Isoelectric focussing instrumentation

Subsequently four different CE modes are described in the sections Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isoelectric Focussing, and Micellar Electrokinetic Chromatography (MEKC), respectively. The fundamental principles of the specific separation modes are briefly explained, using appropriate equations where required. In Table 3 all equations are listed. In addition, the influence of both instrumental parameters and electrolytic solution parameters on the optimization of separations is described. 

Zhou and Johnston [55] reported protein characterization by capillary isoelectric focussing (CIEF) on-hne coupled to RPLC-MS. Direct coupling of CIEF to ESl-MS is limited by interferences by the ampholytes. Inserting RPLC in-between can help removing these interferences. CIEF is performed in combination with a microdialysis membrane-based cathodic cell to remove the ampholyte and to collect protein fractions by stop-and-go CIEF prior to transfer to a 5><0.3-pm-ID C,8 trapping colunm and RPLC separation on a 50><0.3-pm-ID C4 column. The separation is performed using an acetonitrile-water gradient (0.1% acetic acid). ESI-MS is performed on a quadrupole-TOF hybrid (Q-TOF) instrument. 

Zone detection was accomplished with an UV absorption detector which scanned the length of the tube. Kolin( ) described several free zone electrophoresis systems which used flow in serpentine and helical paths to combat thermally driven convection. Catsimpoolas( ) described an instrumental system with scanning detection for following the course of isoelectric focussing in gel-filled tubes. These techniques have not come into widespread use presumably due to their complexity. 

Similarly to gel electrophoresis, a number of modes can be employed that separate analyte mixtures according to different properties. Some of these modes are summarised in Table 3.2 together with their commonly used abbreviations. Depending on the principle of separation, different species can be analysed. After an overview of capillary electrophoretic instrumentation, the different modes of capillary electrophoresis are described in more detail including capillary zone electrophoresis (CZE), capillary isoelectric focussing (CIEF), micellar electro-kinetic chromatography (MEKC) and capillary gel electrophoresis (CGE). 

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