Untitled

Handbook of Thin-Layer
Chromatography
CHROMATOGRAPHIC SCIENCE
SERIES
A Series of Textbooks and Reference Books

Editor: JACK GAZES
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 Novák
6. High-Speed Liquid Chromatography, 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 Cazes
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.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
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 André P.De Leenheer,
Willy E.Lambert, and Marcel G.M.De Ruyter
31. Ion-Pair Chromatography, edited by Milton T.W.Hearn

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 Åke Jönsson
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 Novák
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.Unger
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 Heman
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 Popl, Jan Fähnrich, and Vlastimil
Tatar
54. HPLC in Clinical Chemistry, I.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. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by André 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. ChromatographJc 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, Tim 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 Thiébaut
83. Micellar Liquid Chromatography, Alain Berthod and Celia Garcia-Alvarez-Coque
84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and

Expanded, edited by André 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 Bio-

pharmaceutical Applications, edited byAnurag 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 Ali
Handbook of Thin-Layer
Chromatography
Third Edition, Revised and Expanded
edited by
Joseph Sherma
Bernard Fried
Lafayette College
Easton, Pennsylvania, U.S.A.
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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 István 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 Babić
(“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 Radio-chromatography” by Terry Clark and Otto
Kelin (Chapter 12); “Natural Pigments” by Øyvind M. Andersen and George W.Francis
(Chapter 22); “Pharmaceuticals and Drugs” by Gábor 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
revision—for example, clear line drawings have replaced photographs in some chapters.
As in the past, we welcome comments regarding this edition—notification of errors,
suggestions for improvements in the topics covered, new topics, or new authors.
Joseph Sherma
Bernard Fried
Preface to the First Edition
This book has been designed as a practical, comprehensive laboratory handbook on the
topic of thin-layer 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
Contents
Preface to the Third Edition xi

Preface to the Second Edition xiv
Preface to the First Edition xvi
Contributors xxii
Part I: Principles and Practice of Thin-Layer Chromatography
1. Basic TLC Techniques, Materials, and Apparatus 1

Joseph Sherma
2. Theory and Mechanism of Thin-Layer Chromatography 62
Teresa Kowalska, Krzysztof Kaczmarski, and Wojciech Prus
3. Optimization 106
Claudia Cimpoiu
4. Sorbents and Precoated Layers in Thin-Layer Chromatography 129
Fredric M.Rabel
5. Instrumental Thin-Layer Chromatography (Planar Chromatography) 177
Eike Reich
6. Gradient Development in Thin-Layer Chromatography 200
Wladystaw Gołkiewicz
7. Overpressured Layer Chromatography 229
Emil Mincsovics, Katalin Ferenczi-Fodor, and
8. Detection, Identification, and Documentation 271
Gerda Morlock and Karl-Arthur Kovar
9. Thin-Layer Chromatography Coupled with Mass Spectrometry 311
Kenneth L.Busch
10. Basic Principles of Optical Quantification in TLC 360
Mirko Prošek and Irena Vovk
11. Preparative Layer Chromatography 399
Szabolcs Nyiredy
12. Thin-Layer Radiochromatography 442
István Hazai and Imre Klebovich
13. Applications of Flame Ionization Detectors in Thin-Layer 471
Chromatography
Kumar D.Mukherjee
Part Applications of Thin-Layer Chromatography
II:
14. Amino Acids and Their Derivatives 486
Ravi Bhushan and J.Martens
15. Antibiotics 543
Irena Choma
16. Carbohydrates 579
Mark D.Maloney
17. Enantiomer Separations 611
Kurt Günther and Klaus Möller
18. Herbal Drugs, Herbal Drug Preparations, and Herbal Medicinal 699
Products
Eike Reich and Anne Blatter
19. Hydrocarbons 740
Vicente L.Cebolla and Luis Membrado Giner
20. Hydrophilic Vitamins 770
Fumio Watanabe and Emi Miyamoto
21. Inorganic and Organometallic Compounds 793
Ali Mohammad
22. Lipids 826
Bernard Fried
23. Lipophilic Vitamins 876
A Una Pyka
24. Natural Pigments 911
George W.Francis and Øyvind M.Andersen
25. Nucleic Acids and Their Derivatives 961
Jacob J.Steinberg
26. Peptides and Proteins 981
Ravi Bhushan and J.Martens
27. Pesticides 1005
Marija Kaštelan-Macan and Sandra Babić
28. Pharmaceuticals and Drugs 1055
Szabolcs Nyiredy, Katalin Ferenczi-Fodor, Zoltán Végh, and Gábor
Szepesi
29. Phenols, Aromatic Carboxylic Acids, and Indoles 1126
John H.P.Tyman
30. Steroids 1188
Joseph Sherma
31. Synthetic Dyes 1217
Vinod K.Gupta
32. Toxins (Natural) 1260
W.M.Indrasena
Glossary 1283
Directory of Manufacturers and Suppliers of Plates, Equipment, and 1293
Instruments for Thin-Layer Chromatography
Index 1295
Contributors
Øyvind M.Andersen Department of Chemistry, University of Bergen, Bergen, Norway

Sandra Babić Faculty of Chemical Engineering and Technology, University of Zagreb,
Zagreb, Croatia
Ravi Bhushan Department of Chemistry, Indian Institute of Technology, Roorkee,
Roorkee, India
Anne Blatter CAMAG-Laboratory, Muttenz, Switzerland
Kenneth L.Busch National Science Foundation, Arlington, Virginia, U.S.A.
Vicente L.Cebolla Institute de Carboquímica, CSIC, Zaragoza, Spain
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 Chemical Works of Gedeon Richter Ltd., Budapest, Hungary
George W.Francis Department of Chemistry, University of Bergen, Bergen, Norway
Bernard Fried Department of Biology, Lafayette College, Easton, Pennsylvania, U.S.A.
Wtadyslaw Gołkiewicz Department of Inorganic and Analytical Chemistry, Medical
University, Lublin, Poland
Kurt Günther Industriepark Wolfgang GmbH, Hanau, Germany
Vinod K.Gupta Department of Chemistry, Indian Institute of Technology, Roorkee,
Roorkee, India
István Hazai Department of Pharmacokinetics and Metabolism, IVAX Drug Research
Institute Ltd., Budapest, Hungary
W.M.Indrasena Ocean Nutrition Canada, Halifax, Nova Scotia, Canada
Krzysztof Kaczmarski* Department of Chemistry, Rzeszów University of Technology,
Rzeszów, Poland
Marija Kaštelan-Macan Faculty of Chemical Engineering and Technology, University
of Zagreb, Zagreb, Croatia
Imre Klebovich Department of Pharmacokinetics, EGIS Pharmaceuticals Co. Ltd.,
Budapest, Hungary
Karl-Arthur Kovar Pharmaceutical Institute, University of Tübingen Tübingen,
Germany
Teresa Kowalska Institute of Chemistry, Silesian University, Katowice, Poland
Mark D.Maloney Biology Department, Spelman College, Atlanta, Georgia, U.S.A.
J.Martens FB-Chemie, Universität Oldenburg, Oldenburg, Germany
Luis Membrado Giner Institute de Carboquímica, CSIC, Zaragoza, Spain
Emil Mincsovics OPLC-NIT Ltd., Budapest, Hungary
Emi Miyamoto 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 Möller MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany
Gerda Morlock Scientific Consultant, Stuttgart, Germany
Kumar D.Mukherjee Institute for Lipid Research, Federal Centre for Cereal, Potato and
Lipid Research, Münster, Germany
Szabolcs Nyiredy Research Institute for Medicinal Plants, Budakalász, Hungary
Mirko Prošek Laboratory for Food Chemistry, National Institute of Chemistry,
Ljubljana, Slovenia
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
Fredric M.Rabel EM Science, Gibbstown, New Jersey, U.S.A.
Eike Reich CAMAG-Laboratory, Muttenz, Switzerland
Joseph Sherma Department of Chemistry, Lafayette College, Easton, Pennsylvania,
U.S.A.
*
Current affiliation: Ocean Nutrition Canada, Halifax, Nova Scotia, Canada.
Jacob J.Steinberg Department of Pathology, Albert Einstein College of Medicine and

Montefiore Medical Center, Bronx, New York, U.S.A.
Gábor Szepesi Qualintel Ltd., Budapest, Hungary
Department of Plant Pathophysiology, Plant Protection Institute, Hungarian

Academy of Sciences, Budapest, Hungary
John H.P.Tyman Centre for Environmental Research, Brunel University, Uxbridge,
Middlesex, England
Zoltán Végh 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
1
Basic TLC Techniques, Materials, and
Apparatus
Joseph Sherma
Lafayette 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,
Handbook of thin-layer chromatography 2
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. 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 Ref. 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
Basic TLC techniques, materials, and apparatus 3
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 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 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 24/ 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. Cazes’ 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).
II. THEORY AND FUNDAMENTALS
The basic parameter used to describe migration in TLC is the Rf value, where
Rf values vary from 1 to 0, or from 100 to 0 if multiplied by 100 (hRf).

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,
The capacity factor and Rf are related by the equation
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 equation
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 Rf value 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 coarse-particle 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 µm) than those used for
the usual HP layers (5 µm) 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 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 (Rs) of two zones in TLC by a single ascending
development is
where and 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 (N), which depends linearly on the Rf value. In terms of zone position,
studies have shown that maximum resolution is obtained in the Rf 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.
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. SAMPLING AND SAMPLE PREPARATION
A. 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 into 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.
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 pipet tips, disks,
fixed 96-well plates, flexible 96-well plates, 384-well plates, and large-volume cartridges
and flash Chromatography columns (58). The well plates are compatible with the use of
TLC for drug discovery combintorial chemistry high-throughput applications (59).
The sorbents available from Varian in their Bond Elute columns are illustrative of the
products of other SPE product manufacturers. These include the following.
Nonpolar extraction: C18, octadecyl; C8, octyl; C2, ethyl; CH, cyclohexyl;
PH, phenyl; CNE, end-capped cyanopropyl
Polar extraction: CN, cyanopropyl; 2OH, diol; SI, silica; NH2,
aminopropyl
Cation-exchange extraction: SCX, benzenesulfonic acid (strong); PRS,
propylsulfonic acid (strong); CBA, carboxylic acid (weak)
Anion-exchange extraction: SAX, quaternary amine (strong); PSA,
primary/secondary amine (pKa 10.1, 10.9); NH2, aminopropyl (weak);
DEA, diethylaminopropyl (weak)
Varian also supplies a covalent extraction phase (PBA, phenylboronic acid) for
nucleotides, nucleosides, carbohydrates, and catecholamines and specialty phases for
determination of grease, oils, fats, phenols, PAHs, organic acids, tricyclics,
benzodiazepines, Pharmaceuticals, explosives, pesticides, and neutral, basic, and acidic
drugs. Bond Elute sorbents are supplied in 50 mg to 10 g weights in cartridges up to 60
mL in volume.
Figure 1 shows a Speedisk (J.T.Baker) Positive Pressure Processor for semiautomated
elution of 1,3, and 6 mL SPE columns in batches of 1–48 samples. Totally automated
SPE systems are also available commercially (47).
SPE is used to concentrate solutes from dilute solution, e.g., to collect nonpolar
organic constituents on C18 cartridges. The analytes are recovered by elution from the
column with a few milliliters of an appropriate solvent and spotted for TLC. The
concentration factor obtained for this method, which has been termed “trace enrichment,”
is the ratio of the sample volume to the elution volume. SPE can also be used to purify
concentrated solvent extracts in place of classical large columns that require up to
hundreds of milliliters of elution solvents. A sequence of eluents of increasing strength
can be used to elute compounds with different polarities in different frac-
Figure 1 Speedisk 48 Positive

Pressure Processor for SPE.
(Photograph supplied by Mallinckrodt
Baker Inc.)
tions, and multiple SPE columns can be connected in series for improved cleanup and/or
fractionation.
The basic steps of SPE, illustrated for the most commonly used reversed-phase C18
cartridge, can be summarized as follows:
Conditioning. The cartridge is prepared for receiving the sample by

passing a volume of an appropriate solvent followed by a volume of liquid
similar to the sample matrix. For the C18 cartridge, methanol is passed
through followed by water for extraction of an aqueous sample.
Retention. The sample is applied, and the analyte and other
components with attraction for the sorbent are retained. Non- or weakly
attracted components will pass through, providing the first stage of
cleanup. With the C18 cartridge, the most polar interferences will elute
first, and retention increases as polarity decreases.
Rinsing. One or more solvents with decreasing polarity are passed

through to elute interferences that are more polar than the analyte but keep
the analyte on the column.
Elution. A sufficiently nonpolar eluent is passed to remove the analyte.
Interferences more nonpolar than the analyte will have a greater attraction
for the C18 sorbent and remain uneluted.
The following is an abbreviated guide to the SPE of different classes of sample analytes:
Nonpolar extraction. A polar solution (water, buffers) containing a

