| | Contents | |
| | | |
| |
| 1 | Introduction | 1 |
| 1.1 | What Does TLC Mean? | 1 |
| 1.2 | When Is TLC Used? | 2 |
| 1.3 | Where Is TLC Used? | 3 |
| 1.4 | How Is the Result of a TLC Represented? | 4 |
| 1.4.1 | Retardation Factor | 4 |
| 1.4.2 | Flow Constant | 6 |
| 1.4.3 | Other TLC Parameters | 7 |
| 1.5 | What Kinds of Reference Substances Are Used in TLC? | 8 |
| 1.6 | The Literature on TLC | 9 |
| 1.6.1 | General Literature | 9 |
| 1.6.1.1 | Books and Information Sheets in German | 9 |
| 1.6.1.2 | Books in English | 11 |
| 1.6.1.3 | Book in Another Language | 11 |
| 1.6.2 | Journals | 12 |
| 1.6.2.1 | German Language Journals Containing Articles on TLC (Selection) | 12 |
| 1.6.2.2 | English Language Journals on TLC | 12 |
| 1.6.2.3 | English Language Journals Containing General Articles on Chromatography (Selection) | 12 |
| 1.6.3 | Abstracts | 13 |
| 1.6.4 | Pharmacopoeias | 13 |
| 2 | Precoated Layers | 15 |
| 2.1 | Precoated Layers – Why? | 15 |
| 2.2 | What Are Precoated Layers Produced? | 16 |
| 2.2.1 | Sorbents | 16 |
| 2.2.2 | Supports for Stationary Phases | 19 |
| 2.2.3 | Additives | 20 |
| 2.3 | What Types of Precoated Layers Are There? | 21 |
| 2.4 | What Are the Uses of Precoated Layers? | 27 |
| 2.5 | Criteria for the Selection of Stationary Phases in TLC | 27 |
| 2.5.1 | How Can the Choice of the Stationary Phase be Made? | 28 |
| 2.5.2 | How Can the Recommendations for Stationary Phases Found in Pharmacopoeias be Applied to Precoated Layers? | 28 |
| 2.6 | Effect of the Stationary Phase When Mobile Phases Are Identical | 30 |
| 2.7 | Advice on the Ordering and Storage of Precoated Layers | 31 |
| 2.8 | Problems in the Naming and Arrangement of Precoated Layers | 33 |
| 3 | Before the TLC Development Process | 35 |
| 3.1 | Handling of Precoated Layers | 35 |
| 3.1.1 | Film and Foil | 35 |
| 3.1.2 | Glass Plates | 36 |
| 3.2 | Prewashing | 41 |
| 3.3 | Activation | 43 |
| 3.4 | Conditioning | 44 |
| 3.5 | Impregnation | 46 |
| 3.5.1 | Impregnation by Dipping | 46 |
| 3.5.2 | Impregnation by Spraying | 46 |
| 3.5.3 | Impregnation by Predevelopment | 47 |
| 3.6 | Application of Samples | 50 |
| 3.6.1 | Manual Application of Samples | 51 |
| 3.6.2 | Semiautomatic Application | 56 |
| 3.6.3 | Fully Automatic Application | 61 |
| 3.7 | Positioning of the Samples | 65 |
| 3.8 | Drying Before the Development | 67 |
| 4 | Solvent Systems, Developing Chambers and Development | 69 |
| 4.1 | Solvent Systems | 69 |
| 4.1.1 | Choice of Solvent Systems | 70 |
| 4.1.2 | Preparation and Storage of Solvent Systems | 79 |
| 4.1.3 | Problematical Solvent System Compositions | 82 |
| 4.2 | TLC Developing Chambers | 87 |
| 4.2.1 | What Types of TLC Developing Chambers Are There? | 87 |
| 4.2.1.1 | TLC Chambers for Vertical Development | 88 |
| 4.2.1.2 | TLC Developing Chambers for Horizontal Development | 90 |
| 4.2.2 | Influence of the Chamber Atmosphere | 91 |
| 4.2.2.1 | The Unsaturated N-Chamber | 93 |
| 4.2.2.2 | The Saturated N-Chamber | 94 |
| 4.2.3 | Influence of Temperature in Chromatography | 97 |
| 4.2.4 | Location and Labeling of TLC Developing Chambers | 97 |
| 4.3 | Development of Thin-Layer Chromatograms | 99 |
| 4.3.1 | One-Dimensional Thin-Layer Chromatography | 100 |
| 4.3.1.1 | Vertical Development | 101 |
| 4.3.1.2 | Horizontal Development | 106 |
