Quantitative Thin-Layer Chromatography (eBook)

Preface 6
Acknowledgements 8
Contents 10
Chapter 1: History of Planar Chromatography 17
1.1 History of Paper Chromatography (PC) 17
1.2 History of Thin-Layer Chromatography 23
1.3 The History of Quantitative Planar Chromatography 24
References 26
Chapter 2: Theoretical Basis of Thin Layer Chromatography (TLC) 29
2.1 Planar and Column Chromatography 29
2.2 TLC Capillary Flow 31
2.3 TLC Distribution Equilibrium 34
2.3.1 Adsorption Chromatography 34
2.3.2 Partition Chromatography 36
2.4 The Retardation Factor (Rf) 38
2.4.1 The Empirical Rf Factor 38
2.4.2 The Thermodynamic Rf Factor 40
2.5 Mobile Phase Composition 41
2.6 Transfer of TLC Separations to Columns 43
2.7 The Rm Value 44
2.8 Temperature Dependence of TLC Separations 45
2.9 Advanced Theoretical Considerations 46
2.10 Indices Characterizing Separation and Resolution 53
2.11 Zone Broadening in Planar Chromatography 56
2.11.1 The A term 56
2.11.2 The B term 57
2.11.3 The C term 58
2.11.4 Local Plate Height H 58
2.11.5 The van Deemter Equation 59
2.12 Optimum Separation Conditions in TLC 61
2.13 Separation Number 63
2.14 Real Plate Height 66
References 67
Chapter 3: The Stationary Phase in Thin-Layer Chromatography 69
3.1 Activating and Deactivating Stationary Phases 70
3.2 Snyder´s Adsorption Model 71
3.3 Layer Characteristics 73
3.3.1 Layer Thickness (df) 75
3.3.2 Average Particle Size (dp) 76
3.3.3 Particle Size Distribution 76
3.3.4 Specific Surface Area (Os) 76
3.3.5 Pore Volume (Vp) 77
3.3.6 Average Pore diameter (Pd) 77
3.4 The Most Important Stationary Phases in TLC 78
3.4.1 Aluminium Oxide 78
3.4.2 Magnesium Silicate 78
3.4.3 Silica Gel 79
3.4.4 Chemically Bonded Silica Gel Layers 82
3.4.5 Kieselguhr 83
3.4.6 Cellulose 84
3.4.7 Polyamides 86
3.4.8 Ion Exchange Resins 87
3.4.9 Chiral Phases 88
3.4.10 Layers with Fluorescent Indicators 89
3.4.11 Making Your Own Plates 89
3.5 Light Absorption on Plate Surfaces 90
References 93
Chapter 4: The Mobile Phase in Adsorption and Partition Chromatography 96
4.1 Solvent Characteristics 96
4.2 Solvent Theory for Adsorption Chromatography (According to Snyder) 98
4.2.1 Solvent Strength (epsi0) 100
4.2.2 Solvent Strength of Binary Mixtures 101
4.3 Solvent Theory in Partition Chromatography 103
4.3.1 Solvent Theory (According to Snyder) 104
4.3.2 Other Methods for Characterizing Solvents 107
4.4 Optimizing Solvent Composition 108
4.5 The PRISMA Model (According to Nyiredy) 112
4.6 Solvent Additives 114
4.7 Appendix: Solvent Properties 117
References 118
Chapter 5: Preparing and Applying Samples 119
5.1 Sample Preparation 119
5.1.1 The QuEChERS Approach 119
5.1.2 Solid-Phase Extraction 120
5.1.3 Stir Bar Sorptive Extraction 122
5.2 The Dosage Quality 122
5.3 Choice of Application Position 126
5.4 Practical Application Methods 127
5.4.1 Sample Application via Plate Contact 127
5.4.2 Sample Application Without Plate Contact 128
5.4.3 Sample Application via Contact Spotting 128
5.4.4 Plate Overloading and Incomplete Drying 130
References 131
Chapter 6: Basis for TLC Development Techniques 133
6.1 Influence of the Vapour Phase 133
6.2 Chamber Types for Linear Development 137
6.2.1 N-Chambers (``Trough Chambers´´) 137
6.2.2 S-Chamber (``Small Chamber´´) 138
6.2.3 Vario-KS-Chamber 139
6.2.4 The H-Chamber (``Horizontal Chamber´´) 139
