x 100

L5 S6 T4 4

L5 S7 T5 4

L6 S8 T6 4

L7 S9 T7 4

Rr x 100

L8 S9 T8 5 R*™* x 100

*

*

*

*

*

**

**

**

♦*

*

20 62 — — — — — — — — — — — — — — — — — — 30

51 81 — — — — — — — — — — — — — — — — — — 58

— — — — — — 176 140 148 120 200 152 158 117 173 117 150 104 135 104 —

— — 63 — 100 — — — — — — — — — — — — — — — —

— — 31 35 49 62 — — — — — — — — — — — — — — —

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

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

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

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

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

40 75 —

68 90 —

— — 100

— — —

— — —

— — —

— — —

— — —

— — —

— — —

CRC Handbook of Chromatography

Layer Solvent Technique Reference

146

Table TLC 4 METHYL, ARYL, AND ACETYL DERIVATIVES OF SUGARS

D-Arabinose 2-O-methyl3-O-methyl4 -e-m ethyl­ s'O -m ethyl2,3-di-O-methyl2,4-di-O-methyl2,5-di-O-methyl3,4-di-O-methyl3,5-di-O-methyl2,3,5-tri-O-methylD-Xylose 2-O-methyl3-O-methyl2,3-di-O-methyl2,3,4-tri-O-methylD-Glucose 2-O-methyl3-O-methyl3,6-di-O-methyl2,3,4-tri-O-m ethyl2,3,6-tri-O-m ethyl2,3,4,6-tetra-O-m ethylD -G alactose,2,3,4,6-tetra-0-m ethylD-Mannose 2,3-di-O-methyl2,3,6-tri-O -m ethyl2,3,4,6-tetra-O -m ethyl-

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

24 15 11 40 56 43 81 26 81 100

— — —

— — — —

— — — —

— — — —

— — — —

44 44 68 87

49 49 73 93

68 46 78 84

35 36 51 59

— — — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — —

— — — — — — —

32 32 54 79 77 — 80

33 33 52 80 — — —

53 44 65 79 77 85 70

10 10 28 53 52 79 59

— — — — — — —

— — —

— — —

— — —

— — —

— — —

72 72

— — —

65 73 79

41 43 61

— — —







Rc = m obility relative to that of penta-0-acetyl-/l-D -glucopyranose. R/ira = m obility relative to that o f 2,3, 5-tri-O-m ethyl-D-arabinose.

* Single development. ** Double development. Layer

147

LI = 0.25 mm Silica Gel 60 (M erck® ), dried at 110°C for 2 hr. L2 = 0.25 mm layer, mixture of Silica Gel G 60 (2 parts) and Kieselguhr G (1 part), prepared as for L I . L3 = Silica gel G (M erck® ), air-dried then heated at 110°C for 1 hr.

Carbohydrates

“ b

— — — — — — — — — —

Technique

preparative (0.5 mm) Silica Gel H (M erck® ). cellulose. silica gel buffered with 0.1 M H jB O , activated at 110°C. silica gel. 0.25 mm Silica Gel F 254 (M erck® ). benzene-ethanol ( 2 : 1). benzene-acetic acid-ethanol (2:2:1). 2-butanone-petroleum ether, 40—60° (1:3). chloroform -m ethanol (47:3). benzene-ethanol-water (170:47:15, upper layer). 1-butanol-ethanol-w ater-am m onia (40:10:49:1). ethyl acetate-acetic acid-water (18:7:8). 1-butanol-acetone-water (4:5:1). 2-butanone saturated with 3% aqueous amm onia. ascending development in filter paper-lined glass cham ber. ascending development in saturated cham ber, distance = 15 cm. not specified. double development (12.5 and 12.7 cm), 6.5 hr total time. double developm ent (14.6 and 15.1 cm), 4.5 hr total time. double development (13.6 and 14.4 cm), 2.5 hr total time. double development (15.1 and 13.8 cm), 7 hr total time. single developm ent, standard procedure.

