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Thin-Layer

Chromatography

Fouth Edition, Revised and Expanded

Bernard Fried

Department of Biology Lafayette College Easton, Pennsylvania

Joseph Sherma

Department of Chemistry Lafayette College Easton, Pennsylvania

MARCE L

MARCEL DEKKER, INC.

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K

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Copyright © 1999 by Taylor & Francis Group, LLC

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ISBN: 0-8247-0222-0

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CHROMATOGRAPHIC SCIENCE SERIES

A Series of Monographs

Editor: JACK GAZES

Cherry Hill, New Jersey

1.

Dynamics of Chromatography, J. Calvin Giddings

2.

Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gud- zinowicz

3.

Principles of Adsorption Chromatography: The Separation of Nonionic Or- ganic Compounds, Lloyd R. Snyder

4.

Multicomponent Chromatography: Theory of Interference, Friedrich Helf ferich and Gerhard Klein

5.

Quantitative Analysis by Gas Chromatography, Josef Novak

6.

High-Speed Liquid Chromatography, Peter M. Rajcsanyi and Elisabeth Raj- csanyi

7.

Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzino- wicz, 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

13.

Gerald L. Hawk Liquid Chromatography of Polymers and Related Materials II, edited by

 

14.

Jack Gazes and Xavier Delamare Introduction to Analytical Gas Chromatography: History, Principles, and

 

15.

Practice, John A. Perry Applications of Glass Capillary Gas Chromatography, edited by Walter G.

16.

Jennings Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky

17.

Thin-Layer Chromatography: Techniques and Applications, Bernard Fried

18.

and Joseph Sherma Biological/Biomedical Applications of Liquid Chromatography III, edited by

19.

Gerald L. Hawk Liquid Chromatography of Polymers and Related Materials III, edited by

20.

Jack Gazes Biological/Biomedical Applications of Liquid Chromatography, edited by

21.

Gerald L. Hawk Chromatographic Separation and Extraction with Foamed Plastics and

22.

Rubbers, G. J. Moody and J. D. R. Thomas Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin

Copyright © 1999 by Taylor & Francis Group, LLC

 
   

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. Wittwer, 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

21.

Affinity

Chromatography:

Template

Chromatography

of

Nucleic

Acids

28.

and Proteins, Herbert Schott HPLC in Nucleic Acid Research: Methods and Applications, edited by

 

29.

Phyllis R. Brown Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J.

 

30.

Levy Modern Chromatographic Analysis of the Vitamins, edited by Andre P.

 

31.

De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter Ion-Pair Chromatography, edited by Milton T. W. Hearn

 

32.

Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography,

 

33.

edited by Steven H. Y. Wong Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr

 

34.

and Klaus Pommerening Reaction Detection in Liquid Chromatography, edited by Ira S. Krull

 

35.

Thin-Layer Chromatography: Techniques and Applications. Second Edi-

 

36.

tion, Revised and Expanded, Bernard Fried and Joseph Sherma Quantitative Thin-Layer Chromatography and Its Industrial Applications,

 

37.

edited by Laszlo R. Treiber 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

 

40.

Janca Chromatographic Chiral Separations, edited by Morris Zief and Laura J.

 

41.

Crane Quantitative Analysis by Gas Chromatography: Second Edition, Revised

 

42.

and Expanded, Josef Novak Flow Perturbation Gas Chromatography, N. A. Katsanos

 

43.

44.

Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Nakanishi, and Ryuichi Matsuno

 

45.

Countercurrent Chromatography: Theory and Practice, edited by N. Bhu- shan Mandava and Yoichiro /to Microbore Column Chromatography: A Unified Approach to Chromatog-

46.

raphy, edited by Frank J. Yang Preparative-Scale Chromatography, edited by Eli Grushka

47.

Packings and Stationary Phases in Chromatographic Techniques, edited

48.

by Klaus K. Linger Detection-Oriented Derivatization Techniques in Liquid Chromatography,

49.

edited by Henk Lingeman and Willy J. M. Underberg Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamo-

50.

vics Multidimensional Chromatography: Techniques and Applications, edited

51.

by Hernan Cortes

HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M. Gooding and Fred E. Regnier

Copyright © 1999 by Taylor & Francis Group, LLC

   

52.

Modern Thin-Layer Chromatography, edited by Ne/u Grinberg

ADDITIONAL VOLUMES IN PREPARATION

 

53.

Chromatographic Analysis of Alkaloids, Milan Pop/, Jan Fahnrich, and

 

54.

Vlastimil Tatar HPLC in Clinical Chemistry, /. N. Papadoyannis

 

55.

Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and

 

56.

Bernard Fried 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

 

59.

van der Greef Trace Analysis with Microcolumn Liquid Chromatography, Milos Krejcl

 

60.

Modern Chromatographic Analysis of Vitamins: Second Edition, edited by

 

61.

Andre P. De Leenheer, Willy E. Lambert, and Hans J. Nelis Preparative and Production Scale Chromatography, edited by G. Ganetsos

 

62.

and P. E. Barker Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A.

 

63.

George 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,

 

67.

Revised and Expanded, Bernard Fried and Joseph Sherma Liquid Chromatography for the Analyst, Raymond P. W. Scott

 

68.

Centrifugal Partition Chromatography, edited by A/ain 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

 

72.

Expanded, edited by Joseph Sherma and Bernard Fried Liquid Chromatography of Oligomers, Constantin V. Uglea

 

73.

Chromatographic Detectors: Design, Function, and Operation, Raymond

 

74.

P. W. Scott Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised

 

75.

and Expanded, edited by John A. Adamovics Supercritical Fluid Chromatography with Packed Columns: Techniques

 

76.

and Applications, edited by Klaus Anton and Claire Berger Introduction to Analytical Gas Chromatography: Second Edition, Revised

 

77.

and Expanded, Raymond P. W. Scott Chromatographic Analysis of Environmental and Food Toxicants, edited

78.

by Takayuki Shibamoto Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoen-

79.

makers, and Neil Miller Liquid Chromatography-Mass Spectrometry: Second Edition, Revised and

80.

Expanded, W. M. A. Niessen Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodriguez-D/az,

81.

and Mingde Zhu Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Ber-

 

nard Fried and Joseph Sherma

Copyright © 1999 by Taylor & Francis Group, LLC

 
   

Preface to the Fourth Edition

This new edition of our book has been written to update and expand the topics in the third edition. It provides extensive coverage of qualitative and quantitative thin-layer chromatography (TLC) and high-performance thin-layer chromatogra- phy (HPTLC) and should appeal to a wide range of scientists including biologists, chemists, biochemists, biotechnologists, and medical technologists. We have maintained the same overall organization of the earlier editions, namely, a series of initial chapters on theory and practice (Part I) and a second section of chapters covering applications to important compound classes (Part II). As in the previous editions, practical rather than theoretical aspects of TLC have been emphasized. The book is still intended to serve as an introductory

 

primer. For more detailed coverage of all aspects of TLC, the reader is referred to the 1996 Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded, edited by J. Sherma and B. Fried (Marcel Dekker, Inc., 1996). This edition updates and extends references to the literature. The TLC liter- ature is voluminous and references in this book are necessarily selective. Citations are mainly to the 1985-1997 literature, with retention of some important earlier references. The chapter titles in Part I are unchanged, but some subjects have been reorganized within the chapters and there is a general updating of material and elimination of a number of obsolete instruments. Included among the topics for which new sections or increased coverage is provided are the following: sample preparation by supercritical fluid chromatography (Chapter 3); mobile phases for ion pair and chiral separations (Chapter 4); robotics and automation (Chapter 7);

Copyright © 1999 by Taylor & Francis Group, LLC

 
 

TLC coupled with spectrometry and other analytical methods, video documenta- tion, and computer imaging (Chapter 9); quantification by video densitometry (Chapter 10); validation of quantitative results (Chapter 11); and in situ instru- mental measurement of radioactive zones (Chapter 13). The fourth edition contin- ues to provide extensive coverage of sample preparation in Chapter 3. We believe that this coverage is unique and differs from that found in other available treatises on TLC. Because of the primary importance of commercial precoated plates, detailed instructions for preparing layers were removed (Chapter 3), and descrip- tion of documentation by contact printing has been eliminated (Chapter 9). Part II is concerned with the applications of TLC to various compound classes. It presents a brief and selective overview of the recent literature and various detailed qualitative and quantitative densitometric experiments on or- ganic dyes, lipids, amino acids, carbohydrates, natural pigments, vitamins, nu- cleic acid derivatives, steroids and terpenoids, pharmaceuticals, organic acids, antibiotics, and insecticides. This edition presents new experiments on organic dyes, lipids, and antibiotics, and suggestions for new experiments on lipids, phar- maceuticals, organic acids, and insecticides based on the "TLC Applications" catalog of Macherey Nagel GmbH & Co. KG, Postfach 101352, D-522313 Dueren, Germany. This section of the book should be useful to students and faculty at academic institutions and should also provide useful experimental pro- cedures for the practicing chromatographer. The suggestions for new experiments in this edition should also provide information to chromatographers seeking ad- vanced treatment on applications of TLC to various compounds. Most of the suggestions by reviewers of the third edition have been incorpo- rated into this revision. New line drawings of chromatograms have been added. Some experiments that required the use of plant and animal material that is not easily available were deleted. Corrections were made in Chapter 21 to eliminate minor errors and use of outdated terminology related to steroids. Comments on

 

this edition as well as notification of errors and suggestions for improvements in future editions will be appreciated.

