Capillary Electrophoresis: Principles and Practice

ISBN-13: 9783642780608 / Angielski / Miękka / 2011 / 375 str.

Reinhard Kuhn; Sabrina Hoffstetter-Kuhn

Opis

Capillary electrophoresis (CE) is a brand-new analytical me- thod with the capability of solving many analytical separa- tion problems very fast and economically. This method gives new information about the investigated substances which can- not easily be obtained by other means. CE has become an established method only recently, but will be implemented in almost every analytical laboratory in industry, service units and academia in the near future. The most important fields of CE application are pharmaceutical and biochemical research and quality control. The authors have exhaustive practical experience in the application of CE methods in the pharmaceutical industry and provide the reader with a comprehensive treatment of this method. The main focus is on how to solve problems when applying CE in the laboratory. Physico-chemical theory is only dealt with in depth when necessary to understand the underlying separation mechanisms in order to solve your problems at the analytical bench. An addendum includes tables on the preparation of buffers and recommended further reading.

Spis treści

1 Introduction.- 1.1 Safety Considerations.- 1.2 History.- 1.3 Nomenclature.- 2 Basic Principles.- 2.1 Basic Electrophoretic Separation Modes.- 2.1.1 Zone Electrophoresis.- 2.1.2 Isotachophoresis.- 2.1.3 Isoelectric Focusing.- 2.2 Set-up for Capillary Electrophoresis.- 2.3 Theory of Electrophoretic Migration.- 2.4 Determination of Effective Mobilitiy.- 2.5 Electroosmosis.- 2.6 Performance Criteria.- 2.6.1 Efficiency.- 2.6.2 Resolution.- 3 Factors Influencing Performance.- 3.1 Fundamental Dispersive Effects.- 3.1.1 Diffusion.- 3.1.2 Adsorption.- 3.1.3 Joule Heating.- 3.1.4 Electrophoretic Dispersion.- 3.1.5 Sample Injection Width.- 3.1.6 Comparative Evaluation of the Different Dispersive Effects.- 3.2 Operational Parameters.- 3.2.1 Field Strength.- 3.2.2 Capillary Dimensions.- 3.2.3 Temperature.- 3.3 Electrolyte System.- 3.3.1 Basic Requirements.- 3.3.2 pH.- 3.3.3 Choice of Buffer.- 3.3.4 Ionic Strength.- 3.3.5 Impact of Buffer Composition.- 3.3.6 Complex Formation.- 3.3.6.1 Borate Complexes.- 3.3.6.2 Ion pairs.- 3.3.6.3 Inclusion Complexes.- 3.3.6.4 Metal Complexes.- 3.3.7 Organic Modifiers.- 4 Instrumentation.- 4.1 Injection.- 4.1.1 Hydrodynamic Injection.- 4.1.2 Electrokinetic Injection.- 4.1.3 General Aspects of Injection.- 4.2 Detection.- 4.2.1 General Aspects.- 4.2.2 Evaluation of Detector Performance.- 4.2.3 UV-VIS Absorbance Detection.- 4.2.3.1 Light Sources for UY-VIS Detection.- 4.2.3.2 Optical Layout of a UV-VIS Detector for CE.- 4.2.3.3 Design of the Detection Cell.- 4.2.4 Fluorescence Detection.- 4.2.4.1 Excitation Sources for Fluorescence Detection.- 4.2.4.2 Optical Layout of a Fluorescence Detector.- 4.2.4.3 Derivatization with Fluorescent Tags.- 4.2.4.4 Pre- and Post-Column Derivatization.- 4.2.5 Electrochemical Detection.- 4.2.5.1 Conductometric Detection.- 4.2.5.2 Amperometric Detection.- 4.2.6 Indirect Detection.- 4.2.6.1 General Aspects.- 4.2.6.2 Indirect Absorbance Detection.- 4.2.6.3 Indirect Fluorescence Detection.- 4.2.6.4 Indirect Amperometric Detection.- 4.2.7 Other Spectroscopic Laser-Induced Detection Modes.- 4.2.7.1 Refractive Index Detection.- 4.2.7.2 Thermooptical Absorbance Detection.- 4.2.8 Radiometric Detection.- 4.2.9 Comparison of the Presented Detection Modes for CE.- 4.3 Capillary Column.- 4.4 Sample Collection.- 4.5 Commercial Instruments.- 5 Techniques.- 5.1 Capillary Zone Electrophoresis.