Capillary Electrophoresis: Principles and Practice

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1 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

2 Contents 1 Introduction Safety Considerations History Nomenclature 3 2 Basic Principles Basic Electrophoretic Separation Modes Zone Electrophoresis Isotachophoresis Isoelectric Focusing Set-up for Capillary Electrophoresis Theory of Electrophoretic Migration Determination of Effective Mobilitiy Electroosmosis Performance Criteria Efficiency Resolution 33 3 Factors Influencing Performance Fundamental Dispersive Effects Diffusion Adsorption Joule Heating Electrophoretic Dispersion Sample Injection Width Comparative Evaluation of the Different Dispersive Effects Operational Parameters Field Strength Capillary Dimensions Temperature Electrolyte System Basic Requirements ph Choiceof Buffer Ionic Strength Impact of Buffer Composition Complex Formation 91

3 Vm Contents Borate Complexes Ionpairs Inclusion Complexes Metal Complexes Organic Modifiers 99 4 Instrumentation Injection Hydrodynamic Injection Electrokinetic Injection General Aspects of Injection Detection General Aspects Evaluation of Detector Performance UV-VIS Absorbance Detection Light Sources for UV-VIS Detection Optical Layout ofa UV-VIS Detector for CE Design of the Detection Cell Fluorescence Detection Excitation Sources for Fluorescence Detection Optical Layout of a Fluorescence Detector Derivatization with Fluorescent Tags Pre- and Post-Column Derivatization Electrochemical Detection Conductometric Detection Amperometric Detection Indirect Detection General Aspects Indirect Absorbance Detection Indirect Fluorescence Detection Indirect Amperometric Detection Other Spectroscopic Laser-Induced Detection Modes Refractive Index Detection Thermooptical Absorbance Detection Radiometrie Detection Comparison of the Presented Detection Modes for CE Capillary Column Sample Collection Commercial Instruments Techniques Capillary Zone Electrophoresis General Aspects Capillary Coating Polyacrylamide Coating via Siloxane Bond 165

4 Contents IX Polyacrylamide Coating via Si-C Bond Nonionic Surfactant Coating via Octadecylsilane Diol-Epoxy Coating Polyethylene Glycol Coating PEG Coating via 3-aminopropyltriethoxysilane PEG Coating via Y-glycidoxypropyltrimethylsilane Protein Coating Polyethyleneimine Coating Capillary Gel Electrophoresis Principles of CGE Crosslinked Polyacrylamide Gels (Chemical Gels) General Aspects Preparation of Crosslinked PAA Gel Filled Capillaries Radical Polymerization According to Karger and Cohen Photopolymerization According to Poppe and Coworkers Isotachophoretic Polymerization According to Novotny and Coworkers PhysicalGels AgaroseGels Linear Polyacrylamide Gels Molecular Sieving in Entangled Polymer Solutions of Low Concentration Micellar Electrokinetic Chromatography Principles of MEKC Effect of the Type of Surfactant Effect of Temperature Effect of Buffer ph Effect of Buffer Additives Capillary Isotachophoresis Capillary Isoelectric Focusing Electrochromatography Hyphenated Techniques Capillary Electrophoresis - Mass Spectroscopy (CE-MS) Liquid Chromatography-Capillary Electrophoresis (LC-CE) Capillary Isotachophoresis - Capillary Electrophoresis (CITP-CE) Special Techniques Capillary Affmity Electrophoresis Sample Stacking Qualitative and Quantitative Analysis General Aspects Influence of Injection Method Validation 249

5 X Contents 7 Applications Smalllons Sulphonates and Alkylsulphates Drugs and Natural Products Neutral Substances Herbicides Amino Acids, Peptides and Proteins Carbohydrates and Their Derivatives Nucleotides, Oligonucleotides and Nucleic Acids Chiral Molecules Complex Samples Appendix Buffer Tables Derivatization Procedures (4-Carboxybenzoyl)-2-quinoline Carboxaldehyde (CBQCA) Dansyl Chloride (Dns-Cl) Phenylspiro[furan-2(3H),l'-phthalan]-3,3'-dione (Fluorescamine) Fluorenylmethyl Chloroformate (FMOC) Fluorescein Isothiocyanate (FITC) Preparation of Fluorescein Thiocarbamyl Derivatives Preparation of Fluorescein Thiohydantoin Derivatives Naphthalene-2,3-dicarboxaldehyde (NDA) o-phthaldialdehyde (OPA) Glossary Manufacturers'Directory Further Recommended Reading 341 References 343 Subject Index 371

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