nonpolar analyte is applied to a C18, C8, C2, CNE, CH, PH, or 2OH
column that was preconditioned with methanol followed by water or
buffer (see listing above for abbreviations). The sample must be buffered,
if necessary, to suppress analyte ionization. Polar interferences are
removed by washing with water or buffer or a weak organic-aqueous
solvent that will not elute the analyte [e.g., water (buffer)-methanol (9:1)].
The analyte is eluted with a nonpolar solvent such as methanol,
acetonitrile, tetrahydrofuran (THF), hexane, or methylene chloride.
Polar extraction. A nonpolar solution containing a polar analyte is
applied to an SI, CN, 2OH, or NH2 column that was preconditioned with
the nonpolar solvent in which the analyte is dissolved, such as hexane or
chloroform. Viscous samples are diluted in a non-polar solvent, and water
is removed from the sample, e.g., by filtration through Whatman phase-
separating paper. Nonpolar interferences are removed by washing with a
nonpolar solvent or a polar-nonpolar mixture that is not strong (polar)
enough to elute the analyte. The analyte is recovered by elution with a
polar solvent such as methanol or isopropanol.
Anion-exchange extraction. An aqueous, low ionic strength sample
(water, plasma, diluted urine) containing inorganic or organic anions is
applied to an SAX, NH2, PSA, or DEA column. Both the chosen column
and the analyte must be ionic for exchange to occur. The column is
conditioned with methanol followed by a buffer whose pH is 2 units
above the pKa of the analyte and <7.8 for NH2, PSA, and DEA columns.
The sample pH is adjusted as above for conditioning and applied to the
column. Interferences are removed by washing with the sample buffer and
with an organic solvent such as acetonitrile or methanol, if necessary. The
analyte is eluted with a buffer whose pH is at least 2 units below the
analyte pKa, a buffer whose pH is 2 units above the column pKa, or a
buffer of high ionic strength (>0.1 M). The eluents can be totally aqueous
or aqueous-organic mixtures; addition of an organic modifier such as
methanol may improve analyte recovery.
Cation-exchange extraction. An aqueous, low ionic strength sample
containing inorganic or organic cations is applied to an SCX, PRS, or
CBA column preconditioned with methanol followed by a buffer whose
pH is 2 units below the analyte pKa and >6.8 for the CBA column. The
sample pH is adjusted in the same manner. Interferences are eliminated by
elution with the sample buffer and with an organic solvent, if necessary.
The analyte is eluted with a buffer at least 2 units above the analyte pKa, a
buffer of pH <2.8 for the CBA column, or a buffer of high ionic strength
(>0.1 M). Addition of an organic modifier such as methanol may improve
analyte recovery.
Examples of applications of SPE prior to TLC analysis include analysis for pesticides in
fruits and vegetables according to the official German multimethod S19 using SPE on
silica gel and amino cartridges prior to HPTLC with gradient elution AMD (60);
oxygenated cholesterol derivatives in plasma using silica gel SPE (61); quinoline and
quinuclidine alkaloids in pharmaceutical preparations using cation-exchange SPE (62);
rutin in glycerinic plant extracts using Envi-18 (Supelco) cartridges (63); and aflatoxins
in a variety of foods using phenyl, silica, C18, and Florisil-C18 cartridges (64). A strategy
for choosing SPE cartridge elution solvents based on the PRISMA TLC mobile-phase
optimization procedure was demonstrated for extraction of furocoumarin isomers and
flavonoid glycosides from medicinal and aromatic plants (65).
The use of immunoaffinity columns for sample cleanup is among the newest sample
preparation procedures. Immunoaffinity cleanup was used after methanol extraction for
determination of aflatoxins B-1, B-2, G-1, and G-2 in various food matrices by TLC-
densitometry (66).
Of the current sample preparation methods (46, 48), only SPE (above) and SEE have
had substantial use in combination with TLC. Automated Soxhlet extraction, microwave-
assisted extraction (MAE), and accelerated solvent extraction (ASE) have good potential
for preparing solid samples for TLC analysis, but published methods have not yet
appeared. Stahl first interfaced SEE with TLC in 1977, and there has been increasing
interest in developing new methods in recent years. Examples of SFE-TLC analyses
reported include cyanizine herbicide in soil (67); flavonoids in Scutellariae radix (68);
aloin and aloe-emodin in consumable aloe products (69); semivolatile compounds in
cassia and cinnamon (70); and residues of 20 pesticides of multiple classes in soil (71).
Hydroperoxides in combustion products were separated from solid matrices using SEE
with on-line transfer to TLC plates (72).
6. Additional Sample Preparation Procedures

Additional procedures performed prior to TLC analysis, depending on the sample type,
include drying, grinding, freeze-drying (removal of water), drying of extracts (passage
through a drying column or phase-separating filter paper or addition of a drying agent
such as sodium sulfate), and the steps described below in this section.
Desalting is often required for samples such as urine, serum, and tissue culture media
in order to eliminate streaking and the formation of unresolved zones in the TLC of
amino acids, carbohydrates, and other hydrophilic compounds. Salts are removed from
samples by performing ion exchange, using a desalting column, dialysis, and passage
through a nonpolar sorbent. A simple desalting procedure suitable for 0.1–0.2 mL of
urine, serum, or saline solution is the following. The sample is dried under air at 45°C
and then extracted with 1 mL of 0.5% HC1 in 95% ethanol for 24 h. The extract is
evaporated to dryness and the residue dissolved in 100 µL of ethanolic HC1 prior to
spotting for TLC (73). The ion retardation resin AG 11 A8 (Bio-Rad Laboratories Inc.)
and mixed bed cation/anion-exchange resins (e.g., Bio-Rad AG 501) have been used
successfully for desalting samples prior to TLC.
7. Deproteinization
When proteins may interfere with TLC analysis, they must be removed by
deproteinization procedures. A suitable procedure for an approximate 50 µL sample of
serum involves addition of 100 µL of methanol to precipitate the protein followed by
shaking and centrifugation of the mixture to obtain a clear supernatant. The technique has
been used to deproteinize biological fluids prior to their analysis for drugs (74). Proteins
in samples such as serum, urine, tissue, and milk can be precipitated by addition of
trichloroacetic acid (75), perchloric acid, or sulfosalicylic acid followed by centrifugation
and removal of the supernatant, which may or may not require further cleanup prior to
TLC. Protein removal from various types of samples has also been carried out by pH
modification, denaturation with chaotropic agents or organic solvents, addition of a
compound that competes for binding sites, and the use of restricted-access media.
8. Derivatization
The preparation of derivatives in TLC was reviewed by Edwards (76), who documented
the application of derivatization techniques to a wide range of compounds including
amino acids, steroids, drugs, and environmental pollutants. Fluorescent derivatives for
TLC were reviewed by Wintersteiger (77).
One of the major advantages of TLC is the use of derivatization postchromatography
for the purpose of zone detection. This is normally achieved by spraying the layer with
(or dipping it into) a solution of an appropriate reagent or reagents and then drying or
heating to complete the reaction. Hundreds of such reagents have been described to cause
zones to absorb visible or ultraviolet radiation or to become fluorescent for organic
species in general or to react selectively with particular compound classes (see Sec.
VIII.A). Examples include spraying with ninhydrin reagent to produce purple spots for
amino acids, or with a solution of diazonium reagent (prepared from p-nitroaniline, HCl,
and sodium nitrite) to detect phenols and aromatic amines as orange zones.
Postchromatographic derivatization allows the reaction of all standards and samples
simultaneously under the same conditions, and the separation properties of the solutes are
not changed by the reaction.
Prechromatographic derivatization is advantageous when the parent compound is too
volatile for TLC but the derivative is less volatile, the derivative is easier to separate from
other sample constituents, the derivative has greater stability (e.g., resistance to oxidation
or decomposition), the derivative is more successfully extracted and/or cleaned up, or the
derivative is more sensitively and/or selectively detected. A disadvantage of
prederivatization is that the introduction of usually high molecular weight functional
groups into the derivative may equalize the chromatographic properties of similar
substances and make separation more difficult. In addition, prederivatization of each
sample prior to its application can be tedious and time-consuming, by-products of the
reaction may interfere with the TLC separation, or the presence of excess reagent may
cause a background that interferes with quantification by scanning. It is possible in some

cases to derivatize in situ prior to chromatography. This is usually done by applying a
spot or band of excess reagent to the origin and overspotting the sample while the reagent
zone is still moist, followed by application of heat to accelerate the reaction, if necessary.
Zones of sample and reagent should be chromatographed on adjacent lanes for
comparison. Many different kinds of in situ prechromatographic derivatization have been
reviewed (78).
The following are examples of analyses that include the formation of derivatives prior
to TLC: the formation of fluorescent dansyl derivatives for determination of biogenic
amines in red wine (79) and other foods (80) and of colored thiocarbamoyl derivatives of
biogenic amines (81); the use of p-benzoquinone for derivatization of 2-
(methylamino)ethanol and other primary and secondary amines (82); the separation of p-
dimethylaminobenzaldehyde from p-dimethylamino-cinnamaldehyde after derivatization
with diphenylamine (83); determination of bisoprolol, labetalol, and propafenone as
dabsyl derivatives in pharmaceutical preparations (84); and determination of the toxin
fumonisin B-1 in corn after immunoaffinity column cleanup and derivatization (85). In
many cases, enantiomers have been resolved by TLC after the formation of derivatives,
e.g., amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, and
separated on RP plates (86). The latest methodology involves separation of enantiomers
of compounds such as chiral drugs by TLC without their prior derivatization (87).
9. Evaporation of Solutions
Most sample preparation procedures require concentration or evaporation to dryness of
sample extracts, combined partition solvent batches, or column effluents. It is important
that evaporations be carried out without loss or degradation of the analyte, and studies
may be required to determine which of the available methods is best to use in each
particular situation.
A common method of concentration uses a rotary evaporator with an attached round-
bottomed flask. A helpful variation is to place the solution in a Kuderna-Danish
evaporative concentrator flask with attached lower calibrated tube (Kontes), so that the
concentrate ends up in the tube and can be applied to the layer without transfer.
Nitrogen blowdown is the recommended method for concentration of small volumes
of volatile organic solvents. Gas is supplied to the sample, held in a tube or vial, through
Tygon tubing connected to a glass capillary. The sample is warmed in a 40–60°C water
bath to speed evaporation. Various commercial devices that allow simultaneous
blowdown of multiple samples are available.
10. Reconstitution of Evaporated Residues

It is common practice to evaporate solutions just to dryness and then dissolve the residue
in an exact volume of the same or a different solvent, from which a known aliquot or the
total sample is applied to the layer. The best initial zones on silica gel are obtained if the
solvent is highly volatile and as nonpolar as possible, consistent with complete solubility
and stability of the analyte(s). By use of a nonpolar solvent, purification can be achieved
if some polar impurities in the residue are left undissolved (selective solvation). Solvents
with a high boiling point or polarity are difficult to remove from the sorbent during
application. If a small amount of solvent is retained after application, it can adversely
affect the separation by causing zone spreading or deformation or a different Rf value.
Care must be taken, however, because hot air used to dry solvent at the origin can
decompose labile substances on the surface of an active sorbent. A volatile sample
solvent promotes the production of small, regular initial zones, but containers must be
kept tightly sealed except when filling the sample application device.
IV. SORBENTS AND LAYERS
Sorbent materials and layers are described in Chapter 4 of this Handbook and Chapter 3
of Ref. 1 and in a review paper (88) and an encyclopedia article (89).
A great variety of commercial precoated layers are available for TLC on glass, plastic,
or aluminum foil supports in 20×20 cm size. The most common layer thickness for
analytical TLC is 250 µm, but cellulose and polyamide layers are often 100 µm. For
mechanical stability, 0.1–20% of a gypsum (calcium sulfate), starch, or organic polymer
binder [e.g., poly(acrylic acid)] is added to the sorbent slurry from which the layer is cast.
Plates with gypsum binder, which are known as “soft layers” and are designated with a
G, must be used with greater care than “hard” organic polymer-bound layers to avoid
abrasive conditions. Gypsum binder allows the use of sulfuric acid charring techniques,
and sample zones can be easily scraped from the glass support for subsequent elution of
compounds from the sorbent. Binder-free silica gel plates containing a small amount of
colloidal silica to aid layer adherence are also available. For detection of zones by
fluorescence quenching, plates are impregnated with indicator compounds (e.g.,
manganese-activated zinc silicate) that cause the layer to fluoresce uniformly when
exposed to 254 or 366 nm UV light. Glass is the most inert support material, and its
planarity is advantageous when the layer will be scanned for quantitative analysis.
Procedures and devices for preparing homemade plates are described in Chapter 3 of the
third edition of Fried and Sherma (1). Homemade plates, the quality of which is almost
never equivalent to that of commercial plates, are rarely made except when a needed
layer is not available or cost is a major consideration.
To remove extraneous materials that may be present due to manufacture, shipping, or
storage conditions, it is advisable to preclean plates before use. This has often been done
by predevelopment to the top with dichloromethane-methanol (1:1) or the mobile phase
to be used for the analysis. The following two-step HPTLC plate cleaning method has
been proposed (90) for surface residue removal in critical applications when optimum
sensitivity is required for detection and quantification: Develop the plate to the top with
methanol, air dry for 5 min, totally immerse the plate in a tank filled with methanol, air
dry for 5 min, oven dry for 15 min at 80°C, and cool in a desiccator before use. The
routine activation of adsorbents at 70–80°C for 30 min, or at a higher temperature, is
often proposed in the literature, but this treatment is not usually necessary for commercial
plates unless they have been exposed to high humidity. RP plates do not require
activation prior to use. Suggestions for initial treatment, prewashing, activation, and
conditioning of different types of glass- and foil-backed layers have been published (91).
A. Adsorbents
Silica gel is by far the most frequently used layer material for adsorption TLC. Some
characteristic properties, including porosity, flow resistance, particle size, optimum
velocity, and plate height, have been tabulated for three popular brands of silica gel TLC
and HPTLC plates (38). Separations take place primarily by hydrogen bonding or dipole
interaction with surface silanol groups by using lipophilic mobile phases, and analytes are
separated into groups according to their polarity. Typical properties of TLC silica gel are
a silanol group level of approximately 8 µmol/m2; pore diameter of 40, 60, 80, or 100 Å;
and specific pore volumes of 0.5–2.0 mL (89). Specific differences in the types and
distributions of silanol groups for individual sorbents may result in selectivity
differences, and separations will not be exactly reproducible on different brands of silica
gel layers (25). Other TLC adsorbents include aluminum oxide (alumina), magnesium
oxide [used mostly for carotenoid pigment separations (92)], magnesium silicate
(Florisil) (93), polyamide, and kieselguhr (94).
Alumina (95) is a polar adsorbent that is similar to silica gel in its general
chromatographic properties, but it has an especially high adsorption affinity for carbon-
carbon double bonds and better selectivity toward aromatic hydrocarbons and their
derivatives. The alumina surface is more complex than silica gel, containing hydroxyl
groups, aluminum cations, and oxide anions, and pH and hydration level alter separation
properties (25). It is available in basic (pH 9–10), neutral (7–8), and acid (4–4.5) forms.
The specific surface area of aluminas range from 50 to 250 m2/g (89). The high density of
hydroxyl groups (~13 µmol/m2) leads to a significant degree of water adsorption, and
alumina layers are usually activated by heating for 10 min at 120°C before use (89).
Poly amides 6 (Nylon 6; polymeric caprolactam) and 11 (polymeric undecanamide)
have surface—CO—NH—groups and show high affinity and selectivity for polar
compounds that can form hydrogen bonds with the exposed carbonyl groups. However,
depending on the type of analyte and mobile phase, three separation mechanisms can
operate with poly amide: adsorption, partition (normal- and re versed-phase), and ion
exchange. This has led to separations of compounds from a wide array of chemical
classes such as amino acids, phenols, phenolic compounds, carboxylic acids,
cyclodextrins (96), coumarins, and flavonoids (97). Polyamide has been impregnated
with various metal salts to improve the separation of sulfonamides (98). Separation
numbers for a series of higher fatty acids and alcohols were determined to be 8–12 for
polyamide and 4–9 for cellulose (99).
Homemade mixed sorbent layers have been used by various workers to increase the
resolution of certain samples compared to that obtained on the separate phases. Binary
layers that have been reported include silica gel-alumina (100), kieselguhr-alumina,
alumina-calcium sulfate, mag-nesia-kieselguhr, cellulose-silica gel, poly amide-silica gel,
polyamide-kieselguhr, poly amidecellulose, poly amide-glass powder (similar to silica
gel), silica gel-kieselguhr (101), and alumina-cellulose (102). The properties of these
mixed layers are usually somewhere between those of the two separate phases but are
impossible to predict or explain with certainty. Information on and applications of mixed
layers are mostly contained in older standard TLC texts and reviews.
B. Partition, Preadsorbent, and Impregnated Layers

