Preparative layer chromatography (1)

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Preparative Layer
Chromatography


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CHROMATOGRAPHIC SCIENCE SERIES
A Series of Textbooks and Reference Books
Editor: JACK CAZES

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

19.
20.

Dynamics of Chromatography: Principles and Theory,
J. Calvin Giddings
Gas Chromatographic Analysis of Drugs and Pesticides,
Benjamin J. Gudzinowicz
Principles of Adsorption Chromatography: The Separation
of Nonionic Organic Compounds, Lloyd R. Snyder
Multicomponent Chromatography: Theory of Interference,
Friedrich Helfferich and Gerhard Klein
Quantitative Analysis by Gas Chromatography, Josef Novák
High-Speed Liquid Chromatography, Peter M. Rajcsanyi
and Elisabeth Rajcsanyi
Fundamentals of Integrated GC-MS (in three parts),
Benjamin J. Gudzinowicz, Michael J. Gudzinowicz,
and Horace F. Martin
Liquid Chromatography of Polymers and Related Materials,
Jack Cazes
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
Biological/Biomedical Applications of Liquid Chromatography,
edited by Gerald L. Hawk
Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt
and T. H. Gouw
Biological/Biomedical Applications of Liquid Chromatography II,
edited by Gerald L. Hawk
Liquid Chromatography of Polymers and Related Materials II,
edited by Jack Cazes and Xavier Delamare

Introduction to Analytical Gas Chromatography: History, Principles,
and Practice, John A. Perry
Applications of Glass Capillary Gas Chromatography, edited by
Walter G. Jennings
Steroid Analysis by HPLC: Recent Applications, edited by
Marie P. Kautsky
Thin-Layer Chromatography: Techniques and Applications,
Bernard Fried and Joseph Sherma
Biological/Biomedical Applications of Liquid Chromatography III,
edited by Gerald L. Hawk
Liquid Chromatography of Polymers and Related Materials III,
edited by Jack Cazes
Biological/Biomedical Applications of Liquid Chromatography,
edited by Gerald L. Hawk


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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 Zief
and 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 Naka-nishi, and Ryuichi Matsuno
44. Countercurrent Chromatography: Theory and Practice,

edited by N. Bhushan Man-dava and Yoichiro Ito
45. Microbore Column Chromatography: A Unified Approach
to Chromatography, edited 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 Hernan Cortes


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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 KrejcI
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. Chromatographic Analysis of Pharmaceuticals: Second Edition,
Revised and Expanded, edited by John A. Adamovics
75. Supercritical Fluid Chromatography with Packed Columns: Techniques
and Applications, edited by Klaus Anton
and Claire Berger
76. Introduction to Analytical Gas Chromatography: Second Edition,
Revised and Expanded, Raymond P. W. Scott
77. Chromatographic Analysis of Environmental and Food Toxicants,

edited by Takayuki Shibamoto
78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen,
Peter Schoenmakers, and Neil Miller
79. Liquid Chromatography–Mass Spectrometry: Second Edition,
Revised and Expanded, Wilfried Niessen
80. Capillary Electrophoresis of Proteins, Tim Wehr,
Roberto Rodríguez-Díaz, 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


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83. Micellar Liquid Chromatography, Alain Berthod
and Celia García-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 by
Anurag S. Rathore and Ajoy Velayudhan

89. Handbook of Thin-Layer Chromatography: Third Edition,
Revised and Expanded, edited by Joseph Sherma and Bernard Fried
90. Chiral Separations by Liquid Chromatography and Related
Technologies, Hassan Y. Aboul-Enein and Imran Ali
91. Handbook of Size Exclusion Chromatography and Related Techniques:
Second Edition, edited by Chi-San Wu
92. Handbook of Affinity Chromatography: Second Edition, edited by
David S. Hage
93. Chromatographic Analysis of the Environment: Third Edition,
edited by Leo M. L. Nollet
94. Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis
and Discovery, Paul C.H. Li
95. Preparative Layer Chromatography, edited by Teresa Kowalska
and Joseph Sherma


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Preparative Layer
Chromatography

edited by

Teresa Kowalska
University of Silesia
Katowice, Poland

Joseph Sherma
Lafayette College
Easton, Pennsylvania

Boca Raton London New York

A CRC title, part of the Taylor & Francis imprint, a member of the
Taylor & Francis Group, the academic division of T&F Informa plc.


