Über die Autorin bzw. den Autor

After working for many years for Heumann Pharma (now part of Pfizer), where she was in particular responsible for the training and education of lab technicians, Elke Hahn-Deinstrop is now a freelance consultant for thin-layer chromatography. She is author of numerous papers and frequent lecturer.

Von der hinteren Coverseite

Thin-layer chromatography (TLC) is a powerful, fast and inexpensive analytical method, which has proven its effectiveness in the analysis of pharmaceuticals, food and the environment. This new edition of the practical guide to TLC includes a completely revised chapter on documentation, which now covers the use of digital cameras, while selected new sorbents and instruments are also introduced.

This edition retains the successful features of its predecessor:

• Clear explanation of all steps in the analytical procedure, starting with the choice of a suitable TLC technique and finishing with data evaluation and documentation.
• Special emphasis on the correct choice of materials for TLC. Properties and functions of various materials and the TLC equipment are described, covering precoated layers, solvents and developing chambers, including information on suppliers, among others.
• Many practical hints for troubleshooting.
• Detailed description of using TLC in compliance with GLP/GMP regulations, including the required documentation , allowing readers to easily compile their own standard operating procedures.
• Numerous color illustrations.

From the reviews of the first edition:

"A clear, step-by-step practical guide to TLC, covering every stage of the process from plate selection and sample application to development and quantification. At every stage potential pitfalls are clearly signposted and each chapter contains a profusion of pracitcal tips. Even a seasoned TLC practitioner should find something of use or interest in this book which complements most existing texts."
—The Analyst

Aus dem Klappentext

Thin-layer chromatography (TLC) is a powerful, fast and inexpensive analytical method, which has proven its effectiveness in the analysis of pharmaceuticals, food and the environment. This new edition of the practical guide to TLC includes a completely revised chapter on documentation, which now covers the use of digital cameras, while selected new sorbents and instruments are also introduced.

This edition retains the successful features of its predecessor:

• Clear explanation of all steps in the analytical procedure, starting with the choice of a suitable TLC technique and finishing with data evaluation and documentation.
• Special emphasis on the correct choice of materials for TLC. Properties and functions of various materials and the TLC equipment are described, covering precoated layers, solvents and developing chambers, including information on suppliers, among others.
• Many practical hints for troubleshooting.
• Detailed description of using TLC in compliance with GLP/GMP regulations, including the required documentation , allowing readers to easily compile their own standard operating procedures.
• Numerous color illustrations.

From the reviews of the first edition:

"A clear, step-by-step practical guide to TLC, covering every stage of the process from plate selection and sample application to development and quantification. At every stage potential pitfalls are clearly signposted and each chapter contains a profusion of pracitcal tips. Even a seasoned TLC practitioner should find something of use or interest in this book which complements most existing texts."
—The Analyst

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Applied Thin-Layer Chromatography

John Wiley & Sons

Chapter One

Thin-layer chromatography (TLC) is a very old method of analysis that has been well proven in practice. For more than thirty years, it has occupied a prominent position, especially in qualitative investigations. With the development of modern precoated layers and the introduction of partially or completely automated equipment for the various stages of operation of TLC, not only are highly accurate quantitative determinations now possible, but also the requirement that the work should comply with the GMP/GLP guidelines can be fulfilled.

Following the widespread use of high-performance liquid chromatography (HPLC), the importance of TLC, mainly measured by the work rate of the method, has been forced into the background. This is reflected in the unfavorable treatment of TLC as taught in universities, higher technological teaching establishments, technical colleges and industry. In addition to this, the restructuring of the chemical industry begun some years ago and the consequent job losses have led to considerable loss of specialist know-how in the use of TLC.

For these reasons, it is hoped that the present book will point towards good practical methods of performing TLC. Special attention is paid to possible sources of error. Theoretical aspects are not placed in the foreground, but emphasis is rather placed on the current state of the technology and the scope of modern TLC. The arrangement of the book strictly follows the individual operating steps of TLC, so that the user will be able to locate these various steps with ease.

This book is mainly intended for the younger scientific generation. For teachers it tries to encourage a form of teaching close to practical "real-life" TLC analysis, and the many practical tips also offer invaluable support for the less experienced users in industrial and official laboratories. Last but not least, it can be used by the analyst in a pharmaceutical laboratory as a work of reference.