Compounds that have the same polarity and functional group and migrate together on
silica gel can often be resolved by partition TLC. Crystalline cellulose (AVICEL) or
high-purity fibrous cellulose serves primarily as a support material for the NP liquid-
liquid partition TLC of polar substances, such as amino acids (103), and water-soluble
biopolymers, although adsorption effects cannot be excluded in many cases. The
stationary phase is either water or an impregnated polar liquid such as
dimethylformamide. Cellulose used to prepare thin layers differs from that in
chromatography paper mainly by having shorter fiber length (2–20 µm), resulting in the
same migration sequence for a series of compounds developed with a given mobile phase
but less diffusion and higher efficiency than in paper chromatography.
Kieselguhr (diatomaceous earth) (104) and synthetically prepared silicon dioxide
(Merck silica 50,000) (105) are small surface area, weak adsorbents that are used as the
lower 2–4 cm inactive sample application and concentrating zone in the manufacture of
silica gel and C18 preadsorbent plates. Samples applied to the preadsorbent region usually
develop into sharp, narrow bands at the preadsorbent/sorbent interface, leading to
efficient separations with minimum time and effort in manual application of samples and
possible sample cleanup by retention of interferences in the preadsorbent.
Layers have been impregnated with buffers, chelating agents, metal ions, or other
compounds to aid in the resolution or detection of certain compounds (see Ref. 106 for a
review). If plates are prepared in the laboratory, the reagent is usually added to the
stationary-phase slurry. Reagents are applied to precoated plates by spraying, brushing,
horizontal or vertical dipping, development, or overdevelopment (107). Analtech
precoated plates are available already impregnated with potassium oxalate to facilitate
resolution of polyphosphoinositides, magnesium acetate for phospholipids, 0.1 M NaOH
for organometallics and acidic compounds, silver nitrate for compounds with carbon–
carbon double bonds such as fatty acids (107), and carbomer for mannitol and sorbitol
analysis according to several Pharmacopoeia methods, as well as plates containing
ammonium sulfate for detection of compounds as fluorescent or charred zones after
heating (vapor-phase fluorescence detection). Other reagents that have been added to thin
layers to improve separations include ion-pairing reagents (108), molybdic acid (for
separation of carbohydrates), boric acid (carbohydrates and lipids), poly cyclic aromatic
hydrocarbons (PAHs), (formation of charge transfer complexes with numerous organic
compounds), surfactants (sulfa drugs and substituted pyrazoles) (109), EDTA (reduces
tailing of drugs) (110), urea (wax esters and hydroxybenzenes), ferric ion (carboxy- and
hydroxybenzenes), cupric ion (glucose and sorbitol), caffeine (PAHs), and ammonium
sulfate (surfactants). The separation of amino acids and their derivatives and enantiomers
by impregnated TLC was reviewed by Bhushan and Martens (110a).
C. High-Performance Layers
High-performance (HP) plates (10×10 or 10×20 cm) are produced from sorbents having
narrow pore and particle size distributions and an apparent particle size of 5–7 µm instead
of 8–10 µm for 20×20 cm TLC plates (23). Layer thickness is usually 100–200 µm for
HPTLC plates compared to 250 µm for TLC, but ultrathin (10 µm) layers of monolithic
silica gel have recently been described (110b).
High-performance layers are more efficient, leading to tighter zones, better resolution,
and more sensitive detection. Flow resistance is higher (migration time per centimeter is
slower), but overall development time is shorter because smaller migration distances are
used for HPTLC than for TLC (typically 3–8 cm versus 10–16 cm). The low flow rate
through fine-particle HPTLC plates led to the development of forced-flow methods.
Sample sizes are generally 0.2–1 µL for HPTLC and 1–3 µL for TLC, although the upper
levels of these ranges can be exceeded when spotting with the Linomat instrument or
using preadsorbent layers.
Silica gel is the most widely used type of HP plate, but other HP layers, including
bonded phases, are also commercially available. Among the newest layers are Merck’s
TLC and HPTLC silica gel 60 plates (60 Å pore size) with imprinted identification codes
for use in documentation when analyses are performed according to good manufacturing
practice (GMP) and good laboratory practice (GLP) standards (52). Merck also sells two
new HPTLC layers with spherical silica gel: HPTLC plates with LiChrospher Si60F254S
(0.2 mm layer thickness, 7–8 µm mean particle size), and HPTLC aluminum sheets with
Si60F254S Raman (0.1 mm layer thickness and 3–4 µm particle size). Layers with
spherical particles offer better efficiency, spot capacity, and detection limits than those
with nonspherical particles. The silica gel matrix on the sheets is designed to have the
least possible spectral interference for direct coupling of TLC with Raman spectrometry
(see Sec. VIII.B).
TLC and HPTLC are compared in Chapter 2 of Ref. 1.
D. Bonded Layers
Reversed-phase TLC, in which the stationary phase is less polar than the mobile phase,
was originally carried out on silica gel or kieselguhr layers impregnated with a solution of
paraffin, squalane, silicone oil, octanol, or oleyl alcohol. Analtech sells RP plates with
hydrocarbon liquid phase physically adsorbed onto the surface of a silica gel layer.
Impregnated plates of this kind require the use of aqueous and polar organic mobile
phases saturated with the stationary liquid, and they cannot tolerate the use of nonpolar
organic solvents, which will strip the coating from the support.
Bonded phases with functional groups chemically bonded to silica gel eliminate
stripping of the stationary liquid from the support by incompatible mobile phases.
Alkylsiloxane-bonded silica gel with CH3, C2H5, C8H17, and C18H37 (111) functional
groups are most widely used for RP-TLC of organic compounds (polar and nonpolar
homologous compounds and aromatics), weak acids and bases after ion suppression with
buffered mobile phases, and strong acids and bases using ion-pair reagents. Layers from
different companies but with the same bonded group can have different percentages of
carbon loading and give different results. The hydrophobic nature of the layer increases
with both the chain length and the degree of loading of the groups. Alkylsiloxanebonded
layers with a high level of surface modification are incompatible with highly aqueous
mobile phases and are used mainly for normal-phase separations of low-polarity
compounds (25). Problems of wettability and lack of migration of mobile phases with
high proportions of water have been solved by adding 3% NaCl to the mobile phase
(Whatman layers) or preparing “water-wettable” layers with a slightly larger particle size,
less exhaustive surface bonding, and a modified binder. The latter layers with a low
degree of surface coverage and more residual silanol groups exhibit partially hydrophilic
as well as hydrophobic character and can be used for RP-TLC and NP-TLC. Chemically
bonded phenyl layers are also classified as re versed-phase, but their use has only seldom
been reported in the literature.
Hydrophilic bonded silica gel containing cyano (112), amino (113), or diol (114)
groups bonded to silica gel through a trimethylene chain [—(CH2)3—] are compatible
with aqueous mobile phases and exhibit multimodal mechanisms. Polarity varies as
follows: unbonded silica> diol-silica>amino-silica>cyano-silica>reversed-phase
materials (89). Cyano layers can act as a normal or reversed phase, depending on the
characteristics of the mobile phase, with properties similar to a low-capacity silica gel
and a short-chain alkylsiloxane bonded layer, respectively (25). Amino layers are used in
NP and weak anion-exchange modes. In NP-TLC, compounds are retained on amino
layers by hydrogen bonding as with silica gel, but the selectivity is different. Charged
substances such as nucleotides or sulfonic acids can be separated by ion exchange using
acidic mobile phases. Although there is limited retention in RP-TLC, the separation of
oligonucleotides on amino layers based on differences in hydrophobic properties of the
compounds has been reported. Diol plates can operate with NP- or RP-TLC mechanisms,
depending on the mobile phase and solutes. Polar compounds show reasonable retention
by hydrogen bond and dipole-type interactions in the former mode, and in the RP mode
retention is low but higher than with amino layers. A study of mixed mechanisms on
cyano, amino, and diol layers was reported (115).
E. Layers for Enantiomer Separations

Commercial layers are available for separation of enantiomers by the mechanism of
ligand exchange under the names Chiralplate (Macherey-Nagel) and HPTLC CHIR
(Merck). These con sist of a glass plate coated with a reversed-phase silanized silica gel
and impregnated with the Cu(II) complex of (2S, 4R,2′RS)-N-(2′-hydroxydodecyl)-4-
hydroxyproline. Separation is based on the formation of diasteriomeric chelate complexes
between the central cupric ion, the chiral se lector, and the solute. Enantiometric
resolution is achieved if the antipodes of the chiral solute form complexes of different
stabilities. The history of chiral ligand exchange in TLC and column liquid
chromatography has been reviewed (116).
In addition to ligand exchange, enantiomeric separations have been carried out using
cyclodextrin-containing mobile phases with hydrophobic C18 (117) and cellulose
triacetate (118) layers (inclusion TLC). Chiral selectors have been impregnated into silica
gel layers for NP-TLC enantiomer separations, e.g., the macrocyclic antibiotic
vancomycin for DL-amino acids (199) and L-lysine and L-arginine for β-adrenergic
blocking agents (120). Unmodified cellulose has been used for separation of enantiomeric
amino acids and peptides and other compounds (e.g., 121). Optical isomers can be
derivatized and separated without using impregnated plates or a chiral mobile phase, e.g.,
amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and separated
by RP-TLC (86).
The newest approach is the preparation of molecularly imprinted polymers (MIPs) for
use as chiral stationary phases in TLC. For example, the direct separation of enantiomers
of adrenergic drugs on MIPs of (−)-pseudoephedrine and (−)-norephedrine was
demonstrated as a rapid, sensitive, and reliable method for quality control of these
compounds (121a). Beta-blocking drugs and nonsteroidal anti-inflammatory drugs have
also been separated on molecularly imprinted chiral layers (121b).
Enantiomeric separations by TLC have been reviewed (122–124), and this topic is
covered in Chapter 17 of this Handbook.
F. Miscellaneous Layers
Cellulose has been surface-modified to produce RP (acetylated cellulose), weakly basic
anion-exchange [polyethyleneimine (PEI), aminoethyl (AE), diethylaminoethyl (DEAE),
and ECTEOLA], or weakly acidic cation-exchange [cellulose phosphate (P) and
carboxymethyl-cellulose (CM)] layers. These cellulose exchangers have open structures
that can be penetrated by large hydrophilic molecules such as proteins, enzymes, and
nucleic acids.
Polygram Ionex-25 precoated sheets (Macherey-Nagel) are polyester sheets coated
with a mixture of silica, a polystyrene-based strong acid cation-exchange or strong base
anion-exchange resin, and a binder. The cation exchanger has been used to separate and
identify amino acids in biological samples (125), and both are suited to inorganic ion
separations. A large variety of inorganic ion exchangers, such as titanium(IV) silicate
(126), have been prepared and used mostly for metal ion separations.
Size-exclusion gel TLC has been carried out on dextran (Sephadex) gels with
controlled pore sizes. These layers, which are used to estimate molecular weights and
separate and determine biological macromolecules (e.g., enzymes and serum proteins),
are used in totally swollen condition and developed continuously in the descending
direction.
Combination layers with a C18 strip adjacent to a silica gel layer (Whatman Multi-K
CS5) or a silica gel strip adjacent to a C18 layer (SC5) are available for 2-D TLC with
diverse mechanisms (RP phase and adsorption).
G. Preparative Layers
Preparative silica gel plates are available precoated with a layer thickness of 500–2000
µm. Particle size is typically 5–40 µm with a 25 µm average, but Mallinckrodt-Baker
manufactures a preparative plate with 5 µm spherical particles. Analtech offers a unique
tapered layer for capillary flow preparative separations (see Sec. V.D) and precast
HPTLC silica gel GF rotors (Fig. 2) with 1000–8000 µm nominal thickness for use with
the Cyclograph and Chromatotron centrifugal forced-flow PLC instruments (see Sec. XI).
V. APPLICATION OF SAMPLES
Samples and standards prepared for TLC are dissolved in an appropriate solvent at a
concentration that will allow eventual detection of the solutes of interest. Typically 1–5
µL containing 1 ng to 10 µg of solute is applied in the form of spots or narrow bands to

TLC plates. Because the starting zones should be as small as possible for efficient
separation, larger volumes are spotted by repeated applications of a small volume to the
same origin with solvent evaporation between increments. Evaporation of the sample
solvent must be complete so that the selectivity of the mobile phase and the spot position
in the chromatogram are not altered. This drying is achieved and the sample application
speeded up if a stream of cool or heated air or inert gas is gently blowing across the plate
being spotted. After application, initial zones are usually thoroughly dried with a hair
drier or in an oven. Ideally, the sample should be distributed homogeneously throughout
the starting zone (38).
Sample volumes must be reduced to realize fully the greater efficiency of HPTLC
layers, and 100–200 nL is typically applied. Detection limits are usually 5–10 times
better for HPTLC than for TLC. Optimum initial spot size for HPTLC is about 1–1.5
mm, whereas initial spots for TLC are typically 3–6 mm. Initial zones that are overloaded
with sample will form poorly separated tailed zones during development.
Sample application is one of the main sources of error in quantitative TLC, and great
care should be taken to choose a reliable application device and optimize techniques if
accurate and precise analyses are to be realized. Sample volumes must be accurately
known, and exact posi-
Figure 2 Precast silica gel GF rotor.