REFERENCES 1. H aldorsen, K. M ., Vanadium pentoxide in sulfuric acid, a general chromogenic spray reagent for carbohydrates, J. Chrom atogr., 134, 467, 1977. 2. A udichya, T. D., Thin-layer chrom atography of anomeric aryltetra-O-acetyl-D-glucopyranosides, J. Chrom atogr., 57, 161, 1971. 3. Brennan, P. J., Application of thin-layer chrom atography to the purification and characterisation of some methyl glucosides, J. Chrom atogr., 59, 231, 1971. 4. Sinner, M. Separation of methyl ethers of xylose, glucose and some other sugars by liquid chrom atography, J. Chromatogr., 121,122,1976. 5. Mied, P. A. and Lee, Y. C ., Preparation and separation of methyl ethers of D-arabinose, Anal. Biochem ., 49, 534, 1972.

CRC Handbook of Chromatography

Solvent

L4 = L5 = L6 = L7 = L8 = SI = 52 = 53 = 54 = 55 = 56 = 57 = 58 = 59 = T1 = T2 = T3 = T4 = T5 = T6 = T7 = T8 =

148

Table TLC 4 (continued) METHYL, ARYL, AND ACETYL DERIVATIVES OF SUGARS

Table TLC 5 HIGHER OLIGOSACCHARIDES Malto-oligosaccharides

Isomalto-

Fructans

Oligogalacturonic acids

Type of oligosaccharide LI LI LI LI LI LI LI LI SI S2 S3 S4 SI S2 S3 S4 T1 T1 T1 T1 T2 T2 T2 T2

Layer Solvent Technique Reference

1

1

1

1

1

1

1

1

L2 S5 T3

L2 S6 T4

L2 S7 T5

L2 S7 T6

L2 S7 T7

2

2

2

2

2

Rjr X 100 Degree o f polym erization 1 2

3 4 5 6

7 8

9 10 11

89 92 78 90 60 84 39 77 20 71 — 64 — 57 — 49 — 39 — 31 — 23 — 21 — 19 — 15 — 12 9 7 — —



*

*

93 89 84 78 70 63 55 44 36 30

93 87 89 77 84 61 81 42 77 24 73 14 68 9 62 5 55 3 2 49 42 1 36 — 29 — 24 — 20 — 16

22

17 14 9

6 5 3 2

*







R,W1‘ x 100

R „ 3a * 100 *

*

*

*

*

91 87 83 77 69 61 53 42 33 25 18 17 13 10 7

94 91 87 81 74 65 55 44 33 25 19 14 10 8

93 90

141 119

__

86

100

78

81 75 70 62 53 45 37 31 26 22 17 4 12 10 6 8 5 — 4 6 2 — 5 4

66

53 44 37 31 25 — — — — —

*

*

__

110

107 100

100

100

100

100

86



89 77

93 84 72 62 53

90 85 75 65 55 45 37 30

93

69 52

66*

60 52 45 40 35 30 26 23 20 17



— — —

86

79 72 65 57 48 38 31

__

c

__

68

__

C

110







*

LI LI L4 L4 L5 S9 S10 S ll S12 S ll T9 T9 T10 T10 T il 4 4 5 5 5

__

100

81*

Rjr X 100 *

*

*

**

**

85 78d 52 38 24 15

96

65

100 88

100

52 48 31 19 13

59 48 38 25 13 7 5

8

2

— —





28 —

17

10

7 — —





















































-









-

-





= single development. ** = double development. C = continuous run.

R*. x 100

*

48* 37* 27* 20* 14*



L5 S12 T il 5

9 4 * 58

87 83 77 70 62 55 48 43 38 34 29 23 19 15 12 10 8 7

4 1

80 60 47 27 17 13

76 58 37 22 12 6

3

8



2

4



1









































-





-





-



149

N o te : *

*

L3 S8 T8 3

Carbohydrates

12 13 14 15 16 17 18 19 20

*

L3 S8 T8 3

d '

R m, = m o b il it y relative to m a lt otr io se , calc ulated from published data. R/jVf2 - m o b il it y relative to is o m a lt o s e , calc ulated from published c h r o m a to g r a m .

R om = mobility relative to.a-D-galacturonic acid. Sucrose. Single a-(l-*3) branch.