| 4.3.2 | Two-Dimensional Thin-Layer Chromatography | 108 |
| 4.4 | Drying After Development | 111 |
| 5 | Evaluation Without Derivatization | 113 |
| 5.1 | Direct Visual Evaluation | 113 |
| 5.1.1 | Detection in Daylight | 113 |
| 5.1.2 | Detection with 254-nm UV Light | 113 |
| 5.1.3 | Detection with 365-nm UV Light | 115 |
| 5.2 | Direct Optical Evaluation Using Instruments | 116 |
| 5.2.1 | Principle of Operation of a TLC Scanner | 116 |
| 5.2.2 | Direct Optical Evaluation Above 400 nm | 117 |
| 5.2.3 | Direct Optical Evaluation Below 400 nm | 117 |
| 5.2.4 | Direct Optical Evaluation with 365-nm UV Light (Fluorescence Measurement) | 117 |
| 5.3 | Diode-Array Detection | 120 |
| 5.4 | Coupled Methods for Substance Identification | 122 |
| 5.5 | Documentation Without or Before Derivatization | 123 |
| 6 | Derivatization | 125 |
| 6.1 | Thermochemical Reaction | 126 |
| 6.2 | Irradiation with High-Energy Light | 127 |
| 6.3 | Reaction with Reagents | 129 |
| 6.3.1 | Spraying of TLC Plates | 129 |
| 6.3.1.1 | Manual Spraying of TLC Plates | 129 |
| 6.3.1.2 | Fully Automatic Spraying of TLC Plates | 133 |
| 6.3.2 | Dipping of TLC Plates | 134 |
| 6.3.3 | Vapor Treatment of TLC Plates | 139 |
| 6.3.4 | Coating TLC Plates | 140 |
| 6.4 | Special Cases of Derivatization | 143 |
| 6.4.1 | Prechromatographic Derivatization | 143 |
| 6.4.1.1 | Reaction with Reagents | 143 |
| 6.4.1.2 | Incorporation of Radionuclides | 144 |
| 6.4.2 | Simultaneous Derivatization and Development | 145 |
| 6.4.3 | Reaction Sequences | 146 |
| 6.4.4 | Biological-Physiological Methods of Detection | 147 |
| 6.5 | Further Treatment of Derivatized Chromatograms | 148 |
| 6.5.1 | Effect of Heat | 148 |
| 6.5.2 | Stabilization of Colored and Fluorescent Zones | 150 |
| 7 | Evaluation After Derivatization | 153 |
| 7.1 | Visual Evaluation | 153 |
| 7.1.1 | Visual Qualitative Evaluation | 153 |
| 7.1.2 | Visual Semiquantitative Evaluation | 153 |
| 7.2 | Evaluation Using a TLC Scanner | 154 |
| 7.2.1 | Qualitative Evaluation | 154 |
| 7.2.2 | Quantitative Evaluation | 155 |
| 7.2.2.1 | Absorption Measurement | 156 |
| 7.2.2.2 | Fluorescence Measurement | 161 |
| 7.2.2.3 | Comparison of "Parallel" With "Transverse" Measurement | 166 |
| 7.3 | Evaluation Using a Video System | 174 |
| 7.3.1 | Qualitative Video Evaluation | 175 |
| 7.3.2 | Quantitative Video Evaluation | 176 |
| 7.3.3 | Comparison of the TLC Scanner With Video Evaluation | 177 |
| 7.4 | Evaluation by Flat-Bed Scanner | 178 |
| 7.5 | Evaluation Using a Digital Camera | 178 |
| 8 | Documentation | 181 |
| 8.1 | Description of a Thin-Layer Chromatogram | 181 |
| 8.2 | Documentation by Drawing, Tracing and Photocopying | 182 |
| 8.3 | Photographic Documentation | 183 |
| 8.3.1 | Photography Using the Polaroid Camera MP-4 | 183 |
| 8.3.2 | Photography Using 35-mm Cameras | 183 |
| 8.3.2.1 | Photography in 254-nm UV Light | 184 |
| 8.3.2.2 | Photographs in 365-nm UV Light | 185 |
| 8.3.2.3 | Photographs in White Light | 185 |
| 8.3.3 | Archiving of 35-mm Films | 187 |
| 8.4 | Video Documentation | 188 |
| 8.5 | Documentation With Digital Cameras | 198 |
| 8.6 | TLC Scanner Documentation | 199 |
| 8.7 | Flat-Bed Scanner Documentation | 199 |
| 8.8 | Bioluminescence Measurements | 200 |
| 8.8.1 | Toxicity Screening Using the Bioluminescent Bacteria Vibrio fischeri | 200 |