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Published in 2006 by
CRC Press
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© 2006 by Taylor & Francis Group, LLC
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International Standard Book Number-10: 0-8493-4039-X (Hardcover)
International Standard Book Number-13: 978-0-8493-4039-0 (Hardcover)
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Library of Congress Cataloging-in-Publication Data
Preparative layer chromatography / edited by Teresa Kowalska and Joseph Sherma.
p. cm. -- (Chromatographic science series ; 95)
Includes bibliographical references and index.
ISBN 0-8493-4039-X (alk. paper)
1. Preparative layer chromatography. I. Kowalska, Teresa. II. Sherma, Joseph. III. Chromatographic
science ; v. 95.
QD79.C52P74 2006
543'.84--dc22

2005052138

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Preface
This book has been designed as a practical, comprehensive source of information
on the field of classical preparative layer chromatography (PLC). It is organized in
two parts, the first of which covers the theory and up-to-date procedures of PLC
(Chapter 1 to Chapter 8), while the second (Chapter 9 to Chapter 16) includes
applications to a selection of the most important compound classes and samples
types. Overall, the topics covered in the 16 chapters are evidence for the versatility
and wide use of PLC at the current time. We have designed this first book ever
published on PLC to be valuable for scientists with a high degree of experience in
the separation sciences, but because most chapters include considerable introductory
and background information, it is also appropriate for the relatively inexperienced
chromatographer.
The contributors to the book are experts on the topics about which they write
and include many of the best known and most knowledgeable workers in the field
of thin-layer chromatography and PLC throughout the world. Rather than attempting
to adopt a uniform style, we have allowed chapter authors the freedom to present
their topics in a way that they considered most effective. They have used figures
and tables as needed to augment the text, and selective reference lists include the
most important new literature, as well significant older references, to set the basis
of their chapters.
We had great cooperation from the authors in submitting their chapters in a
timely fashion, so that the book has been completed about six months sooner than
anticipated. None of the chapters was unduly delayed, so all are equally up to date
in their coverage. The authors represent laboratories in Germany, Poland, Romania,
Norway, Canada, Japan, India, and the U.S. and, therefore, have provided a global
perspective for the book.
We hope that this book will be valuable for practitioners and teachers in diverse

scientific fields that make use of chromatographic methods and that it will promote
better understanding of the field and lead to its even wider utilization.
Teresa Kowalska
Joseph Sherma


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About the Editors
Teresa Kowalska is currently a professor in the Department of the Physicochemical
Basis of Chromatography at the University of Silesia (Katowice, Poland). Her
scientific interests include the physicochemical foundations of liquid chromatography and gas chromatography, with special attention focused on modeling of planar
chromatography both in its analytical and preparative mode. Over the past 37 yr Dr.
Kowalska has directed the programs of over 70 M.Sc. degree students who have
carried out their research on the theory and practice of different chromatographic
and hyphenated techniques. She has also supervised the research in the separation
science of 8 Ph.D. students. Dr. Kowalska is the author of more than 200 scientific
papers, more than 300 scientific conference papers, and a vast number of the book
chapters and encyclopedia entries in the field of chromatography. It is perhaps
noteworthy that she has authored (and then updated) the chapter on “Theory and
Mechanism of Thin-Layer Chromatography” for all three editions of the Handbook
of Thin-Layer Chromatography, edited by professors J. Sherma and B. Fried, and
published by Marcel Dekker.
Dr. Kowalska has acted as editor of Acta Chromatographica, the annual periodical published by the University of Silesia (Katowice, Poland) and devoted to all
chromatographic and hyphenated techniques, right from its establishment in 1992.
Acta Chromatographica appears as a hard copy journal and also online in the digital
format. Its contributors originate from an international academic community, and it

