Handbook of Thin-Layer ChromatographyThird Edition, Revised and Expandededited by

Joseph Sherma Bernard FriedLafayette College Easton, Pennsylvania, U.S.A.

MARCEL DEKKER, INC.

NEW YORK BASEL

Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-0895-4 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

CHROMATOGRAPfflC SCIENCE SERIESA Series of Textbooks and Reference Books Editor: JACK CAZES

1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings 2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz 3. Principles of Adsorption Chromatography: The Separation of Nonionic Organic Compounds, Lloyd R. Snyder 4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and Gerhard Klein 5. Quantitative Analysis by Gas Chromatography, Josef Novak 6. High-Speed Liquid Chromatogrsjphy, Peter M. Rajcsanyi and Elisabeth Rajcsanyi 7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz, Michael J. Gudzinowicz, and Horace F. Martin 8. Liquid Chromatography of Polymers and Related Materials, Jack Gazes 9. GLC and HPLC Determination of Therapeutic Agents (in three parts), Part 1 edited by Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3 edited by Kiyoshi Tsuji 10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk 11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw 12. Biological/Biomedical Applications of Liquid Chromatography II, edited by Gerald L. Hawk 13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes and Xavier Delamare 14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice, John A. Perry 15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings 16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky 17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried and Joseph Sherma 18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald L. Hawk 19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack Cazes 20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk 21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers, G. J. Moody and J. D. R. Thomas 22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin 23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey 24. High-Performance Liquid Chromatography in Forensic Chemistry, edited by Ira S. Lurie and John D. Witiwer, Jr. 25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca 26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock and James T. Sparrow 27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins, Herbert Schott 28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R. Brown 29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy 30. Modern Chromatographic Analysis of the Vitamins, edited by Andre P. De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter 31. Ion-Pair Chromatography, edited by Milton T. W. Heam 32. Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, edited by Steven H. Y. Wong

33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr and Klaus Pommerening 34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull 35. Thin-Layer Chromatography: Techniques and Applications. Second Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 36. Quantitative Thin-Layer Chromatography and Its Industrial Applications, edited by Laszlo R. Treiber 37. Ion Chromatography, edited by James G. Tarter 38. Chromatographic Theory and Basic Principles, edited by Jan Ake Jonsson 39. Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca 40. Chromatographic Chiral Separations, edited by Morris Ziefand Laura J. Crane 41. Quantitative Analysis by Gas Chromatography, Second Edition, Revised and Expanded, Josef Novak 42. Flow Perturbation Gas Chromatography, N. A. Katsanos 43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Nakanishi, and Ryuichi Matsuno 44. Countercurrent Chromatography: Theory and Practice, edited by N. Bhushan Mandava and Yoichiro Ito 45. Microbore Column Chromatography: A Unified Approach to Chromatography, edi ted by Frank J. Yang 46. Preparative-Scale Chromatography, edited by Eli Grushka 47. Packings and Stationary Phases in Chromatographic Techniques, edited by Klaus K. Linger 48. Detection-Oriented Derivatization Techniques in Liquid Chromatography, edited by Henk Lingeman and Willy J. M. Underberg 49. Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics 50. Multidimensional Chromatography: Techniques and Applications, edited by Neman Cortes 51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M. Gooding and Fred E. Regnier 52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg 53. Chromatographic Analysis of Alkaloids, Milan Pop/, Jan Fahnrich, and Vlastimil Tatar 54. HPLC in Clinical Chemistry, /. N. Papadoyannis 55. Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and Bernard Fried 56. Gas-Liquid-Solid Chromatography, V. G. Berezkin 57. Complexation Chromatography, edited by D. Cagniant 58. Liquid Chromatography-Mass Spectrometry, W. M. A. Niessen and Jan van der Greef 59. Trace Analysis with Microcolumn Liquid Chromatography, Milos Krejcl 60. Modem Chromatographic Analysis of Vitamins: Second Edition, edited by Andre P. De Leenheer, Willy E. Lambert, and Hans J. Nelis 61. Preparative and Production Scale Chromatography, edited by G. Ganetsos and P. E. Barker 62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George 63. Handbook of Affinity Chromatography, edited by Toni Kline 64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman 65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto 66. Thin-Layer Chromatography: Techniques and Applications: Third Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 67. Liquid Chromatography for the Analyst, Raymond P. W. Scott 68. Centrifugal Partition Chromatography, edited by Alain P. Foucault 69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu 70. Techniques and Practice of Chromatography, Raymond P. W. Scott 71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried 72. Liquid Chromatography of Oligomers, Constantin V. Uglea 73. Chromatographic Detectors: Design, Function, and Operation, Raymond P. W. Scott

74. Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised and Expanded, edited by John A. Adamovics 75. Supercritical Fluid Chromatography with Packed Columns: Techniques and Applications, edited by Klaus Anton and Claire Berger 76. Introduction to Analytical Gas Chromatography: Second Edition, Revised and Expanded, Raymond P. W. Scott 77. Chromatographic Analysis of Environmental and Food Toxicants, edited by Takayuki Shibamoto 78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers, and Neil Miller 79. Liquid Chromatography-Mass Spectrometry: Second Edition, Revised and Expanded, Wilfried Niessen 80. Capillary Electrophoresis of Proteins, T7m Wehr, Roberto Rodriguez-Diaz, and Mingde Zhu 81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thiebaut 83. Micellar Liquid Chromatography, Alain Berthod and Celia Garcia-Alvarez-Coque 84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and Expanded, edited by Andre P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer 85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio, and Raymond P. W. Scott 86. Current Practice of Gas Chromatography-Mass Spectrometry, edited by W. M. A. Niessen 87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded, edited by Karen M. Gooding and Fred E. Regnier 88. Scale-Up and Optimization in Preparative Chromatography: Principles and Biopharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan 89. Handbook of Thin-Layer Chromatography: Third Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried

ADDITIONAL VOLUMES IN PREPARATION Chiral Separations by Liquid Chromatography and Related Technologies, Hassan Y. Aboul-Enein and Imran AH

To President Arthur J. Rothkopf and Provost June Schlueter in appreciation of the continuing support of Lafayette College for our research and publication activities as emeritus professors

Preface to the Third Edition

Contributing authors in the third edition of the Handbook of Thin-Layer Chromatography were asked by the editors to cover new advances in their fields and delete old technologies and obsolete information. The authors expanded chapters when necessary to cover topics adequately. The result is chapters that describe the state-of-the-art of each subject, with updated references. The same overall organization of the second edition was adopted. Part I contains chapters on the theory, principles, practice, and instrumentation of thin-layer chromatography (TLC). Part II chapters cover applications of TLC to a variety of compound classes. A subject index, an expanded glossary of important terms, and a list of sources of supplies and equipment are included. Within the two parts of the book, some changes in topics have occurred, and some contributors have been replaced. In Part I, new contributing authors wrote Chapter 3 ("Optimization" by Claudia Cimpoiu), Chapter 4 ("Sorbents and Precoated Layers in Thin-Layer Chromatography" by Fredric M. Rabel), Chapter 5 ("Instrumental Thin-Layer Chromatography" by Eike Reich), and Chapter 12 ("Thin-Layer Radiochromatography" by Istvan Hazai and Imre Klebovich). Automation and robotics were covered in Chapter 14 of the second edition, but a chapter on this topic is not included in this edition because of a lack of sufficient new information. Part II contains chapters on two new compound classes: hydrocarbons (Chapter 19 by Vicente Cebolla and Luis Membrado) and herbals (Chapter 18 by Eike Reich and Anne Blatter). The following are new authors of chapters in Part II: Irena Choma ("Antibiotics," Chapter 15), Mark D. Maloney ("Carbohydrates," Chapter 16), Fumio Watanabe and Emi Miyamoto ("Hydrophilic Vitamins," Chapter 20), Alina Pyka ("Lipophilic Vitamins," Chapter 23), Marija Kastelan-Macan and Sandra Babic ("Pesticides," Chapter 27), Joseph Sherma ("Steroids," Chapter 30), and W. M. Indrasena ("Toxins [Natural]," Chapter 32). No topics were eliminated from Part II. Throughout the book, practical aspects are emphasized in order to help those in university, government, industrial, and independent testing laboratories understand the principles of TLC and apply it to their analyses. This book is a useful reference volume for chemists, biochemists, biologists, laboratory technicians, laboratory managers, medical technologists, biotechnologists, forensic scientists, veterinary toxicologists, pharmaceutical analysts, environmental scientists, and attendees of workshops or short courses on TLC. It is also a useful reference for graduate and undergraduate students in chemistry, biochemistry, biology, and related programs, particularly those in quantitative analysis, instrumental analysis, and separation science. Whenever possible, suggestions by reviewers of the second edition were incorporated in this edition. We would be pleased to receive comments, notification of errors, and suggestions for deletion of topics, new topics, or new authors for the next edition. Joseph Sherma Bernard Fried