(Photograph supplied by Analtech.)
tioning of initial zones is critical when measurements are to be made by scanning.
Automated sample application is preferred for best results in quantitative TLC.
A. Choice of the Sample Solvent

Samples and standards are best prepared in a solvent that dissolves the analytes
completely, is volatile, has low viscosity, wets the sorbent layer, and is a weak
chromatographic solvent for the analytes. In practice, it may be impossible to find a
solvent with all of these properties. For silica gel TLC, it is important to use the weakest
(least polar) solvent that allows quantitative dissolution and spotting of the sample, so
that preliminary development and separation within the initial spot at the origin does not
occur, resulting in significant loss in separation efficiency. The Rf of the compounds of
interest should be <0.1 in this solvent. The choice of a weak solvent is more difficult for
RP layers because solvents that wet the layer (e.g., acetonitrile, methanol) may be strong
(nonpolar) enough to cause predevelopment of the spot. If all or part of the sample is
solidified or adsorbed onto the layer surface, a slow dissolution effect can cause
significant tailing of the spots.
B. Application of Spots
Instruments and techniques for a sample application are described in Chapter 5 of this
Handbook and Chapters of Ref. 1.
Samples and standards are applied to the layer as small round spots by using one of a
variety of application devices, for example, a wooden stick with flattened end, glass
capillary pipet, or syringe with a 90° needle tip. Drummond microcap micropipets,
available in virtually any size between 0.1 and 200 µL (Fig. 3), and 10–50 µL digital
microdispensers (Fig. 4) are highly recommended for manual applications for both
qualitative and quantitative TLC. For linear or circular HPTLC, initial zone diameter
should not exceed 1.5 mm for maximum resolution. Spots for HPTLC can be applied to
an exact layer position using a 100 or 200 nL Pt-Ir pipet held in a mechanical device that
electromagnetically brings the pipet into reproducible contact with the layer without
surface damage (Camag Nanomat). Camag and Desaga also supply completely
automated devices with which selectable volumes of samples and standards are applied
from vials
Figure 3 Application of initial zones to

a glass-backed 20×20 cm channeled
preadsorbent silica gel plate using a
Microcap micropipet. (Photograph
supplied by Analtech.)
Figure 4 Twenty-five microliter digital

microdispenser. (Photograph supplied
by Drummond Scientific.)
as spots or bands in any order to specified layer positions through a capillary pipet that is
rinsed with solvent between applications.
C. Formation of Bands
Bands or streaks of sample are applied manually, are applied automatically with the
Camag Linomat, are formed automatically during development by use of plates with a
preadsorbent or concentrating area (see Sec. IV.B), or are produced by predevelopment
on conventional plates. Manual application essentially involves placing a contiguous
series of spots from a syringe or micropipet side by side. Even with practice, it is difficult
to do this uniformly and reproducibly on a conventional plate. The Linomat, which is
based on movement of the plate underneath a fixed syringe from which a nitrogen
atomizer sprays the sample onto the origin at a constant rate, is advantageous because
larger sample volumes [40 µL or more (127)] can be concentrated during the application
process compared to other HPTLC devices, and variable volumes of the same standard
solution can be applied for calibration in densitometry.
In using preadsorbent plates, samples are spotted or streaked onto the preadsorbent
area, and narrow, accurately aligned, homogeneous bands form automatically at the
interface with the sorbent upon development. When laned or channeled plates are used,
the length of the band is confined within the channel. Sample application is fast and
simple for relatively large volumes (up to~50 µL for TLC and 25 µL for HPTLC). High
efficiency can be obtained for HPTLC by spotting larger volumes of dilute solutions
rather than nanoliter volumes of highly concentrated solutions. Crude samples can be
directly spotted, and salts and other impurities may be retained in the preadsorbent and
not interfere with sample resolution or detection. Figure 5 shows the sequence of zone
separation in various stages of development on a preadsorbent TLC plate.
The final method for forming initial bands is to concentrate a large spot into a line by
partial predevelopment of the layer with a strong solvent in which all components move
with the solvent front. After the plate is dried, it is developed with the mobile phase
needed to provide resolution.
It has been shown that bands give sharper separations and lower detection limits than
spots and are advantageous for densitometry because the length of the scanner light beam
can be made shorter than the length of the band (one-half to two-thirds of the original
band length). This method of aliquot scanning minimizes the need to exactly position the
zone within the beam.
D. Sample Application for Preparative Layer Chromatography

The amount of sample that can be applied to a preparative layer depends on the mixture
to be separated, the layer, and the development method. Adsorption TLC permits the
application of
Figure 5 Sequence of development

stages on a preadsorbent layer.
larger samples than partition TLC. Capacity increases roughly as a function of the
thickness of the layer.
Samples for preparative layer chromatography are applied as bands across thicker
layers. This can be done manually by allowing sample to flow from a pipet drawn along
the edge of a ruler. The Camag Linomat, described above, is among the special
instruments designed to apply samples more uniformly.
Analtech Preparative Uniplate-T wedge-shaped layers are tapered from a thickness of
1700 µm at the top to 300 µm at the bottom and have a 700 µm thick preadsorbent area at
the bottom for manual or instrumental sample application. Sample concentration occurs
in the preadsorbent zone, and low Rf bands tend to separate better in the thinner lower
layer region.
VI. SELECTION AND OPTIMIZATION OF THE MOBILE PHASE
Selection of mobile phases is discussed in Chapter 6 of Ref. 1 and in Ref. 25. Criteria and
strategies for optimization of mobile phases are covered in Chapter 3 of this Handbook,
Chapter 3 of the previous edition of this handbook written by a different author, and Refs.
25, 128, 129, and 130. Solvent systems for different compound classes are given in the
respective applications chapters in Part II of this Handbook and in the 29 CRC Handbook
of Chromatography volumes edited by J.Sherma and G.Zweig between 1972 and 1993.
The flexibility of TLC relative to HPLC is enhanced by the greater choice of solvents
available for preparing TLC mobile phases. The choice of solvents for HPLC is limited
by the re-quirements for their chemical and physical properties imposed by the nature of
the method. HPLC is a closed system operated under high pressure with on-line
detection, most often using a UV monitor, and the column is continually reused. Solvent
components with high vapor pressure (e.g., ethyl ether) or UV absorbance (benzene) or
those that might degrade the column (NaOH) are difficult to use in HPLC but are readily
applicable to TLC.
Single-development, capillary flow TLC typically produces <5000 theoretical plates
and a zone capacity for baseline-resolved peaks of 10–14 (38). Therefore, selectivity,
which is established by the choice of layer and mobile phase, is the most critical
parameter in achieving the required separation. Mobile phases for TLC are selected in
relation to the nature of the layer and mixture to be separated. The strength (polarity) of
the mobile phase influences the Rf range of the solutes, while the chemical classification
of the solvent components determines the interactions and selectivity of the system.
Single solvents and solvent mixtures have been classified according to elution strength in
relation to a particular sorbent. These “eluotropic series” are used along with knowledge
of the solubility (polarity) characteristics of the mixture to select the chromatographic
system to be used. As polarity increases, a solvent becomes stronger (increases Rf values)
in normal-phase TLC, whereas solvents that are strong for RP-TLC are less polar.
Retention in liquid chromatography is a complex process involving solute interactions in
both the mobile and stationary phases. Assorted models of varying complexity have been
proposed to attempt to explain and predict retention and separations, but the exact nature
of the mechanisms is still incompletely understood (see Sec. 4.5 in Ref. 131 for an
excellent discussion). Because of the similarity of results in comparable TLC and HPLC
systems (Sec. XIII), analogous retention mechanisms are probably operative in the two
methods.
Mobile phases are most often selected by consulting literature sources to find those
that were previously used for separation of the compounds of interest or similar
compounds. This is followed by a trial-and-error approach to modify the mobile phase for
the particular layer and other local conditions being used, if necessary.
Systematic and computer-assisted approaches to mobile-phase selection and
optimization have been developed based on solvent strength and selectivity parameters.
Mixtures of solvents that differ in their interaction mechanisms and selectivity effects are
used in these procedures, ranging from simple binary solvent combinations to mixtures of
three solvents with a fourth weak, non-selective strength-adjusting solvent. Snyder has
arranged solvents in eight selectivity groups and within a selectivity triangle to simplify
the systematic design of mobile-phase mixtures (see Ref. 25 for descriptions). Some of
the strategies for solvent optimization are the PRISM A method (37, 132), the mixture
design approach with the solvation parameter model (25, 133), a thermodynamic model
(134), the Snyder—Soczewinski model (134), simplex (37), quality factor (37), window
diagrams (25), overlapping resolution maps (25), and iterative procedures (25). The
PRISMA model, which is a three-dimensional geometrical design that correlates the
solvent strength and selectivity of the mobile phase, is the most successful and most
widely used. It involves selection of three to five solvents for construction of the model
plus a low, constant concentration of a modifier to improve the separation and reduce
tailing, if necessary. A structured trial-and-error approach is used that is described in
detail for use in TLC in Ref. 135. A fully automatic method for selection of mobile
phases for silica gel TLC was described for tetrahydroisoquinolines and corresponding 1-
ones using LSChrom software based on the Snyder theory (135a).
A. Solvents for NP-TLC

Poole and Dias (26) identify the following primary solvents for initial use in TLC mobile-
phase optimization: hexane, toluene, methyl tert-butyl ether, dichloromethane,
chloroform, ethyl acetate, acetone, pyridine, triethylamine, acetonitrile, methanol, 2-
propanol, 2,2,2-trifluoroethanol, acetic acid, and water. All of Snyder’s selectivity groups
are represented by these solvents, which ensures exploration of the full range of solvent
selectivity possibilities.
For silica gel TLC, the four solvents used most often are hexane as the diluent, with
methylene chloride (selectivity group V, dipole interactions), chloroform (group VIII,
proton donor), and methyl tert-butyl ether (MTBE) or diethyl ether (group I, proton
acceptor or basic). The latter three solvents are located at the apexes of the selectivity
triangle and are therefore most likely to enhance resolution. The systematic approach
(136) is to prepare binary mixtures of each solvent with hexane in such proportions that
Rf values are within the useful 0.15–0.7 range (0.3 optimal). If none of these gives the
required separation, then equal-strength ternary mixtures of hexane with two of the other
solvents are prepared, and, finally, a quaternary mixture of all four solvents, if necessary.
In each case, solvent strength is controlled by the amount of hexane used, and selectivity
is varied by changing the ratios of the other three solvents. Preparation of mixtures is
facilitated by consulting an eluotropic series listing relative solvent strengths [P′ values
(137)] and calculating solvent strengths of mixtures as the sum of the volume fraction
multiplied by the solvent strength for each component (138). Additional selectivity can
be achieved by exploring other basic solvents in place of MTBE, e.g., triethylamine,
pyridine, THF, or dimethylsulfoxide (DMSO). If the required separation is still not
obtained, a change from acidic silica gel to amino-bonded silica (basic) or cyano-bonded
silica (dipole-interacting) with four-solvent optimization should be tried next. If none of
these normal-phase systems is successful, a reversed-phase system may be needed.
B. Solvents for Reversed-Phase TLC

Although the retention mechanisms on chemically bonded reversed phases are not clearly
elaborated, Rf values for a series of solutes are usually reversed compared to the sequence
on silica gel if water constitutes a large proportion of the RP mobile phase. It is also
possible to obtain excellent separations on RP plates by using entirely organic mixtures
as the mobile phase.
Two-solvent mixtures composed of water plus an alcohol, acetonitrile, acetone,
dioxane, or an ether are widely used, with methanol-water (8:2) a convenient first-try
solvent for C8 or C18 layers. Solvent strength is varied by changing the water/organic
modifier ratio, and selectivity by using a different modifier or modifier mixture. A more
polar sample requires a weaker solvent (relatively more water). In general, small changes
in mobile-phase strength have less effect on Rf in RP-TLC than in normal-phase TLC,
making mobile-phase selection easier in the former case.
A four-solvent, seven-mixture optimization approach similar to that described in
Section IV.A but based on Snyder’s RP solvent strength (S) values was illustrated by
Sherma and Charvat (140) for C18 RP-TLC using methanol, acetonitrile, and THF, with
water as the weak, strength-adjusting carrier. Additional solvent modifiers recommended
for RP-TLC include isopropanol, dimethylformamide, and DMSO.
Buffered weak acid and basic mobile phases or ion-pair reagents (strong acids and
bases) are used to separate ionic and easily ionized compounds by RP-TLC (25).
Hydrophilic quinolines were separated by adsorption-ion association on diol-silica layers
by using solutions of the ion-pairing reagent di(2-ethylhexyl)orthophosphoric acid in
polar solvents as mobile phases (141).
C. Solvents for Ion-Exchange and Gel TLC

Mobile phases for ion-exchange TLC are usually aqueous solutions with specified pH
and ionic strength. pH determines the degree of exchange by controlling the ionization of
solute and exchanger functional groups, whereas increasing ionic strength generally
reduces the retention of solutes. Organic solvents have been added to increase selectivity
in some ion-exchange systems.
Mobile phases for gel TLC must dissolve the sample and allow the gel network to
swell. For gel permeation TLC on organic polymer layers (e.g., styrene-divinylbenzene),
organic solvents such as THF are common, whereas aqueous buffers are employed for gel
filtration chromatography on layers such as dextran.
VII. DEVELOPMENT TECHNIQUES
Layer development techniques are covered in Chapter 7 of Ref. 1.