is meant to promote the development in separation sciences. It apparently serves its
purpose well, as can be judged from a wide readership, abundant citations throughout
the professional literature, and also from the ISI ranking quota.
Last but not least, in the course of the past almost 30 yr Dr. Kowalska has
been active as organizer (and in recent years as a cochairperson, also) of the annual
all-Polish chromatographic symposia with international participation, uninterruptedly held each year (since 1977) in the small mountain resort of Szczyrk in South
Poland. Integration of an international community of chromatographers through
these meetings has been regarded by Dr. Kowalska as a specific yet important
contribution to chromatography.
Joseph Sherma is John D. and Frances H. Larkin Professor Emeritus of Chemistry
at Lafayette College, Easton, Pennsylvania. He is author or coauthor of over 500
scientific papers and editor or coeditor of over 50 books and manuals in the areas
of analytical chemistry and chromatography. Dr. Sherma is coauthor, with Bernard
Fried, Kreider Professor Emeritus of Biology at Lafayette College, of Thin Layer
Chromatography (editions 1 to 4) and coeditor with Professor Fried of the Handbook
of Thin Layer Chromatography (editions 1 to 3), both published by Marcel Dekker,
Inc. He served for 23 yr as the editor for residues and trace elements of the Journal


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of AOAC International and serves currently on the editorial advisory boards of the
Journal of Liquid Chromatography and Related Technologies, the Journal of Environmental Science and Health (Part B), the Journal of Planar Chromatography–Modern TLC, Acta Chromatographica, and Acta Universitatis Cibiniensis, Seria
F. Chemia. Dr. Sherma received his Ph.D. degree (1958) from Rutgers State University, New Brunswick, New Jersey.


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Contributor List
Dr. Simla Basar

Institute of Organic Chemistry
University of Hamburg
Hamburg, Germany
Prof. Jan Bladek
/¸
Institute of Chemistry
Military University of Technology
Warsaw, Poland
Dr. Virginia Coman
Raluca Ripan Institute for Research in
Chemistry
Cluj-Napoca, Romania
Dr. Tadeusz H. Dzido
Faculty of Pharmacy
Medical Academy of Lublin
Lublin, Poland
Dr. Monika Fabianska
´
Faculty of Earth Sciences
The University of Silesia
Sosnowiec, Poland
Prof. George W. Francis
Department of Chemistry
University of Bergen
Bergen, Norway

Dr. Weerasinghe Indrasena
Ocean Nutrition Canada Ltd.
Halifax, Nova Scotia, Canada
Grzegorz Józwiak

´
Faculty of Pharmacy
Medical University of Lublin
Lublin, Poland
Prof. Krzysztof Kaczmarski
Faculty of Chemistry
Technical University of Rzeszów
Rzeszów, Poland
Dr. Angelika Koch
Frohme Apotheke
Hamburg, Germany
Prof. Teresa Kowalska
Institute of Chemistry
The University of Silesia
Katowice, Poland
Dr. Emi Miyamoto
Department of Health Science
Kochi Women’s University
Kochi, Japan

Dr. Michal/ /L. Hajnos
Faculty of Pharmacy
Medical University of Lublin
Lublin, Poland

Dr. Ali Mohammad
Department of Applied Chemistry,
Faculty of Engineering and Technology
Aligarh Muslim University
Aligarh, India

Dr. Heinz E. Hauck
LSA/R&D
Merck KGaA
Darmstadt, Germany

Dr. Gertrud E. Morlock
University of Hohenheim
Institute of Food Chemistry
Stuttgart, Germany


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Dr. Beata Polak
Faculty of Pharmacy
Medical University of Lublin
Lublin, Poland
Dr. Wojciech Prus
University of Bielsko-Biala
Faculty of Textile Engineering and
Environmental Protection
Department of Chemistry
Bielsko-Biala, Poland
Rita Richter
Institute of Organic Chemistry
University of Hamburg,
Hamburg, Germany
Dr. Mieczyslaw
/

