HYDROGEL POLYMER-COATINGS FOR CAPILLARY ELECTROPHORETIC SEPARATION OF PROTEINS

Three hydrogel polymers, poly(2-hydroxypropylmethacrylate), poly(2-hydroxyethylmethacrylate), and hydroxypropylcellulose, were coated and immobilized on fused silica capillary surfaces using three different methods, all involving modification of the surfaces before polymerization and/or cross-linking. Since the hydrogel polymers swelled in aqueous solution and allowed permeation of low-molecular-weight compounds, an equilibrated state was attained between the hydrogel polymer layers and the buffer solution, which eliminated the interactions between proteins and the capillary surface, and allowed highly efficient and reproducible capillary electrophoretic separations of both basic and acidic proteins. The experimental results indicated that silanol groups on the capillary surface were covered and phosphate anions were trapped in the coating layers, which produced defined, but reduced, electroosmotic flows when compared to uncoated capillaries. High resolution separations of ribonuclease B variants and cytochrome c proteins from different sources were obtained.