- 5.1.1 General Aspects.- 5.1.2 Capillary Coating.- 5.1.2.1 Polyacrylamide Coating via Siloxane Bond.- 5.1.2.2 Polyacrylamide Coating via Si-C Bond.- 5.1.2.3 Nonionic Surfactant Coating via Octadecylsilane.- 5.1.2.4 Diol-Epoxy Coating.- 5.1.2.5 Polyethylene Glycol Coating.- 5.1.2.5.1 PEG Coating via 3-aminopropyltriethoxysilane.- 5.1.2.5.2 PEG Coating via ?-glycidoxypropyltrimethylsilane.- 5.1.2.6 Protein Coating.- 5.1.2.7 Polyethyleneimine Coating.- 5.2 Capillary Gel Electrophoresis.- 5.2.1 Principles of CGE.- 5.2.2 Crosslinked Polyacrylamide Gels (Chemical Gels).- 5.2.2.1 General Aspects.- 5.2.2.2 Preparation of Crosslinked PAA Gel Filled Capillaries.- 5.2.2.2.1 Radical Polymerization According to Karger and Cohen.- 5.2.2.2.2 Photopolymerization According to Poppe and Coworkers.- 5.2.2.2.3 Isotachophoretic Polymerization According to Novotny and Coworkers.- 5.2.3 Physical Gels.- 5.2.3.1 Agarose Gels.- 5.2.3.2 Linear Polyacrylamide Gels.- 5.2.3.3 Molecular Sieving in Entangled Polymer Solutions of Low Concentration.- 5.3 Micellar Electrokinetic Chromatography.- 5.3.1 Principles of MEKC.- 5.3.2 Effect of the Type of Surfactant.- 5.3.3 Effect of Temperature.- 5.3.4 Effect of Buffer pH.- 5.3.5 Effect of Buffer Additives.- 5.4 Capillary Isotachophoresis.- 5.5 Capillary Isoelectric Focusing.- 5.6 Electrochromatography.- 5.7 Hyphenated Techniques.- 5.7.1 Capillary Electrophoresis — Mass Spectroscopy (CE-MS).- 5.7.2 Liquid Chromatography — Capillary Electrophoresis (LC-CE).- 5.7.3 Capillary Isotachophoresis — Capillary Electrophoresis (CITP-CE).- 5.8 Special Techniques.- 5.8.1 Capillary Affinity Electrophoresis.- 5.8.2 Sample Stacking.- 6 Qualitative and Quantitative Analysis.- 6.1 General Aspects.- 6.2 Influence of Injection.- 6.3 Method Validation.- 7 Applications.- 7.1 Small Ions.- 7.2 Sulphonates and Alkylsulphates.- 7.3 Drugs and Natural Products.- 7.4 Neutral Substances.- 7.5 Herbicides.- 7.6 Amino Acids, Peptides and Proteins.- 7.7 Carbohydrates and Their Derivatives.- 7.8 Nucleotides, Oligonucleotides and Nucleic Acids.- 7.9 Chiral Molecules.- 7.10 Complex Samples.- 8 Appendix 331.- 8.1 Buffer Tables.- 8.2 Derivatization Procedures.- 8.2.1 3-(4-Carboxybenzoyl)-2-quinoline Carboxaldehyde (CBQCA).- 8.2.2 Dansyl Chloride (Dns-Cl).- 8.2.3 4-Phenylspko[furan-2(3H),l?-phthalan]-3,3?,-dione I (Fluorescamine).- 8.2.4 9-Fluorenylmethyl Chloroformate (FMOC).- 8.2.5 Fluorescein Isothiocyanate (FITC).- 8.2.5.1 Preparation of Fluorescein Thiocarbamyl Derivatives.- 8.2.5.2 Preparation of Fluorescein Thiohydantoin Derivatives.- 8.2.6 Naphthalene-23-dicarboxaldehyde (NDA).- 8.2.7 o-Phthaldialdehyde (OPA).- 8.3 Glossary.- 8.4 Manufacturers’ Directory.- 8.5 Further Recommended Reading.- References.

O autorze

Capillary electrophoresis (CE) is a brand-new analytical method with the capability of solving many analytical problems very fast and economically. CE will be implemented in almost every analytical laboratory in industry, service units and academia in the near future, the most important fields of CE application being pharmaceutical and biochemical research and quality control. The authors have exhaustive practical experience in the application of CE methods in the pharmaceutical industry and provide the reader with a comprehensive treatment of this method. The main focus is on how to apply the method and how to solve problems in the laboratory. Physico-chemical theory is dealt with in depth where necessary to understand the underlying separation mechanisms or the influence of different instrumental parameters. An addendum includes tables on the preparation of buffers and recommended further reading.