Sajewicz
Institute of Chemistry
The University of Silesia
Katowice, Poland
Michael Schulz
LSA/R&D
Merck KGaA
Darmstadt, Germany

Dr. Joseph Sherma
Department of Chemistry
Lafayette College
Easton, Pennsylvania
Prof. Bernd Spangenberg
Environmental Techniques Section
University of Applied Sciences
Offenburg, Germany
Anna Szymanczyk
´
Institute of Chemistry
Military University of Technology
Warsaw, Poland
Prof. Monika Waksmundzka-Hajnos
Faculty of Pharmacy
Medical University of Lublin
Lublin, Poland
Prof. Fumio Watanabe
Department of Health Science
Kochi Women’s University
Kochi, Japan

Prof. Teresa Wawrzynowicz
Faculty of Pharmacy
Medical University of Lublin
Lublin, Poland


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Contents
SECTION I
Chapter 1

Introduction ..........................................................................................3

Teresa Kowalska and Joseph Sherma
Chapter 2

Adsorption Planar Chromatography in the Nonlinear Range:
Selected Drawbacks and Selected Guidelines...................................11

Krzysztof Kaczmarski, Wojciech Prus, Mieczyslaw
/
Sajewicz, and Teresa Kowalska
Chapter 3

Sorbents and Precoated Layers in PLC.............................................41

Heinz E. Hauck and Michael Schulz
Chapter 4

Selection and Optimization of the Mobile Phase for PLC ...............61

Virginia Coman
Chapter 5

Sample Application and Chromatogram Development .....................99

Gertrud E. Morlock
Chapter 6

On Methodical Possibilities of the Horizontal Chambers
in PLC ..............................................................................................131

Tadeusz H. Dzido and Beata Polak
Chapter 7

Location of Separated Zones by Use of Visualization Reagents,
UV Absorbance on Layers Containing a Fluorescent Indicator,
and Densitometry .............................................................................163

Bernd Spangenberg
Chapter 8

Additional Detection Methods and Removal of Zones from
the Layer ..........................................................................................177

Joseph Sherma


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SECTION II
Chapter 9

Medical Applications of PLC ..........................................................193

Jan Bladek
/¸
and Anna Szymanczyk
´
Chapter 10 PLC of Hydrophilic Vitamins ..........................................................237
Fumio Watanabe and Emi Miyamoto
Chapter 11 Preparative Layer Chromatography of Natural Mixtures ...............251
Monika Waksmundzka-Hajnos, Teresa Wawrzynowicz, Michal/ L.
/ Hajnos,
and Grzegorz Józwiak
´
Chapter 12 Application of Preparative Layer Chromatography to Lipids.........299
Weerasinghe M. Indrasena
Chapter 13 The Use of PLC for Separation of Natural Pigments.....................325
George W. Francis
Chapter 14 Application of PLC to Inorganics and Organometallics .................347
Ali Mohammad
Chapter 15 PLC in a Cleanup and Group Fractionation of Geochemical
Samples: A Review of Commonly Applied Techniques .................369
Monika J. Fabianska
´
Chapter 16 The Use of PLC for Isolation and Identification of Unknown
Compounds from the Frankincense Resin (Olibanum):
Strategies for Finding Marker Substances.......................................391

Angelika Koch, Rita Richter, and Simla Basar
Index......................................................................................................................413


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Section I


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1

Introduction
Teresa Kowalska and Joseph Sherma

CONTENTS
1.1 Chromatography Background ..........................................................................3
1.2 Basics of PLC ..................................................................................................4
1.3 Principles and Characteristics of PLC.............................................................5
1.4 Organization of the Book ................................................................................8
1.5 Epilogue .........................................................................................................10
References................................................................................................................10

1.1 CHROMATOGRAPHY BACKGROUND
The invention of chromatography can be traced to the milestone paper published in
1906 by the Russian botanist and plant physiologist Mikhail Semyonovitch Tswett