Preface to the Second Edition

The second edition of the Handbook of Thin-Layer Chromatography updates and expands the coverage of the field of TLC and HPTLC in the first edition. The same overall organization of the first edition has been maintained: an initial series of chapters on theory, practice, and instrumentation and a second section of chapters concerned with applications to important compound types. The literature has been updated to as recently as 1995 in most chapters. A number of changes have occurred in the topics covered, and several of the chapters have been written by new contributing authors: "Optimization" by Qin-Sun Wang (Chapter 3); "Basic Principles of Optical Quantitation in TLC" by Mirko Prosek and Marko Pukl (Chapter 10); "Thin-Layer Radiochromatography" by Terry Clark and Otto Kelin (Chapter 12); "Natural Pigments" by 0yvind M. Andersen and George W. Francis (Chapter 22); "Pharmaceuticals and Drugs" by Gabor Szepesi and Szabolcs Nyiredy (Chapter 24); "Nucleic Acids and Their Derivatives" by Jacob J. Steinberg, Antonio Cajigas, and Gary W. Oliver, Jr. (Chapter 26); and "Hydrophilic Vitamins" by John C. Linnell (Chapter 30). These changes resulted from either the inability of the original authors to contribute to the second edition or our desire to change the emphasis of coverage of certain topics. The separate chapter on photographic documentation of thin-layer chromatograms in the first edition (Chapter 9) has been eliminated and the subject is now covered in Chapter 8 ("Detection, Identification, and Documentation" by K.-A. Kovar and Gerda E. Morlock). A new chapter titled "Automation and Robotics in Planar Chromatography" by Eric P. R. Postaire, Pascal Delvordre, and Christian Sarbach (Chapter 14) has been added. A chapter on polymers and oligomers was not included in this edition because of a lack of sufficient new information on this topic. Suggestions made by reviewers of the first edition have been incorporated into this revisionfor example, clear line drawings have replaced photographs in some chapters. As in the past, we welcome comments regarding this editionnotification of errors, suggestions for improvements in the topics covered, new topics, or new authors. Joseph Sherma Bernard Fried

VII

Preface to the First Edition

This book has been designed as a practical, comprehensive laboratory handbook on the topic of thinlayer chromatography (TLC). It is divided into two parts, the first of which covers the theories and general practices of TLC (Chapter 1-13), while the second (Chapters 14-31) includes applications based mainly on compound types. The book will be a valuable source of information for scientists with a high degree of expertise in the separation sciences, but because most chapters include considerable introductory and background material, it is also appropriate for the relatively inexperienced chromatographer. Contributors to the book are recognized experts on the topics they have covered and include many of the best-known and most knowledgeable workers in the field of TLC throughout the world. As much as possible, we attempted to adopt a uniform style for each chapter while still allowing authors the latitude to present their topics in what they considered to be the most effective way. Consequently, in the applications chapters (14-31), most authors have included the following sections: introduction, sample preparation, layers and mobile phases, chromatographic techniques, detection, quantification, and detailed experiments. Authors were encouraged to use many figures and tables and to be as practical as possible except for the chapters devoted to theory (2, 3, and 10). The literature covered by most authors includes mainly the period from 1975 to 1989. Some of the more significant older literature has also been covered, but many authors refer to the earlier comprehensive treatises by Stahl and Kirchner for this material. Authors have been selective in their choice of references and present TLC methods that are most suitable for laboratory work. It is important to point out that the Handbook of Thin-Layer Chromatography has a comprehensive, organized plan and, unlike many recent books in the field, is not a random collection of chapters on "advances" or papers from a symposium. An earlier laboratory handbook on TLC was written by Egon Stahl in 1965. We hope that our handbook may have at least a small fraction of the impact in the near future that this classic work had on the development and growth of TLC during the past 25 years. If the book is well accepted and contributors cooperate, we hope to update coverage of all important aspects of TLC with regular later editions. Joseph Sherma Bernard Fried

IX

Contents

Preface to the Third Edition Preface to the Second Edition Preface to the First Edition Contributors Part I: Principles and Practice of Thin-Layer Chromatography 1. 2. 3. 4. 5. 6. 1. 8. 9. 10. Basic TLC Techniques, Materials, and Apparatus Joseph Sherma Theory and Mechanism of Thin-Layer Chromatography Teresa Kowalska, Krzysztof Kaczmarski, and Wojciech Prus Optimization Claudia Cimpoiu Sorbents and Precoated Layers in Thin-Layer Chromatography Fredric M. Rabel Instrumental Thin-Layer Chromatography (Planar Chromatography) Eike Reich Gradient Development in Thin-Layer Chromatography Wladystaw Golkiewicz Overpressured Layer Chromatography Emil Mincsovics, Katalin Ferenczi-Fodor, and Ernd Tyihdk Detection, Identification, and Documentation Gerda Morlock and Karl-Arthur Kovar Thin-Layer Chromatography Coupled with Mass Spectrometry Kenneth L. Busch Basic Principles of Optical Quantification in TLC Mirko Prosek and Irena Vovk

v vii ix xv

1 47 81 99 135 153 175 207 239 277

XI

xii 11. Preparative Layer Chromatography Szabolcs Nyiredy Thin-Layer Radiochromatography Istvdn Hazai and Imre Klebovich Applications of Flame lonization Detectors in Thin-Layer Chromatography Kumar D. Mukherjee

CONTENTS 307

12.

339

13.

361

Part II: Applications of Thin-Layer Chromatography 14. Amino Acids and Their Derivatives Ravi Bhushan and J. Martens Antibiotics Irena Choma Carbohydrates Mark D. Maloney Enantiomer Separations Kurt Gunther and Klaus Moller Herbal Drugs, Herbal Drug Preparations, and Herbal Medicinal Products Eike Reich and Anne Blatter Hydrocarbons Vicente L. Cebolla and Luis Membrado Giner Hydrophilic Vitamins Fumio Watanabe and Emi Miyamoto Inorganic and Organometallic Compounds AH Mohammad Lipids Bernard Fried Lipophilic Vitamins A Una Pyka Natural Pigments George W. Francis and 0yvind M. Andersen Nucleic Acids and Their Derivatives Jacob J. Steinberg Peptides and Proteins Ravi Bhushan and J. Martens Pesticides Marija Kastelan-Macan and Sandra Babic 373

15.

417

16.

445

17.

471

18.

535

19.

565

20.

589

21.

607

22.

635

23.

671

24.

697

25.

733

26.

749

27.

767

CONTENTS 28. 29. 30. 31. 32. Pharmaceuticals and Drugs Szabolcs Nyiredy, Katalin Ferenczi-Fodor, Zoltdn Vegh, and Gdbor Szepesi Phenols, Aromatic Carboxylic Acids, and Indoles John H. P. Tyman Steroids Joseph Shernia Synthetic Dyes Vinod K. Gupta Toxins (Natural) W. M. Indrasena

xiii 807 865 913 935 969

Glossary Directory of Manufacturers and Suppliers of Plates, Equipment, and Instruments for Thin-Layer Chromatography Index

987 995 997

Contributors

0yvind M. Andersen Department of Chemistry, University of Bergen, Bergen, Norway Sandra Babic Croatia Ravi Bhushan India Anne Blatter Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb,

Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee, CAMAG-Laboratory, Muttenz, Switzerland National Science Foundation, Arlington, Virginia, U.S.A. Institute de Carboquimica, CSIC, Zaragoza, Spain

Kenneth L. Busch Vicente L. Cebolla Irena Choma

Marie Curie Sklodovska University, Lublin, Poland

Claudia Cimpoiu Faculty of Chemistry and Chemical Engineering, "Babes-Bolyai" University, Cluj-Napoca, Romania Katalin Ferenczi-Fodor George W. Francis Bernard Fried Chemical Works of Gedeon Richter Ltd., Budapest, Hungary

Department of Chemistry, University of Bergen, Bergen, Norway

Department of Biology, Lafayette College, Easton, Pennsylvania, U.S.A. Department of Inorganic and Analytical Chemistry, Medical University,

Wladyslaw Golkiewicz Lublin, Poland Kurt Giinther Vinod K. Gupta India

Industriepark Wolfgang GmbH, Hanau, Germany Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee,

Istvan Hazai Department of Pharmacokinetics and Metabolism, IVAX Drug Research Institute Ltd., Budapest, Hungary W. M. Indrasena Ocean Nutrition Canada, Halifax, Nova Scotia, CanadaXV

xvi Krzysztof Kaczmarski* Rzeszow, Poland Marija Kastelan-Macan Zagreb, Zagreb, Croatia Imre Klebovich Hungary

CONTRIBUTORS Department of Chemistry, Rzeszow University of Technology,

Faculty of Chemical Engineering and Technology, University of

Department of Pharmacokinetics, EGIS Pharmaceuticals Co. Ltd., Budapest,

Karl-Arthur Kovar Teresa Kowalska Mark D. Maloney J. Martens

Pharmaceutical Institute, University of Tubingen Tubingen, Germany Institute of Chemistry, Silesian University, Katowice, Poland Biology Department, Spelman College, Atlanta, Georgia, U.S.A.