1.1 What Does TLC Mean?

Chromatography means a method of analysis in which a mobile phase passes over a stationary phase in such a way that a mixture of substances is separated into its components. The term "thin-layer chromatography", introduced by E. Stahl in 1956, means a chromatographic separation process in which the stationary phase consists of a thin layer applied to a solid substrate or "support". For some years, TLC has also been referred to as planar chromatography. However, apart from the fact that paper chromatography, which is also a planar method, is now hardly used, I do not think that this term will ever be widely accepted because the abbreviation PC could easily be confused with the abbreviation for personal computer.

1.2 When Is TLC Used?

An essential precondition is that the substances or mixtures of substances to be analyzed should be soluble in a solvent or mixture of solvents.

TLC is used if

the substances are nonvolatile or of low volatility

the substances are strongly polar, of medium polarity, nonpolar or ionic

a large number of samples must be analyzed simultaneously, cost-effectively, and within a limited period of time

the samples to be analyzed would damage or destroy the columns of LC (liquid chromatography) or GC (gas chromatography)

the solvents used would attack the sorbents in LC column packings

the substances in the material being analyzed cannot be detected by the methods of LC or GC or only with great difficulty

after the chromatography, all the components of the sample have to be detectable (remain at the start or migrate with the front)

the components of a mixture of substances after separation have to be detected individually or have to be subjected to various detection methods one after the other (e.g. in drug screening)

no source of electricity is available

1.3 Where Is TLC Used?

Pharmaceuticals and Drugs

Identification, purity testing and determination of the concentration of active ingredients, auxiliary substances and preservatives in drugs and drug preparations, process control in synthetic manufacturing processes.

Clinical Chemistry, Forensic Chemistry and Biochemistry

Determination of active substances and their metabolites in biological matrices, diagnosis of metabolic disorders such as PKU (phenylketonuria), cystinuria and maple syrup disease in babies.

Cosmetology

Dye raw materials and end products, preservatives, surfactants, fatty acids, constituents of perfumes.

Food Analysis

Determination of pesticides and fungicides in drinking water, residues in vegetables, salads and meat, vitamins in soft drinks and margarine, banned additives in Germany (e.g. sandalwood extract in fish and meat products), compliance with limit values (e.g. polycyclic compounds in drinking water, aflatoxins in milk and milk products).

Environmental Analysis

Groundwater analysis, determination of pollutants from abandoned armaments in soils and surface waters, decomposition products from azo dyes used in textiles.

Analysis of Inorganic Substances

Determination of inorganic ions (metals).

Other Areas

Electrolytic technology (meta-nitrobenzoic acid in nickel plating baths).

A graphical representation of the distribution of TLC publications among the most important fields of application during the years 1993 and 1994 is given in Fig. 1. However, this diagram does not give any indication of the distribution of the actual use of the technique. Reliable information on this subject is difficult to obtain. Information on quantities of materials used for TLC must mainly come from the manufacturers, but they are unwilling to release this on grounds of industrial secrecy. Our own research in northern and southern Germany has revealed that 40% of the precoated layers go to universities and other higher educational establishments for use in the areas of pharmacy, medicine and biology, while a further ca. 40% are used in the pharmaceutical industry, including use by pharmacists, and the remainder is divided between official investigative organizations (e.g. food monitoring, police and customs) and private institutions. This leads us to conclude that the majority of TLC users work in the area of pharmaceutical investigations. Recent polls confirm this distribution.

1.4 How is the Result of a TLC Represented?

Please do not expect a profound treatment of chromatographic parameters at this point. As beginners in TLC you should not be frightened off at the very beginning of this book. Any reader interested in the theory of TLC should read books devoted to this subject, the two by Geiss being especially recommended.

The subject of TLC has its own special parameters and concepts, the most important of these for practical purposes being described below.

1.4.1 Retardation Factor

The position of a substance zone (spot) in a thin-layer chromatogram can be described with the aid of the retardation factor [R.sub.f]. This is defined as the quotient obtained by dividing the distance between the substance zone and the starting line by the distance between the solvent front and the starting line (see Fig. 3):

[R.sub.f] = [Z.sub.S] / [Z.sub.F] - [Z.sub.0]

where

[R.sub.f] = retardation factor...