EXPERIMENTAL ASPECTS OF CAPILLARY ISOELECTRIC-FOCUSING WITH ELECTROOSMOTIC ZONE DISPLACEMENT

The investigation of different parameters affecting separation and resolution in capillary isoelectric focusing (CIEF) with electroosmotic zone displacement is reported. Focusing is performed in an uncoated, open-tubular fused-silica capillary of length 50-100 cm and 75 mum I.D. An experiment proceeds by first filling the entire capillary with the catholyte containing the neutral polymer(s). Sample composed of carrier ampholytes and proteins is introduced at the anodic capillary end, the initial zone length being 10-50% of the effective capillary length. After application of power, two electrokinetic effects occur concurrently, the formation of a longitudinal pH gradient with the separation of proteins (isoelectric focusing) and, owing to the negative surface charge of untreated fused silica, the displacement of the entire zone pattern towards the cathode (electroosmosis). Basic proteins reach the detector prior to neutral and acidic compounds. The concentrations of the polymers, different protein solubilizing additives, carrier ampholytes, anolyte and catholyte, and also the initial sample zone length, the applied voltage, the capillary length and the rinsing procedures are shown to affect focusing in CIEF. A knowledge of the effects of these parameters leads to optimization of protein separation and resolution and provides an insight into the pros and cons of this method.