6.3 Controlling Separations via the Vapour Phase 140
6.3.1 Solvent Composition During Separation 140
6.3.2 Plate Pre-loading via the Vapour Phase 144
6.4 Circular Separations 147
6.5 Solvent Gradients 148
6.5.1 Theory of Solvent Gradients 148
6.5.2 Evaporation-Controlled Gradient Elution 154
6.5.3 Multiple Development in TLC 156
6.5.4 Automated Multiple Development (AMD) 157
6.6 Normal Phase Separations with Water-Containing Solvents 159
6.7 Plate Development with Forced Flow 161
6.7.1 Rotation Planar Chromatography (RPC) 161
6.7.2 Over-pressure Layer Chromatography (OPLC) 161
6.8 Two Dimensional TLC (2D TLC) 162
6.8.1 Development in Orthogonal Directions 162
6.8.2 Grafted TLC 163
6.8.3 Stability Test and SRS Technique 165
6.9 Drying the Plate 167
References 167
Chapter 7: Specific Staining Reactions 169
7.1 Chemical Reactions Prior to Separation (Pre-chromatographic Derivatization) 171
7.1.1 Sample Enrichment by Pre-chromatographic Derivatization 171
7.1.2 Pre-chromatographic In Situ Derivatization 173
7.1.2.1 Oxidation 174
7.1.2.2 Reductions 175
7.1.2.3 Hydrolyses 175
7.1.2.4 Halogenations 175
Chlorination 175
Bromination 176
Iodination 176
7.1.2.5 Nitrations 176
7.1.2.6 Diazotizations 176
7.1.2.7 Esterification 176
7.1.3 Pre-chromatographic Staining 177
7.1.3.1 Reactions with Carbonyl Compounds 177
7.1.3.2 Reactions with SH-, NH- and OH Groups 178
7.1.3.3 Derivatization of Carboxylic Acids 180
7.1.3.4 Reactions with Alcohols and Amines 180
7.1.4 Reagents in the Mobile Phase 181
7.2 Post-chromatographic Reactions (Derivatization After Development) 182
7.2.1 Fluorescence Enhancer 184
7.2.2 pH and Redox Indicators 185
7.2.3 Universal Reagents (Charring Reagents) 186
7.2.4 Aldehyde Reagents 187
7.2.5 CH- and NH-Reacting Reagents 190
7.2.5.1 4-Aminoantipyrine Reagent (Emerson reagent) 191
7.2.5.2 MBTH Reagent (Besthorn Reagent) 191
7.2.5.3 2,6-Dibromochinone-4-chlorimide (Gibbs´ Reagent) 192
7.2.5.4 1,2-Naphthoquinone-4-sulphonic Acid Reagent (Folins´ Reagent) 193
7.2.5.5 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazol Reagent (NBD-Chloride Reagent) 194
7.2.6 Boron-Containing Reagents 194
7.2.6.1 Natural Product Reagent (Neu Reagent) 194
7.2.6.2 Diphenylboric Acid Anhydride-Salicylic Aldehyde Reagent (DOOB Reagent) 195
7.2.6.3 Tetraphenylborate Reagent 196
7.2.7 Alkaline Reagents 196
7.2.8 Chloramine-T Reagent 197
7.2.9 Diazotization Reactions 198
7.2.10 Iodine-Starch and Wursters Reagents 199
7.2.10.1 Wursters Red Reagent 199
7.2.10.2 Wursters Blue Reagent 200
7.2.10.3 Iodine-Starch Reagent 200
7.2.11 Reactions with Metal Reagents 201
7.2.11.1 Phosphate Detection Using Ammonium Molybdate 201
7.2.11.2 Dragendorff Reagent 201
7.2.11.3 Antimony(III) Chloride Reagent (Carr-Price Reagent) 202
7.2.11.4 Silver Nitrate Reagent 202
7.2.11.5 Aluminium Chloride Reagent 203
7.2.11.6 Iron(III) Chloride Reagent 203
7.2.11.7 Iodoplatinate Reagent 203
7.2.11.8 Palladium Dichloride Reagent 204
7.2.12 Reagents for Metal Cations 204
7.2.12.1 Alizarin Reagent 204
7.2.12.2 8-Hydroxyquinoline Reagent 205
7.2.12.3 Selenium Determination Using 2,3-Diaminonaphthalene 205
7.2.12.4 Rubeanic Acid Reagent 205
7.3 Reactions via the Gas Phase 205
7.3.1 Ammonium Bicarbonate Reagent 206
7.3.2 Tin(IV) Chloride Reagent 206
7.3.3 Formic Acid Reagent 207
