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Thin-layer Chromatography

A Modern Practical Approach


RSC Chromatography Monographs
Series Editor: R.M. Smith, Loughborough University of Technology, UK
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C.F. Simpson, London, UK, G.G. Wallace, Wollongong, Australia
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Edited by J Pawliszyn, University of Waterloo, Waterloo, Ontario, Canada
Capillary Electrochromatography
Edited by K D Bartle and P Myers, University of Leeds, UK
Chromatographic Integration Methods, Second Edition
N Dyson, Dyson Instruments, UK

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,
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Packed Column SFC


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Thin-layer Chromatography
A Modern Practical Approach

Peter E. Wall
VWR International Ltd., Poole, Dorset
ISBN 0-85404-535-X

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Preface

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

Chapter 1 Introduction and History 1


1 Introduction to Thin-layer Chromatography 1
2 History of TLC 2
3 References 4

Chapter 2 Sorbents and TLC Layers 6


1 Sorbent Selection 6
1.1 Introduction 6
1.2 Silica Based Sorbents 7
1.2.1 Silica Gel 7
1.2.2 Silica Gel Bonded Phases 12
1.3 Non-silica Sorbents 35
1.3.1 Cellulose 35
1.3.2 Cellulose Bonded Phases 37
1.3.3 Aluminium Oxide 37
1.3.4 Kieselguhr 38
1.3.5 Polyamide 39
1.3.6 Miscellaneous Stationary Phases 39
1.3.7 Mixed Stationary Phases 40
1.3.8 Dual Phases 40
2 Preparation of TLC Sheets and Plates 40
2.1 ‘‘Home made’’ TLC Plates 40
2.2 Pre-coated TLC/HPTLC Sheets and Plates 41
3 Cutting TLC/HPTLC Sheets and Plates 41
4 Humidity Effects with TLC Plates 42
5 Pre-washing TLC Plates 43
6 Use of Phosphorescent/Fluorescent Indicators 44
7 Channelled TLC Layers 44
8 Concentration Zone TLC/HPTLC Plates 45

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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

Chapter 3 Sample Pre-treatment 59


1 Introduction 59
2 Extractions from Solid Samples 60
3 Solid Phase Extraction Systems (SPE) 61
3.1 Diatomaceous Earth 61
3.2 Bonded Silica Gels and Polymers 61
3.3 Sorbent Conditioning 63
3.4 Sample Injection 63
3.5 Cleaning 63
3.6 Recovery of Analytes 63
4 References 64

Chapter 4 Sample Application 65


1 Introduction 65
2 Selecting the Sample Solvent 65
3 Theoretical Determination of Spot Size 66
3.1 Spot Size and Loading Equations 66
3.2 Spot Capacity Equation 68
4 Sample Loading 72
4.1 Layer Damaging Effects 74
4.2 Humidity Considerations 75
5 Sample Application Methods 75
5.1 Manual Methods 76
5.2 Instrumental Methods 77
5.2.1 Manual Instrumental Techniques 77
5.2.2 Automated Techniques 78
5.3 Large Sample Application 80
5.3.1 Contact Spotting 80
5.3.2 Use of Concentration Zone
TLC/HPTLC 81
6 Choice of Band or Spot Application 82
7 References 85
Contents ix

Chapter 5 Development Techniques 86


1 Introduction 86
2 The Theory of Solvent Migration 86
3 Mechanisms of TLC Separation 90
3.1 Introduction 90
3.2 Adsorption Separations 90
3.3 Partition Separations 91
3.4 Ion-exchange Separations 91
4 Solvent Selection 92
4.1 Eluotropic Series and Solvent Optimisation 93
4.1.1 Selectivity Triangles 96
4.1.2 PRISMA Model 99
5 Development Chambers 100
5.1 N-Chambers and Chamber Saturation Effects 100
5.2 Twin-trough Chamber 105
5.3 Sandwich, Horizontal and U-chambers 105
5.4 Automatic Developing Chamber (ADC) 108
5.5 Forced-flow Development (OPLC) 109
5.6 Vario Chambers 110
6 Multiple Development Techniques 112
6.1 Single Mobile Phase Multiple and Continuous
Development 112
6.2 Two-dimensional Development 115
6.3 Manual Gradient Development 118
6.4 Automated Multiple Development (AMD) 118
7 Mobile Phase Additives 121
7.1 Use of Buffers and pH 121
7.2 Acid or Base Additives 124
7.3 Ion-pairing Reagents 124
7.4 Chiral Additives 126
7.4.1 Cyclodextrins 127
7.4.2 Bovine Serum Albumin (BSA) 129
7.4.3 Other Chiral Selectors 129
7.5 Argentation (Silver Nitrate) TLC 132
8 References 132

Chapter 6 Detection and Visualisation 134


1 Introduction 134
2 Non-destructive Techniques 135
2.1 Visible Detection 135
2.2 Ultra-violet Detection 135
2.3 Reversible Reactions 137
2.3.1 Iodine Vapour 137
2.3.2 Ammonia Vapour 138
x Contents
2.4 Non-reversible Reactions 138
2.4.1 Fluorescent Dyes 138
2.4.2 pH Indicators 139
3 Destructive Techniques 139
3.1 Charring Reactions 140
3.2 Thermochemical Activation 140
4 Derivatisation Reactions 141
4.1 Post-chromatographic Visualisation 141
4.1.1 Universal Chemical Reagents 143
4.1.2 Group Specific Reagents 146
4.1.3 Sequencing Reactions 149
4.2 Pre-chromatographic Visualisation 151
5 References 153

Chapter 7 Quantification and Video Imaging 154


1 Introduction 154
2 Densitometry 155
2.1 Mode of Operation of Spectrodensitometers 155
2.2 Theory of Spectrodensitometry 158
3 Video Imaging and Densitometry 160
4 Future Trends – Spectrodensitometry or Video Imaging? 163
5 References 164

Chapter 8 TLC Coupling Techniques 166


1 Introduction 166
2 TLC/HPTLC and HPLC 167
3 TLC and MS 167
4 TLC Coupled with FTIR 171
4.1 On-line TLC – FTIR 172
4.2 Off-line TLC – FTIR 172
5 TLC and Raman Spectroscopy 173
6 References 174

Subject Index 177

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