Capillary Electrophoresis: Methods and Potentials

ISBN-13: 9783642858567 / Angielski / Miękka / 2012 / 215 str.

Heinz Engelhardt; Wolfgang Beck; Thomas Schmitt

Opis

Capillary electrophoresis (CE), also designated by the acronym HPCE for "High Per- formance Capillary Electrophoresis" is a rapidly growing analytical separation method. It unites the separation technique of classical electrophoresis on plates with the instrumental methods of chromatography with respect to direct detection of the solutes separated in the capillary and their ready identification and quantification. The initial problems of inadequate reproducibility in quantitative analysis, due to the necessity of handling extremely small volumes, have largely been solved in the sec- ond generation commercial instruments. Hence, a rapid and reliable separation sys- tem is available for ionic compounds from the smallest cation (the lithium ion) up to poly anions with molecular weights ranging in the millions (such as DNA molecules). The methods of gel electrophoresis and isoelectric focusing can be readily extended to separation techniques carried out in a capillary. For nonionic compounds an addi- tional separation method is available in the form of micellar electrokinetic chroma- tography (MEKC). This involves a true chromatographic separation process because the distribution of the analytes between the buffer and the micelles is superimposed on the electrophoretic migration, which contributes substantially to the selectivity.

Spis treści

1 Introduction.- 2 Principles of Capillary Electrophoresis.- 3 Theoretical Foundations and Their Influence on the Analytical Results.- 3.1 Electrophoretic Migration.- 3.2 Conductivity.- 3.3 Electroosmotic Flow.- 3.4 Band Broadening.- 3.4.1 Efficiency losses through diffusion.- 3.4.2 Efficiency loss through temperature effects.- 3.4.3 Loss in efficiency through electrodispersion.- 3.4.4 Efficiency losses through wall adsorption.- 3.4.5 Efficiency losses through overloading of the separation system.- 3.4.6 Efficiency losses through superimposition of flow profiles.- 3.4.7 Summary.- 4 Instrumentation.- 4.1 Power Supply.- 4.2 Capillaries.- 4.3 Sample Introduction.- 4.3.1 Pressure injection.- 4.3.2 Hydrostatic injection.- 4.3.3 Electrokinetic injection.- 4.3.4 Sample-split systems.- 4.3.5 Enrichment effects in sample introduction: sample stacking.- 4.4 Thermostating.- 4.5 Detection.- 4.5.1 UV detection.- 4.5.2 Fluorescence detection.- 4.5.3 Conductivity detection.- 4.5.4 Other detection methods.- 4.5.5 Derivatization reactions.- 4.6 Special Problems of Quantitative Analysis in CE.- 5 Capillary Zone Electrophoresis (CZE).- 5.1 Principles of Optimization in CZE.- 5.1.1 Effect of pH.- 5.1.2 Effect of buffer concentration.- 5.1.3 Buffer selection.- 5.1.4 Applications.- 5.2 Indirect Detection Methods in CE.- 5.2.1 Principles of indirect detection techniques.- 5.2.2 Separation of cations with indirect UV detection.- 5.2.3 Separation of anions with indirect UV detection.- 5.2.4 Analysis of cations and anions with indirect fluorescence detection.- 5.3 Capillary Zone Electrophoresis of Proteins.- 5.3.1 Separations in uncoated capillaries.- 5.3.1.1 Selection of the pH.- 5.3.1.2 Addition of salts to the buffer.- 5.3.1.3 Use of buffer additives for the separation of proteins.- 5.3.1.4 Dynamic coating of capillaries.- 5.3.2 Protein separations with surface-modified capillaries.- 5.3.2.1 Coatings for capillary electrophoresis.- 5.3.3 Overview of important chemical coatings for protein separation.- 5.3.3.1 Conventional coatings.- 5.3.3.2 Polymeric coatings.- 5.3.4 Summary.- 6 Micellar Electrokinetic Chromatography (MEKC).- 6.1 Fundamentals of MEKC.- 6.2 Optimization of Resolution.- 6.3 Selection of the Detergent.- 6.4 Separations by MEKC.- 7 Separation of Enantiomers by CE.- 7.1 Enantiomeric Separations with Cyclodextrins as Chiral Selectors.- 7.1.1 Neutral cyclodextrins.- 7.1.2 Ionic cyclodextrins.- 7.2 Other Separation Systems.- 8 Capillary Gel Electrophoresis (CGE).- 8.1 Acrylamide-based Gels.- 8.1.1 Preparation and manipulation of gel-filled capillaries.- 8.1.2 Crosslinked Polyacrylamide gels.- 8.1.3 Linear Polyacrylamide gels (LPA).- 8.1.3.1 Separation of DNA fragments with LPA.- 8.1.3.2 SDS PAGE (Polyacrylamide gel electrophoresis) of proteins.- 8.2 Polysaccharide-based Gels and other Polymers.- 8.3 Migration Models of Biopolymers in Polymer Solutions.- 9 Isoelectric Focusing in Capillaries (CIEF).- 10 Other Separation Techniques in CE.- 10.1 Isotachophoresis (ITP).- 10.2 Electrochromatography (EC).- 11 A Troubleshooting Guide to CE.- 11.1 Determination of the Problem Source.- Test 1: Recording a breakthrough curve under flushing pressure.- Test 2: Recording a breakthrough curve with the injection pressure.- Test 3: Checking the potental source.- 11.2 Scenarios of Problems: “What to do if…”.- 12 Literature Index.- 12.1 Literature Cited.- 12.2 Additional Literature Sources.- 13 Acknowledgement.