A. Linear Development
Development of a TLC plate is most often carried out in the ascending mode by
immersing the lower edge of the plate in the mobile phase in a rectangular glass chamber
(N-chamber) (see A and B in Fig. 6). Chambers made from pressed glass, as shown in
Fig. 6, or lightweight sheet glass are available commercially. “Saturated” or
“unsaturated” chamber conditions can be used for development. In the former case, the
mobile phase is poured into a chamber lined with a filter paper sheet or saturation pad,
and the chamber is covered for a period of time (typically 15 min) to allow vapor
equilibration. The chamber is quickly opened, the plate with applied initial zones is
inserted, and the tank is covered again. For development under unsaturated conditions,
mobile phase is poured into a chamber containing no paper liner, the plate is inserted, and
the chamber is covered immediately. The chamber becomes progressively more saturated
with increasing duration of the separation. Unsaturated chambers usually result in higher
Rf values and lower efficiency (38).
Conditions inside TLC chambers during development with solvent mixtures are
complicated because of the presence of three phases: the layer, liquid mobile phase, and
vapor. Evaporation and condensation processes continually occur, and mobile-phase
gradients are formed because the more polar components will be sorbed preferentially by
the hydrophilic layer, causing the remaining solvent to be depleted in this solvent (solvent
demixing). These gradients, which are not deliberately chosen or controlled as are
mobile-phase gradients in HPLC (and occasionally in TLC; see Chap. 6), are detrimental
to reproducibility of analyses but cause areas of different selectivity along the length of
the layer that can be exploited for improving separations. Development times,
separations, reproducibilities, and Rf values can be very different for the same systems in
saturated and unsaturated N-chambers. Different types and sizes of developing chambers
and small changes in the mobile-phase composition and/or temperature and relative
humidity during development may cause dramatic changes in the retention characteristics
of the compounds to be separated (40). Development conditions must be controlled and
recorded if reproducible results are to be obtained from day to day in one laboratory or
between laboratories. Procedures for standardizing TLC results have been described (142,
143). The most reproducible ascending capillary flow development conditions for TLC
and HPTLC plates are achieved using the Camag Automatic Developing Chamber
(ADC).
The bottom of the Camag twin-trough N-chamber is bisected by a glass ridge running
along its center. It is used as a conventional saturated or unsaturated chamber by placing
mobile phase only on the side where the plate will be inserted (4–20 mL is used
depending on the plate size). In a second mode, one side is filled with mobile phase and
the plate is placed into the other, empty, side. After equilibration of the chamber space
and layer with vapors, development is started
Figure 6 Assorted rectancular (A and

B) and cylindrical (C-I) flat-bottomed
glass chambers for development of
TLC and HPTLC plates of different
sizes. (Photograph supplied by
Analtech.)
without removing the cover by carefully tipping the chamber to transfer mobile phase to
the side with the plate. In a third mode, a different solvent or humidity-controlling
sulfuric acid-water mixture is put on one side and the plate and mobile phase on the other
to provide gas-phase conditioning during development. Desaga sells a thermostatted
chamber for control of temperature during development (118) (see Sec. VILG).
Sandwich chambers (S-chambers), which have a second glass plate placed about 1 mm
from the surface of the layer and therefore a very small gas space, can be used
unsaturated, or saturated through the presence of a mobile-phase-soaked counter layer or
filter paper sheet or pad. S-chambers today are mostly horizontal, and the Camag Linear
Development Chamber and Desaga H-Separation Chamber are examples. The Camag
chamber allows development of 70 samples simultaneously from both ends toward the
center on a 20×10 cm HPTLC plate, or one-half that number of samples can be developed
over the full length of the plate from one end only. Ascending and horizontal S-chambers
have been described (144).
The Camag HPTLC Vario System is a horizontal chamber that has a wide variety of
operational modes and applications. The plate is placed layer down over a tray with
various compartments, which can hold different mobile phases, humidity controlling
liquids, and volatile acids and bases whose vapors will impregnate and condition or
preload the layer. Developing solvent is in a separate tray and is transferred to the layer
by a wick. The Vario chamber can be used to test six mobile phases side by side on one
plate for solvent optimization, to determine if layer pre-equilibration (preloading) is
advantageous, to ascertain if S- or N-chamber configuration is best, and to test different
humidity conditions.
Two new techniques involving horizontal development have been described. In
“halfway development,” a new horizontal sandwich chamber is used to apply mobile
phase to any part of a plate in order to redevelop a separated zone. This chamber can also
be used for relay development of an overlength plate, programmed multiple development,
gradient development, band application, concentration, and micropreparative separation

(144a). Flow TLC (FTLC) involves sample injection into mobile phase flowing over a
horizontal layer with continuous optical detection at a fixed layer position. Mobile phase
is evaporated from the end of the plate to maintain constant mobile-phase velocity
depending on capillary effects (144b). Practical applications of these new techniques
have not yet been demonstrated.
Ascending development of TLC sheets has been carried out in plastic bags for quality
screening of pharmaceuticals under field conditions (145).
Displacement TLC uses three different mobile phases (carrier, displacer, and
regenerant) and three main steps (loading the sample; development of the displacement
train, collecting the fractions of the separated bands; and regeneration). The displacement
system is generated in situ, when the mixture of carrier and displacer is separated as a
result of the carrier running faster than the displacer-carrier mixture. The principles,
techniques, and possibilities of displacement TLC have been described (146), but few
practical applications have been reported.
B. Circular and Anticircular Development

Circular or radial development was first carried out in a Petri dish containing mobile
phase and a wick that touches the layer, supported on top of the dish, at its central point.
The Camag Li-Chamber and Anticircular U-Chamber have been used for circular and
anticircular development, respectively, but these instruments are no longer listed in the
Camag catalog. Except for the very occasional publication of research employing circular
development (e.g., Refs. 147, 148), the method is almost never used currently for
analytical TLC. However, circular chromatograms are produced by use of the modern
preparative methods, e.g., multilayer OPLC (ML-OPLC) and micropreparative U-RPC
(149).
C. Multiple Development
Thin-layer chromatography with multiple development often allows separation of
complex mixtures or closely related substances not resolvable with a single development.
The plate is repeatedly developed in the same direction, with the mobile phase dried
between runs. Each subsequent development achieves zone reconcentration as the trailing
edges of the zones move closer to the fronts, resulting in narrower bands and greater
efficiency, resolution, and sensitivity. The classic multiple development method involves
repeated development with the same mobile phase for the same distance. As an example
of its use, double development was required for silica gel HPTLC assay of potassium
salicylate in diuretic tablets and capsules (150).
Multiple development can also be performed with a change in the solvent composition
and/ or migration distance for each step in order to optimize the separation of certain
mixture components. Compounds that are difficult to separate require a large number of
developments with a selective solvent that initially produces low Rf values; maximum
zone center separation has been shown to occur when the zones have migrated 63.2% of
the development distance (151). Quantitative measurement can be made at the end of any
development stage when the different elements are separated optimally. The zone-
refocusing effect of multiple development has been illustrated by a densitogram of the

HPTLC separation of 10 PTH-amino acid derivatives (13).
An apparatus comprising an N-type chamber with connections for adding and
removing solvents and gas phases is available from Camag for AMD. AMD involves the
use of a stepwise gradient of different mobile phases of decreasing strength in 10–30
successive developments in the same direction, increasing in length by 1–5 mm, to
separate complex mixtures with a wide polarity range. The initial solvent, which is
strongest, focuses the zones during the first short run, and the mobile phase is changed
for each, or most, of the following cycles. Between runs the mobile phase is completely
removed from the developing chamber and the layer is dried and activated by vacuum
evaporation and then conditioned with a controlled atmosphere of vapors prior to the next
development. The combination of the focusing effect and gradient elution results in high
resolution and improved detection limits. Widths of separated zones are approximately
constant at 2–3 mm, and separation capacity for baseline-resolved components is 30–40
(13). A typical universal gradient for a silica gel layer involves 25 steps with methanol,
dichloromethane or tert-butyl ether, and hexane as solvents (152). A theoretical model
has been presented for computer-aided optimization of AMD separations (153), and
philosophies for method development in AMD have been discussed (38). Examples of
recent analyses using AMD include sugars on diol layers with a 15-step acetonitrile–
water gradient decreasing linearly from 35% to 15% water (154), beet and cane molasses
in the sugar industry (155), and different lipid classes (156). AMD is described in
Chapters 5 and 6 of this Handbook.
D. Continuous Development
Continuous development is another technique that increases separating power relative to
conventional ascending unidimensional development. The top end of the plate is
extended outside of the chamber so that mobile phase evaporates and its flow is
continuous. Weak solvents are used to increase selectivity, and development distances are
kept short so that development time does not become excessive. The method has been
used mostly with HPTLC plates, for which Regis makes the Short Bed/Continuous
Development (SB/CD) Chamber.
It has been shown that minimum analysis time is always shorter for continuous
development than for conventional development when conditions are analyzed (157).
Optimum conditions for the continuous TLC separation of steroids in terms of analysis
time, plate length, and mole fraction of a binary mobile phase were determined using the
overlapping resolution maps technique (158).
Although the SB/CD chamber is specified for use in the latest edition of the U.S.
Pharmacopeia (USP 24/NF 19, p. 1917), the method has little current use.
E. Two-Dimensional Development
In 2-D TLC, a sample is spotted in the corner of a layer and developed sequentially at
right angles using two mobile phases that provide complementary retention mechanisms,
with drying between the runs. With the correct choice of mobile phases, sample
components will be distributed over the entire surface of the layer, increasing resolving
power by almost the square of that obtained in a one-dimensional system. The zone
capacity will increase from 10–20 for capillary flow TLC to 100–250 for capillary flow
2-D TLC (30). Predicted zone capacity for 2-D TLC with forced flow or AMD is
approximately 1500 (13), but this has not been tested. If the same mobile phase is used in
both directions, or different mobile phases that result in differences in intensity rather
than true orthogonality, the zones become distributed along the diagonal between the two
development directions rather than over the whole surface, leading to a resolution factor
of only because of the increased migration distances for the sample. The use of
bilayer plates and chemically bonded layers that separate according to different
mechanisms depending upon the nature of the mobile phase (both described above), as
well as other types of specialized layers (see below), are advantageous for 2-D TLC.
Disadvantages of the 2-D method include difficulty with interpretation of results,
reduced reproducibility compared to one-dimensional TLC, poorer detection sensitivity
because of greater diffusion during two developments, and the inability to carry out
reliable in situ quantification because standards cannot be developed together in both
directions and slit-scanning densitometers are designed to measure zones in a single layer
track. Electronic scanning with a video densitometer is a possible solution, but routine,
accurate, and precise quantitative analyses of 2-D chromatograms have not been
demonstrated.
Computer-assisted methods were described for mobile-phase selection and
optimization of mixtures of eight pesticides (159) and 10 antihistamines (160) in 2-D
TLC.
Recent applications of 2-D TLC include the following separations: penicillium fungal
extract on a cyano-derivatized silica gel layer (161); opiates on HPTLC silica gel (162);
PAHs on C8 and diol mixed phases (163); fatty acids on urea- and silver nitrate-
impregnated silica gel for the first and second dimensions, respectively (164); parabens
and carboxylic acid additives in pharmaceutical formulations on silica gel, with paraffin
impregnation after the first run (165); and saponins on mechanically connected silica gel
and C18 plates (166). Overpressured development has been occasionally used in 2-D TLC
(167), and mass spectrometry to identify the separated zones (168).
Two-dimensional TLC along with multiple unidimensional, programmed multiple,
and automated multiple development were covered in an encyclopedia article (169).
F. Forced-Flow Planar Chromatography