7.3.4 Hydrogen Chloride Reagent 207
7.3.5 Trichloroacetic Acid Reagent 207
7.3.6 Nitric Acid Reagent 208
7.4 Thermal Treatment of TLC Plates 208
7.5 Activity Analysis Using Chemical Reagents 208
7.5.1 Folin-Ciocalteu Reagent 209
7.5.2 Checking for Free Radical Scavenger Activity Using DPPH Reagent 209
7.5.3 Nucleophilic Reaction Ability 210
References 211
Chapter 8: Bioeffective-Linked Analysis in Modern HPTLC 215
8.1 Principle of the Method 216
8.1.1 Contaminant Analysis in the Environment and Food and the Principle of Bioactivity-Based Analysis 216
8.1.2 Aims and Fundamental Aspects of Bioeffective-Linked Analysis by Thin-Layer Chromatography 217
8.1.3 HPTLC as a Method for Bioeffective-Linked Analysis 217
8.2 General Rules for the Analysis of Bioeffective Compounds 219
8.3 Enzyme Tests 220
8.3.1 Urease-Inhibition Test for Heavy Metals 220
8.3.2 Analysis Using Redox Enzymes 221
8.3.3 The Detection of Cholinesterase Inhibitors 222
8.3.3.1 The Physiological Importance of Cholinesterase 222
8.3.3.2 The Molecular Mechanism of Cholinesterase Inhibition 222
8.3.3.3 HPTLC-Cholinesterase Assay Procedures 223
Cholinesterase Enzyme Solution 224
Different Reagent Solutions 225
8.3.3.4 Quantitative Analysis of HPTLC-cholinesterase Assays 229
8.4 Inhibition of Photosynthesis by Herbicides 230
8.4.1 Reagent Preparation 230
8.4.2 Hill Reaction 231
8.4.3 Detection Using Algae 231
8.5 Detecting Bioeffective Compounds with Photobacteria 232
8.5.1 Practical Use of Photobacteria 232
8.5.2 Reaction Time Optimization 234
8.5.3 Applications of Photobacteria 235
8.6 Detection of Fungicidal- and Antibiotical-Active Substances in Environmental Samples 236
8.6.1 Determining Fungicides 236
8.6.2 Screening of Pesticides in Food and Surface Water 236
8.6.3 Detection of Compounds by Antibiotic Activity 237
8.7 Yeast Estrogen Screen 239
References 241
Chapter 9: Planar Chromatography Detectors 244
9.1 Transmittance Measurements in Thin-Layer Chromatography 244
9.1.1 The Lambert-Beer Law 245
9.2 Reflectance Measurements in TLC and HPTLC 246
9.2.1 The Kubelka-Munk Equation 247
9.2.2 Reflectance Measurements with a Diode-Array Scanner 250
9.2.3 Spatial Resolution on the Plate 252
9.2.4 Spectral Distribution on HPTLC Plates 253
9.2.5 Spectral Evaluation Algorithm 255
9.2.6 Video-Densitometric Measurements 258
9.3 Infrared and Raman Detection in Thin-Layer Chromatography 260
9.3.1 Analysis of Thin-Layer Chromatograms by Diffuse Reflectance Infrared Fourier Transformation 260
9.3.2 Analysis of Thin-Layer Chromatograms by Near-Infrared FT-Raman Spectroscopy 261
9.3.3 Analysis of Thin-Layer Chromatograms by Surface-Enhanced Raman Scattering Spectrometry 262
9.4 Mass Spectrometric Detection in TLC 263
9.4.1 Direct Plate Extraction (SSSP) 263
9.4.2 MALDI Techniques (MALDI-MS) 265
9.4.3 Atmospheric Pressure Mass Spectrometry 265
9.5 Thin-Layer Radiochromatography (TL-RC) 267
9.5.1 Direct Radioactivity Measurements on TLC Plates 267
9.5.2 Phosphor Imaging 269
References 270
Chapter 10: Diffuse Reflectance from TLC Layers 274
10.1 The Lambert Cosine Law 274
10.2 Theory of Diffuse Reflectance 276
10.2.1 Special Case a: The Reversal Reflectance Formula 280
10.2.2 Special Case b: The Fluorescence Formula 280
10.2.3 Special Case c: The Kubelka-Munk Expression 281
10.3 Mass-Dependent Reflection 283
