Capillary Isoelectric Focusing cIEF

2.3.1. Mechanism

Molecules that carry both positively and negatively charged groups exhibit, at a specific pH, an equal number of positive and negative charges. At this pH, known as the isoelectric pH or pI, the molecule, although charged, behaves as if it is neutral because its positive and negative charges cancel each other. The molecule, therefore, has no tendency to migrate in an electrical field. In iso-electric focusing, special reagents called ampholytes are used to create a pH gradient within the capillary. These ampholytes are mixtures of buffers with a range of pKa values. In an electrical field, ampholytes will arrange themselves in order of pKa; this gradient is trapped between a strong acid and a strong base. Analytes introduced into this gradient will migrate to the point where the pH of the gradient equals their pI. At this point the analyte, having no net charge, ceases to migrate. It will remain at that position so long as the pH gradient is stable, typically as long as the voltage is applied.

2.3.2. Applications

Capillary isoelectric focusing (cIEF) is used almost exclusively for the separation of closely related protein species. Hemoglobin can be separated into several bands by this technique, whereas separation by SDS-CGE usually results in a single form being identified. In this application, the protein sample is mixed with the ampholyte solution and the mixture is pumped into the capillary. When voltage is applied, the proteins and the ampholytes migrate to their appropriate positions in the gradient. When focusing is complete, the proteins in the mixture are distributed throughout the length of the capillary. In order to detect the proteins it is necessary to mobilize them so that they pass by the detector in turn. There are two ways to accomplish this mobilization. Pressure mobilization utilizes positive pressure applied to one end of the capillary to drive the entire fluid column through the window. In order to prevent band distortion, this pressure must be applied carefully, preferably while the voltage is still being applied. Chemical mobilization requires that one end of the capillary be transferred to a salt solution after focusing has taken place. Upon application of voltage, salt will migrate into the capillary, disrupting the pH gradient and allowing the proteins to migrate past the detector by electrophoresis.

cIEF is also widely used for examining the distribution of carbohydrate isoforms of glycoproteins. In other techniques, such as SDS-CGE or CZE, these proteins tend to move as diffuse bands and to generate broad peaks. cIEF can often resolve these bands into peaks that differ by as little as one charged sugar group.

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