FB-Chemie, Universitat Oldenburg, Oldenburg, Germany Institute de Carboquimica, CSIC, Zaragoza, Spain

Luis Membrado Giner Emil Mincsovics Emi Miyamoto

OPLC-NIT Ltd., Budapest, Hungary Department of Health Science, Kochi Women's University, Kochi, Japan

Ali Mohammad Department of Applied Chemistry, Zakir Husain College of Engineering and Technology, Aligarh Muslim University, Aligarh, India Klaus Moller Gerda Morlock MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany Scientific Consultant, Stuttgart, Germany

Kumar D. Mukherjee Institute for Lipid Research, Federal Centre for Cereal, Potato and Lipid Research, Miinster, Germany Szabolcs Nyiredy Mirko Prosek Slovenia Research Institute for Medicinal Plants, Budakalasz, Hungary

Laboratory for Food Chemistry, National Institute of Chemistry, Ljubljana,

Wojciech Prus Textile Engineering and Environmental Protection, University of Technology and the Arts, Bielsko-Biala, Poland Alina Pyka Faculty of Pharmacy, Silesian Academy of Medicine, Sosnowiec, Poland EM Science, Gibbstown, New Jersey, U.S.A.

Fredric M. Rabel Eike Reich

CAMAG-Laboratory, Muttenz, Switzerland Department of Chemistry, Lafayette College, Easton, Pennsylvania, U.S.A. Ocean Nutrition Canada, Halifax, Nova Scotia, Canada.

Joseph Sherma ^Current affiliation:

CONTRIBUTORS

xvii

Jacob J. Steinberg Department of Pathology, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, U.S.A. Gabor Szepesi Qualintel Ltd., Budapest, Hungary

Erno Tyihak Department of Plant Pathophysiology, Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary John H. P. Tyman England Zoltan Vegh Centre for Environmental Research, Brunei University, Uxbridge, Middlesex,

Chemical Works of Gedeon Richter Ltd., Budapest, Hungary

Irena Vovk Laboratory for Food Chemistry, National Institute of Chemistry, Ljubljana, Slovenia Fumio Watanabe Department of Health Science, Kochi Women's University, Kochi, Japan

Glossary

Absorption Retention of a solute by partitioning into a liquid or liquidlike stationary phase coated on or bonded to a surface. Accuracy The agreement between an experimental result (a single measurement or the mean of several replicate measurements) and the true or theoretical value. Activation The process of heating an adsorbent layer to drive off moisture and convert it to its most attractive or retentive state. Activity The relative strength of the surface of a sorbent such as silica gel or alumina in adsorption chromatography. Activity is reduced by adding water or another polar modifier that is attracted by hydrogen bonding to the active sites. Activity grades (Brockmann activity grades) A standard grading system for the activity (adsorptivity) of alumina based on deactivation with water. Grade I is anhydrous alumina and has the highest activity. Grades II, III, IV, and V contain 3%, 6%, 10%, and 15% (by weight) water, respectively. Additive A substance added to the mobile phase to improve solute separation or detection. Adsorption The attraction between the surface atoms of a solid and an external molecule by intermolecular forces such as hydrogen bonds, London forces, electrostatic forces, and charge transfer forces. The adsorbent is the stationary phase for adsorption TLC. Alumina layer An aluminum oxide layer that is available with basic, neutral, or acid modifications and is used in normal-phase adsorption TLC. Amino layer A propylamino layer used in normal bonded phase TLC or as a weak anion exchanger. Analyte A solute that is to be identified or, more often, quantitatively determined by thin-layer chromatography or other method. Analytical TLC Thin-layer chromatography performed on 100-250 /mi layers for the purpose of separation, identification, or quantification of substances. Anion exchange The mode of TLC that uses a layer with a structure, such as a bonded amino group, that can separate anions. Anticircular TLC Development of a layer from an outer circle of initial zones toward the center. Argentation TLC Thin-layer chromatography using a layer, usually silica gel, impregnated with silver nitrate for the purpose of improving the separation of certain compounds. Ascending development The usual mode of thin-layer chromatography development in which the mobile phase moves upward by capillary action of the sorbent, as opposed to circular, descending, or horizontal development. Azeotrope (azeotropic mixture) A liquid mixture of two or more substances that behaves like a single substance in that the vapor produced by partial evaporation of the liquid has the same composition as the liquid. Band Chromatographic zone, usually in the shape of a horizontal line rather than a round spot.987

Copyright 2003 by Taylor & Francis Group LLC

988

GLOSSARY

Binary mobile phase A mobile phase with two components such as solvents, acids, bases, or buffers. Binder Any chemical added to a sorbent to improve the stability or hardness of the layer. Bonded phase A stationary phase chemically bonded to (as opposed to mechanically deposited on) a support material. Capacity factor (') A measure of sample retention by a layer: k' = (1 - Rf/Rf). Cation exchange The mode of TLC that uses a layer with a structure, such as a sulfonic acid functional group, that can separate cations. Centrifugal layer chromatography Analytical or preparative chromatography in which solvent is driven through the layer by centrifugal force in circular, anticircular, or linear modes. Chamber The tank, jar, or vessel of other type in which thin-layer chromatographic development is carried out. Chamber saturation Equilibration of the chamber or tank with mobile phase vapor before the plate is developed. Chiral layers Layers that can separate enantiomeric mixtures. Chlorosilane A chemical reagent used to prepare siloxane bonded phases such as C-18 used in bonded-phase TLC. Chromatogram The series of zones on or in the layer after development, or the densitometric scan of the zones. Chromatographic solvent Solvent or mixture of solvents used as the mobile phase. Chromatographic system The combination of the solvent, sorbent, and the sample mixture. The interactions of the Chromatographic system determine the selectivity of the separation. Chromatography A method of analysis in which the flow of mobile phase promotes the separation of substances by differential migration from a narrow initial zone in a porous, sorptive, stationary medium. Chromatostrips An early term used for narrow thin-layer plates. Chromogenic A reagent or reaction causing a solute to become colored. Cochromatography Development of a mixture prepared by adding a known standard to a sample thought to consist of or contain that substance. Formation of two zones from the mixture indicates the nonequivalence of the standard and sample, whereas the inability to separate the mixture is one piece of evidence for confirmation of identity. Column chromatography Chromatography carried out by passing a liquid (or gaseous) mobile phase through a stationary phase packed in a column (see Open-column chromatography and HPLC). Confirmation An ancillary qualitative analysis that proves the identity of a constituent in a sample with greater certainty than the original TLC analysis, usually by means of some type of on-line or off-line spectrometry. Quantitative results can also be confirmed by reanalysis of the sample using an independent method. Conjugate The combined form in which drugs or pesticides can be found in plant or human samples, e.g., glucuronide or sulfate. Conjugates usually require hydrolysis by acid, base, or enzymes to free the analyte prior to TLC analysis. Continuous development Development of a layer for a greater distance than the actual length; also called overrun development. Deactivation Adding moisture to an adsorbent layer to lower its retention of polar solutes. Demixing Separation of mobile phase components during development, leading to the formation of one or more solvent fronts along the layer. Densitometry Quantification of a zone directly on the layer with an instrument that measures color, absorption of ultraviolet light, fluorescence, or radioactivity. Deproteinization A sample preparation procedure involving removal of protein, e.g., by precipitation with a reagent. Derivatization Reaction of solutes before chromatography or directly on the layer for the purpose of facilitating separation or detection. Desalting A sample preparation procedure involving removal of salts by some procedure such as ion exchange or dialysis.

