Comparison of different capillary isoelectric focusing methods—use of “narrow pH cuts” of carrier ampholytes as original tools to improve resolution

Abstract

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of “narrow pH cuts” of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.