High-Performance Thin-Layer Chromatography (HPTLC) (eBook)

About the Book 6
About the Editor 8
Preface 10
Contents 12
List of Contributors 14
Part I Introduction 18
Chapter 1:An Overview of HPTLC: A Modern Analytical Technique with Excellent Potentialfor Automation, Optimization, Hyphenation,and Multidimensional Applications 19
Journey of Thin-Layer Chromatography 22
Recent Developments 23
Over-Pressured Layer Chromatography 23
Planar Electrochromatography 23
Image Analysis 24
Two-Dimensional Separations 25
High-Performance Thin-Layer Chromatography (HPTLC) 25
HPTLC: Separation and Resolution 27
HPTLC: Validation Process 28
Selectivity 28
Sensitivity 29
Precision 29
Accuracy 29
Ruggedness 29
Limit of Detection 30
Stability 30
HPTLC: Optimization and Process Control 30
HPTLC: Automation 31
HPTLC: Hyphenation 32
Liquid Chromatography-Thin-Layer Chromatography (LC-TLC) 33
High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS) 33
High-Performance Thin-Layer Chromatography-Infrared Spectroscopy (HPTLC-IR) 34
HPTLC: Laser 35
HPTLC: Multidimensional Applications 36
Notes 36
Suggested Readings 37
Part II Fundamentals, Principle and Advantages of HPTLC 41
Chapter 2: Fundamentals and Theory of HPTLC-Based Separation 42
Planar Chromatography 43
Theoretical Considerations 43
Separation Efficiency 43
Partition Coefficient 45
Retention/Retardation Factor 46
Capacity Factor 48
Spot Capacity 49
Plate Height 49
Resolution 51
Selectivity 52
Notes 53
References 53
Chapter 3: Experimental Aspects and Implementation of HPTLC 55
HPTLC Methodology 56
Stationary Phase 59
Layer Prewashing 59
Mobile Phase 60
Sample Preparation and Application 60
Development of Chromatogram 61
Detection 61
Derivatization 62
Immersing 62
Spraying 62
Heating 62
Quantification 62
Documentation 63
Validation of Method 63
Specificity 64
Sensitivity 64
Linearity and Calibration Curve 64
Accuracy 65
Precision 65
Repeatability 65
Robustness 65
Retardation Factor 65
Peak Purity 66
Notes 67
References 67
Chapter 4: High-Performance Thin-Layer Chromatography: Excellent Automation 69
Automation of TLC to HPTLC 69
Protocol 71
Preparation of Plate 71
Plate Size 71
Sample Application Position 71
Chromatogram Development 72
Derivatization 72
Image Documentation 72
Auto Spray Loading 72
Automated Development 73
Digital Camera-Based Image Documentation 76
Software-Induced Scanning 76
Notes 79
References 79
Part III Applications of HPTLC Separation 80
Chapter 5: Multidimensional and Multimodal Separations by HPTLC in Phytochemistry 81
Multiple Development Techniques 83
Unidimensional Multiple Development 85
Incremental Multiple Development 87
Gradient Multiple Development 88
Bivariant Multiple Development and its Automated Version: Automated Multiple Development 91
Multidimensional Thin-Layer Chromatography 92
Comprehensive 2D Chromatography on One Adsorbent 92
Two-Dimensional Separations on Polar Adsorbents: Silica, Cellulose, Polyamide 93
Two-Dimensional Separations on Polar Chemically-Bonded Stationary Phases: CN-Silica, DIOL-Silica, and NH2-Silica 95
2D-TLC on Bilayer Plates and Coupled-Layer 2D-TLC 96
Combination of MD-PC Methods: Hyphenated Techniques 99
Notes 101
References 101
Chapter 6: Stability-Indicating HPTLC Determination of Imatinib Mesylate in Bulk Drug and Pharmaceutical Dosage 105
Method Validation 107
Precision 107
Robustness 107
Detection and Limit of Quantification 107
Specificity 107
Recovery Studies 108
Analysis of Imatinib Mesylate 108
Forced Degradation of Imatinib Mesylate 109
Acid- and Base-Induced Degradation 109
Hydrogen Peroxide-Induced Degradation 109
Dry Heat Degradation Product 110
Optimum Mobile Phase 111
Calibration Curves 111
Validation 111
Precision 111
Robustness of the Method 112
LOD and LOQ 112
Specificity 112
Recovery Studies 113
Analysis of Prepared Formulation 113
Stability-Indicating Property 113
Notes 113
References 114
Chapter 7: HPTLC Fingerprint Analysis: A Quality Control for Authentication of Herbal Phytochemicals 116
Chromatographic Fingerprints 118
Techniques Used for Fingerprint Analysis of Herbal Medicines 119
Thin-Layer Chromatography 119
Gas Chromatography 119
High-Performance Liquid Chromatography 120
Hyphenated Procedures 120
Traditional Medicines and Chromatographic Fingerprints 121