(1872–1919) [1,2]. In the experiments reported in this paper, Tswett separated
chloroplast pigments from leaves in a column of precipitated chalk washed with
carbon disulfide mobile phase. During the 20th century and in the new millennium,
chromatography has become an indispensable separation tool that is very widely
used in natural and life science laboratories throughout the world.
Tswett’s initial column liquid chromatography method was developed, tested,
and applied in two parallel modes, liquid–solid adsorption and liquid–liquid partition. Adsorption chromatography, based on a purely physical principle of adsorption,
considerably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This
remains true today in thin-layer chromatography (TLC), for which silica gel is by
far the major stationary phase. In column chromatography, however, reversed-phase
liquid chromatography using chemically bonded stationary phases is the most popular method.
Preparative layer chromatography (PLC) was apparently first reported by F.J.
Ritter and G.M. Meyer in 1962 [3]. They used layers of 1-mm thickness. Earlier
preparative work, e.g., that reported by J.M. Miller and R.G. Kirchner (the inventors
of TLC as it is performed today by development of the layer in a closed tank,
analogous to ascending paper chromatography) in 1951 and 1952 [4,5], was termed
TLC but was carried out on adsorbent bars used as columns or on analytical layers
after column chromatography. In his classic TLC laboratory handbook, originally
published in German in 1962 and translated to English in 1965 [6], Egon Stahl made
only a few statements about the method he called “micropreparative TLC.” Layers
3


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4

Preparative Layer Chromatography

of 0.5- and 0.7-mm thickness prepared with a spreading device were recommended

for this method by Stahl, as well as streak (band) application of larger quantities of
mixture using a “specially designed instrument” (described in the Ritter and Meyer
paper [3]) or a “microspray gun” (used in Stahl’s laboratory).

1.2 BASICS OF PLC
PLC is used to separate and isolate amounts of material (e.g., 10 to 1000 mg) larger
than those used in analytical TLC. The purpose of PLC is to obtain pure compounds
for further chromatographic or spectrometric analysis or for determination of biological activity. “Classical” PLC (CPLC), involving mobile phase migration by
capillary action, requires relatively simple and inexpensive equipment, but thorough
comprehension of the relevant chromatographic principles and techniques is critical.
The required information for performing successful PLC is provided in this book.
The sample dissolved in a weak (nonpolar for silica gel), volatile solvent is applied
as a narrow band across the plate. Manual application can be achieved with a pipet
or syringe guided by a ruler, or round spots can be placed close together, side by side,
in a line. Sample application instruments are available commercially, e.g., a mechanical streaker from Analtech and an automated spray-on apparatus from CAMAG.
Plates with 0.5- to 2-mm layer thickness are normally used for increased loading
capacity. Layers can be self-made in the laboratory, or commercially precoated
preparative plates are available with silica gel, alumina, cellulose, C-2 or C-18
bonded silica gel, and other sorbents. Resolution is lower than on thinner analytical
layers having a smaller average particle size and particle size range. Precoated plates
with a preadsorbent or concentrating zone facilitate application of sample bands.
The mobile phase is usually selected by trial-and-error guided by prior experience
or by performing preliminary analytical separations of the sample in a saturated
chamber. PLC separations will be inferior to analytical TLC separations using the
same mobile phase because of the thicker layer, larger particle size, and overloaded
sample conditions used for PLC. A good general rule is that analytical TLC should
achieve separations with least 0.1 Rf value difference if the PLC separations are to be
adequate with the transferred mobile phase. Isocratic development is usually used, but
gradient development has been applied in certain situations for increased resolution.
Rectangular glass tanks (N chambers) with inner dimensions of 21 ¥ 21 ¥ 9 cm