Layer

Solvent

Technique

LI L2 L3 L4 L5 SI 52 53 54 55 56 57 58 59 S l€ 5 11 512 T1 T2 T3 T4 T5 T6 T7 T8 T9

= 0.25 mm Kieselguhr G (M erck® ). = precoated silica gel high-perform ance TLC plates (M erck® , Cat. No. 5633). = precoated 0.25 mm silica gel plates (M erck® , Cat. No. 5721). = Cellulose MN-300, Polygram Cel-300 plates. = Eastm an E-13255 cellulose plates. = 1-butanol-pyridine-water (13:4:3). = 1-butanol-pyridine-water (6:4:3). = 1-butanol-pyridine-water (5:4:2). = 1-butanol-pyridine-water (20:19:11). = 2 -propanol-acetone-water (8:7:5). = 2-propanol-water (7:3). = ethanol-acetone-w ater (9:6:5). = 1-propanol-nitrom ethane-w ater (5:2:3). = 1-propanol-ethyl acetate-water (4:5:1). = 1-propanol-ethyl acetate-water (3:1:1). = ethyl acetate-acetic acid-water (2 : 1 :2 ). = ethyl acetate-acetic acid-water (4:2:3). = plates 5 x 20 cm; oligosaccharides in 90% dimethylsulfoxide solution, applied as narrow band 1 cm long; ascending development. = plates 20 x 45 cm; oligosaccharide mixtures applied as narrow band 16 cm long. = oligosaccharides, dissolved in 70% aqueous ethanol, spotted on plate; developed for 30 min at 60°C. = developed for 2 hr at 60°C. = developed for 30 min at 26°C. = developed for 30 min at 40°C. = developed for 30 min at 58°C. = continuous development for 19 hr at 30°C. = single, ascending development (12 cm) at ambient tem perature.

CRC Handbook of Chromatography

“ b 1

150

Table TLC 5 (continued) HIGHER OLIGOSACCHARIDES

T10 = single, ascending development (95 min) at 24°C. T 1 1 = double developm ent (2.5 hr each) at 24°C. REFEREN CES 1. Shannon, J. C. and Creech, R. G ., Thin-layer chrom atography of malto-oligosaccharides, J. C hrom atogr.,44, 307, 1969. 2. N urok, D. and Zlatkis, A ., Separation of m alto-oligosaccharides by high-perform ance thin-layer chrom atography at m oderate tem perature, J. Chrom atogr., 142,449, 1977. 3. Covacevich, M. T. and Richards, G. N., Continuous quantitative thin-layer chrom atography of oligosaccharides, J. Chrom a­ togr., 129,420, 1976. 4. Collins, F. W. and C handorkar, K. R., Thin-layer chrom atography of fructo-oligosaccharides, J. Chrom atogr., 56, 163, 1971. 5. Liu, Y. K. and Luh, B. S., Preparation and thin-layer chrom atography of oligogalacturonic acids, J. Chrom atogr., 151, 39, 1978.

C a r b o h y d r a te s

151

CRC Handbook of Chromatography

152

Table TLC 6 GLYCOSAMINOGLYCANS, GLYCOPEPTIDES, AND GLYCOLIPIDS Layer Solvent Detection Technique Reference

LI SI D1 T1

LI SI D1 T2

L2 S2 D2 T3

1

1

2

Rf x 100

C om pound

L3 S3 D3 T4 3

L4 S4 D4 T5 4

R cas° ^ 100

Ribbx 100

Glycosaminoglycans C hondroitin 6 -sulfate Chondroitin 4-sulfate M„ 13,000 M„ 31,000 M„ 61,000 Derm atan sulfate Heparin N-Acetylheparan sulfate Keratan sulfate

32 31

35 36





















60 80







100

15 14

23 16



21

22



72 55 —

43

75



— — — — — —

— —

Glycopeptides Glucosylgalactosylhydroxylysine Ovalbumin G P IV, M„ 1,500 Ovalbumin GP III, M„ 1,900 Transferrin G P, M„ 2,600 Fetuin GP, M„ 3,300

































50 56 58 65









68

73 63 54 38 29

— — — — —

— — — — —

16 6 1

— — —

— —

Glycolipids Brain gangliosides G m3 G w2 G mi G d3 Goia G D16 Gn G qi

— — — — — — — —

— — — — — — — —

Note: G P = glycopeptide “ b

R 0.5 = mobility relative to chondroitin 4-sulfate, M„ 61,000, calculated from published data R£fc = mobility relative to a-lactalbum in CNBr peptide I.