Authentication of the Species Prone to Confusion 121
Monitoring the Dynamic Change Due to Interaction of Mixed Herbal Drugs during Extraction 122
Quality Evaluation of the Crude Drugs 124
Adulterants and the Authentic Sample 124
Notes 125
References 126
Chapter 8: HPTLC in Herbal Drug Quantification 128
Protocol Used for Drug Analysis 129
Selection of Chromatographic Layer 129
Sample and Standard Preparation 129
Activation of Precoated Plates 130
Application of Sample and Standard 130
Selection of Mobile Phase 130
Preconditioning (Chamber Saturation) 130
Chromatographic Development and Drying 131
Detection and Visualization 131
Quantification 131
Validation of Analytical Method 132
Simultaneous Estimation of Androgrpholide and Wadelolactone 132
Quantification of Eugenol, Luteoline, Ursolic Acid and Oleanolic Acid in Black and Green Varieties of Ocimum Sanctum 132
Determination of Phyllanthin and Hypophyllanthin 133
Determination of Cucuminoids 134
Micro Analytical Technique for Determination of Podophyllotoxin by RP-HPTLC 135
HPTLC in Morphological and Geographical Variations in the Herbal Raw Material 135
HPTLC Studies of Various Indian Herbal Formulations 136
Sennoside Content 136
Andrographolide in Extracts and Formulations 137
Determination of Hyperforin in Hypericum perforatum 139
Determination of Corosolic Acid 140
Evaluation and Validation of a Polyherbal Formulation 140
Quantitative Analysis of Alkaloids in Hardin Grass (Phalaris aquatical) 142
Densitometric Determination of Hecogenin from Agave americana Leaf 143
Determination of Triterpenoid Acids (Arjungenin, Arjunolic Acid) from Terminalia arjuna Stem Bark 144
Quantification of Valerenic Acid in Valeriana jatamansi and Valeriana officinalis 144
Determination of Rutin in Amaryllis belladonna L. Flowers 145
Determination of Quercetin, Rutin, and Coumaric Acid in Flowers of Rhododendron arboreum 146
Determination of Rutin in Amaranthus spinosus Linn 146
Determination of Lycorine in Amaryllidaceae Plants Extracts 148
Notes 149
References 149
Chapter 9: HPTLC Determination of Artemisinin and Its Derivatives in Bulk and Pharmaceutical Dosage 151
Artemisinin Derivatives 152
Analytical Methods 152
Chromatographic Conditions 153
Calibration Curve 154
Validation of the Method as per ICH Guideline 154
Precision 154
Robustness 154
Limit of Detection and Limit of Quantification 155
Recovery Studies 157
Analysis of Artemisinin and Artsunate in Marketed Formulations 158
Analysis of Artemether and Arteether in Marketed Formulations 158
Notes 159
References 159
Chapter 10: TLC/HPTLC in Biomedical Applications 161
Chromatographic Condition in Biomedical Application 162
Retention Parameter in TLC 163
Chromatographic Systems 164
Stationary Phases 164
Mobile Phases 164
Amino Acids and Proteins 167
Carbohydrates 168
Lipids 170
Bile Acids 172
Drugs 174
Vitamins 177
Porphyrins 181
Notes 182
References 182
Chapter 11: Analytical Aspects of High Performance Thin Layer Chromatography 189
Qualitative Analysis 190
Quantitative Analysis 194
External and Internal Standardization Methods 194
Standard Addition Method 195
Reporting the Results of Analyses 195
Validation Method of Analysis 196
Specificity 198
Linearity of the Response 200
Accuracy 202
Precision 204
Detection Limit, Quantification Limit 205
Range 206
Robustness/Ruggedness 207
Stability for Bioanalytical Method 208
Application of HPTLC 208
Notes 209
References 209
Chapter 12: Quantitative Analysis and Validation of Method Using HPTLC 212
Preference of HPTLC over Column Chromatography 212
Quantification 214
Stability of the Analyte 215
Stability of the Sample in Solution 215
Stability on the Absorbent Layer Prior to Development 215
Stability during Development 216
Stability after Chromatographic Development 216
Purity of Reagent and Solvents 217
Densitometry 217
Optimization of Wavelength 217
Purity of the Separated Band 218
Calibration Curve 218
Single Level Calibrations 218
Multilevel Calibration 220
Classical Internal Standard Method 221
Validation of the Method 222
Principles of Method Validation 222
Selectivity 222
Specificity 223
Linearity and Range 224
Precision 224
Repeatability (Precision on Replication) 224
Reproducibility (Precision of Comparison) 224
Detection and Limit of Quantitation 225