are used most frequently for the ascending, capillary-flow development of PLC plates,
which usually measure 20 ¥ 20 cm. The tank is lined with thick chromatography
paper (e.g., Whatman 3 MM) soaked in the mobile phase and allowed to equilibrate
with the mobile phase vapor for up to 2 h prior to development over a maximum
distance of 18 cm. A saturated chamber provides faster capillary flow of the mobile
phase, more uniform bulk and alpha solvent fronts, and higher separation efficiency.
A plate angle of 75° from horizontal is recommended for the fastest development
with minimum zone distortion. Special taper plates (Analtech) with layer thicknesses
ranging from 300 mm (bottom) to 1700 mm (top) provide increased mobile phase
velocity compared to plates with uniform layer thickness. Circular, multiple, and
two-dimensional development, as well as development at temperatures other than
the ambient, are also used for PLC in special applications.


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Introduction

5

Zones containing separated compounds can be detected nondestructively after
plate development and evaporation of the mobile phase by their natural color in
white light, natural fluorescence under 254-nm or 366-nm ultraviolet (UV) light, or
absorption of UV light on layers containing a fluorescent indicator (phosphor). This
method, termed fluorescence quenching, gives dark zones on a fluorescent background. Postchromatographic chromogenic or fluorogenic reagents can be used to
detect compounds that are not naturally visible or fluorescent and do not quench
fluorescence. One of the most widely used reagents is iodine vapor, which reversibly
detects many types of compounds as brown zones. Destructive chromogenic or
fluorogenic reagents must be applied only to the side edges of the layer (the rest of
the layer is covered with a glass plate) to locate the areas from which to recover the

separated compounds.
The zones containing the desired compounds are scraped from the plate backing,
the compounds are eluted with a strong solvent, any remaining sorbent particles are
separated, and the solution is concentrated.
All of these steps are described in greater detail in the chapters in Section I of
this book.

1.3 PRINCIPLES AND CHARACTERISTICS OF PLC
Successful separations in adsorption chromatography (adsorption TLC included)
are due to the difference in the energies of adsorption between the two separated
species. The phenomenon of adsorption on a solid–liquid interface can be characterized best by the empirical adsorption isotherm, which is unique for a given
compound with a particular stationary phase–mobile phase combination. There is
one feature, however, that all mixture components share in common, namely, the
general nature of their adsorption isotherms. Each empirical isotherm consists of
a linear and nonlinear part. The linear part corresponds to the stepwise saturation
of active sites on the adsorbent surface, prior to its complete saturation. For a
compound deposited on the adsorbent surface within the linear range of the isotherm, a circular TLC zone shape is expected and the densitometrically measured
mass distribution (i.e., the concentration profile of the zone) should be regular
(Gaussian). In the case of mass overload, the system operates within the nonlinear
sector of the isotherm, with an oval zone shape and tailing of the skewed (nonGaussian) concentration profile.
There is no rigid demarcation line between adsorption TLC operating within the
linear or nonlinear range for the following reasons: (1) each individual solute is
characterized by its own adsorption isotherm, (2) for most analytes in most chromatographic systems, the respective adsorption isotherms remain unknown, and (3)
there is no need for steady control of the isotherm sector that is utilized in an
experiment. When separating a compound mixture, it often happens that some of
the constituents are in the linear range of their adsorption isotherms, whereas others
are in the nonlinear (i.e., mass-overload) range.
However, mostly because of the intuitive, trial-and-error approach of thin-layer
chromatographers, long ago the technique split into two subtechniques, one benefiting from the linear range of the adsorption isotherm and the other utilizing the

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Preparative Layer Chromatography