Forced-flow development is accomplished by use of centrifugal force (RPC) or pressure
(OPLC). Analytical or preparative forced-flow planar chromatography (FFPC) can be
carried out off-line (starting with a dry layer), on-line with a stationary phase previously
equilibrated with mobile phase as in HPLC, or a combination of the two (e.g., off-line
sample application and on-line separation and detection).
Rotation planar chromatography (RPC) is mainly a preparative method that has been
described in Ref. 149. It uses U (ultramicro), M (micro), or N (normal) chambers, which
differ mostly in the size of the vapor space. RPC in M or U chambers is applicable to a
wide range of substance classes and polarities. Continuous development is possible with
different types of the RPC techniques if an outer ring is scraped from the stationary phase
and the development is stopped and the compound of interest scraped off when it reaches
this ring. From analytical TLC separations in saturated or unsaturated tanks, mobile
phases can be transferred via analytical ultramicro U-RPC and M-RPC (separation
distance 8 cm, average layer particle size 11 µm) to preparative U-RPC and M-RPC (10
cm, 14–15 µm), respectively, if the mobile-phase strength and selectivity are kept
constant. The sample is applied as a circle in the center of the layer. Preparative RPC is
covered in Chapter 11 of this Handbook.
Overpressured layer chromatography (OPLC) (see Chap. 7 in this Handbook) was
invented to overcome the changing velocity of the mobile phase in the plate and to
eliminate the vapor phase present in capillary development TLC. The process is as
follows (5): Samples are applied to the dry layer, which is placed into the pressurized
development chamber. The layer is tightly covered and is sealed on its sides by an elastic
membrane (plastic sheet), which is pressurized by an inert gas or water cushion. The
mobile phase is delivered directly to the layer by pumping at constant velocity through a
slit in the membrane.
Modes of OPLC include linear, two-directional, long-distance, and circular. The
delivery point of the mobile phase is varied for each of these. Use of commercial plates
with sealed edges prevents the mobile phase from running off the plate in the linear
mode. On-line operation for analysis of one sample involves mobile-phase flow from the
OPLC apparatus directly to an HPLC detector; the plate is dried after development and is
then scanned separately in off-line linear analysis of multiple samples. On-line OPLC is
most similar to HPLC, but linear off-line OPLC of up to 18 samples with a run distance
of 18 cm on a 20×20 cm plate is most commonly used. Two-directional OPLC is used
with less complex samples for separation of up to 70 samples over an 8 cm run distance.
Samples are applied on two parallel, vertical origin lines in the center of the layer, and
mobile phase enters from a channel in between and simultaneously develops the samples
toward the sides of the plate. Long-distance OPLC extends the migration distance by
employing three to five stacked plates with slits to direct mobile phase from one plate to
the next. Samples are spotted in a radial pattern around the center of the plate for circular
OPLC. Mobile phase enters at the center, and low Rf compounds are especially well
resolved in this mode. A statistical method of mobile-phase selection was developed
specifically for OPLC (170), and the PRISMA method can also be used with unsaturated
chambers for the initial trial-and-error experiments.
The latest instrument for analytical and semipreparative OPLC is the Personal Basic
System (BS) 50 (OPLC-NIT Engineering Ltd., Budapest, Hungary) (171). It consists
basically of a separation chamber and a liquid delivery system. The separation chamber
contains a holding unit, hydraulic unit, layer cassette, and drain valve, and there is a
pumping system to deliver the mobile phase and hydraulic liquid. The entire apparatus
and development process are computer-controlled, and external pressure (up to 50 bar),
mobile-phase flow rate and volume, and development time can be automatically
programmed. With this OPLC apparatus, minimum values of reduced plate height are
2.1–3.5, depending on the operating conditions and layer properties. The corresponding
range for a good HPLC column is 2.0–2.5, so efficiencies are comparable under optimum
conditions (39).
The principles, techniques, and instrumentation for OPLC are reviewed in Refs. 39
and 172.
G. Gradient Development
The three types of gradients that have been used the most in TLC are mobile phase,
stationary phase, and temperature. Planned mobile-phase gradients must be distinguished
from the natural, uncontrolled gradients resulting from solvent demixing during
development. AMD, mentioned in Section VII.C, involves development in a commercial
instrument with a “universal gradient” starting with the most polar (strongest) mobile
phase and becoming increasingly weaker in order to form focused, well-resolved zones.
The horizontal DS-Chamber (Chromdes, Lublin, Poland) is a Teflon sandwich
chamber that is often used with stepwise gradient development (increasing mobile-phase
strength) (173). The use of mobile-phase gradients was reported for separations of
pigments by RP- and NP-TLC (174, 175); phenolic acids on silica, propylamine, and diol
layers (176); and alkaloids on sodium bicarbonate-impregnated silica gel (177).
Strategies for computer-aided optimization of gradient elution programs were published
(178, 179).
Stationary-phase gradients involve a continuous or discontinuous change of sorbent
composition, and they are used much less than mobile-phase gradients. Discontinuous
gradients are produced by treating different layer areas with different reagents to alter the
separation mechanism or by casting layers with different sorbent regions. The latter can
be commercial bilayer plates (described above) or layers made by using a modified
sorbent spreader. As an example, 2-D TLC with a sorbent gradient (C18 and silica gel)
was applied to the analysis of saponins (166).
Most TLC is performed at room temperature in nonthermostatted chambers, but recent
use of temperature-controlled TLC by one research group has been reported. These
workers have described homemade and commercial devices that provide a temperature
gradient for separations of chiral and nonchiral compounds (180), technical problems
associated with temperature-controlled TLC (181), and studies of the interactions
between native cyclodextrins and n-alcohols (182) and the retention and separation of
cholesterol and bile acids (183) using thermostatted RP-TLC.
Gradient development in TLC is described in Chapter 6 of this Handbook.
VIII. DETECTION AND QUALITATIVE IDENTIFICATION OF

ZONES
Detection and qualitative evaluation are covered in Chapters 8 and 9, respectively, of Ref.
1, in Ref. 184, and in Chapter 8 of this Handbook.
A. Detection (Visualization) of Zones

After development, the plate is dried with a hair drier, in an oven, or on a programmable
plate heater (Desaga or Camag) to evaporate the mobile phase. Compounds are detected
on layers by their natural color, natural fluorescence under UV light, or quenching of
fluorescence on a phosphor-containing layer or as colored, UV-absorbing, or fluorescent
zones after reaction with an appropriate reagent. Additional methods are based on
radiochemical detection (e.g., autoradiography, scintillation counting, and direct in situ
scanning) and on biological properties (e.g., enzyme inhibition, bioautography, and
immunostaining techniques). A significant advantage of TLC over HPLC is that detection

is static rather than dynamic or on-line. This eliminates the time constraints of detection
on the fly and permits flexibility through the utilization of a variety of compatible
detection techniques and reactions in combination.
Chromogenic reagents typically have detection limits ranging from low nanograms to
several micrograms; fluorogenic reagents, from high picograms to high nanograms; and
enzyme inhibition, from low picograms to low nanograms. The selectivity of the latter
two reagent types allows detection and confirmation at low parts-per-billion (ppb)
concentrations in many samples, at which level most chromogenic reagents would not be
effective. Certain compound classes lacking convenient chromophores are especially
amenable to TLC analysis because of the ease of using postchromatographic chemical
reactions for their detection; these include organic acids, lipids, carbohydrates, amino
acids and peptides, and surfactants (38).
Descriptions of more than 450 detection reagents are available in Volume II of the
Handbook of Chromatography (185). Reagents for specific compounds are found in the
data tables of Volume I and later volumes of this series devoted to different chemical
classes. The chapters in Part II of this Handbook contain descriptions of many detection
reagents applicable to particular types of compounds. New detection reagents are updated
biennially in the Analytical Chemistry review of TLC written by Sherma (11). Books
have been published on physical and chemical detection methods in TLC (186, 187).
Compounds that are naturally colored are detected on layers directly, whereas
compounds with native fluorescence are viewed under UV light (Fig. 7). In some cases,
fluorescence is visible on a wetted plate only, so the layer is impregnated with a
nonvolatile liquid. Certain compounds fluoresce only when the layer is adjusted to a
specific pH value, e.g., quinine at low pH. Compounds that absorb UV light can be
detected on layers containing an indicator (phosphor) that fluoresces upon excitation with
254 nm or 366 nm UV light. When irradiated, absorbing compounds diminish (quench)
the uniform layer fluorescence and are visualized as dark zones on a bright (usually
green) background. One of the most common fluorescent indicators is manganese-
activated zinc sulfate, which is excited by 254 nm UV radiation.
Color, UV absorbance, or fluorescence can be induced by application of a detection
reagent that chemically reacts with the compound(s) to be visualized. Less commonly,
detection methods utilize the biological activity of the separated compounds. The most
active current area of biological detection involves immunostaining (Western blotting).
For example, glucuronides of glycyrrhetic acid were detected after silica gel TLC by
transfer to a poly(vinylidene difluoride) (PVDF) membrane and treatment of the
membrane with sodium periodate solution followed by bovine serum albumin (BSA),
resulting in glucuronides of glycyrrhetic acid-BSA conjugate. Individual spots were
stained with monoclonal antibody against glycyrrhizin (188). Steroidal alkaloid
glycosides have also been detected by TLC immunostaining (189). TLC immunostaining
has been combined with TLC blotting/secondary ion mass spectrometry (SIMS) in a
number of analyses, e.g., the analysis of glycosphingolipids (190). In situ enzymatic
reactions have been used for direct detection and quantification of toxicologically active
compounds at low levels. As an example, 20–80 ng of pentachlorophenol could be
quantified using a slit-scanning or video densitometer based on the degree of the
compound’s cholinesterase inhibition (191).
Figure 7 Viewing cabinet for

inspecting TLC plates under 254 nm or
366 nm UV light. (Photograph
supplied by Camag.)
The detection reagent solution is usually applied postchromatography by spraying or
dipping the layer. Alternatively, the reagent may be preimpregnated into the layer prior to
spotting and mobile-phase development, or it can be included in the mobile phase for
impregnation during the development step. Volatile reagents may be applied by exposing
the plate to their vapors. This is the case for iodine, a “universal” detection reagent for
many organic compounds, and induced vapor-phase fluorescence (192). Charring with
concentrated sulfuric acid is another general detection method. Many reagents are
specific for compounds with certain functional groups. Reagent-free detection can be
achieved with some compound-layer combinations by simply heating the plate
(thermochemical reaction). Sugars have been detected as fluorescent zones by heating on
amino layers and creatine as a quenched zone on silica gel F254 layers (193).
For quantitative TLC, it is essential that the detection reagents be applied uniformly.
Manual spraying is used most often, but a more homogeneous distribution of reagent in
the layer may be achieved if the reagent can be applied by use of a manual dipping device
(Desaga). Camag’s plate immersion device, which effects vertical plate movement at a
uniform rate for precise dip application of reagents, and Desaga’s automatic, computer-
controlled ChromaJet DS 20 spray apparatus have been used for performing quantitative
analyses with improved accuracy and precision.
The high carbon content of RP layers hinders some common detection methods. Most
charring reagents and those that do not wet the layer cannot be used. Reagents that have
been successfully employed on C18 layers include iodine, 10% phosphomolybdic acid in
ethanol with heating at 120°C (for lipids), 10% sulfuric acid in ethanol with heating for
2–3 min (general reagent), and fluorescamine (amino acids).
B. Qualitative Identification of Zones

A major use of TLC is to identify unknown sample components or to confirm the identity
of compounds initially detected by GC or HPLC. Qualitative identification is based on
characteristic colors produced by a selective detection reagent combined with Rf values.
Identification can be aided by using more than one detection reagent, often applied in
sequence to a single chromatogram. To have the highest confidence in the identification
procedure, Rf values of sample zones should be compared to standards in more than one
type of TLC system with different mechanisms, e.g., adsorption, normal- and reversed-
phase bonded layers, and ion-exchange layers (194). Alternatively, multiple mobile
phases can be used with one type of layer. For example, principal component analysis of
standardized Rf values of 443 drugs and their metabolites chromatographed on silica gel
in four mobile phases was employed for identification of unknown drugs in cases of
overdose intoxication or poisoning (195).
In general, at least one spectroscopic method must be used in addition to TLC results
in order to make a valid statement of identity (152). TLC has been combined off-line and
on-line with UV-visible (UV-vis), fluorescence, NMR, IR, Raman, photoacoustic, line
narrowing, and MS for compound identification. See Refs. 196 and 197 for reviews of
these coupled methods.
Modern densitometers allow in situ measurement of visible and UV absorption and
fluorescence excitation and emission spectra. Sample and standard spectra from the same
layer should be directly compared for identification purposes. Lack of a match is definite
proof that the compounds in the zones are different, but an apparent match may not be
sufficient proof that the compounds are the same if the spectra are not characteristic of
the total molecular structure (38). If the corresponding standards are not available for
comparison with presumptive sample zones, UV-vis and fluorescence spectra are usually
not sufficiently characteristic to identify unknowns by making structural assignments
through spectral interpretation. Recording UV-vis spectra of separated zones both before
and after pre- or postchromatographic derivatization increases the probability of correct
zone identification (152). Novel library searching software was introduced for improved
identification of compounds in autopsy urine samples by use of in situ UV spectra (198).
Virtually all combinations of TLC and NMR spectrometry for separated substance
characterization have involved zone removal and extraction of the analyte with a solvent.
A preliminary paper on high resolution magic-angle-spinning solid-state NMR for in situ
compound identification was published (199), but no subsequent reports on the use of this
approach have been found.
HPLC effluents have been deposited on a silica gel layer, which was not used for
chromatography but acted as a substrate for analyte identification by fluorescence line-
narrowing spectrometry (FLNS) (200). Off-line coupling of TLC and FLNS for high-
resolution, low-temperature spectral characterization was reviewed (201). FLNS and PEI-
cellulose TLC were coupled for low-picomole detection of DNA adducts using
fluorescence background subtraction, time-resolved detection, and a new synchronous
scanning procedure (202).
TLC is combined with Fourier transform infrared (FTIR) spectrometry off-line by