Layer

Solvent

Detection

LI L2 L3 L4 SI 52 53 54 D1

= 0.10 mm M N-Polygram Cellulose 400 (Machery-Nagel). = precoated silica gel high-perform ance plates (Merck). = 0.5 mm Sephadex G-200, Superfine (Pharm acia). = 1.25 mm Sephadex G-50, Superfine (Pharm acia). = 0.04 M am m onium form ate-m ethanol (9:11), containing 1.1 m M com plexone-III. = methyl acetate-2-propanol-0.033 MKC1 (9:6:4). = 2 M N aC l. = 6 M guanidine hydrochloride-0.1 M phosphate buffer (pH 7.0). = Azure A dye (50 mg) in acetone-m ethanol-2% acetic acid (20:60:20); after treatm ent for 2—3 min, plate washed with l°7o acetic acid. D2 = plate sprayed with orcinol-hydrochloric acid. D3 = plate covered with filter paper; paper dried and stained with 0.1 % toluidine blue in 2% acetic acid for 30 min, then washed with 2°7o acetic acid.

Carbohydrates

153

Table TLC 6 (continued) GLYCOSAMINOGLYCANS, GLYCOPEPTIDES, AND GLYCOLIPIDS

Technique

D4 = paper replica of plate stained with fluorescamine in acetone (2 m g/m I), heated at 60°C for 5 min and examined under U.V. T1 = plates impregnated with solution containing lead acetate (3 g) in 80 m i 96% ethanol, with 20 m i 40% form alin + 1 m i glacial acetic acid added; development in presaturated cham ber for 3 hr. T2 = as T 1; development for 24 hr. T3 = gangliosides dissolved in chloroform -m ethanol (2:1) at concentrations 0.1 m g/m i sialic acid each; 1 \il applied in 5 mm streak; development for 30 min. T4 = plate inclined at 15°, with filter paper bridge to solvent; solvent allowed to flow for at least 24 hr before application of samples (1—3 \it, containing 50— 100 /ug), absorbed from filter paper strips laid on gel surface; development continued until Blue Dextran 2 000 m arker migrated 10 cm (2—3 hr). T5 = plate inclined at about 8 ° in Pharm acia TLG apparatus; samples dissolved in solvent at least 2 hr before application (in volume 5 y.1, containing 75— 100 y.g)\ cytochrome c as m arker; development for 16— 18 hr. REFERENCES

1. Havass, Z. and Szabo, L ., Thin-layer chrom atographic separation of glycosaminoglycans, J. Chromatogr., 71, 580—584, 1972. 2. Z anetta, J.-P ., Vitiello, F ., and Robert, J., Thin-layer chrom atography of gangliosides, J. Chromatogr., 137, 481, 1977. 3. Taniguchi, N ., Thin-layer gel filtration of urinary mucopolysaccharides in H unter’s syndrome and in norm al individuals, Clin. Chim. Acta, 30, 801, 1970. 4. Hung, C .-H ., Strickland, D. K., and Hudson, B. G ., Estim ation of molecular weights of peptides and glycopeptides by thin-layer gel filtration, Anal. Biochem ., 80, 91, 1977.

Carbohydrates

155

S e c tio n I.V

ELECTRO PHO RETIC DATA T h i s s e c t i o n is i n c l u d e d b e c a u s e p a p e r e l e c t r o p h o r e s i s s e r v e s a s a u s e f u l c o m p l e m e n t t o p a p e r a n d t h i n - l a y e r c h r o m a t o g r a p h y in e x a m i n a t i o n o f s u g a r s a n d d e r i v a t i v e s . C o m p r e h e n s i v e d a t a o b t a i n e d b y t h i s m e t h o d a r e p r e s e n t e d in T a b l e s E L 1 t o E L 3. T h e a p p lic a tio n to c a r b o h y d r a te s o f o th e r e le c tro p h o r e tic te c h n iq u e s (g lass-fib er, c e l l u l o s e a c e t a t e a n d g e l e l e c t r o p h o r e s i s ) is b r i e f l y r e v i e w e d a t t h e e n d o f th i s s e c t i o n (T a b le s E L 4 to E L 6 a n d a c c o m p a n y i n g text).