advantages of the nonlinear range. Much less attention has been given to nonlinear
TLC, and no monograph covering this subject has ever appeared on the market until
now. The remainder of this section will be focused on this very important, yet
considerably underestimated, method.
Linear TLC is well suited to perform traditional analytical tasks. With its use,
one can successfully separate mixtures consisting of a limited number of analytes
and, using more or less sophisticated supplementary techniques, identify the constituents of such mixtures on the layer. With more complex samples, linear TLC can
at least help to fractionate samples into the separate classes of compounds. Owing
to the proportionality between the amount of solute contained in zones and their in
situ densitometric scan areas, a standard calibration plot that is useful for quantification of an analyte in a sample can be established. In summary, linear TLC conforms
very well with many separation-, identification-, and quantification-oriented analytical strategies that are focused on the individual chemical species. Owing to its
flexibility, economy, and relative simplicity, linear TLC sometimes outperforms
certain other more expensive and less user-friendly analytical techniques. Optimization of an analytical result of linear TLC can be attained easily with a number of
simple semiempirical, or even purely empirical, retention models (e.g., the MartinSynge or Snyder-Soczewinski approaches); in more difficult cases, a variety of more
advanced chemometric approaches are also available. Knowledge of the operating
principles, techniques, and applications of linear TLC can be learned from books
published in many languages throughout the world, and also from a selection of
international scientific journals devoted exclusively, or partially, to the chromatographic sciences.
Contrary to the linear mode, nonlinear adsorption TLC was conceived, and to
a large extent remained, an “unofficial” and almost “underground” separation technique. Among chromatography practitioners, it is known as PLC (or PTLC [preparative thin-layer chromatography] or PPC [preparative planar chromatography]), and
it is generally accepted that this otherwise very useful separation tool does not serve
an analytical purpose, at least in the sense discussed in the previous paragraph. As
described in the last section, in PLC much higher amounts of the mixtures are applied

to be separated than in linear TLC. Therefore, the layer has to be considerably
thicker, and the optimization rules borrowed from the linear mode are either obeyed
to a much lesser extent or do not hold at all. Thus, a simple statement that PLC
lacks a theoretical basis of its own is essentially true. There is no book published
in English or any other language from which this technique can be learned. In most
cases, it is individually “discovered” by those who need a simple, rapid, inexpensive,
and low-scale (i.e., microgram or milligram) separation tool and are fortunate enough
to be familiar with the more common analytical TLC. The basic disadvantage of all
of these “amateur discoveries,” no matter how inquisitive and inventive their authors
might be, is that they are usually made in an unorganized trial-and-error manner
(sometimes aided by advice from a slightly more experienced colleague), and they
eventually prove far less beneficial than they could be if properly introduced in book
form by an expert, or group of experts.
All researchers involved in natural and life sciences who wish to isolate or purify
microgram or milligram quantities of a given compound but, for whatever reason,


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7

cannot make use of a column technique would benefit from the correct use of PLC.
For many scientists worldwide, the fully automated column approach can prove to
be too expensive, too complex, or both. Nonpressurized open preparative column
chromatography can be chosen, but this is not very effective with low levels of
compounds, particularly not with difficult separations (i.e., in the case of closely
migrating or partially overlapping peak profiles).
PLC is well suited for micropreparative separations. For example, consider the

simple scenario of someone involved in a multistage organic synthesis and needing
rapid spectrometric (e.g., mass, infrared, nuclear magnetic resonance, or x-ray)
confirmation of each step of the synthetic procedure (available today for trace
amounts of the compounds). In such cases, optimization of compound isolation with
an instrumental technique will almost certainly prove to be much more time consuming and expensive than the planar mode, e.g., using short, narrow strips of
aluminum- or plastic-backed adsorbent layers or microscope-slide-sized glassbacked plates in a pilot procedure.
Manufacturers of TLC materials and accessories are well prepared to satisfy the
needs for professionally performed PLC. High-quality precoated preparative plates
are available from a number of commercial sources. Alternatively, less expensive or
specialty preparative plates can be “homemade” in the laboratory, and loose sorbents
and coating devices can be purchased for this purpose. More-or-less-automated
devices can also be purchased for band application of higher quantities of sample
solutions to preparative layers. At least for some users, sophisticated densitometric
and other instrumental techniques are available as nondestructive tools for preliminary detection and identification of separated compounds in order to enhance the
efficiency of their isolation. The only aid still missing, and maybe the most important
of all, is a comprehensive monograph on PLC that might encourage and instruct
many potential users on how to fully benefit from this very versatile, efficient,
relatively inexpensive, and rather easy to use isolation and purification technique.
This book was planned to fill that void.
The oldest, simplest, and most frequently employed method for feeding the
mobile phase to the layer in PLC is with the aid of capillary forces in the ascending
direction. Forced-flow development can also be used for analytical TLC and PLC.
The most popular forced-flow modes are overpressured layer chromatography
(OPLC; sometimes termed optimum performance laminar chromatography), in
which the mobile phase flow is due to mechanical force (pressure), and rotation
planar chromatography (RPC), for which the transport of the mobile phase occurs
due to a centrifugal force. In this book, coverage is restricted to CPLC, which
operates with capillary flow and is accessible in all laboratories without the purchase
of quite expensive and rather complex instrumentation needed for preparative OPLC
and RPC. CPLC is technically compatible with capillary flow analytical TLC, the