Copyright 2003 by Taylor & Francis Group LLC

GLOSSARY

989

Descending chromatography Chromatography in which the mobile phase moves downward through the layer. Destructive detection A detection method that irreversibly changes the chemical nature of the solute, e.g., sulfuric acid charring. Detection The process of locating a separated substance on a chromatogram, whether by physical methods, chemical methods, or biological methods (visualization). Development The movement of the mobile phase through the layer to form the chromatogram. This term does not mean detection of the zones. Development solvent The mobile phase. Diatomaceous earth A naturally occurring fine white powder formed from the skeletons of microscopic marine organisms (kieselguhr). Diethylaminoethyl (DEAE) A weak anion-exchange layer material. Diol A hydrophilic phase having two OH groups on adjacent carbon atoms of an aliphatic chain. The layer can function with a normal- or reversed-phase mechanism. Displacement TLC Mode of TLC in which the mobile phase contains a "displacer component" that has a higher affinity for the stationary phase than any of the solutes to be separated. This is in contrast to the usual elution mode of TLC, in which the mobile phase has a weaker affinity than the analytes for the sorbent. In displacement chromatography, development leads to formation of a "displacement train" of zones, all moving with the same velocity in reverse order of affinity for the sorbent and having higher concentration than in linear elution chromatography. Displacement TLC has been used to scout for optimum displacers and determine solute distribution for displacement column HPLC, which is used mostly for preparative separations. (J Bariska, T Csermely, S Furst, H Kalasz, M Bhatori. Displacement thin layer chromatography. J Liq Chromatogr Relat Technol 23:531-549, 2000.) Distribution constant (K) See Partition Coefficient. Drop chromatography Application of a drop of mixture solution to a layer, followed by applications of drops of a solvent on top of the sample to develop it into concentric rings. Efficiency The narrowness of a peak compared with the length of time the component is in contact with the stationary phase. Efficiency is measured by plate number N or height equivalent to a theoretical plate, H. Eluent A solvent used to remove or elute a substance away from the sorbent during recovery for preparative or quantitative TLC. Also, the mobile phase used to perform column chromatography, but an improper term for the mobile phase used for development in thin-layer chromatography. Eluotropic series A series of solvents arranged in order of increasing ability to displace a solute, i.e., increasing polarity in adsorption chromatography. Elution The removal of a solute from a sorbent by passage of a suitable solvent. The eluent is the solvent, and the effluent or eluate is the liquid flowing from the sorbent (the eluent the solute). Elution development is defined as the development of a small amount of sample through a column or flat bed of sorbent with a liquid less strongly sorbed than the sample. The terms elution development and chromatography are generally used synonymously; the term displacement development is used when the developing solvent is more strongly sorbed than the sample, and the term frontal analysis is used when the sample is passed continuously through the sorbent. Enrichment The concentration effect obtained during sample preparation. If a compound contained in 1 L of water is collected on a solid-phase extraction column and is subsequently eluted with 2 mL of a solvent, the enlightment factor is 500. Flatbed (or planar) chromatography Common term for paper chromatography and TLC. Fluorogenic A reagent or reaction causing a solute to become fluorescent. Forced-flow planar chromatography (FFPC) Planar chromatography in which solvent migrates through the layer under the influence of external pressure (overpressured layer chromatography, OPLC) or centrifugal force (rotation planar chromatography, RPC). Front The visible boundary at the junction of the mobile phase wetted layer and the "dry" layer.

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990

GLOSSARY

FT spectroscopy Fourier transform. A type of spectroscopy in which the intensity or radiant power of many wavelengths are simultaneously measured as a function of time, and the resultant time-domain spectrum is converted by use of a computer to a conventional frequency-domain spectrum by Fourier analysis (a mathematical way to decompose a signal into its component wavelengths). GC Gas chromatography, most often gas-liquid partition chromatography (GLC). Gel filtration (gel permeation) Size-exclusion chromatography using a polymer gel layer or clean-up column. Gel filtration uses a polydextran gel, highly cross-linked polymer, silica gel, or other porous medium and an aqueous mobile phase. Biopolymers and water-soluble polymers are usually separated. Gel permeation uses a styrene-divinylbenzene type of gel and an organic mobile phase to separate organic species and polymers. Gradient The change in layer of mobile-phase composition made to improve separations. H (HETP) See Plate number. Hard layer An abrasion-resistant sorbent layer bound to the backing by an organic polymer. Homolog A member of a homologous series, which is a related succession of compounds each containing one or more carbon atoms and two more hydrogen atoms than the one before it in the series. The alcohols methanol, ethanol, and propanol are homologs. HPLC High-performance column liquid chromatography, performed under high pressure with narrow-bore columns containing small-diameter packings and continuous-flow detectors. HPTLC High-performance thin-layer chromatography, performed on layers with small, uniform, densely packed sorbent particles. hRf 100 X Rf. Hydrophilic Substances that are soluble in water or other polar solutions. Hydrophobic Substances that are soluble in nonpolar hydrocarbon solvents and insoluble in water. Impregnated layer A layer containing a liquid or solid chemical or mixtures of chemicals added to aid separation or detection of solutes. In situ Occurring "in place," directly on the layer. Ion exchange A process whereby ions of the same charge sign replace one another in a given phase. In chromatography, the term usually refers to systems in which the stationary phase is made up of an ionic polymer. This can be a synthetic resin, a cellulose or Sephadex-type exchanger, or various types of inorganic materials. IR Infrared. Isomer One of two or more compounds having the same molecular weight and formula but differing with respect to the arrangement or configuration of the atoms. Examples of isomers are ortho-(l,2)-, meta-(l,3)-, and para-(l,4)-dichlorobenzene. Kieselguhr A weakly adsorptive diatomaceous earth used in TLC mostly as a preadsorbent material and also used as a sample clean-up medium. Ligand exchange TLC A technique in which chelating ligands are added to the mobile phase and undergo sorption onto the layer. These sorbed molecules act as chelating agents with certain solutes to enhance their separation. Chelating stationary phases in which chelating groups are chemically bonded to the sorbent can also be used. Lipophilic See Hydrophobic. Liquid chromatography The type of chromatography in which the mobile phase is a liquid and the stationary phase is a layer or column. Liquid-liquid TLC See Partition TLC. Mass spectrometry A method for structure determination based on ionization of the compound and the sorting out of ions in a mass analyzer. Mass transfer Movement of a solute between the stationary and mobile phases. Metabolite The product(s) resulting from one or more chemical reactions that change or break down a compound such as a drug or pesticide in a biological system (e.g., human or animal body or plant) or the environment. Microgram (/ug) 10 6 g or 100 ng. Migration Travel of a solute through the layer in the direction of the mobile phase flow.

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GLOSSARY

991

Mobile phase The moving liquid phase used for development, often called the development solvent or solvent. Multimodal layer A layer, such as cyano- or amino-bonded silica gel, that can operate with two or more separation mechanisms depending on the choice of mobile phase. Multimodal separation A separation involving two distinct techniques, such as GC and TLC or HPLC and TLC. TLC is normally the second of the two techniques used. The coupling of two separation techniques is also called "multidimensional chromatography." Multiple development Repeated development of the chromatogram in the same direction with one mobile phase or different mobile phases for the same or different distances. N-Chamber A square or rectangular tank or chamber, or a cylinder, with (saturated) or without (unsaturated) a paper liner and covered with a top, for development of TLC or HPTLC plates. Nanogram (ng) 10~9 g or 0.001 /zg. nm Nanometer or 10~9 m. NMR (or Nuclear magnetic resonance spectrometry) A method for structure determination of organic compounds based on the magnetic properties of different nuclei. Nondestructive detection Detection of a substance on a chromatogram by a process that will not permanently change the chemical nature of the substances being detected. Normal-phase chromatography Adsorption or partition TLC in which the stationary phase is polar in relation to the mobile phase. Octadecylsilane The most popular re versed-phase sorbent in TLC. Abbreviated in bonded silica layer names as C-18. Open-column chromatography Liquid-column chromatography performed in the classical manner in a relatively large bore, usually glass, column under gravity or low-pressure flow. Used as a clean-up method for samples prior to TLC. Origin The location of the applied sample; also, the starting point for chromatographic development of the applied sample. Overrun development Continuous development beyond the top edge of the layer. Partition coefficient or ratio (Kd) The ratio of concentrations of solute in each phase: Kd = CJ Cm, where Cs and Cm are the concentrations in the stationary and mobile phases, respectively. Partition TLC Separation of solutes based on differential distribution between a stationary liquid supported on the layer and the liquid mobile phase. In normal-phase partition, the stationary liquid is more polar than the mobile phase, whereas in reversed-phase partition, the stationary liquid is the less polar. The term is also used for TLC on bonded phases in some cases. PC Paper chromatography. Also used as an abbreviation for planar chromatomagraphy in some cases. Phenyl layer A layer composed of a nonpolar bonded phase prepared by reaction of dimethylphenylchlorosilane or alkoxysilane with silica gel. Some literature sources indicate that it has selective affinity for aromatic compounds. Picogram (pg) 10~12 g or 10~3 ng. Planar (or Flatbed) chromatography Common term for thin-layer or paper chromatography. Plate height (HETP or H) The length of the layer divided by the number of plates (N). Small plate heights result in better resolution. Plate number (N) N = 16(dr/W)2, where dr is the distance the spot has migrated and W is its width (size in the direction of development). N can be measured using either the spots on the plate or their densitometric scans. PMD Programmed multiple development. The repeated development of a TLC plate with the same mobile phase or different mobile phases in the same direction for gradually increasing distances, using an automated commercial instrument. Also termed automated multiple development (AMD). Polarity The effect of the combined interactions between a functional group and a layer or mobile phase, i.e., dispersion, dipole, and/or hydrogen bonding forces, is designated "polarity." In chromatographic systems, benzene is described as a more polar solvent than toluene, although only the latter has a true dipole moment, the classical requirement for polarity. In chromato-