PAPER ELECTROPHORESIS T a b le s E L 1 to E L 3 h a v e b een c o m p ile d m a in ly fr o m d a ta o rig in ally p u b lish e d by D r . H . W e i g e l ( R o y a l H o l l o w a y C o l l e g e , U n i v e r s i t y o f L o n d o n ) in

bohydrate Chemistry,

Advances in Car­

18, 6 1 — 9 7 , 1 9 6 3 . T h e s e d a t a a r e r e p r i n t e d b y p e r m i s s i o n o f D r .

W eig el a n d A c a d e m ic P re s s . T h e li s t o f p a p e r s , e l e c t r o l y t e s , t e c h n i q u e s , a n d r e f e r e n c e s a p p l i c a b l e t o a ll t h r e e t a b l e s w ill b e f o u n d a f t e r T a b l e E L 3.

156

CRC Handbook o f Chromatography Table EL 1 PAPER ELECTROPHORESIS OF SUGARS”

Paper Electrolyte Technique Reference Com pound Aldoses D-Erythrose L-Threose L-Arabinose D-Xylose D-Lyxose D-Ribose D-Glucose D-Galactose D-Mannose D-Allose D-Altrose D-Gulose L-ldose D-Talose L-Rhamnose L-Fucose 2 -Acetam ido- 2 -deoxyD-glucose 2 -Acetamido- 2 -deoxyD-galactose D -g/yceroL -g/ucoH eptose D-glycero-D-galactoHeptose D-glycero-L-galactoHeptose D-glyceroL-m annoHeptose D- glycero- D-aifo-Heptose D-g/ycero-D-gu/o-Heptose D-glycero D-id o H eptose Ketoses D- eryfhroPentulose D-f/jreo-Pentulose D-Fructose L-Sorbose D-Tagatose D-Psicose L-ga/acto-Heptulose D-g/uco-Heptulose D-m anno-Heptulose Uronic acids D-Glucuronic acid D-Galacturonic acid D-M annuronic acid L-Guluronic acid

PI El T1

P2 E2 T2

1

2

P3 E3 T3 3

P4 E4 T4 4

P4 E5 T5 5

P2 E6 T6

P2 E7 T2

2

2

M*‘ x 100

M cbx 100

— — 96

— — 91

100

101

— 77

71 75

100

100

100

93 72 — — — — — 52 89 23

93 69 83 97 82

— — — — —

— — 150 140 190



210

— — 240 180 230 470

100

100

130 140 180 — — — — 130 —

180

87 49 83' —

— — — — — — — — — —

10

— 580 — — — 50 — —

35









104









110

P5 E8 T7 6,7

P5 E9 T7 7,8

M / x 100

— — 7 8

0

30

110

104'

100

100

40

20

16 28 35 75 77 53 115 119 32

6

0

0

10

0

0

41 33

90 60

110 '

— — 30 17 42

5 0

0

90'

0

110 '





10

0

31 42

110

110

— —



28

— 70 60'

22

6

0

0























23

20

0











40

94'













40

21















80

100 '















90

48















110

98'















100

73

— — 90 95

90 75 89 97 95 76

— — —

— — 210 200



240

209 194 75 73 103 188

73 41



— — 930 850 860

— — 50 30 105

— — 25' 20 ' 110 '

— —

102

110

7

22

16 65 91'

110 '































100



87













40



120



89 —





















126 104





















88

















75













157

Carbohydrates Table EL 1(continued) PAPER ELECTROPHORESIS OF SUGARS'* Paper Electrolyte Technique Reference

PI El T1 1

C om pound

P4 E4 T4 4

P3 E3 T3 3

P4 E5 T5 5

24 69 69 32 23 69 75 38 80 —

18 28

P2 E6 T6 2

x

Mobx 100

Oligosaccharides Sophorose Nigerose Lam inaribiose Maltose Cellobiose Isomaltose Gentiobiose Lactose Melibiose Turanose Sucrose Raffinose u Explanation of terms and h Mc = mobility relative to 1 M w = mobility relative to d M v = mobility relative to ' Streaks.