mode that is most widely used. This means that for the two methods (analytical
and preparative), virtually the same laboratory equipment and procedures can be
used (e.g., sorbent type, coating device [if commercial precoated plates are not
used], chamber, sample application device, mobile phase, development mode
[ascending], reagent sprayer, etc.), and personnel can easily switch from one method
to the other or perform both in a parallel manner at the same time. This technical


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Preparative Layer Chromatography

compatibility of the two modes and the ease of switching between them represent
significant cost-saving advantages that for most laboratories throughout the world
are difficult to overestimate.
Another reason for choosing CPLC as the sole option discussed in this book is
that the chromatographic behavior of compounds in this method resembles most
closely that observed in capillary flow analytical TLC. This is particularly important
in view of the fact that, so far, the theoretical background of PLC has been practically
nonexistent, while the semiempirical rules valid for capillary flow analytical TLC
can be approximated relatively well for use in CPLC. This means that an experienced
chromatographer well acquainted with the separation potential of capillary flow
analytical TLC has a good chance to (intuitively, at least) select working parameters
for CPLC that will produce a close to optimum result. The same goal cannot,
however, be so easily reached with preparative OPLC and RPC. For example, the
forced flow that operates in OPLC elevates quite drastically the chromatographic
activity of the adsorbent layer [7]. This results from the mobile phase being pushed
through the narrow pores of the layer, which are impenetrable when capillary forces

alone are at play. The elevation of adsorbent activity is equivalent to a corresponding
lowering of the mobile phase strength, leading to a considerable change in retention
of solutes in a given layer-mobile phase system and making an intuitive, experiencebased system optimization virtually impossible.

1.4 ORGANIZATION OF THE BOOK
Section I of this book includes chapters on the principles and practice of PLC. After
this introductory Chapter 1, Chapter 2 provides information on efforts undertaken
to date in order to establish the theoretical foundations of PLC. With growing
availability and popularity of modern computer-aided densitometers, separation
results can be obtained in digital form as a series of concentration profiles that can
be relatively easily assessed and processed. From these, relevant conclusions can be
drawn in exactly the same manner as in automated column chromatographic techniques. Efforts undertaken to build a theoretical foundation of PLC largely consist
of adaptation of known strategies (with their validity confirmed in preparative column liquid chromatography) to the working conditions of PLC systems.
Chapter 3 through Chapter 8 deal with the basic aspects of the practical uses of
PLC. Chapter 3 describes sorbent materials and precoated layers for normal or
straight phase (adsorption) chromatography (silica gel and aluminum oxide 60) and
partition chromatography (silica gel, aluminum oxide 150, and cellulose), and precoated layers for reversed-phase chromatography (RP-18 or C-18). Properties of the
bulk sorbents and precoated layers, a survey of commercial products, and examples
of substance classes that can be separated are given.
Chapter 4 discusses the selection and optimization of mobile phases for successful separations in PLC. Chapter 5 details procedures for sample application
and development of layers, and Chapter 6 complements Chapter 5 by dealing
specifically with the use of horizontal chambers for the development of preparative
layers, including linear, continuous, two-dimensional, gradient, circular, and anticircular modes.