Practical Thin-Layer Chromatography

A Multidisciplinary Approach

Bernard Fried, Joseph Sherma

• Publisher: CRC Press
• Year: 2017
• Print ISBN: 9780367411572
• eBook ISBN: 9781351422413
• Topic: Physical Sciences
• Subtopic: Industrial & Technical Chemistry

Practical Thin-Layer Chromatography provides thorough coverage of the principles, practices, and applications of thin-layer chromatography (TLC) for important sample and compound types. This information is directed specifically at workers in the most active scientific fields.

Chapter 1

INTRODUCTION TO TECHNIQUES AND INSTRUMENTATION

Tibor Cserháti and Esther Forgács

CONTENTS

I.	Introduction
II.	Theoretical Aspects of Thin-Layer Chromatography A. Stationary Phases 1. Silica and Surface Modified Silicas 2. Other TLC Sorbents 3. Impregnation of Sorbents B. Mobile Phases C. Retention Parameters in TLC D. Retention Mechanism
III.	Practical Considerations A. Sample Preparation B. Sample Application C. Development and Developing Chambers 1. Linear Ascending Development 2. Linear Horizontal Development 3. Circular Development Methods 4. Anticircular Development D. Detection of Solutes 1. Derivatization E. Qualitative and Quantitative Evaluation F. Sensitivity and Reproducibility in TLC G. TLC as a Pilot Method for HPLC H. Determination of Molecular Parameters with Adsorptive and Reversed-Phase TLC
References

I. INTRODUCTION

Thin-layer chromatography (TLC) was developed more than 30 years ago for the separation and semi-quantitative determination of the individual components of more or less complicated mixtures. In the last decade, the application of various TLC methods for the separation and quantitative determination of a wide variety of organic and inorganic substances has considerably increased. This increase is probably due to the improved instrumentation and automation of the various steps of TLC analysis (gradient and forced flow methods, centrifugal development, circular rotation planar chromatography, high-pressure planar liquid chromatography, densitometry, among others. In addition, coupled spectroscopic methods (TLC-UV-VIS, TLC-MS, and TLC-FTIR) have been developed, considerably enhancing the reproducibility of TLC. TLC methods have been successfully used in many fields of research and development such as in clinical medicine,¹ forensic chemistry, biochemistry, pharmaceutical analysis for the estimation of impurity profiles of drugs and related materials,² drug screening, toxicology, environmental pollution studies,^3,4 as well as – to a lesser extent – in cosmetology, foodstuff analysis,⁵ in the analysis of metal ions,⁶ explosives and their biodegradation products,⁷ and in petroleum analysis.⁸

The widespread application of TLC is also due to the fact that it is a simple and rapid analytical procedure; it is extremely flexible because a great variety of mobile and stationary phases and detection reagents can be tested and used for the solution of any separation problem.

The general aspects and practical applications of TLC have been discussed in detail in excellent books⁹ and in reviews¹⁰ dealing with the various fields of application such as the analysis of inorganic pollutants¹¹ and pesticide residues.¹² The new advances in the theoretical and practical applications of various TLC methods are presented and discussed every 2 years in Analytical Chemistry.^13,14

The objectives of this chapter are to give a general overview of the theory and practice of TLC and to offer a brief summary of the new advances in this rapidly developing field of chromatographic separation science for researchers and serious students interested in any of the topics covered.

II. THEORETICAL ASPECTS OF THIN-LAYER CHROMATOGRAPHY

TLC separation includes the interaction of the compounds to be separated (solutes) with both a stationary and a mobile phase.

A. STATIONARY PHASES

Stationary phases are generally chemically well–defined inorganic (sometimes organic) materials with porous structures and with relatively high specific surface areas. TLC plates are prepared from these materials by binding them to a support (glass, aluminium, plastic foil) with the help of various organic (polyvinylalcohols with various molecular masses) or inorganic (gypsum) binders. The presence of binders modifies, only to a small extent, the retention capacity and selectivity of the original stationary phase. The performance of a TLC sorbent depends considerably on its specific surface area and pore volume, the mean pore diameter and the pore size distribution, and the particle size and particle size distribution. Smaller particle size and narrower particle size distribution enhances separation efficiency and improves resolution, decreases analysis time, and increases detection sensitivity. The average particle size of the traditional TLC sorbents is between 10 and 50 µm, with a fairly wide size distribution. High performance TLC (HPTLC) sorbents have an average particle size of about 5 µm, with a narrower particle size distribution. It has been proven many times that the performance of HPTLC plates is superior to that of normal TLC plates. However, in the analysis of dye components contained in hair color formulas the performance of HPTLC layers sometimes was inferior to that of normal TLC layers.¹⁵

