R. Kuhn S. Hoffstetter-Kuhn Capillary Electrophoresis: Principles and Practice With 90 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo HongKong Barcelona Budapest
Contents 1 Introduction 1 1.1 Safety Considerations 1 1.2 History 1 1.3 Nomenclature 3 2 Basic Principles 5 2.1 Basic Electrophoretic Separation Modes 5 2.1.1 Zone Electrophoresis 6 2.1.2 Isotachophoresis 7 2.1.3 Isoelectric Focusing 8 2.2 Set-up for Capillary Electrophoresis 10 2.3 Theory of Electrophoretic Migration 11 2.4 Determination of Effective Mobilitiy 19 2.5 Electroosmosis 22 2.6 Performance Criteria 29 2.6.1 Efficiency 30 2.6.2 Resolution 33 3 Factors Influencing Performance 37 3.1 Fundamental Dispersive Effects 37 3.1.1 Diffusion 38 3.1.2 Adsorption 40 3.1.3 Joule Heating 43 3.1.4 Electrophoretic Dispersion 50 3.1.5 Sample Injection Width 58 3.1.6 Comparative Evaluation of the Different Dispersive Effects.. 59 3.2 Operational Parameters 62 3.2.1 Field Strength 62 3.2.2 Capillary Dimensions 65 3.2.3 Temperature 70 3.3 Electrolyte System 74 3.3.1 Basic Requirements 74 3.3.2 ph 74 3.3.3 Choiceof Buffer 82 3.3.4 Ionic Strength 82 3.3.5 Impact of Buffer Composition 87 3.3.6 Complex Formation 91
Vm Contents 3.3.6.1 Borate Complexes 93 3.3.6.2 Ionpairs 94 3.3.6.3 Inclusion Complexes 95 3.3.6.4 Metal Complexes 98 3.3.7 Organic Modifiers 99 4 Instrumentation 103 4.1 Injection 103 4.1.1 Hydrodynamic Injection 103 4.1.2 Electrokinetic Injection 105 4.1.3 General Aspects of Injection 107 4.2 Detection 109 4.2.1 General Aspects 109 4.2.2 Evaluation of Detector Performance 110 4.2.3 UV-VIS Absorbance Detection 114 4.2.3.1 Light Sources for UV-VIS Detection 115 4.2.3.2 Optical Layout ofa UV-VIS Detector for CE 116 4.2.3.3 Design of the Detection Cell 116 4.2.4 Fluorescence Detection 123 4.2.4.1 Excitation Sources for Fluorescence Detection 124 4.2.4.2 Optical Layout of a Fluorescence Detector 125 4.2.4.3 Derivatization with Fluorescent Tags 128 4.2.4.4 Pre- and Post-Column Derivatization 131 4.2.5 Electrochemical Detection 133 4.2.5.1 Conductometric Detection 134 4.2.5.2 Amperometric Detection 136 4.2.6 Indirect Detection 142 4.2.6.1 General Aspects 142 4.2.6.2 Indirect Absorbance Detection 144 4.2.6.3 Indirect Fluorescence Detection 145 4.2.6.4 Indirect Amperometric Detection 147 4.2.7 Other Spectroscopic Laser-Induced Detection Modes 147 4.2.7.1 Refractive Index Detection 148 4.2.7.2 Thermooptical Absorbance Detection 149 4.2.8 Radiometrie Detection 150 4.2.9 Comparison of the Presented Detection Modes for CE 150 4.3 Capillary Column 152 4.4 Sample Collection 156 4.5 Commercial Instruments 157 5 Techniques 161 5.1 Capillary Zone Electrophoresis 161 5.1.1 General Aspects 162 5.1.2 Capillary Coating 162 5.1.2.1 Polyacrylamide Coating via Siloxane Bond 165
