CAPILLARY ISOELECTRIC-FOCUSING OF PROTEINS IN UNCOATED FUSED-SILICA CAPILLARIES USING POLYMERIC ADDITIVES

Isoelectric focusing performed in the capillary format (CIEF) represents a powerful separation technique for the analysis of proteins. To date, all CIEF separations have been performed in coated capillaries, which essentially eliminate electroendosmotic flow. Thus, focused protein zones were eluted with a technique called salt mobilization. Here we demonstrate the ability to perform CIEF in uncoated capillaries through the use of polymeric additives in the sample/ampholyte mixture. These additives can then act as dynamic coatings of the silica wall, thereby covering many of the possible adsorption sites to which proteins may adsorb. We also demonstrate that through the judicious choice of additive concentration, electroendosmotic flow may be controlled, but not totally eliminated, obviating the need for performing salt mobilization of focused protein zones. That is, maintaining some flow allows for the mobilization of focused zones past the UV detection window during the focusing step. The developed methodology is shown applicable to basic, neutral, and acidic proteins. The main disadvantage is broadened peaks for very acidic proteins. The final optimized conditions are evaluated in terms of reproducibility and qualitative capability.