10.4 Simplifying the Expression 287
References 287
Chapter 11: Fluorescence in TLC Layers 289
11.1 Theory of Fluorescence and Phosphorescence 289
11.2 Fluorescence Enhancement 292
11.3 Quantification in TLC by Fluorescence 294
11.3.1 Low Sample Concentration Fluorescence in Light Scattering Media 295
11.3.2 High Sample Concentration Fluorescence in Light Scattering Media 296
11.4 Contour Plots for Fluorescence Evaluation 297
11.5 TLC Plates Containing a Fluorescent Dye 297
References 300
Chapter 12: Chemometrics in HPTLC 301
12.1 Calculation of RF Values 301
12.2 Compound Identification Using UV-Visible and Fluorescence Spectra 303
12.3 Correlation Spectroscopy 305
12.3.1 Theory of Correlation Spectroscopy 305
12.3.2 Combination of RF and UV-Visible Spectral Library Search 308
12.3.3 Zone Purity Check 308
12.4 Selection of the Measurement Wavelength 310
12.5 Statistical Photometric Error (Detector Variance) 313
12.5.1 Reciprocal Model 313
12.5.2 Absorbance Model 314
12.5.3 Kubelka-Munk Model 315
12.5.4 Fluorescence model 316
12.5.5 Minimizing the Statistical Photometric Error 317
12.6 Diode Bundling and Data Smoothing 317
12.7 Signal Integration: Area or Height Evaluation? 319
12.8 Deconvolution of Overlapping Peaks 320
12.9 New Visualization Methods for Plots 322
References 325
Chapter 13: Statistics for Quantitative TLC 327
13.1 The Mean Value 327
13.2 Variance and Precision 328
13.2.1 Definition of Variance 328
13.2.2 Relative Variance 330
13.2.3 Quantification of Relative Variance 331
13.3 Trueness, Precision, and Accuracy 332
13.4 The Gauss Distribution 333
13.4.1 Area of the Gauss Distribution 334
13.4.2 Quantiles of the Gauss Distribution 334
13.4.3 Test for a Normal Distribution 336
13.5 Student´s Distribution (t Distribution) 337
13.6 Error Propagation 338
13.7 Calibration Methods 340
13.7.1 The Linear Calibration Function 341
13.7.2 Estimating Xc 342
13.7.3 Estimating a and Yc 343
13.7.4 Estimating the variances of a and Yc 345
13.8 The F-Test 346
13.9 The Linear Regression Variance 346
13.10 The Analytical Function of Linear Regression 347
13.11 Quantitative Analysis Using External Standards 350
13.12 Second-Order Calibration Function 351
13.13 Analytical Function of the Second-Order Calibration 355
13.14 Risk of Systematic Error 357
13.14.1 Constant Systematic Error 357
13.14.2 Proportional Systematic Error 357
13.15 Use of Internal Standards 358
13.16 Standard Addition Method 359
13.17 Mean t Test 362
References 363
Chapter 14: Planning an Analysis and Validation in TLC 365
14.1 Terms Used in Validation 365
14.2 Method Validation 366
14.2.1 Testing Specificity 366
14.2.2 Quantifying Analytes 369
14.2.2.1 General Quantification Remarks 369
14.2.3 General Aspects of Calibration 370
14.2.4 Linearity and Working Range 371
14.2.4.1 Linearity in HPTLC 371
14.2.4.2 Working Range in HPTLC 372
14.2.5 Choosing a Calibration Function 372
14.2.6 External Standard Calculation 374
14.2.7 Optimized Calibration Methods 374
14.2.8 Precision 375
14.2.9 Accuracy 376
14.2.10 Confidence Interval 379
14.2.11 Limit of Detection and Limit of Quantification 379
14.2.11.1 Limit of Detection 380
14.2.11.2 Limit of Quantification (Quantitation Limit) 380
14.2.12 Robustness 382
14.3 Control Charts as Quality Indicators in Routine Analysis 384
References 385
Index 386