scraping off the zone and eluting the compound onto a KBr pellet or on-line by
subtracting a silica gel layer background spectrum from a spectrum of the zone, both
measured in situ using diffuse reflectance FTIR (DRIFT) spectrometry (175). The silica
gel TLC on-line method has been used to identify hydrocarbons in wastewater (203), the
major marijuana metabolite in urine (on-line TLC-UV was also used) (204), impurities in
chlordiazepoxide bulk drug powder and tablets (205), and impurities in flurazepam bulk
drug powder and capsules (on-line TLC-UV and a specialized plate containing 50%
magnesium tungstate were also used) (206). The development, techniques, and
applications of HPTLC-FTIR on-line coupling have been reviewed (207).
Photoacoustic spectrometry was applied to the characterization of TLC plates with
respect to surface and in-depth distribution of different compounds inside the sorbent
(208), as well as for qualitative and quantitative analysis of compounds separated by
TLC, including mapping techniques (see Ref. 209 for a review).
Surface-enhanced Raman scattering spectrometry (SERS) can be used to characterize
nanogram to picogram amounts of solutes on silver-treated HPTLC plates using a visible
(Ar ion, 514.5 nm) or near-IR (NIR) (Nd-YAG) laser for excitation. Methods for silica
gel layer preactivation include dipping the plate into a solution of silver oxalate and
subsequently pyrolyzing it to form silver particles on the layer (210), vacuum evaporation
(211), and citrate reduction (212). It was found that the type of plate and the development
procedure (traditional vs. OPLC) influenced significantly the quality of SERS spectra
obtained (213). NIR-SERS was shown to be useful for both qualitative and quantitative
analysis in a single step (214).
Thin-layer chromatography coupled with mass spectrometry (TLC/MS) is covered in
Chapter 9 of this Handbook, and the following MS methods combined with TLC have
been reviewed: fast atom or ion bombardment, matrix-assisted laser desorption ionization
(MALDI), surface assisted laser desorption ionization (SALDI), and the electrospray
(ES) interface (215).
Recent applications of off-line TLC/MS, which involves scraping of separated zones
from the layer and extraction of the analyte from the sorbent, include analyses of organic
reactions by TLC/MALDI time-of-flight (TOF) MS (216); the major ganglioside from
crucian carp liver by TLC/ES-MS (217); deramciclane metabolites by OPLC/MS (218),
fast atom bombardment (FAB) tandem mass spectrometry (MS-MS) (219), and digital
autoradiography (DAR); numerous drugs and metabolites by TLC/electron impact (EI)-
MS (220); caffeine in soft drinks by TLC/SPE-atmospheric pressure chemical ionization
(APCI)-MS (221); and lipopolysaccharides from bacteria by MALDI (222).
On-line TLC/MS analysis without elution from the layer was reported for
benzodiazepines by TLC/FAB-MS and MS-MS (223), for impurities in a newly
synthesized drug by TLC/MALDI-TOF-MS (224), and for picogram levels of nucleotides
(225) and pesticides (226) by TLC/ MALDI-MS. The application of SALDI was
demonstrated for a wide range of organic compounds including peptides using silica gel
with the surface covered by activated carbon particles and added glycerol (227). A hybrid
plate for TLC/MALDI-MS that recovered about 100% of the analytes consisted of a
silica gel layer and a MALDI layer configured adjacently on a common backing; after
separation on silica gel, the plate was rotated 90° and the analytes eluted onto the MALDI
layer (228). Plates are prepared for TLC/MALDI-TOF-MS by brushing them with a
supersaturated solution of matrix or electrospraying the matrix solution (229). TLC/MS

was the subject of an encyclopedia article (230).
The following criteria for identification of an analyte by TLC or HPTLC were
recommended (230a) after study by a board of European experts. The Rf value of the
analyte should agree within ±3% compared to the standard material under the same
conditions. The visual appearance of the analyte should be indistinguishable from that of
the standard material. The center of the spot nearest that due to the analyte should be
separated from it by at least half the sum of the spot diameters. For identification,
additional cochromatography in the TLC step is mandatory. As a result, only the spot
presumed to be due to the analyte should be intensified; a new spot should not appear. If
full-spectrum detection is possible, the maximum absorption wavelength in the spectrum
of the analyte should be the same as that of the standard material, within a margin
determined by the resolution of the detection system. The spectrum of the analyte should
not be visually different from that of the standard material.
IX. DOCUMENTATION OF TLC RESULTS
Methods for documentation of TLC results are described in Chapter 9 of Ref. 1 and
Chapter 8 of this Handbook. Procedures were described for true color
photodocumentation of 254 nm and 366 nm UV-irradiated chromatograms (231) and
optimized black-and-white and color photography of colored, fluorescent, and
fluorescence-quenched zones (232). A video image archival system made use of
integrating cameras (233). A system comprising a computer, digital image scanner, and
black-and-white and color printers was described for documenting visible TLC spots but
not those detected under UV light (234). Commercial photographic and video
documentation instrumentation for colored, fluorescent, and quenched zones is available
from commercial sources, including Camag (235, 236). Various paper brands were tested
for printing and color stability characteristics and the effects on archiving of
chromatograms produced from the Camag video system using an ink jet printer (237).
X. QUANTIFICATION
Quantitative TLC is the subject of Chapter 10 of Ref. 1. The theory and techniques of
densitometric TLC are elaborated in Chapter 10 of this Handbook, and instrumental
aspects of quantification are presented in Chapter 5. Quantification of lipids and
hydrocarbons and some other types of compounds has been carried out on rods of silica
gel or other sorbent with direct interfacing to a flame ionization detector (FID) (the
Chromarod or Iatroscan system). TLC-FID is covered in Chapter 13 and Chapter 19 on
hydrocarbons in Part II of this Handbook but not in this chapter.
A. Introduction
Visual comparison of the spot intensity of a definite sample aliquot with the intensities of
simultaneously developed reference spots containing known weights of analyte is a
simple, direct method for quantitative analysis. The method is only semiquantitative, with
accuracy and precision in the 10–30% range, but this level is often adequate for the
purpose intended. Visual comparison works best if amounts near the detection limit are
applied and the sample is closely bracketed by standards. Visual estimation is specified in
various pharmacopoeias for purity testing of both drug active raw materials and
formulated products (238).
B. Zone Elution
The zone elution method involves the following steps: drying the layer, locating the
separated analyte zone, scraping the portion of layer containing the analyte, collecting the
sorbent, eluting the analyte from the sorbent, and measuring against standards by an
independent microanalytical method such as solution absorption or fluorescence
spectrometry, GC, HPLC, or voltammetry.
The chromatogram is dried to remove the mobile phase, components of which might
intefere in the determinative step. The conditions of drying must not cause loss of the
analyte by volatility or decomposition. Zones are located by direct observation for
compounds that are naturally colored or fluorescent or those that quench fluorescence on
phosphor-containing layers. Other compounds must be located by application of a
visualizing reagent to samples that are chromatographed simultaneously on outside lanes
of the layer to serve as a guide for the areas of the layer that are removed by scraping.
The zones are scraped and transferred carefully to a suitable container. The analyte is
eluted from the sorbent using a solvent that provides complete, or at least reproducible,
recovery.
The zone elution quantification method is tedious and time-consuming and is likely to
be inaccurate because of difficulties in locating the exact zone boundaries, loss of sorbent
during scraping and collection, nonreproducible or incomplete elution from the sorbent,
and background interferences due to eluted impurities from the sorbent. These errors are
minimized if standards and samples are chromatographed, scraped, and eluted together as
consistently as possible, and if an equal-size blank area of layer is scraped and eluted in
the same way. Prewashing the layer by development with an appropriate solvent will help
to minimize the blank value.
An apparatus was described to facilitate sample elution without transfer of the solid; a
total solvent volume of only 60 µL was used, and recoveries were >90% (239). Camag
sold the Eluchrom automatic elution instrument for a number of years, but it has been
discontinued.
Despite its inconvenience, the basic TLC elution method, usually combined with UV-
vis absorption or fluorescence spectrometry, is used advantageously to separate and
quantify a great variety of analytes in laboratories not equipped with a densitometer. The
spot elution method continues to be prescribed in some assay methods in the U.S.
Pharmacopeia.
C. Slit-Scanning Densitometry
In situ measurement of zones with a densitometer is the preferred method for quantitative
TLC with maximum accuracy, precision, selectivity, and sensitivity. Densitometry in
TLC is reviewed in Ref. 240.
Substances separated by TLC or HPTLC are quantified by in situ measurement of
absorbed visible or UV light or emitted fluorescence upon excitation with UV light.
Absorption of UV light is measured either on regular layers or on layers with
incorporated phosphor, the latter resulting in dark zones on a fluorescent background
(fluorescence quenching). Only those substances that have absorption spectra overlapping
the excitation spectrum of the phosphor will be detected and quantifiable by this method.
Although it has been claimed in the literature that better quantitative results are obtained
for direct measurement of UV absorption, more analyses have been reported based on
fluorescence quenching (e.g., 52).
Scanning densitometers manufactured by different companies (e.g., Desaga) (Fig.8)
have many common features. Halogen or tungsten lamps are used to provide light for the
visible region, deuterium lamps for the UV region (absorption measurement), and
mercury or xenon arc sources or a laser (241, 242) for fluorescence excitation. Filters or
monochromators (prism or grating) are employed for wavelength selection, and a
photomultiplier tube or photodiode detector for signal measurement. The plate, mounted
on a movable stage controlled by stepping motors, is scanned with a fixed beam of
monochromatic light in the form of an adjustable rectangular slit, the height of which is
matched to the width of the largest spot or band. Most reported analyses have involved
HPTLC plates and single-wavelength, single-track linear reflectance scanning, but
transmission scanning is sometimes used (243). Signal diminution (absorbance) or
increase (fluorescence) between the zone and a blank area of the layer is the measurement
upon which quantitative analysis is based. The use of laned plates causes the initial and
developed zones to be present in accurately known track locations, which is
advantageous for setting up and operating both manual and automatic scanners.
Single-beam scanners may produce chromatograms with drifting baselines due to
irregular or impure layers. Double-beam scanners are able to eliminate general plate
background effects, but these are no longer manufactured or used to any extent. In dual
wavelength scanning (175, 244), two monochromators alternately furnish the sample lane
with a reference wavelength (minimal absorbance by the analtye zone) and a sample
wavelength (maximum absorbance by the analyte). The reference wavelength cancels out
background interferences contributed by the sample and corrects for layer irregularities.
Zigzag (flying spot) scanning (244, 245), which uses a spot of light that moves over the
zones with swings that correspond to the length of the slit, provides more reproducible
readings for zones with irregular shapes or nonuniform concentration distributions. It has
been claimed that simultaneous measurement of transmission and reflection will also
diminish the effects of noise arising from an inhomogeneous plate background and
improve sensitivity, but use of this procedure is seldom reported. A rapid, high-resolution
fiber-optic diode array HPTLC scanner was described recently (246).
Computer-controlled densitometers can perform a number of functions: data
acquisition by scanning over an entire plate following a preselected geometric pattern
with control of all scanning parameters; automated peak searching and optimization of
scanning for each fraction located; multiple-wavelength scanning to find, if possible, a
common wavelength for all substances to be
Figure 8 Photograph of the Desaga

Densitometer CD 60 with a
superimposed schematic diagram of
the light path including (right to left)
the source lamp, two mirrors, grating
monochromator, mirror, beam splitter,
plate with chromatogram to be
scanned, reference and measuring
detectors above the plate (reflection),
and detector below the plate
(transmission). (Photograph supplied
by Desaga.)
quantified, to optically resolve fractions incompletely separated by TLC, and to identify
fractions by comparison of spectra with those of standards cochromatographed on the
same plate or stored in a spectrum library through pattern recognition techniques;
baseline location and correction; computation of peak areas and/or heights of samples and
codeveloped standards and processing of the analog raw data to quantitative digital
results, including calculation of calibration curves by linear or polynomial regression,
interpolation of sample concentrations, statistical analysis of reproducibility, and
presentation of a complete analysis report; and storage of raw data for later reintegration,
calibration, or evaluation with different parameters without rerunning the chromatogram.
A high quality chromatogram with compact, regularly shaped and well-resolved zones
will lead typically to relative standard deviation (RSD) values in the range of 0.5–3% in
quantitative HPTLC using a modern commercial computer-controlled densitometer.
The ability to spot unknowns and standards on the same plate and subject them to the
same chromatographic conditions (in-system calibration) is an inherent advantage of
quantitative TLC compared to sequential elution column chromatography. Systematic
errors are minimized, an internal standard is less often required, and accuracy and
precision values compare very favorably to those of GC and HPLC. Automatic
instruments for sample application are necessary for the highest precision and accuracy in
quantitative TLC. Because signal response is related to spot size for a fixed weight of
analyte (247), it is usually recommended to apply a fixed volume of the sample and
standard solutions of different concentrations to produce zones of constant size but
varying intensity. However, the application of constant volumes appears not to be
necessary for good results on laned preadsorbent plates if the developed bands spread
across the width of each lane or when narrow bands are directly applied with the Linomat
spray-on apparatus. In these cases, different volumes of a single standard solution can be
applied.
Compared to absorption, fluorescence densitometry (248) has the advantages of higher
sensitivity (often low picogram levels), calibration curves with a wider linear range (102–
103), and improved selectivity because few compounds fluoresce and characteristic
excitation and emission wavelengths can be chosen. Enhanced sensitivity has been
obtained by impregnation of the dry plate with an antioxidant to reduce quenching from
oxidation reactions or with a fluorescence-enhancing liquid such as paraffin, glycerol, or
Triton X prior to scanning. Nonfluorescent compounds can often be converted to
fluorescent compounds by pre- or postchromatographic derivatization with a fluorogenic
reagent (e.g., dansyl chloride) or by treatment with ammonium hydrogen carbonate
vapors at 100–150°C for 1–12 h to produce a reproducible fluorescent product. One of
the most successful areas for application of fluorodensitometry is the quantification of
toxins, e.g., deoxynivalenol in cereal products (249) (see Chapter 32 on toxins in Part II
of this Handbook).
Absorption of light by zones on TLC plates is not described adequately by the
LambertBeer law that is usually applied to solution spectrometry, because of the diffuse
reflectance (scattering) by the sorbent particles. The Kubelka-Munk equation, which
includes both light absorption and scattering coefficients, is usually applied as the basis
of in situ TLC quantification, especially when reflected light is employed. This equation
predicts a nonlinear relationship between the detector signal for reflectance
measurements (peak area or height) and the amount of analyte (weight or concentration).
Calibration curves obtained in practice using the reflectance or transmission scanning
mode are unique for each analyte, have an intercept greater than (0, 0), and are essentially
linear at low weights but tend to curve toward the weight axis at higher weights. If the
calibration plot obtained by linear regression does not have a sufficiently high correlation
coefficient (r value), then application of lower standard weights can be tried or the
calibration curve can be fit to a polynomial function using the densitometer software. The
external standardization method, with interpolation of the weight or concentration of
unknowns from the calibration curve, has been used most often for quantitative
densitometry. The internal standardization quantification method is used only
occasionally, and the standard addition method not at all. The data pair sample
application scheme is preferred by some analysts, especially when maximum precision

