Capillary Isoelectric Focussing CIEF

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. 

Instrumentation for CIEF is not much different from other CE methods. The capillary is filled with an ampholyte mixture. An electric field of about 400 to 600 Vcm is applied and fhepH gradient develops inside the capillary. The sample can be placed together with the ampholyte mixture into the capillary and focussed whilst the pH gradient is developing. It is essential to coat the capillaries to prevent EOF as this may sweep the analytes past the detector before focussing has finished. 

Detection in CE occurs at a fixed point and not over the whole medium as in gel electrophoresis. Hence, it is necessary to mobilise the focussed bands and move them past the detection window. This can be achieved by chemical and hydro-dynamic flow mobilisation in coated capillaries or by electroosmotic mobilisation in uncoated or partially coated capillaries. 

In hydrodynamic mobilisation, focussing and mobilisation are carried out as two separate steps. After focussing is completed, the capillary is connected to apressure pump or vacuum while the focussing voltage is still applied. The focussed bands are thus, moved and pass the detector. The pressure driven flow results in a parabolic flow profile, which can compromise the resolution due to band broadening. An advantage of this method is that very basic and very acidic proteins, which are focused at the extremities of the capillaries, can be mobilised easily. 

Alternatively, imaging along the whole length of the capillary can eliminate the need for mobilisation. 

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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. 

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). 

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