1. Silica and Surface Modified Silicas

Many sorbents have been tested for TLC applications; however, silicas with various surface characteristics and silicas with covalently bonded organic ligands (amino, diol, cyano, chiral phases, silanized silica, C₂-, C₈-, and C₁₈- alkyl bonded silicas) on the surface are used the most frequently for TLC separations. A wide variety of TLC and HPTLC ready–made plates are available, facilitating the solution of many separation problems. The polarity order of the ready–made TLC plates based on silica is approximately silica > amino silica > cyano silica > octadecyl silica. It must be noted that the polarity order listed above is only of limited value, depending considerably on the chemical character of the solutes and the composition of the eluent system. The stability and wetting of the old octadecylsilica plates were insufficient in eluents containing a considerable ratio of water. The mechanical stability of the layers can be increased by adding sodium chloride or any other neutral salt to the eluent; however, the salts can influence the retention of solutes (salting–in or salting–out effects), sometimes reducing separation efficiency. The effect of salt is higher when the solute has one or more dissociable polar substructures. A new generation of RP-18 plates overcomes this difficulty; they can be used in aqueous systems, conserving their mechanical stability and providing good mobility, even for water. These plates are generally marked as “W.”

The medium–polar amino, diol, and cyano layers can be used both in the adsorption and reversed-phase separation mode, depending on the composition (polarity) of the mobile phase. Although these supports exhibit good separation capacitiy, they are not frequently used in practice. Diol layers have been used for the enantiomeric separation of metoprolol, propanolol, and alprenolol using N-benzyloxycarbonylglycil-L-proline as chiral selector,¹⁶ as well as for the separation of glycine, sulfate, and glucuronide conjugates.¹⁷ Amino–bonded layers have been used for the detection of catecholamine in biological materials,¹⁸ for the separation of all substrates of the 7,8-dihydropteroate synthase in the reversed-phase mode,¹⁹ for the quantitation of 5-methylcytosine,²⁰ and for the separation of some steroid hormones.²¹ Silanized silica plates were used for quantitative analysis of quaternary ammonium antiseptics.²² Until recently, chiral plates (reversed-phase silica modified with Cu²⁺ and a chiral agent) have found only limited application for enantiomer separation. The overwhelming majority of chiral separations have been carried out by adding various chiral selectors to the eluent. Silica plates with concentrating zones allow the application of a relatively high volume of a dilute sample. Their application is preferable when the concentration of the solute or solutes in the sample cannot be enhanced by other methods.

2. Other TLC Sorbents

Other inorganic and organic sorbents such as alumina (neutral and acidic), magnesium silicate, diatomaceous earth (kieselguhr), celluloses and cellulose derivatives, and polyamides have found only limited application in TLC, although their separation capacities differ from those of traditional adsorption and reversed-phase layers based on silica. Alumina layers have been used for the separation of nonylphenyl ethylene oxide oligomers using acetonitrile–chloroform mixtures of various compositions as eluents,²³ and for the separation of a new type of plant regulator, jas-monates.²⁴ Cellulose layers have been used recently for the separation of some organomercurial antiseptics in 1M NaCl as eluent.²⁵ An excellent separation of alpha–carotene was achieved on magnesium oxide layers. The pigments were extracted with acetone, then transferred into petroleum ether by adding saturated NaCl solution.²⁶ Phenyldimethylsiloxane–treated high performance thin-layer chromatographic plates were used for the separation of flavonoids (baicalein, baicalin, wogonin, and oroxylin–A) in Scutellaria radix. It was found that the separation was better than on C₁₈ plates.²⁷ Zirconia is being used more and more frequently in HPLC; however, until now it has found only limited applications in TLC.²⁸ Water–insoluble β-cyclodextrin polymer beads have been used as TLC sorbent.²⁹ It has been established that this sorbent shows retention characteristics deviating from those of traditional adsorptive an...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Chapter 1 Introduction to Techniques and Instrumentation
• Chapter 2 Thin-Layer Chromatography in Bacteriology
• Chapter 3 Thin-Layer Chromatography in Plant Sciences
• Chapter 4 Thin-Layer Chromatography in Parasitology
• Chapter 5 Thin-Layer Chromatography in Studies in Entomology
• Chapter 6 Thin-Layer Chromatography of Skin Secretions in Vertebrates
• Chapter 7 Thin-Layer Chromatography in Clinical Chemistry
• Chapter 8 Thin-Layer Chromatography in Environmental Analysis
• Chapter 9 Thin-Layer Chromatography in Food Analysis
• Chapter 10 Thin-Layer Chromatography in Forensic Toxicology
• Chapter 11 Thin-Layer Chromatography in Pharmaceutical Analysis
• Chapter 12 Thin-Layer Chromatography in Veterinary Toxicology
• Index