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992

GLOSSARY

graphic behavior, hydrocarbons and halogens are of low polarity, esters and ketones have intermediate polarity, and alcohols and amines are highly polar. The polarity of a silica gel surface is reduced by impregnation or chemical bonding with a hydrocarbon. Precision The agreement or lack of scatter among replicate determinations or analyses, without regard to the true answer. Precoated plates Commercial plates or sheets sold with the layer already formed and ready for use. Preloading Sorption of gaseous molecules by the layer prior to development with the mobile phase; also called layer conditioning or preadsorption. Preparative TLC Thin-layer chromatography of larger quantities of material on thick layers for the purpose of isolating separated substances for further analysis or use. The term preparative layer chromatography (PLC) is sometimes used instead. Qualitative analysis A TLC analysis that identifies the constituents present in a sample. Quantitative analysis A TLC analysis that determines the weight or concentration of a constituent of a sample. Rf The ratio of the distance of migration of the center of a zone divided by the distance of migration of the solvent front, both measured from the origin. Rm A value used in relating chromatographic behavior to chemical structure: Rm = \og(l/Rf 1). Rx The same as Rf except that the distance of solvent front migration is replaced by the distance of migration of some reference compound x. Radial (circular) development Development of a layer in such a manner as to form circular or arc-shaped zones. Some workers differentiate circular and radial TLC by the use of "circular" for the case where one initial zone is developed into circular zones and "radial" for development of a series of initial zones, spotted in a circular pattern, into arcs. Raman spectroscopy A technique in which a beam of intense monochromatographic radiation, such as from a laser, is focused on a sample, and scattering produces radiation that is shifted slightly in frequency by an increment of energy corresponding to a natural transition of the molecule (Raman lines). The method complements IR absorption spectroscopy for measurement of molecular vibrations. Relative standard deviation (RSD) The standard deviation of a series of replicate analyses is divided by the mean, and the resulting quotient is multipled by 100. Also called coefficient of variation. Resolution The ability to separate two zones. The mathematical description of resolution is R = 2(Rfl Rf2)/(Wi + W2), where Rf[ and Rf2 are the Rf values of any two zones and Wl and W2 are their respective zone lengths (in the direction of development). Migration distances can be used instead of Rf values in this equation. Resolution is the result of the combined contributions of efficiency (zone compactness), selectivity (separation of zone centers), and capacity (average Rf value of the pair of substances to be separated). Retention factor Another name for capacity factor. Re versed-phase chromatography Liquid-liquid partition TLC in which the stationary phase is nonpolar compared to the mobile phase. The layer can be impregnated or bonded. Rod TLC TLC carried out on sintered glass rods, usually in conjunction with a scanning flame ionization detector. Sandwich chamber (S-chamber) A developing chamber formed from the plate itself, a spacer, and a layered or nonlayered cover plate that stands in a trough containing the mobile phase, or some other type of small-volume chamber in which vapor-phase saturation occurs quickly. Saturated The condition of a chamber that is lined with paper and equilibrated with mobile phase vapors before chromatographic development is begun. Secondary front An additional solvent or mobile phase front below the primary front, that occurs because the mobile phase components demix. Selectivity The ability of a chromatographic system to produce different Rf values for the components of a mixture, i.e., to separate the zone centers. Sensitivity The ability to detect or measure a small mass of analyte.

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GLOSSARY

993

Separation number A measure of the separating power of a TLC system: The number of substances completely separated (resolution =1) between Rf = 0 and Rf = 1 by a homogeneous mobile phase (no solvent gradients in the direction of development). Silanol An SiOH group on the surface of silica gel. Silica gel The most common layer material, used unmodified for adsorption TLC, as a support for partition and bonded-phase TLC, and with different pore sizes for size-exclusion TLC. It has an amorphous, porous structure with siloxane and silanol groups on the surface. Siloxane SiOSi groups on the surface of silica gel. Soft layer A sorbent layer prepared without binder or with gypsum binder (see Hard layer). Solid-phase extraction (SPE) An alternative to traditional separatory funnel extraction in which an analyte is extracted from a liquid sample by use of a solid packed in a small column, cartridge, or disk. The sample is forced through the solid, which is an adsorbent or bonded phase, with the aid of vacuum or pressure; the analyte is retained on the solid, and it is subsequently eluted with a small volume of a strong solvent, usually resulting in significant enrichment. The latest format for SPE is placement of the packing (usually 20-40 />im diameter particle size) in the wells of a 96-well flow-through plate. Solute A general term for the compounds or ions being chromatographed. Solvent The liquid used to dissolve the sample for application to the layer. Sometimes used to refer to the mobile phase or to the liquid used to elute chromatographed zones from scraped layer material. Solvent front The farthest point of movement of the mobile phase during development. Solvent strength () A measure of the polarity of a solvent for liquid-solid adsorption chromatography. It is based on the free energy of adsorption onto a standard surface. Values for common solvents range from 0.00 (pentane) to 0.95 (methanol). Sorbent The layer material used in TLC. Sorption A general term for the attraction between a layer and a solute, without specification of the type of physical mechanism (i.e., adsorption, partition, ion exchange) or mixed mechanism involved. Sorbent is a related general term referring to the layer itself. Spectroscopy An analytical technique based on the interaction of electromagnetic radiation with matter. Also called spectrometry. Spot Used synonymously with zone, but usually meant to indicate a round or elliptical shape. Spot capacity Same as separation number. Stationary phase The solid sorbent layer, with or without any impregnation agent, preloaded vapor molecules, or immobilized mobile phase component(s). Stepwise development Development using a mobile phase whose composition is changed using discontinuous, stepped gradients, in contrast to continuously variable gradient elution. Straight-phase chromatography Another name for normal-phase chromatography. Streak An initial zone in the shape of a narrow horizontal line at the origin. Streaking See Tailing. Supercritical fluid extraction (SFE) A technique for extracting analytes from sample matrices by using a dense gas. Carbon dioxide, which becomes a supercritical fluid when used above its critical pressure (1070 psi) and temperature (31C), is the most widely applied extraction medium for SFE because it is nontoxic and nonflammable and facilitates extractions at low temperatures in a nonoxidizing environment. Tailing Formation of a zone with an elongated rear portion, often leading to incomplete resolution. Throughput A term used mostly in the context of "sample throughput," which indicates the number of samples that can be analyzed by a particular method in a given period of time. TLC has high sample throughput because multiple samples can be applied to a single plate. "Solvent throughput" is a term that designates the amount of solvent passing through the layer. For example, there is higher solvent throughput with an unsaturated N-chamber than with a saturated N-chamber because of the greater amount of solvent that passes through the layer to replace the solvent that has evaporated from it. TLC Thin-layer chromatography.

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994

GLOSSARY

TLG Thin-layer gel chromatography, in which separations are based mainly on solute sizes. Trailing See Tailing. Two-dimensional development Successive development of a chromatogram with the same solvent or different solvents in directions at a 90 angle to each other. Unsaturated The condition of a chamber that has the mobile phase and plate added together so that equilibration with the vapors is occurring during chromatographic development. UV Ultraviolet. Validation The process of determining the suitability of a given TLC method for an intended application, such as qualitative identification, assays, semiquantitative limit tests, or quantitative determination of impurities in pharmaceutical analysis. The characteristics tested can include accuracy, precision, specificity, detection limit, quantification limit, linearity, and robustness. Visualization Detection of the zones on a chromatogram. Zone The area of distribution on the layer containing the individual solutes or mixture before, during, or after chromatography. The initial zone is the applied sample prior to development. Band, zone, and spot are often used more or less interchangeably, but spot usually denotes a round zone, and band a flat, horizontally elongated zone.

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1Basic TLC Techniques, Materials, and ApparatusJoseph ShermaLafayette College, Easton, Pennsylvania, U.S.A.

I.

INTRODUCTION AND HISTORY

The purpose of this chapter is to present an overview of all important aspects of thin-layer chromatography (TLC). It briefly reviews information and provides updated references on topics covered in the remaining chapters in Part I and refers readers to the specific chapters. It treats topics that are not covered in separate chapters, such as sampling and sample preparation and the more classical procedures of TLC, in more detail. A suggested source of additional information, both basic and advanced, on the practice and applications of TLC is the primer written by Fried and Sherma (1). A. Introduction to TLC

Thin-layer chromatography and paper chromatography comprise "planar chromatography." TLC is the simplest of all the widely used chromatographic methods to perform. A suitable closed vessel containing solvent and a coated plate are all that are required to carry out separations and qualitative and semiquantitative analysis. With optimization of techniques and materials and the use of available commercial instruments, highly efficient separations and accurate and precise quantification can be achieved. Planar chromatography can also be used for preparative-scale separations by employing specialized layers, apparatus, and techniques. Basic TLC is carried out as follows. A small aliquot of sample is placed near one end of the stationary phase, a thin layer of sorbent, to form the initial zone. The sample is then dried. The end of the stationary phase with the initial zone is placed into the mobile phase, usually a mixture of two to four pure solvents, inside a closed chamber. If the layer and mobile phase were chosen correctly, the components of the mixture migrate at different rates during movement of the mobile phase through the stationary phase. This is termed development of the chromatogram. When the mobile phase has moved an appropriate distance, the stationary phase is removed, the mobile phase is rapidly dried, and the zones are detected in daylight or under ultraviolet (UV) light with or without the application of a suitable visualization reagent. Differential migration is the result of varying degrees of affinity of the mixture components for the stationary and mobile phases. Various separation mechanisms are involved, the predominant forces depending upon the exact properties of the two phases and the solutes. The interactions involved in determining chromatographic retention and selectivity include hydrogen bonding, electron-pair donor/electron-pair acceptor (charge transfer), ion-ion, ion-dipole, and van der Waals interactions. Among the latter are dipole-dipole (Keesom), dipole-induced dipole (Debye), and instantaneous dipole-induced dipole (London) interactions. Sample collection, preservation, and purification are problems common to TLC and all other chromatographic methods. For complex samples, the TLC development will usually not completely resolve the analyte from interferences unless a prior purification (cleanup) is carried out.

