Thin Layer..Chromatography

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Thin-layer ChromatographyA Modern Practical ApproachRSC Chromatography MonographsSeries Editor: R.M. Smith, Loughborough University of Technology, UKAdvisory Panel: J.C. Berridge, Sandwich, UK, G.B. Cox, Indianapolis, USA,I.S. Lurie, Virginia, USA, P.J. Schoenmakers, Eindhoven, The Netherlands,C.F. Simpson, London, UK, G.G. Wallace, Wollongong, AustraliaOther titles in this series:Applications of Solid Phase MicroextractionEdited by J Pawliszyn, University of Waterloo, Waterloo, Ontario, CanadaCapillary ElectrochromatographyEdited by K D Bartle and P Myers, University of Leeds, UKChromatographic Integration Methods, Second EditionN Dyson, Dyson Instruments, UKCyclodextrins in ChromatographyBy T Cserhati and E Forgacs, Hungarian Academy of Sciences, Budapest, HungaryElectrochemical Detection in the HPLC of Drugs and PoisonsR J Flanagan, Guys and St Thomas NHS Foundation Trust, London, UK, D Perrett,Queen Marys School of Medicine and Dentistry, London, UK and R Whelpton,University of London, London, UKHPLC: A Practical GuideT Hanai, Health Research Foundation, Kyoto, JapanHyphenated Techniques in Speciation AnalysisEdited by J Szpunar and R Lobinski, CNRS, Pau, FrancePacked Column SFCT A Berger, Hewlett Packard, Wilmington, Delaware, USASeparation of Fullerenes by Liquid ChromatographyEdited by Kiyokatsu Jinno, Toyohashi University of Technology, JapanValidation of Chromatography Data Systems: Meeting Business and RegulatoryRequirementsR D McDowall, McDowall Consulting, Bromley, Kent, UKHow to obtain future titles on publication:A standing order plan is available for this series. A standing order will bringdelivery of each new volume upon publication. For further information pleasecontact:Sales and Customer Care, Royal Society of Chemistry, Thomas Graham House,Science Park, Milton Road, Cambridge, CB4 0WFTelephone: 144(0) 1223 420066, Fax: 144(0) 1223426017, Email: sales@rsc.orgThin-layer ChromatographyA Modern Practical ApproachPeter E. WallVWR International Ltd., Poole, DorsetISBN 0-85404-535-XA catalogue record for this book is available from the British Library# The Royal Society of Chemistry 2005All rights reservedApart from fair dealing for the purposes of research for non-commercial purposes or for private study,criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyrightand Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, inany form or by any means, without the prior permission in writing of The Royal Society of Chemistry, orin the case of reproduction in accordance with the terms of licences issued by the Copyright LicensingAgency in the UK, or in accordance with the terms of the licences issued by the appropriateReproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the termsstated here should be sent to The Royal Society of Chemistry at the address printed on this page.Published by The Royal Society of Chemistry,Thomas Graham House, Science Park, Milton Road,Cambridge CB4 0WF, UKRegistered Charity Number 207890For further information see our web site at www.rsc.orgTypeset by Alden Bookset, Northampton, UKPrinted by Athenaeum Press Ltd, Gateshead, Tyne and Wear, UKPrefaceThin-layer chromatography (TLC) is without doubt one of the most versatile andwidely used separation methods in chromatography. Commercially, many sorbentson a variety of backings are now available. Most stages of the technique are nowautomated (can now be operated instrumentally) and modern HPTLC (Highperformance thin-layer chromatography) allows the handling of a large number ofsamples in one chromatographic run. Speed of separation (development time), highsensitivity and good reproducibility all result from the higher quality ofchromatographic layers and the continual improvement in instrumentation. Inaddition TLC has remained relatively inexpensive and one can easily see why it isstill popular today. It has found a use in a wide range of application areas as theconcept of TLC is so simple and samples usually require only minimal pre-treatment. It is often thought of only in terms of its use in pharmaceutical analysisand production and in clinical analysis, but many standard methods in industrialchemistry, environmental toxicology, food chemistry, water, inorganic andpesticide analysis, dye purity, cosmetics, plant materials, and herbal analysis relyupon TLC as the preferred approach. In its simplest form, TLC costs little, but evenincluding the more sophisticated instrumentation, it still remains less expensive persample analysis than, for example HPLC. Thin-layer chromatography continues tobe an active technique in research with about 500700 publications appearing eachyear during the 1980s and 1990s.It is the purpose of this book to describe the advances made, particularly in thelast two to three decades, which have revolutionised TLC and transformed it intoa modern instrumental technique. All aspects of TLC have been affected, fromthe sorbent layer technology, through the spotting devices and developingequipment, to the nal detection and quantication. Computers too, now play animportant role in the control of equipment and in the computation of the vastamount of data obtained from scans of the developed TLC layer. This has resultedin the ability to store and retrieve images of chromatograms and physical data onactual separation results and conditions for future use. Instrumental planarchromatography is now capable of handling samples with minimal pre-treatment,detecting components at low nanogram sensitivities and with relative standarddeviations of about 1%. It is the opinion of the author that these developmentsvdemonstrate that the previous image of low sensitivity, poor resolution andreproducibility can be discarded and that TLC is now truly a modern contemporaryof HPLC and GC. Modern TLC has become a powerful, reliable and cost efcientmethod for qualitative and quantitative analysis.The chapters in this book have been designed in such a way that the readerfollows each step of the planar chromatographic process in logical order. Hence thechoice of sorbent is followed by preparation of sample for application and themethods of application. The subject of chromatogram development logically comesnext with detection, quantication and/or video imaging usually being the nalsteps. However, sometimes further instrumental analysis is necessary, hence thenal chapter on hyphenated techniques. Any theory or necessary mathematicalequations or expressions are introduced when required within the text of eachsubject. As the objective of this book is to provide a publication or manual that canbe used by the practising chromatographer, the depth of theory reects only whatis required to explain why certain practical steps are taken. It is the intention ofthe author that this book will be of practical value and use to those who arecontemplating using TLC for the rst time, and also to those who have been planarchromatographers for some time. With these points in mind, the practical examplesof chromatographic separations reect the eld-tested procedures available. Thebook concentrates on the basic steps involved in TLC, providing practical guidanceto achieve superior separations on a TLC/HPTLC sorbent layer. For this reasonthere are some techniques that are not covered, such as sorbent coated quartz rodsand thin-layer radiochromatography. However, the basic principles for optimisedseparations described in the various chapters will still apply in these relatedtechniques. Recommendations made throughout the text to obtain acceptableand often high quality results are made on the basis of many years of practicalexperience in planar chromatography by the author.Numerous commercial products are referred to in this book as would benecessary with any publication that discusses instrumental TLC/HPTLC. Thereferences made are based on the experiences of the author with these products,and are denitely not meant to imply that they are superior to comparable productsfrom other manufacturers.vi PrefaceContentsChapter 1 Introduction and History 11 Introduction to Thin-layer Chromatography 12 History of TLC 23 References 4Chapter 2 Sorbents and TLC Layers 61 Sorbent Selection 61.1 Introduction 61.2 Silica Based Sorbents 71.2.1 Silica Gel 71.2.2 Silica Gel Bonded Phases 121.3 Non-silica Sorbents 351.3.1 Cellulose 351.3.2 Cellulose Bonded Phases 371.3.3 Aluminium Oxide 371.3.4 Kieselguhr 381.3.5 Polyamide 391.3.6 Miscellaneous Stationary Phases 391.3.7 Mixed Stationary Phases 401.3.8 Dual Phases 402 Preparation of TLC Sheets and Plates 402.1