Synopsis
Thin-layer chromatography (TLC) is a powerful, fast and inexpensive analytical method. It has proven its usefulness in pharmaceutical, food and environmental analysis. This new edition of the practical TLC guide features a completely revised chapter on documentation, now including the use of digital cameras. Selected new sorbents and instruments are also introduced.
Why has the prior edition been successful? All steps of the analytical procedure are clearly explained, starting with the choice of a suitable TLC technique and ending with data evaluation and documentation. Special emphasis is put on the proper choice of materials for TLC. Properties and functions of various materials and the TLC equipment are described, covering e.g. precoated layers, solvents and developing chambers, including information on suppliers. Many practical hints for trouble shooting are given. All this is illustrated with numerous coloured figures.
How to use TLC in compliance with GLP/GMP regulations is described in detail, including the required documentation. Therefore the reader can very easily compile his own standard operating procedures.
"synopsis" may belong to another edition of this title.
About the Author
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.
From the Back Cover
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
From the Inside Flap
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
Excerpt. © Reprinted by permission. All rights reserved.
Applied Thin-Layer Chromatography
Best Practice and Avoidance of MistakesJohn Wiley & Sons
Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimAll right reserved.
ISBN: 978-3-527-31553-6
Chapter One
Introduction
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
[Z.sub.S] = distance of the substance zone from the starting line [mm]
[Z.sub.F] = distance of the solvent front from the solvent liquid level [mm]
[Z.sub.0] = distance between the solvent liquid level and the starting line [mm]
From this formula, one obtains an "observed" [R.sub.f] value, which describes the position of a spot in the chromatogram in a simple numerical way. It gives no information about the chromatographic process used or under what other "boundary conditions" this result was obtained. This calculated [R.sub.f] is always [less than or equal to] 1. As it has been found to be inconvenient in routine laboratory work always to write a zero and a decimal point, the [R.sub.f] value is multiplied by 100, referred to as the h[R.sub.f] value, quoted as a whole number, and used for the qualitative description of thin-layer chromatograms.
In the calculation of hRf values as described in the literature, the distance [Z.sub.S] is measured from the starting line to the mid-point of the substance zone. In general, this is correct and is also accurate enough for small spots. However, in purity tests on pharmaceutical materials, amounts of substance up to and even exceeding 1000 g/spot are used, and this can lead to hRf value ranges up to ca. 18. If, in addition, limit-value amounts of at least 0.1% of the same substance are applied and chromatographed on the same plate, these ideally lie exactly in the calculated central point of the main spot. However, this does not always happen. They are more likely to deviate from this position and be distributed over the whole hRf value range. Here, the term "hRf value range" means the imaginary hRf value range from the beginning to the end of a substance spot. In Fig. 2a-c, the chromatogram of purity tests of three active substances are given in which the position of the small amount of substance is respectively at the top end, approximately in the center, and at the bottom end of the hRf range.
* Figure 2: see Photograph Section.
[??] Practical Tip for calculation of the hRf values:
In purity tests, always quote hRf values as a range extending from the beginning to the end of a substance spot.
Figure 3 gives a graphical representation of the parameters and terms used in this book to describe a thin-layer chromatogram. Explanations of other terms are given in Section 1.4.3.
Because of the often poor reproducibility, especially when TLC plates prepared in-house are used and the conditions necessary for a good chromatographic result are in consequence not complied with, the so-called [R.sub.St] value, based on a standard substance, was formerly often also given. This is defined as
[R.sub.St] = [Z.sub.S] / [Z.sub.St]
where
[Z.sub.S] = distance from the substance zone to the starting line [mm]
[Z.sub.St] = distance of the standard substance from the starting line [mm]
According to Geiss, it is not a good principle to quote [R.sub.St] values as they are practically worthless and only give the appearance of certainty. In pharmacopoeias also, the still common linking of samples to standard substances with known [R.sub.f] values has been shown to be of doubtful value as routine laboratory practice. Therefore, only the hRf value is used to evaluate results in this book.
1.4.2 Flow Constant
The flow constant or velocity constant (kappa) is a measure of the migration rate of the solvent front. It is an important parameter for TLC users and can be used to calculate, for example, development times with different separation distances, provided that the sorbent, solvent system, chamber type and temperature remain constant. The flow constant is given by the following equation:
kappa = [Z.sub.[F.sup.2]] / t
where
kappa = flow constant [[mm.sup.2]/s]
[Z.sub.F] = distance between the solvent front and the solvent level [mm]
t = development time [s]
The following example illustrates the usefulness of the flow constant in laboratory work. In a TLC, if the development time for a migration distance of 10 cm was 30 min and the [Z.sub.0] distance is 5 mm, the kappa value is 6.125 [mm.sup.2]/s.