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This is most often done by selective extraction and column chromatography. In some cases substances are converted, prior to TLC, to a derivative that is more suitable for separation, detection, and/or quantification than the parent compound. TLC can cope with highly contaminated samples, and the entire chromatogram can be evaluated, reducing the degree of cleanup required and saving time and expense. The presence of strongly adsorbed impurities or even particles is of no concern, because the plate is used only once (2). Detection is simplest when the compounds of interest are naturally colored or fluorescent or absorb UV light. However, application of a detection reagent by spraying or dipping is required to produce color or fluorescence for most compounds. Absorption of UV light is common for most aromatic and conjugated compounds and some unsaturated compounds. These compounds can be detected simply by inspection under 254 nm UV light on layers impregnated with a fluorescence indicator (fluorescence quench detection). Compound identification in TLC is based initially on a comparison of Rf values to authentic reference standards. Rf values are generally not exactly reproducible from laboratory to laboratory or even in different runs in the same laboratory, so they should be considered mainly as guides to relative migration distances and sequences. Factors causing Rf values to vary include dimensions and type of chamber, nature and size of the layer, direction of the mobile-phase flow, volume and composition of the mobile phase, equilibration conditions, humidity, and sample preparation methods preceding TLC. See Chapter 11 in Ret. 1 for a discussion of reproducibility in TLC. Confirmation of identification can be obtained by scraping the layer and eluting the analyte followed by infrared (IR) spectrometry, nuclear magnetic resonance (NMR) spectrometry, mass spectrometry (MS), or other spectrometric methods if sufficient compound is available. These methods can also be used to characterize zones directly on the layer (in situ).

B.

History of TLC

The history of liquid chromatography, which dates back to the first description of chromatography by Michael Tswett (3) in the early 1900s, was reviewed by Sherma (4). Recent reviews of TLC were written by Ettre and Kalasz (5), Sherma (6), Kreuzig (7), and Berezkin (8). TLC is a relatively new discipline, and chromatography historians usually date the advent of modern TLC from 1958. A review by Pelick et al. (9) tabulates significant early developments in TLC and provides translations of classical papers by Izmailov and Schraiber and by Stahl. In 1938, Izmailov and Schraiber separated certain medicinal compounds on unbound alumina or other adsorbents spread on glass plates. Because they applied drops of solvent to the plate containing the sample and sorbent layer, the procedure was termed drop chromatography. Meinhard and Hall in 1949 used binder to adhere alumina to microscope slides, and these layers were used in the separation of certain inorganic ions with the use of drop chromatography; this method was called surface chromatography. In the 1950s, Kirchner and colleagues at the U.S. Department of Agriculture performed TLC as we know it today. They used silica gel held on glass plates with the aid of a binder, and plates were developed with the conventional ascending procedures used in paper chromatography. Kirchner coined the term "chromatostrips" for his layers, which also contained fluorescence indicator for the first time. Stahl introduced the term "thin-layer chromatography" in the late 1950s. His major contributions were the standardization of materials, procedures, and nomenclature and the description of selective solvent systems for resolution of important compound classes. His first laboratory manual (10) popularized TLC, and he obtained the support of commercial companies (Merck, Desaga) in offering standardized materials and apparatus for TLC. Quantitative TLC was introduced by Kirchner et al. in 1954 when they described an elution method of determination of biphenyl in citrus fruits. Densitometry in TLC was initially reported in the mid-1960s using commercial densitometers such as the Photovolt and Joyce Loebl Chromascan. Plates with uniform, fine-particle layers were produced commercially in the mid-1970s and provided impetus for the improvements in theoretical understanding, practice, and instrumentation that occurred in the late 1970s and 1980s and led to the methods termed high-performance

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BASIC TECHNIQUES, MATERIALS, APPARATUS

3

thin-layer chromatography (HPTLC) and instrumental HPTLC. Centrifugally accelerated preparative layer chromatography (PLC) and overpressured layer chromatography (OPLC), which are the major forced-flow planar chromatographic techniques, were introduced in the late 1970s. These and other high-performance and quantitative methods caused a renaissance in the field of TLC that is reflected in this Handbook. Although the major use of TLC will probably continue to be as a general low-cost and low-technology qualitative and screening method in laboratories worldwide, there is no doubt that TLC will continue to evolve and grow in the new millennium as a highly selective, sensitive, quantitative, rapid, and automated technique for analysis of all varieties of samples and analytes and for preparative separations. To keep abreast of this inevitable progress in TLC, the biennial reviews of advances in theory, practice, and applications by Sherma, the most recent of which was published in 2002 (11), are indispensable.

C.

Comparisons of TLC to HPTLC and Column Liquid Chromatography (HPLC)

Detailed comparisons of TLC to other chromatographic methods, especially HPLC, and of TLC to HPTLC are presented in Chapters 1 and 2 of Ref. 1. TLC involves the concurrent processing of multiple samples and standards on an open layer developed by a mobile phase. Development is performed, usually without pressure, in a variety of modes, including simple one-dimensional, usually in ascending or horizontal mode; multiple; circular (rarely); and multidimensional. Zones are detected statically, with diverse possibilities. Paper chromatography, which was invented by Consden, Gordon, and Martin in 1944, is fundamentally very similar to TLC, differing mainly in the nature of the stationary phase. Paper chromatography has lost favor compared to TLC because the latter is faster and more efficient, allows more versatility in the choice of stationary and mobile phases, and is more suitable for quantitative analysis. High-performance TLC layers are smaller; contain sorbent with smaller, more uniform particle size; are thinner; and are developed for a shorter distance compared to TLC layers. These factors lead to faster separations, reduced zone diffusion, better separation efficiency, lower detection limits, less solvent consumption, and the ability to apply more samples per plate. However, smaller samples, more exact spotting techniques, and more reproducible development techniques are required to obtain optimal results. High-performance liquid chromatography involves the elution under pressure of sequential samples in a closed on-line system, with dynamic detection of solutes, usually by UV absorption. The predominant mode of HPLC is reversed phase (RP) on bonded silica columns, whereas for TLC normal phase (NP) on silica gel is most widely used. This makes the two methods complementary for compound separation and identification. A paper by Sherma (12) offers a detailed review of the relationship of TLC to other chromatographic methods, especially HPLC. TLC is the most versatile and flexible chromatographic method for separation of all types of organic and inorganic molecules that can be dissolved and are not volatile. It is rapid because precoated layers are usually used without preparation. Even though it is not fully automated as is possible for HPLC, TLC has the highest sample throughput because up to 30 individual samples and standards can be applied to a single plate and separated at the same time. The ability to separate samples simultaneously in parallel lanes is important in applications that require high sample throughput, e.g., surveillance programs to detect food containing unacceptable levels of drug residues, to ensure a safe drinking water supply, to control the use of recreational and performance-enhancing drugs, and similar screening applications (13). Modern computer-controlled scanning instruments and automated sample application and development instruments allow accuracy and precision in quantification that are in many cases equivalent to those obtained with HPLC and gas chromatography (GC). There is a wide choice of layers and developing solvents (acidic, basic, completely aqueous, aqueous-organic). Solvents that can interfere with HPLC UV detection can be used in TLC because the mobile phase is

