Cyclodextrins in Chromatography
By T Cserháti and E Forgács, Hungarian Academy of Sciences, Budapest, Hungary
Electrochemical Detection in the HPLC of Drugs and Poisons
R J Flanagan, Guy’s and St Thomas’ NHS Foundation Trust, London, UK, D Perrett,
Queen Mary’s School of Medicine and Dentistry, London, UK and R Whelpton,
University of London, London, UK
HPLC: A Practical Guide
T Hanai, Health Research Foundation, Kyoto, Japan
Peter E. Wall
VWR International Ltd., Poole, Dorset
ISBN 0-85404-535-X
A catalogue record for this book is available from the British Library
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and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in
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stated here should be sent to The Royal Society of Chemistry at the address printed on this page.
Thin-layer chromatography (TLC) is without doubt one of the most versatile and
widely used separation methods in chromatography. Commercially, many sorbents
on a variety of backings are now available. Most stages of the technique are now
automated (can now be operated instrumentally) and modern HPTLC (High
performance thin-layer chromatography) allows the handling of a large number of
samples in one chromatographic run. Speed of separation (development time), high
sensitivity and good reproducibility all result from the higher quality of
chromatographic layers and the continual improvement in instrumentation. In
addition TLC has remained relatively inexpensive and one can easily see why it is
still popular today. It has found a use in a wide range of application areas as the
concept of TLC is so simple and samples usually require only minimal pre-
treatment. It is often thought of only in terms of its use in pharmaceutical analysis
and production and in clinical analysis, but many standard methods in industrial
chemistry, environmental toxicology, food chemistry, water, inorganic and
pesticide analysis, dye purity, cosmetics, plant materials, and herbal analysis rely
upon TLC as the preferred approach. In its simplest form, TLC costs little, but even
including the more sophisticated instrumentation, it still remains less expensive per
sample analysis than, for example HPLC. Thin-layer chromatography continues to
be an active technique in research with about 500–700 publications appearing each
year during the 1980s and 1990s.
It is the purpose of this book to describe the advances made, particularly in the
last two to three decades, which have revolutionised TLC and transformed it into
a modern instrumental technique. All aspects of TLC have been affected, from
the sorbent layer technology, through the ‘‘spotting’’ devices and developing
equipment, to the final detection and quantification. Computers too, now play an
important role in the control of equipment and in the computation of the vast
amount of data obtained from scans of the developed TLC layer. This has resulted
in the ability to store and retrieve images of chromatograms and physical data on
actual separation results and conditions for future use. Instrumental planar
chromatography is now capable of handling samples with minimal pre-treatment,
detecting components at low nanogram sensitivities and with relative standard
deviations of about 1%. It is the opinion of the author that these developments
v
vi Preface
demonstrate that the previous image of low sensitivity, poor resolution and
reproducibility can be discarded and that TLC is now truly a modern contemporary
of HPLC and GC. Modern TLC has become a powerful, reliable and cost efficient
method for qualitative and quantitative analysis.
The chapters in this book have been designed in such a way that the reader
follows each step of the planar chromatographic process in logical order. Hence the
choice of sorbent is followed by preparation of sample for application and the
methods of application. The subject of chromatogram development logically comes
next with detection, quantification and/or video imaging usually being the final
steps. However, sometimes further instrumental analysis is necessary, hence the
final chapter on hyphenated techniques. Any theory or necessary mathematical
equations or expressions are introduced when required within the text of each
subject. As the objective of this book is to provide a publication or manual that can
be used by the practising chromatographer, the depth of theory reflects only what
is required to explain why certain practical steps are taken. It is the intention of
the author that this book will be of practical value and use to those who are
contemplating using TLC for the first time, and also to those who have been planar
chromatographers for some time. With these points in mind, the practical examples
of chromatographic separations reflect the field-tested procedures available. The
book concentrates on the basic steps involved in TLC, providing practical guidance
to achieve superior separations on a TLC/HPTLC sorbent layer. For this reason
there are some techniques that are not covered, such as sorbent coated quartz rods
and thin-layer radiochromatography. However, the basic principles for optimised
separations described in the various chapters will still apply in these related
techniques. Recommendations made throughout the text to obtain acceptable
and often high quality results are made on the basis of many years of practical
experience in planar chromatography by the author.
Numerous commercial products are referred to in this book as would be
necessary with any publication that discusses instrumental TLC/HPTLC. The
references made are based on the experiences of the author with these products,
and are definitely not meant to imply that they are superior to comparable products
from other manufacturers.
Contents
vii
viii Contents
9 HPTLC Pre-coated Plates 45
9.1 HPTLC Spherical Silica Gel 60 48
10 Improving Resolution with Buffers and Complexing
Reagents 49
10.1 Impregnation of Sorbent before Layer Coating 49
10.2 Impregnation of the Ready Coated Layer 50
11 Silver Nitrate Impregnation 50
12 Charge Transfer TLC 51
13 Preparative Layer Chromatography (PLC) 54
14 References 55