Optimization of capillary zone electrophoresis and capillary isoelectric focusing methods for high-resolution protein separation and analysis

Abstract

Capillary electrophoresis techniques offer powerful alternatives to traditional chromatographic methods for protein separation, yet achieving optimal resolution requires careful parameter optimization. This research systematically evaluated capillary zone electrophoresis and capillary isoelectric focusing parameters to maximize separation efficiency for complex protein mixtures. Experiments were conducted at the Monterrey Institute of Analytical Sciences between February 2023 and October 2023 using a commercial capillary electrophoresis system equipped with photodiode array detection. For CZE, optimization of buffer composition (phosphate, borate, Tris-glycine), concentration (20-100 mM), pH (2.5-10.0), and applied voltage (10-30 kV) demonstrated that 50 mM borate buffer at pH 9.2 with 25 kV provided optimal resolution (Rs=4.8±0.3) for a five-protein standard mixture. The cIEF optimization evaluated ampholyte concentration (2-6%), pH gradient range, focusing time, and mobilization conditions. Peak capacity increased from 45±8 with 2% ampholyte to 98±6 with 5% ampholyte using narrow-range (pH 4-7) carrier ampholytes. Theoretical plate counts exceeded 450,000 plates/m under optimized CZE conditions and 320,000 plates/m for cIEF, representing substantial improvements over standard protocols. Method validation demonstrated linearity (r²>0.998), precision (RSD