OPTIMIZING SEPARATION PARAMETERS IN CAPILLARY ISOELECTRIC-FOCUSING

Several modifications have been developed for capillary isoelectric focusing (IEF) to improve separation and detection of proteins. A basic compound was incorporated into the ampholyte mixture to block the segment between the monitor point and capillary end during focusing, enabling detection of basic proteins during mobilization. Use of a low-pI zwitterionic mobilization agent increased mobilization efficiency of the acidic region of the pH gradient, improving detection of acidic proteins. Mobilization with a neutral-pI zwitterion selectively mobilized neutral and basic proteins with improved resolution. Observation of colored proteins in glass capillaries mounted on thermosensitive liquid crystal was used to determine the heat generation patterns along the capillary and the effect of salt on the IEF process. The presence of salt in the sample resulted in long focusing and mobilization times. Incorporation of a non-ionic detergent in the sample plus ampholyte mixture reduced precipitation and improved reproducibility in capillary IEF of gamma-globulins.