 

Bernard Fried

 

Joseph Sherma

Copyright © 1999 by Taylor & Francis Group, LLC

 
 

Preface to the First Edition

The purpose of this book is to acquaint the reader with the principles, practices, and applications of thin-layer chromatography (TLC). The book is divided into two parts, the first of which is concerned mainly with general practices of TLC and the second with applications based on compound types. Although we feel this book is a valuable source of practical information for any scientist currently using or contemplating the use of TLC, it was written with special consideration for those without extensive training in analytical chemistry. This includes the majority of biology students and graduate biologists, for whom we feel this book will be especially valuable. After reading our book these people should be better equipped to examine critically the more comprehensive texts already published

 

on TLC. The authors of this book have had considerable experience in both qual- itative and quantitative TLC. One author (J.S.), an analytical chemist, has had widespread experience in the separation sciences. The other author (B.F.), an experimental parasitologist, has recently become involved in TLC analyses of hydrophilic and lipophilic excretions of parasitic worms. Both authors have had considerable experience teaching TLC at Lafayette College and at short courses sponsored by Kontes Co., Inc., Vineland, New Jersey, and the Center for Profes- sional Advancement, East Brunswick, New Jersey. Additionally, the authors have advised scientists from academia, government, and industrial research on princi- ples, practices, and applications of TLC. At Lafayette, which is an undergraduate teaching institution, we have ad- vised colleagues on TLC. For instance, we helped botanists with TLC procedures

Copyright © 1999 by Taylor & Francis Group, LLC

 
 

for the separation of plant pigments and zoologists with separation problems related to hydrophilic and lipophilic substances from animal tissues and fluids. Several of our colleagues have recently introduced one or more TLC experiments in their courses. The aim of this book, in part, is to aid teachers in introducing TLC experiments in courses. The second part presents various detailed TLC ex- periments that can usually be performed in one or several 3-hr lab periods. Most experiments utilize inexpensive materials that are readily available. Our experiments are based on TLC separations on silica and cellulosesor- bents. Chapter 3, however, discusses less widely used sorbents such as alumina, Sephadex, ion-exchange materials, polyamides, and others. Since TLC is used mainly as a separation tool for small-molecular-weight, nonionized, lipophilic and hyrophilic substances, our detailed experiments emphasize TLC on silica and cellulose. We have referred to numerous commercial products in this book. These references are based mainly on our combined experiences with these products and are not meant to imply that any product is better than others not mentioned. Throughout this book the following conventions have been adopted: the terms "solvent" and "mobile phase" are used more or less interchangeably; "spot" and "zone" are used interchangeably with no designation of a certain shape; mobile phases are designated in volume proportions unless otherwise stated.

Bernard Fried

Joseph Sherma

Copyright © 1999 by Taylor & Francis Group, LLC

 
 

Contents

Preface

to the Fourth Edition

Preface

to the First Edition

PART I: GENERAL PRACTICES OF TLC

1. Introduction and History

2. Mechanism and Theory

3. Sorbents, Layers, and Precoated Plates

4.

Obtaining Material for TLC and Sample Preparation

 

5.

Application of Samples

 

6.

Solvent Systems

 

7.

Development Techniques

 

8.

Detection and Visualization

 

9.

Qualitative Evaluation and Documentation

 

10.

Quantification

 

11.

Reproducibility and Validation of Results

 

Copyright © 1999 by Taylor & Francis Group, LLC

 
   

12. Preparative Layer Chromatography

13. Radiochemical Techniques

Contents

PART II: APPLICATIONS OF TLC TO DIFFERENT COMPOUND TYPES

14. Basic TLC Design and TLC of Organic Dyes

15. Lipids

16. Amino Acids

17. Carbohydrates

18. Natural Pigments

19. Vitamins

20. Nucleic Acid Derivatives

21. Steroids and Terpinoids

22. Pharmaceuticals

23. Miscellaneous Applications

Directory of Manufacturers

and Sources of Standards,

Sample

Preparation Supplies, and TLC Instruments, Glossary

Plates, and Reagents

Copyright © 1999 by Taylor & Francis Group, LLC