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removed from the plate prior to detection. Every sample is separated on a fresh layer, so that problems involved with carryover and cross-contamination of samples and sorbent regeneration procedures are avoided. Mobile-phase consumption is low, minimizing the costs of solvent purchase and disposal. Because layers are normally not reused, sample preparation methods are less demanding, and complex, impure samples can be applied to the layer without concern for the extra (ghost) peaks and noneluting compounds that shorten the life of HPLC columns. Simultaneous sample cleanup and separation of target compounds are often achieved with TLC (13). The wide choice of development methods and pre- or postchromatographic detection reagents leads to unsurpassed specificity in TLC, and all components in every sample, including irreversibly sorbed substances, can be detected. There is no need to rely on peaks drawn by a recorder or to worry about sample components possibly remaining uneluted on a column. Because it is an off-line method, the various steps of the procedure are carried out independently. Examples of the advantages of this approach include the ability to apply compatible detection methods in sequence and to scan zones repeatedly with a densitometer using different parameters that are optimum for individual sample components. HPLC can generally provide a higher separation power than TLC, but most HPLC separations do not require high efficiency, so the methods are quite comparable in such applications. The pyramidal screening approach, in which TLC is used as a screening step followed by HPLC confirmation and quantification of only positive samples, can result in less analytical time and lower cost than when all samples are analyzed by HPLC (13). Abjean (14) showed that 300 meat samples could be analyzed for sulfonamide drugs by a single analyst in 12 days using TLC screening and HPLC analysis of positive samples compared to 50 days for HPLC multiresidue analysis alone. The cost was 80% less, and confirmation of residue identity was more reliable because two independent methods were used. The simultaneous identification of chloramphenicol, nitrofurans, and sulfonamides in pork or beef is an example of TLC multiclass screening (15). The drugs were identified by homogenization and extraction from 1 g of tissue with ethyl acetate, cleanup of the extract on a silica gel solid-phase extraction (SPE) cartridge, and separation by TLC. Spraying with pyridine detected nitrofurans, and subsequently fluorescamine detected chloramphenicol and sulfonamides. Twenty samples could be analyzed per day per analyst for three residue classes by a single method. The determination of antibiotics in milk (16) and of poly cyclic aromatic hydrocarbons (PAHs) in soil (17) are other TLC screening methods that have demonstrated advantages in terms of simplicity, time, and cost compared to HPLC. D. The Literature on TLC The literature of TLC has been reviewed biennially by Sherma since 1970 (latest review, Ref. 11). The major journals for papers on TLC are Journal of Planar Chromatography-Modern TLC, Journal of Liquid Chromatography & Related Technologies, and Acta Chromatographica. Other chromatographic journals such as Chromatographia, Journal of Chromatographic Science, and Journal of Chromatography, A, and B and general analytical journals such as Journal of AOAC International, Analytical Biochemistry, Analytical Chemistry, and The Analyst contain some articles on TLC. The Camag Bibliography Service (CBS) regularly abstracts TLC papers and is available in paper and CD-ROM versions. Books that have appeared since the publication of the second edition of this Handbook are those by Kaiser et al. (18) (a random collection of chapters on techniques and applications in German), Hahn-Deinstrop (19) (a practical book focused on pharmaceutical analysis), and Fried and Sherma (20) (the only TLC book organized by discipline). Special issues on thin layer chromatography of the Journal of Liquid Chromatography & Related Technologies, edited by Sherma and Fried, were published as Issues 1 and 10 of Volume 22/1999 and Issue 10 of Volume 247 2001. Book chapters (21,22) and an encyclopedia article (23) covering TLC, several general review articles (13,24,25), and a guide to method development (26) were published within the last seven years. Gazes' Encyclopedia of Chromatography (27) contains 30 articles on methods and applications of TLC. The IUPAC Commission on Analytical Nomenclature published a list of approved terms and definitions for planar Chromatography in 1993 (28).Copyright 2003 by Taylor & Francis Group LLC

BASIC TECHNIQUES, MATERIALS, APPARATUS II. THEORY AND FUNDAMENTALS

5

The basic parameter used to describe migration in TLC is the Rf value, where distance moved by the solute distance moved by mobile phase front Rf values vary from 1 to 0, or from 100 to 0 if multiplied by 100 (hR{). The capacity factor, k', is the ratio of the quantities of solute distributed between the mobile and stationary phases, or the ratio of the respective times the substance spends in the two phases, , ts tm retention time in stationary phrase retention time in mobile phase

The capacity factor and Rf are related by the equationk' =

Rf

The classic Van Deemter equation and its modifications have been used to describe zone spreading in GC and HPLC in terms of eddy diffusion, molecular diffusion, and mass transfer. The efficiency of a zone in HPTLC is given by the equationWb

where N is the number of theoretical plates, Zf is the distance of solvent migration, and Wb is the diameter of the zone (29). In contrast to column chromatography, in which all solutes move the same distance, separated components migrate different distances in TLC, and their zones are broadened to varying degrees. Therefore, N is dependent on the substance migrating as well as on the migration distance, and efficiency must be reported in terms of a compound with a specific /Rvalue such as 0.5 or 1.0. Separation efficiency and capacity in TLC were discussed by Poole (13). Efficiency is limited by less than optimal velocity of the mobile phase driven by capillary forces, leading to zone broadening that is largely dominated by molecular diffusion. Mobile-phase velocity decreases approximately quadratically with migration distance, resulting in the migration of zones through regions of varying efficiency and the need to specify plate height for the layer as an average value. For sorbents with narrow particle size range, solvent front velocity is greater for coarseparticle layers than for layers with fine particles (30). It has also been shown that for RP layers with bonded long-chain alkyl groups, mobile phases with larger percentages of water will ascend very slowly, requiring plates to be prepared from particles with a larger diameter (10-13 pm) than those used for the usual HP layers (5 fjim) or from sorbents with a lower degree of surface modification. Polar-bonded sorbents, such as cyano or amino, are wetted by aqueous solvents (30). Guiochon and coworkers (31-35) showed that for capillary flow TLC on fine-particle (HP) layers, zone broadening is controlled by the size of the sorbent particles for short migration distances and molecular diffusion for long migration distances. For large-particle sorbent layers, the packing and slow mass transfer processes can both contribute to broadened, irregularly shaped zones. High plate numbers can be generated on layers with relatively large particles only with long migration distances, especially for solutes with large diffusion coefficients. HPTLC layers produce the highest efficiency for short migration distances of 5-6 mm, and efficiency eventually is poorer than for TLC as the migration distance increases and molecular diffusion overtakes zone center separation to become the limiting factor. Longer solvent front migration distances require layers with a larger particle size to obtain a reasonable range of mobile-phase velocities and total number of theoretical plates (13,24). The results of these studies indicate that HPTLC plates can produce more compact zones in a shorter development distance, increasing the speed and detection limits of the zones. About 5000 theoretical plates can be obtained for a 5-7 cm development on HPTLC plates, whereas a development distance of approximately 15 cm is needed to obtain this

Copyright 2003 by Taylor & Francis Group LLC

6

SHERMA

number of plates for a layer with larger particles (30). The experimental zone capacity for baseline separated peaks in a chromatogram resulting from capillary controlled flow is about 12-14, and this is not strongly dependent on the average particle size of the layer (13). Zone capacity for forced-flow development is 30-40; for capillary controlled flow automated multiple development (AMD), 30-40; and for two-dimensional (2-D) capillary flow, approximately 100. An equation (36) for resolution (/?,) of two zones in TLC by a single ascending development is

*.=

'2) - 1][1 - Rr_]

where k\ and k'2 are the capacity factors for the two solutes to be separated and N is the number of theoretical plates. The subscript 2 refers to the zone with the higher Rf value. As in the analogous resolution equation for HPLC, this equation includes terms related to the efficiency of the layer, the selectivity of the TLC system, and the capacity of the system (the zone positions on the layer). Resolution increases with the square root of the layer efficiency (TV), which depends linearly on the Rf value. In terms of zone position, studies have shown that maximum resolution is obtained in the R, range of 0.2-0.5 (30). The most effective means for increasing resolution on a TLC or HPTLC layer with the usual capillary flow, one-dimensional single development is to improve selectivity by variation of the mobile phase, the choice of which is aided by systematic optimization methods such as simplex, PRISMA, and others that have been developed (37) (see Chap. 3). Other approaches for increasing resolution include the use of capillary flow with multiple or two-dimensional development or forced-flow development. The foregoing discussion applies to capillary flow TLC, in which the migration velocity of the mobile phase through the layer is controlled by capillary forces and decreases as development distance increases (38). The optimum velocity necessary for maximum efficiency is not realized in capillary flow TLC. In forced-flow planar chromatography, the mobile phase is driven by centrifugal force [rotation planar chromatography (RPC)] or by a pump (OPLC) (see Chap. 7) through a layer enclosed by a polymeric or metal membrane under external pressure. RPC is used mainly for PLC (see Chap. 11), whereas many applications of OPLC for analytical separations have been reported. RPC never reaches an overall mobile-phase velocity that would give the highest separation efficiency, because the radial velocity of solvent migration diminishes from the center to the circumference of the plate (39). In OPLC, mobile-phase velocity can be controlled at a predetermined constant close to optimal value so that solvent front migration is a linear function of time (30). As a result, average plate height is approximately independent of migration distance and is most favorable for HPTLC plates, zone broadening by diffusion is minor even over long migration distances, plate number increases linearly with migration distance, and resolution continues to increase as migration distances increases (30,38). The time required for the mobile phase to cover the same distance in OPLC is typically five- to tenfold shorter than in TLC, depending on the surface tension, viscosity, and the ability to wet the layer. Separation time is further reduced because the number of theoretical plates needed to achieve a separation is generated in a shorter time because of the near-optimal mobile-phase flow rate (39). Poole (13) showed that for a development distance of 18 cm, forced-flow development can produce 8000 theoretical plates in 9 min. Increased efficiency is obtained by use of longer bed lengths (e.g., serial coupling of stacked, connected layers) over longer times. Electro-osmotic flow caused by applying an electric field across a wet layer containing both ionized silanol groups and mobile ions is an additional mechanism for moving the mobile phase through the layer. Nurok (39) reported that separation of six pyrimidines on silica gel with acetonitrile mobile phase was 12 times faster than with conventional TLC and that separation in the RP mode is two to three times faster depending on the mobile phase. Only preliminary studies of this approach have been carried out to date, and Poole (13) reports that the mobile-phase velocity declined with migration distance and showed only moderate increase compared to capillary flow, and that the demonstrated improved performance with electro-osmotic flow has been below that predicted by theory.