and accuracy are required in pharmaceutical assays. In the data pair method, all the
standard and sample solutions are applied twice, with duplicates not next to each other
but separated by half the width of the plate (19). The principles of quantitative analysis in
TLC and HPTLC are reviewed in Ref. 250.
As an aid in method development, quantitative TLC has been treated in detail from the
theoretical and practical viewpoints, including a description of protocols for sample
calibration; for establishing resolution, sensitivity, detectability, and optimum scan rate;
and for comparing the performance characteristics of different slit-scanning
densitometers (247).
The development of formal validation [quality assurance (QA)] procedures for TLC
has been addressed in the literature mostly in relation to pharmaceutical analyses, e.g.,
HPTLC assay of theophylline in an effervescent tablet (251). Guidelines formulated by
the International Conference on Harmonization (ICH) for analytical procedures were
adapted for TLC for use at different levels, i.e., qualitative identity testing, assay of active
ingredients, semiquantitative limit tests, and quantitative determination of impurities, and
described by Ferenczi-Fodor et al. (252). Basic acceptance criteria for evaluation of
validation experiments, based on the authors’ previous practical experience (253–255),
were proposed for accuracy, precision (repeatability and intermediate precision),
specificity, detection limit, quantification limit, linearity, and range, and selected
parameters for robustness testing of given procedures and QA of quantitative TLC testing
by control charts were described. Szepesi and Nyiredy (238) describe rules for validation
of pharmaceutical analyses that include scanning every zone in triplicate to establish
instrumental error, spotting the same volume of test solution in triplicate, and spotting
three bracketing calibration standards that contain a known relationship to the expected
test solution value, e.g., 80%, 100%, and 120%. As a recent example outside of
pharmaceutical analysis, an OPLC method for determination of aflatoxins in wheat was
validated fully, including robustness testing (256). Validation of data is necessary for
analyses performed under the good laboratory practice (GLP) guidelines (38).
D. Image Analysis (Video Densitometry)

Video camera systems are available from several manufacturers for documentation and
densitometric quantification of TLC plates. As an example, the Camag VideoScan
instrument consists of a lighting module with short- and longwave UV and visible
sources upon which the layer is placed, a charge-coupled device (CCD) camera with
zoom and long-time integration capability, and a personal computer (PC) under MS
Windows control with frame grabber, monitor, and printer. The available software for
quantitative evaluation, such as Camag VideoScan software (257), allows display of the
tracks of the chromatographic image acquired with the video camera as analog curves
and calculation of their peak properties (Rf, height, area, height percent, and area percent).
For quantification, the computer creates a standard curve from the areas or heights of the
standards and interpolates unknown values from the curve. In addition to commercial
instruments, laboratory-assembled computer-controlled CCD camera systems have been
described for HPTLC quantification (257a).
Video scanners have certain advantages, including rapid data collection over the entire
layer surface, simple design with virtually no moving parts, and unique software
approaches for archiving and comparing chromatograms (258). However, current
instruments are not capable of illuminating the plate uniformly with monochromatic light
of selected wavelength, are less sensitive, and provide lower resolution for chromatogram
recording than slit-scanning densitometers. Video densitometers can measure visible
spots that are colored, fluorescent, or quench fluorescence on F-layers (259) in
transmittance and reflectance modes (260), but they cannot perform spectral analysis. In
addition to a CCD camera, video densitometry has been carried out using a flat-bed
scanner and commercial software (261). With improvements in electronic scanning, it is
very possible that video densitometers will replace mechanical scanning densitometers at
some time in the future, with especially great potential for evaluation of 2-D
chromatograms (13).
XI. PREPARATIVE LAYER CHROMATOGRAPHY
Preparative layer chromatography (PLC) is used to isolate 10–500 mg or more of

material on layers thicker than those used for analytical TLC. As examples of commercial
preparative layers, Whatman, Analtech, and EM Science (Merck) offer 0.5–2 mm layers
composed of silica gel, alumina, cellulose, and C18 bonded silica gel. Preparative layers
of other sorbents and thicknesses may be available from other manufacturers and have
been made in the laboratory. The presence of a fluorescent indicator facilitates
nondestructive detection. The term “micropreparative layer chromatography (MPLC)”
has been used for separations of up to 10 mg on a 0.25 mm analytical layer.
Samples are applied as streaks, either manually or with an instrument such as the
Linomat, or by using a solid-phase sample application (SPSA) device (149). Ascending
and horizontal development have been used most often, and the mobile phase is often
chosen after preliminary tests on analytical plates. Incompletely separated bands are
scraped and eluted and rechromatographed on a second plate (262). Multiple
development and gradient elution have been applied for complex mixtures (263), and
RPC (Fig. 2) and OPLC have also been used for PLC and MPLC. A recent application of
centrifugal PLC is the fractionation of moderate molecular weight polysiloxanes for use
as secondary standards for column gel permeation chromatography (GPC) (263a).
If the compounds to be recovered are not colored or fluorescent and do not absorb UV
light, a detection reagent must be applied to the edges of the plate to locate the zones to
be recovered. Plates with prescored edges facilitate this process. Pure compounds are
recovered by scraping and elution with a suitable solvent (Fig. 9).
Layers for PLC are discussed in Section IV.G, and application of zones in Section
V.D.
Procedures and apparatus for PLC are described in Chapter 12 of Ref. 1, in Chapter 11
of this Handbook, and in Ref. 149.
XII. RADIOCHEMICAL TECHNIQUES
Radio-TLC techniques are described in Chapter 12 of this Handbook and Chapter 13 of

Ref. 1.
Thin-layer radiochromatography (TLRC), or radio-TLC, is used for separation,
identification, and measurement of radioisotopes. The principal methods employed are
autoradiography, zonal analysis, and the use of radiation detectors. An important
application of TLRC is in the quality control and development of radiopharmaceuticals
such as Tc-99m (264).
In autoradiography, an X-ray or photographic film is exposed to emissions from
radioactive zones on the layer to produce an image on the film. The exposed film is
developed by the usual photographic methods to reveal spots of varying darkness at the
locations of the separated zones. The darkness, which is related to the amount of
radioactivity, can be quantified by densitometry using a calibration curve prepared from a
film exposed to zones of radioactive standards. A disadvantage is the long exposure
period required for certain weakly radioactive isotopes. Two variations of direct-exposure
autoradiography are direct exposure with an intensifying screen (plates coated with
inorganic phosphors) and fluorography (impregnation of a scintillator into the layer
followed by direct exposure).
For zonal analysis, zones are removed by scraping, the sorbent is transferred into vials,
scintillation fluid is added, and the light emitted due to interaction of the radioactive
nuclei with the fluid is measured with a scintillation counter. The study of bile acids in
humans by liquid scintillation counting coupled with densitometry is an example of an
application (265).
A variety of radiation detectors have been used for TLRC, including spark chambers,
radioscanners, linear analyzers, a radioanalytic imaging system (Wallac Ambis),
multiwire proportional counters, and phosphor imaging analyzers. All of these have been
described in Chapter 12 of the first and second editions of this Handbook, but use of the
latter two has been reported most often since the second edition was published and is
discussed below.
The DAR is a two-dimensional position-sensitive multiwire proportional counter that
measures all radioactive zones simultaneously on a 20×20 cm plate. The metabolism of
the anxiolytic compound deramciclane was studied by using DAR after separation by
conventional TLC (266) and in combination with TLC/FAB-MS-MS (219) and
OPCL/MS (218, 267, 268). TLC and DAR were also coupled for the analysis of neutral
C14 lipids neosynthesized by the human sebaceous gland (269).
Phosphor or bioimaging analyzers operate by using a phosphor imaging plate made of
fine crystals of BaF:Eu2+ (Fugi Photo Co., Ltd.) to store emitted beta energy from the
layer; scanning
Figure 9 Prescored preparative plate

with scraped zone. Edges are snapped
off, detection reagent is applied, and
the edges are realigned to locate the
zones to be recovered by scraping.
the plate with a laser in the reading unit; and measuring the resulting luminescence,
which is proportional to the recorded radiation intensity, with a phototube. Radio-TLC
with this detector has been used for detection as well as reliable quantification (270) of
metabolites in pharmaco-kinetic studies (271) and of taxol and its metabolites in
microsomal samples (272).
XIII. TRANSFER OF RESULTS FROM TLC TO HPLC
Two approaches for using TLC data as a pilot method for mobile-phase optimization in
HPLC are correlation between retention parameters and thermodynamic description of
adsorption systems with mixed mobile phases (273). The first method was used in a study
involving silica gel, C18, C8, diol, NH2, and CN layers, 62 pesticide standards, and a real
sample by correlating k′p retention values from TLC to k′c values for HPLC by linear
regression. Good results of transfer were obtained except with silica gel and diol, with
which some restrictions were needed because the HPLC and TLC sorbents were not of
equal activity (274).
Multistep gradient elution RP-TLC on paraffin-impregnated silica gel of the colored
pigments in red wine was shown to accurately predict the mobile-phase gradient for the
same separation in C18-HPLC (275). Successful transfer studies were also published for
phenol and its methyl and chloro derivatives on bonded amino, cyano, and diol stationary
phases (276) and for choosing binary gradient programs for HPLC of raw products in a
synthesis research laboratory (277).
XIV. MULTIMODAL SEPARATION METHODS
The term “murtimodal” has been used in two ways, to designate layers such as cyano-
derivatized silica gels that can operate with two or more mechanisms (278) (see Sec.
IV.D) and, in the context of this section, to specify multidimensional separations that are
performed by on-line coupling of TLC, HPTLC, or OPLC with another technique in
order to improve the separation capacity available from either of the individual methods.
In the past, reports of the direct coupling of GC, SFE, and supercritical fluid
chromatography to TLC have appeared, but recent publications are limited to combining
HPLC and TLC.
Although the TLC-HPLC combination has been performed off-line by scraping and
eluting TLC zones followed by injection into the HPLC instrument (279) or by TLC of
collected column fractions (280), the most reported and most advantageous approach is
when the two methods are coupled on-line using TLC as the second stage. This sequence
allows utilization of the unique advantages of TLC, including further separation by a
diverse mechanism, static detection with multiple methods, and storage on the layer of all
zones in the column effluent fractions for evaluation without time constraints. A spray jet
aerosol sample applicator (13, 281, 282) has been most commonly used to deposit
column effluent onto the layer origin. As an example of an application, iprodione
residues in vegetables have been determined by RP-HPLC followed by TLC-AMD (283).
XV. TLC DETERMINATION OF LIPOPHILICITY
The use of RP-TLC quantitative structure-activity studies aimed at determining

lipophilicity has been reviewed (284, 285) but is not discussed in detail in this book. This
parameter is important because it governs molecular properties such as penetration of
bioactive compounds through hydrophobic cell membranes and uptake by target organs
or organisms (i.e., biological activity).
Lipophilicity is evaluated on the basis of the linear relationship between Rm values and
the concentration of organic solvent in the aqueous mobile phase in accordance with
well-known TLC equations. The Rm0 value, which is the theoretical Rm at 0% organic
solvent or pure water, is determined by extrapolation (286, 287). Paraffin oil-impregnated
silica gel (288) and C18 bonded silica gel (289) layers are used in most cases, and the
nature of the organic solvent has been found not to affect the measurement (290).
Principal component analysis has also been used to evaluate lipophilicity (291, 292).
Many compound types have been studied, including 1,2,4-triazole (293) and furan (294)
derivatives.
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Handbook of Thin-Layer

A Series of Textbooks and Reference Books

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

4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and

5. Quantitative Analysis by Gas Chromatography, Josef Novák

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

7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J.Gudzinowicz, Michael

8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes

10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald

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

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

18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald

20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L.

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.

25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca

26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S.Hancock

27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins,

29. Pyrolysis and GC in Polymer Analysis, edited by S.A.Liebman and E.J.Levy

31. Ion-Pair Chromatography, edited by Milton T.W.Hearn

34. Reaction Detection in Liquid Chromatography, edited by Ira S.Krull

35. Thin-Layer Chromatography: Techniques and Applications. Second Edition, Revised

36. Quantitative Thin-Layer Chromatography and Its Industrial Applications, edited by

37. Ion Chromatography, edited by James G.Tarter

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

42. Flow Perturbation Gas Chromatography, N.A.Katsanos

43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Nakanishi,

44. Countercurrent Chromatography: Theory and Practice, edited by N.Bhushan Mandava

45. Microbore Column Chromatography: A Unified Approach to Chromatography, edi

46. Preparative-Scale Chromatography, edited by Eli Grushka

47. Packings and Stationary Phases in Chromatographic Techniques, edited by Klaus

48. Detection-Oriented Derivatization Techniques in Liquid Chromatography, edited by

49. Chromatographic Analysis of Pharmaceuticals, edited by John A.Adamovics

51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M.

52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg

54. HPLC in Clinical Chemistry, I.N.Papadoyannis

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

56. Gas–Liquid–Solid Chromatography, V.G.Berezkin

57. Complexation Chromatography, edited by D.Cagniant

59. Trace Analysis with Microcolumn Liquid Chromatography, Milos Krejcl

60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by André P.

61. Preparative and Production Scale Chromatography, edited by G.Ganetsos and P.

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

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

71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded,

72. Liquid Chromatography of Oligomers, Constantin V.Uglea

74. ChromatographJc Analysis of Pharmaceuticals: Second Edition, Revised and