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JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 27 instrumental liquid chromatograph y a practical manual on high-performance liquid chromatographic methods second, completely revised edition This Page Intentionally Left Blank JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 27 instrurnental liquid chromatography a practical manual on high-performance liquid chromatographic methods second, completely revised edition NA. Parris E.I. du Pont de Nemours & Company, Biomedical Products Department, Research and Development Division, Experimental Station Laboratory, Wilmington, D E 19898, U.S.A. ELSEVl ER Amsterdam - Oxford - New York - Tokyo 1984 ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 21 1,1000 AE Amsterdam, The Netherlands Distributors for the United States and Ceneda: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 52, Vanderbilt Avenue New York, NY 10017 First edition, first impression second impression Second, completely revised edition, first impression second impression 1976 1979 1984 1985 ISBN 044442061 4 (Vol. 27) ISBN 044441 61 6-1 (Series) 0 Elsevier Science Publishers B.V., 1984 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V./Science & Technology Division, P.O. Box 330,1000 AH Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including phocopying outside of the USA, should be referred to the publisher. Printed in The Netherlands Contents ............................... ix Preface to the First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi ................................... xiii Journal of Chromatography Library Preface to the Second Edition FUNDAMENTALS AND INSTRUMENTATION 1 . Introduction and historical background . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................... 3 6 2. Basic principles and terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General resolution equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calculation of optimum column length . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 17 17 21 3 . The chromatographic support and column . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of band broadening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of particle size in LC columns . . . . . . . . . . . . . . . . . . . . . . . . . . . Porous layer supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Totally porous (microparticulate) supports . . . . . . . . . . . . . . . . . . . . . . Dependence of column efficiency on operating conditions . . . . . . . . . . . Columns for high-pressure LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column efficiency and internal diameter . . . . . . . . . . . . . . . . . . . . . . . Methods of packing chromatographic columns . . . . . . . . . . . . . . . . . . . Microbore columns . . . . . . . . . .