Copyright 2003 by Taylor & Francis Group LLC

BASIC TECHNIQUES, MATERIALS, APPARATUS

7

The classic book by Geiss (40) is recommended as an excellent source of information on the fundamentals of TLC. Although the book is highly theoretical and mathematical, numerous practical summaries and suggestions can be found throughout its chapters to guide anyone working with TLC. Especially useful in better understanding TLC is Chapter 6 in Geiss (40), on the role of the vapor phase. It explains and distinguishes chamber saturation (saturation of the chamber atmosphere), sorptive saturation (preloading of the layer from the atmosphere), and capillary saturation (saturation of the layer through the rising mobile phase) and the results caused by different chamber types and solvent mixtures. It is safe to say that few practitioners of TLC clearly understand these complicated effects that occur during development. The Geiss book also contains a discussion and a decision flow chart for optimization of separations of two closely related substances or a wide polarity range multicomponent mixture with the use of different mobile phases, development approaches, chamber types, and layers. Readers are directed to Chapter 2 of this Handbook and to Ref. 41 for discussions of the physicochemical theory and mechanism of TLC. Reference 42 covers studies of quantitative structure-retention relationships, one of the more important theoretical fields of TLC. III. A. SAMPLING AND SAMPLE PREPARATION Sampling for TLC Analysis

One of the most important steps in analysis is that of obtaining an appropriate sample of the material to be analyzed. If a nonrepresentative sample is taken, the analytical result will be unreliable no matter how excellent the procedure and laboratory work. As an example, the purity of a bottle of 100 analgesic tablets should not be determined by analyzing one tablet, which might be nonrepresentative of the average tablet. A better plan is to grind together 10 tablets to form a homogeneous powder and take a sample weight equivalent to the average weight of one tablet for the analysis. In this way, the composition of the laboratory sample has a much higher probability of accurately representing the average composition of the entire contents of the bottle. The sample should not change or be lost as a result of storage prior to TLC analysis. The integrity of most samples can be maintained by storage in a freezer. However, with some samples, freezing and thawing or the introduction of the common fixatives formalin or ethanol can affect the results of subsequent analyses (43). The storage container should be airtight to prevent volatilization of the sample or introduction of air, water, or other vapors. The container should be constructed from a material chosen such that impurities are not leached into the sample from the inside surface and analyte cannot be lost by adsorption on the inside surface. Plastic is a common choice for storage of samples to be analyzed for metals, and glass for samples with organic analytes. A detailed discussion of sampling procedures for different types of gas, liquid, solid, and bulk samples is beyond the scope of this chapter. Chapter 4 in Ref. 1 contains information on obtaining and storing human, warm- and cold-blooded animal, microbial organism, and plant material samples for TLC. Most college textbooks on quantitative analysis and instrumental analysis contain sections or chapters on the theory and practice of sampling (e.g., Ref. 44). B. Sample Preparation

Sample preparation for TLC is covered in Chapter 4 of Ref. 1 with an emphasis on biological samples. The only chapter on sample preparation specifically for TLC was written by Sherma (45), but because of its date it does not contain modern methods. A review paper on sample preparation for chromatographic analysis of plant material (46) and two reports on instruments for sample preparation (47,48) contain information on the newest methods. Sections on sample preparation related to specific compound types will be found in most of the applications chapters in Part II of this Handbook. If the analyte is present in low concentration in a complex sample such as biological or plant material, then extraction, isolation, and concentration procedures must usually precede TLC. Because layers are not reused, it is often possible to spot cruder samples than could be injected intoCopyright 2003 by Taylor & Francis Group LLC

an HPLC column, including samples containing irreversibly sorbed impurities. On the other hand, any impurities that would comigrate with the analyte and adversely affect its detection or cause a distorted or trailing analyte zone must be removed prior to TLC. Isolation and/or preconcentration procedures for TLC are similar to those used for GC and HPLC and include Soxhlet extraction (49), sonication extraction (50), supercritical fluid extraction (SFE), and SPE. Purification of extracts is accomplished by methods such as solvent partitioning, column chromatography, desalting, and deproteinization. 1. Direct Spotting of Samples Certain samples can be successfully analyzed by direct spotting without extraction or cleanup. The applied volume must give a detectable zone with a scan area that can be bracketed by the scan areas of a series of standard concentrations if densitometric quantification is desired. Impurities must not retain the compound at the origin, distort its shape (cause tailing), or alter the Rf value of the zone. The quantification of benzoic and sorbic acid preservatives in beverages directly applied onto a plate with a preadsorbent spotting strip is an example (51). The preadsorbent facilitated the analysis because samples could be quickly and easily applied over a large area, the initial zone was automatically concentrated at the layer interface upon development, and the kieselguhr strip retained sample impurities. Unpurified urine and serum samples have also been applied successfully to preadsorbent layers for determination of amino acids, drugs, and lipids. 2. Direct Application of Sample Solutions or Extracts For determination of macro constituents in relatively pure matrices, samples can be dissolved in an appropriate volume of pure solvent followed by spotting of an aliquot of solution on the layer. This approach has been used for HPTLC assay of active ingredients of many pharmaceutical dosage forms, e.g., cimetidine in acid reduction tablets (52). Natural or synthetic vanilla flavors were determined in chocolate by slurrying the sample with 95% ethanol, sonication, filtering to remove solid material, and direct application to the layer (38). Fillers and other inert ingredients in samples such as foods and pharmaceuticals often remain undissolved. This will cause no problem if the analyte is dissolved completely and the insoluble material is filtered or centrifuged into a pellet or allowed to settle to the bottom of the sample container prior to spotting clear test solution. Extracts of trace constituents in some types of adequately pure samples can also be spotted directly after concentration of an extract to a suitable volume. Any coextracted impurities must be resolved from the analyte by the TLC separation step or not detected by the visualization method used. To minimize the amount of coextractives, the least polar analyte that will quantitatively extract the analyte should be used, leaving as many polar impurities as possible unextracted. Direct spotting of extracts was used to determine hydrocarbons in wastewater extracted with heptane by means of a microseparator (53) and the pesticide dichlorvos in minced visceral tissue extracted with ethyl acetate (54). 3. Cleanup of Extracts by Solvent Partitioning Extracts that are too impure for direct spotting can be cleaned up by partitioning with immiscible solvents. The principle of differential partitioning is to leave impurities behind in one solvent layer while extracting the analyte into the other layer. Acids are converted into salts that are soluble in aqueous solutions at high pH but are un-ionized and extractable into organic solvents at low pH. Basic compounds are extracted into organic solvents at high pH and into water in their salt forms at low pH. In practice, the pH should be at least two units below the pKa of an acid and two units above the pKa of a base in order to have a large enough fraction of uncharged molecules to allow efficient extraction into organic solvents. As an example, the mycotoxin patulin was determined in apples, apple concentrate, and apple juice by extraction with ethyl acetate, cleanup by partition with 1.5% sodium carbonate solution, and silica gel TLC-densitometry (55). Other uses of liquid-liquid extraction in sample preparation are to remove oils, fats, and lipids from samples if these compounds will interfere with subsequent TLC and to concentrate sample solutions prior to spotting.Copyright 2003 by Taylor & Francis Group LLC

BASIC TECHNIQUES, MATERIALS, APPARATUS

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4. Cleanup of Extracts by Column Chromatography Chromatography on gel permeation, silica gel, alumina, Florisil, and carbon columns, among others, has been very widely used for cleanup of samples, often after preliminary purification by solvent partitioning. Examples are the TLC determination of uracil herbicides in roots of Echinacea angustifolia Moench (Asteraceae) after acetone extraction, partitioning with cyclohexane and then chloroform, and purification on a Florisil R column eluted with dichloromethane-acetone (9:1) (56) and 12 dyes in food extracts after elution from an XAD-2 column with acetone, methanol, and water (57). Column chromatographic cleanup, which usually employs large volumes of solvents to elute fractions of the sample, has been largely replaced by SPE in order to speed up and simplify extraction and cleanup and save on the cost of purchasing and disposing of solvents. 5. Modern Sample Preparation Systems The field of sample preparation has moved increasingly toward the use of disposable microcolumns and cartridges in order to speed up and simplify extraction and cleanup. These sample preparation systems are of two basic types. Columns packed with diatomaceous earth and designed for efficient liquid-liquid extractions in place of separatory funnels are available with capacities ranging from 0.3 to 300 mL of sample (e.g., Chem Elute Hydromatrix columns from Varian). The packing is either unbuffered or buffered at pH 4.5 and 9.0 for extraction of acidic and basic compounds, respectively. The aqueous sample is poured into the column, and after a 5 min wait, organic extracting solvent is poured into the column. The eluent containing the analyte is collected, evaporated to dryness under nitrogen flow, reconstituted in an appropriate solvent, and spotted for TLC analysis. Extraction columns of this type are used for screening drugs of abuse in urine (e.g., Extube Tox Elute 10 and 20 mL columns from Varian). The second method, SPE, uses sorbent phases with a variety of mechanisms and formats. The most common formats are microcolumns or cartridges with 100-500 mg of sorbent packed in 1-5 mL syringe barrels. Other SPE formats include