Contents IX 5.1.2.2 Polyacrylamide Coating via Si-C Bond 166 5.1.2.3 Nonionic Surfactant Coating via Octadecylsilane 167 5.1.2.4 Diol-Epoxy Coating 169 5.1.2.5 Polyethylene Glycol Coating 170 5.1.2.5.1 PEG Coating via 3-aminopropyltriethoxysilane 170 5.1.2.5.2 PEG Coating via Y-glycidoxypropyltrimethylsilane 171 5.1.2.6 Protein Coating 172 5.1.2.7 Polyethyleneimine Coating 172 5.2 Capillary Gel Electrophoresis 173 5.2.1 Principles of CGE 173 5.2.2 Crosslinked Polyacrylamide Gels (Chemical Gels) 176 5.2.2.1 General Aspects 176 5.2.2.2 Preparation of Crosslinked PAA Gel Filled Capillaries 179 5.2.2.2.1 Radical Polymerization According to Karger and Cohen 182 5.2.2.2.2 Photopolymerization According to Poppe and Coworkers 184 5.2.2.2.3 Isotachophoretic Polymerization According to Novotny and Coworkers 185 5.2.3 PhysicalGels 186 5.2.3.1 AgaroseGels 186 5.2.3.2 Linear Polyacrylamide Gels 188 5.2.3.3 Molecular Sieving in Entangled Polymer Solutions of Low Concentration 190 5.3 Micellar Electrokinetic Chromatography 191 5.3.1 Principles of MEKC 191 5.3.2 Effect of the Type of Surfactant 198 5.3.3 Effect of Temperature 200 5.3.4 Effect of Buffer ph 202 5.3.5 Effect of Buffer Additives 203 5.4 Capillary Isotachophoresis 205 5.5 Capillary Isoelectric Focusing 206 5.6 Electrochromatography 212 5.7 Hyphenated Techniques 217 5.7.1 Capillary Electrophoresis - Mass Spectroscopy (CE-MS) 218 5.7.2 Liquid Chromatography-Capillary Electrophoresis (LC-CE).. 222 5.7.3 Capillary Isotachophoresis - Capillary Electrophoresis (CITP-CE) 225 5.8 Special Techniques 227 5.8.1 Capillary Affmity Electrophoresis 227 5.8.2 Sample Stacking 232 6 Qualitative and Quantitative Analysis 243 6.1 General Aspects 243 6.2 Influence of Injection 246 6.3 Method Validation 249
X Contents 7 Applications 251 7.1 Smalllons 251 7.2 Sulphonates and Alkylsulphates 260 7.3 Drugs and Natural Products 261 7.4 Neutral Substances 275 7.5 Herbicides 275 7.6 Amino Acids, Peptides and Proteins 278 7.7 Carbohydrates and Their Derivatives 304 7.8 Nucleotides, Oligonucleotides and Nucleic Acids 313 7.9 Chiral Molecules 320 7.10 Complex Samples 322 8 Appendix 331 8.1 Buffer Tables 331 8.2 Derivatization Procedures 333 8.2.1 3-(4-Carboxybenzoyl)-2-quinoline Carboxaldehyde (CBQCA). 333 8.2.2 Dansyl Chloride (Dns-Cl) 333 8.2.3 4-Phenylspiro[furan-2(3H),l'-phthalan]-3,3'-dione (Fluorescamine) 333 8.2.4 9-Fluorenylmethyl Chloroformate (FMOC) 333 8.2.5 Fluorescein Isothiocyanate (FITC) 334 8.2.5.1 Preparation of Fluorescein Thiocarbamyl Derivatives 334 8.2.5.2 Preparation of Fluorescein Thiohydantoin Derivatives 334 8.2.6 Naphthalene-2,3-dicarboxaldehyde (NDA) 334 8.2.7 o-phthaldialdehyde (OPA) 335 8.3 Glossary 335 8.4 Manufacturers'Directory 338 8.5 Further Recommended Reading 341 References 343 Subject Index 371