Robustness 225
Accuracy 226
Examples for the Successful Usage of HPTLC Method for Quantification 226
Notes 228
References 229
Chapter 13: Quantification of Low Molecular Mass Compounds Using Thermostated Planar Chromatography 231
Temperature Effects 231
Approach to Temperature-Controlled Planar Chromatography 234
Temperature-Sensitive Quantification of Target Compounds 247
Notes 249
References 249
Part IV HPTLC and Its Future to Combinatorial Approach 253
Chapter 14: Basic Principles of Planar Chromatography and Its Potential for Hyphenated Techniques 254
Principles of Planar Chromatography 256
Modern Chambers for TLC 256
Conventional Chambers (N-Chambers) 257
Horizontal Chambers for Linear Development 258
Horizontal Chambers for Radial (Circular and Anticircular) Development 261
Sample Application 261
Chromatogram Development 261
Mobile Phases Applied in TLC 262
Classification of the Modes of Chromatogram Development 262
Sample Preparation 266
Acid Hydrolysis 267
Method for Sulfur-Containing Amino Acids 267
Detection and Identification of Biomolecules in TLC 268
Quantitative Determination of Biomolecules 270
Hyphenated Techniques 271
Combined Thin Layer Chromatography/Fourier Transform Infrared Spectroscopy (TLC-FTIR) and Nuclear Magnetic Resonance (TLC-NMR) 271
Thin Layer Chromatography with Diode Array Scanning (TLC-DAD) 273
Thin Layer Chromatography/Mass Spectrometry (TLC-MS) 282
Automated Multiple Development of Biomolecules 287
Multidimensional Planar Chromatography (MD-PC) Methods in Biomolecules Analysis 292
Comprehensive Two-Dimensional (2D) Chromatography on One Adsorbent 293
Two-Dimensional Thin-Layer Chromatography (2D-TLC) 295
Graft Thin-Layer Chromatography 297
Combination of Multidimensional Planar Chromatography (MDPC) Methods - Hyphenated Techniques 299
Future Aspects of Multidimensional Planar Chromatography (MD-PC): Combined MD-PC-DAD with HPLC-DAD 305
Notes 306
References 306
Chapter 15: HPTLC-MS Coupling: New Dimension of HPTLC 318
Introduction 319
Hyphenation 321
Scanning of TLC/HPTLC PLATE for Quantification or Finger Printing 325
Slit Scanning 325
Video Scanning 326
HPTLC-MS 326
Type of MS Techniques Used for TLC/HPTLC-MS Analysis 327
Electrospray Ionization 327
Matrix-Assisted Laser Desorption Ionization 329
Desorption Electrospray Ionization 330
Direct Analysis in Real Time 331
Secondary Ion Mass Spectrometry 332
Fast Atom Bombardment 333
Electrospray-Assisted Laser Desorption Ionization/Fused Droplet 334
Tandem Mass Spectrometry 335
Present Challenge for Drug Discoveries 336
Notes 336
References 337
Chapter 16: TLC/HPTLC with Direct Mass Spectrometric Detection: A Review of the Progress Achieved in the Last 5 Years 341
Chromatographic Methods and Product Identification 341
Directly on the TLC Plate with and Without Any Derivatization 342
Analysis by Reelution from the TLC Plate 343
Spectroscopic Analysis Directly on the TLC Plate 343
NMR Spectroscopy 344
IR and Raman Spectroscopy 344
Mass Spectrometry 345
MS Methods Based on Previous Extraction of the Analytes 345
MS Desorption Methods 346
TLC/MALDI Combination 346
How Does MALDI-TOF MS Work? 348
Quantitative MALDI-TOF MS 351
Characterization Using Direct TLC/MALDI Coupling 351
Small Proteins and Peptides 351
DNA and Nucleic Acids 353
Carbohydrates 353
Pharmaceuticals 355
Glycolipids 357
Phospholipids 359
Notes 363
References 364
Chapter 17: Scanning Diode Laser Desorption Thin-Layer Chromatography Coupled with Atmospheric Pressure Chemical Ionization Mass Spectrometry 370
Desorption from a TLC Plate by a Continuous Wave Near Infrared Diode Laser 372
Experimental Arrangement for Laser Desorption 372
Evaluation with Reserpine as Sample 373
Separated Process of Desorption and Ionization 375
Use of Glycerol 376
Dependence on Laser Power 378
Full Plate Detection System 379
Experimental Arrangement for the Full Scanning System 380
Influence of the Power Density 382
Rapid Screening for Specific Substances 384
Internal Standard 385
Notes 386
References 387
Chapter 18: HPTLC Hyphenated with FTIR: Principles, Instrumentation and Qualitative Analysis and Quantitation 389
Principle, Instrumentation and Data Presentation 390
Qualitative and Quantitative Analysis 395
Salient Features of HPTLC-FTIR 395
Notes 396
References 397
Index 399