Capillary isoelectric focussing

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. 

Fractionation by capillary isoelectric focussing (CIEF) prior to LC-MS was reported by several groups (Ch. 17.5.6). Retention time from the RPLC and pl-data from the CIEF separation were applied to validate potentially identified peptides from the SEQUEST search [27]. The initial number of 7629 identified peptides was rednced to 1837 identified and 1130 likely hits by pi and retention time validation. 

Keywords Capillary electrophoresis Capillary gel electrophoresis Capillary isoelectric focussing Polyelectrolytes Polystyrenesulfonates Polyvinylpyridines Polycarboxybetaines Polyampholytes... 

Fig. 30 Capillary isoelectric focussing of polyampholytes PA1-PA4. Separation conditions capillary PVA 40/50 cm, i.d. 75 pm BGE 300 ppm sample in MilliQ+2% Servalyt anolyte H3P04, catholyte NaOH /=+30.0 kV focussing time 12 min mobilization 30 kV+0.1 psi T=25 °C detection at 280 nm...

Hempe JM, Graver RD. Separation of hemoglobin variants with similar change by capillary isoelectric focussing value of isoelectric point for identification of common and uncommon hemoglobin variants. Electrophoresis 2000 21 738-43. 

Mario N, Baudin B, Aussel C, Giboudeau J. Capillary isoelectric focussing and high-performance cation-exchange chromatography compared for qualitative and quantitative analysis of hemoglobin variants. Clin Chem 1997 43 2137-42. 

Sugano M, Hidaka H, Yamauchi K, Nakabayashi T, Higuchic Y, Fujita K, et al. Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focussing method. Electrophoresis 2000 21 3016-9. 

Wehr, T., R. Rodriguez-Diaz, and M. Zhu, Recent advances in capillary isoelectric focussing. Chromatographia Suppl., 2001, 53, S47-S58. 

Capillary Isoelectric Focussing CIEF isoelectric point, pi zwitter-ions... 

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). 

Isoelectric focussing as outlined in section 3.2.4 for gels can also be performed in a capillary containing a free solution of electrolyte. Capillary isoelectric focussing, (CIEF) can be advantageous over lEF. The process is readily automated. Due to higher electric fields, the focussing times are very short often the separation is finished within minutes rather than hours. Only minute sample quantities are required, a few ijlL in the sample reservoir and a few nL in the capillary. Quantification is also easier and more reproducible in CIEF. 

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