P2 E2 T2 2













30 22

— —









37 77 64 16 26



130 110 40 30 90 100 70 140

— — —













7 10

15 15













— —

10 40







— —

P5 E9 T7 7,8

M," x 100





P5 E8 T7 6,7

100









P2 E7 T2 2

24 32 30 14 25

13 10 7 4 7

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 10 0 —



references at end of Table EL 3. D-glucose. D-ribose. D-glucitol.

Table EL 2 PAPER ELECTROPHORESIS OF ALDITOLS, CYCLITOLS, AND REDUCED D I S A C C H A R I D E S '* Paper Electrolyte Technique Reference

Pi El T1 1

P4 E4 T4 4

Mo x 100

Com pound Alditols Erythritol L-Threitol D-Arabinitol L-Arabinitol Xylitol Ribitol D-Glucitol Galactitol D-Mannitol Allitol D-Altritol L-Iditol D-Talitol L-Rhamnitol D-glycero-D-gfucoH eptitol D -glyceroL-gluco-Hept\to\ D-glycero-D-gafacfo-Heptitol D-glycero-D-altro- Heptitol

P2 E2 T2 2

75

M „b x 100

75 75 87

100









79 85 83 97 91 90

180 170 120 190 210 190













81 89









90

— —

98 90



92 100 —

88 95 98 92

P4 E5 T5 5

— —







10 30 —

60 90 30 130 100 100 — —

140 —

80 —













P2 E6 T6 2, 7

P2 E7 T2 2

x 100

Mu x 100

3 11 14

100 50 110

25 4 47 32 23 9

110 110 100 100 100 94 99

53 96 124 —

155 76 161 145 130 92 —

173 138 —

171 176 140 144

P5 E9 T7 7,8

P5 E8 T7 6 ,7 ,9





57 17 —

53c 59c 51c 27



90c 24 104 —



104 103 100 100 100 97c 97c 100

























158

CRC Handbook of Chromatography

Table EL 2 (continued) PAPER ELECTROPHORESIS OF ALDITOLS, CYCLITOLS, AND REDUCED D IS A C C H A R ID E S ” Paper Electrolyte Technique Reference

PI El T1

P4 E4 T4 4

Mc x 100

Com pound

“ h c d

2

1

D-glycero-D- talo-Heptitol D-g7yceroD- ido- Heptitol (meso)-glycero-ido- Heptitol (meso)-glycero-gulo- Heptitol (meso)-glycero- a77o-Heptitol Cyclitols a//o-Inositol cis- Inositol epAInositol (+ )-Inositol (-)-Inositol muco- Inositol myo- Inositol n e oinositol scy77o-Inositol Reduced disaccharides D-glucitol 2- O-a-D-gluco-pyranosyl2- O-ft-D-gluco-pyranosyl3-O-a-D-gluco-pyranosyl3- O-ft-D-gluco-pyranosyl4- O-a-D-gluco-pyranosyl4- 0-/3-D-gluco-pyranosylD-glucitol 6 - O-a-D-gluco-pyranosyl6 - 0-/3-D-gluco-pyranosyl6 - O-a-D-galacto-pyranosyl4- 0-/3-D-galacto-pyranosylD-mannitol 2- O-a-D-manno-pyranosyl3- O-a-D-manno-pyranosyl2- O-a-D-gluco-pyranosylL-gulitol 1- O-a-D-galacto-pyranosyl3- 0-/LD-galacto-pyranosyl1- O-a-D-gluco-pyranosyl1- O-ft-D-gluco-pyranosyl2- O-a-D-gluco-pyranosyl3- O-a-D-gluco-pyranosyl3- O-ft-D-gluco-pyranosyl-

P2 E2 T2

— — — —

88 —

73 63 63 96 53 —

93 85 78 85 95

P4 E5 T5 5 M m6 x 100







100









50 40 29







180

180

100

0

2









70 —

20

0 — —



23 36 16 43 7













11





62 116 74c





20

41 75c 64

40 110 0 —





d

0

0

76 73











120





69 70





















120

110 —





















150 150





0

0





0

0









40 40





















80 80 80 40

60

0 —





0

20



39

100 0

0



10

8 —





P5 E9 T7 7, 8

P5 E8 T7 6 , 7, 9 M 5 x 100

M r X 100 34 71c 79c 12c