| 8.8.2 | Detecting Bioluminescence With the BioLuminizerTM | 200 |
| 9 | GMP/GLP-Conforming Operations in TLC | 203 |
| 9.1 | Validation of TLC Methods | 207 |
| 9.2 | Use of Qualified/Calibrated Equipment | 211 |
| 9.3 | GMP/GLP-Conforming Raw Data Sheets | 214 |
| 9.4 | Examples of GMP/GLP-Conforming Testing Procedures (TPs) | 223 |
| 9.4.1 | Identity and Purity of a Bulk Pharmaceutical Chemical and Determination of the Limit Values of Related Compounds | 224 |
| 9.4.2 | Identity and Purity of Various Flavonoid-Containing Plant Extracts | 224 |
| 9.4.3 | Content of a Pharmaceutical Chemical in a Tablet | 225 |
| 10 | Effects of Stress | 241 |
| 10.1 | Controlled Stress on a Substance | 241 |
| 10.2 | TLC-Sensitive Substances | 242 |
| 10.2.1 | Interactions With Sorbents | 242 |
| 10.2.2 | Effect of Elevated Temperature | 242 |
| 10.2.3 | Effect of Light | 243 |
| 10.2.4 | Oxidative Effects | 245 |
| 11 | Special Methods in TLC | 247 |
| 11.1 | AMD – Automated Multiple Development | 247 |
| 11.2 | OPLC – Overpressured Layer Chromatography | 248 |
| 11.3 | HPPLC – High Pressure Planar Liquid Chromatography | 249 |
| 11.4 | TLC-FID/FTID – Combination of TLC and Flame-Ionization Detector or Flame-Thermionic Ionization Detector | 249 |
| 11.5 | TLC-NDIR | 250 |
| 11.6 | RPC – Rotation Planar Chromatography | 252 |
| 12 | Appendix | 253 |
| 12.1 | CHROMart | 253 |
| 12.2 | References | 255 |
| 12.3 | Abbreviations | 263 |
| 12.4 | Acknowledgements | 267 |
| 12.5 | Market Overview | 268 |
| | Photograph Section | 271 |
| | Subject Index | 309 |
| | List of Tables | |
| | Table 1: Types of sorbents and supports for precoated layers | 18 |
| | Table 2: Meanings of code letters and numbers in product designations | 19 |
| | Table 3a: Important commercially available precoated layers and examples of typical applications | 22 – 23 |
| | Table 3b: New precoated layers | 23 |
| | Table 4: Peppermint oil (Oleum menthae piperitae) | 24 |
| | Table 5: Production of constant humidity in closed vessels | 44 |
| | Table 6: Greater Celandine (Chelidonium majus L.) | 49 |
| | Table 7: Semiautomatic application using the Linomat IV | 59 |
| | Table 8: Eluotropic Series | 71 |
| | Table 9: Carbamazepine | 75 |
| | Table 10: Birch leaves (Betulae folium) | 76 |
| | Table 11: Primula root (Primulae radix) | 77 |
| | Table 12: Liquorice root (Liquiritiae radix) | 78 |
| | Table 13: Solvent requirements for different TLC separation chambers | 81 |
| | Table 14: Dry extract of nettle root (Urticae radix) | 83 |
| | Table 15: Spironolactone and furosemide | 85 |
| | Table 16: Theophylline, theobromine, caffeine | 86 |
| | Table 17: Influence of the chamber atmosphere as shown with different samples of greater celandine | 93 |
| | Table 18: Sugar | 102 – 103 |
| | Table 19: Coneflower (Echinacea) | 136 – 137 |
| | Table 20: Comparison of parallel measurement with transverse | 166 |
| | Table 21: Comparison of semiquantitative visual and quantitative video evaluation of Figure 69 | 176 |
| | Table 22: Assay of Caffeine [%] in various samples of coffee and tea | 177 |
| | Table 23: Photographic documentation using single lens reflex cameras | 184 |
| | Table 24: Fundamental guidelines according to the ICH | 207 |
| | Table 25: Qualities of solvents | 224 |
| | Table 26: Benchmarking between HPLC and HPTLC for assay of theophylline tablets | 225 |