Question: How much time is required to develop a 15-cm migration distance if the sorbent, solvent system, [Z.sub.0] and laboratory temperature remain constant?
Answer: 65.4 min.
This means that more than twice the development time is required for a migration distance which is only 5 cm longer!
It should be mentioned here that the flow constant is influenced by other effects also, e.g. the surface tension and viscosity of the solvent system. In general, the greater the viscosity and the smaller the surface tension of the solvent system, the smaller is the migration rate of the front.
1.4.3 Other TLC Parameters
In the TLC literature, different terms are often used for the same characteristic values and parameters. As this can lead to confusion, especially for beginners, the most commonly used terms are listed below, those used in this book being in bold type.
Solvent system Developing solvent, mobile phase, eluent (only used in OPLC) Migration distance Run distance, run height, developing distance Developing time Run time Derivatization reagent Detection reagent
Other terms commonly used in TLC are:
Fluorescence quenching. If a TLC plate has a layer which contains a fluorescence indicator, UV-active substances cause the fluorescence to be totally or partially extinguished and can be seen as dark spots on a bright background (see also Section 2.2.3 "Additives").
Separation efficiency describes the spread of the spots caused by chromatographic effects in the chosen system.
System suitability. This is an expression used in the German Pharmacopoeia (Deutsche Arzneibuch, DAB), and describes a method of testing a system whereby two or more substances have to be separated from each other on a TLC plate prepared in-house, in order to establish whether samples under investigation can in fact be analyzed using the system.
Selectivity describes the varying strengths of the interactions between the sample substances to be separated and the stationary phase ([DELTA]hRf) in the chosen TLC system.
1.5 What Kinds of Reference Substances Are Used in TLC?
Because of their great importance to TLC, the various types of reference substances are described in the following Section. These are often known as "standards" and must only be used if they are of suitable quality for the intended application. These levels of quality are of especial importance in the field of pharmacy. All the relevant requirements must therefore be controlled in an SOP (standard operating procedure, see Chapter 9 "GMP/GLP-Conforming Operations in TLC").
1. Pharmacopoeia Substance (PS)
This term indicates a commercially available substance that meets the requirements of the relevant pharmacopoeia. For example, the American pharmacopoeia is indicated by the suffix USP, the British by BP, and the European by CRS. The possible use of a PS is specified by the relevant institutions, and is terminated by a change in the LOT number in the suppliers' catalogs. The Commission of the USP lists so-called "official distributors", of which the company LGC Promochem is a member (see Section 12.5 "Market Overview"). Care must be taken when ordering a substance listed in a pharmacopoeia to use the precise term for the substance. Although it is extremely rare, it does happen that the related compounds (rel. c.) of a substance have different names in the DAB and CRS lists.
It is especially confusing if, for example, the "rel. c. A" of a substance (e.g. ranitidine HCl) in the USP list appears as "rel. c. B" in the BP list, "rel. c. A" in the BP list bears the name "rel. c. C" in the USP list, and the "rel. c. B" in the USP list does not appear at all in the BP list.
2. Primary Reference Substance (PRS)
This term denotes a substance referred to as a Class 1 Standard by suppliers (analysis certificate with, e.g., at least two assays performed by different methods) or defined by the user's own tests without reference to other substances.
3. Secondary Reference Substance (SRS)
A tested and accepted batch of a substance which, after comparison with a PS or PRS, is declared as a "house standard". Can also be termed a working standard.
4. Related Compound (rel. c.)
This is usually a substance obtained from a supplier, but may be a substance produced by the user for purity testing, which is not a PS or a PRS and does not require information about its concentration. Such substances are in most cases decomposition products or intermediates in the synthesis process, and can be linked to a particular active substance.
(Continues...)
Excerpted from Applied Thin-Layer Chromatography Copyright © 2007 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.
"About this title" may belong to another edition of this title.
Other Popular Editions of the Same Title
Search results for Applied Thin-Layer Chromatography: Best Practice and...
Stock Image
Applied Thin-Layer Chromatography: Best Practice and Avoidance of Mistakes
Seller: Mispah books, Redhill, SURRE, United Kingdom
Seller rating 3 out of 5 stars
Hardcover. Condition: Very Good. Very Good. book. Seller Inventory # ERICA82935273155356
Buy Used
US$ 414.47
Shipping:
US$ 32.88
From United Kingdom to U.S.A.
Quantity: 1 available