Separation of Basic Proteins in Bare Fused-Silica Capillaries with Diethylentriamine Phosphate Buffer as the Background Electrolyte Solution

Abstract

The effectiveness of diethylentriamine (DIEN) phosphate buffer at separating basic proteins in bare fused-silica capillaries at various pH values was examined. Such a buffer consists of an aqueous solution of DIEN titrated to the desired pH value with phosphoric acid. The study was conducted by investigating the effect of DIEN phosphate buffer on the electrophoretic mobility and efficiency of four basic proteins at various pH values within the ranges over which the phosphate salts of DIEN are effective at controlling the protonic equilibrium on the basis of the acidic pKa values of either diethylentriamine or phosphoric acid. The ranges were taken as one pH unit below and above the acidic pKa values of either DIEN or phosphoric acid. Electrophoretic separations of the four basic proteins performed at the selected pH values, ranging from pH 3.0 to pH 8.0, showed well-resolved efficient and symmetric peaks, demonstrating the capability of DIEN phosphate buffer at inhibiting untoward interactions of basic proteins with the active sites on the inner wall of bare fused-silica capillaries. Such effect is ascribed to the absorption of DIEN ions at the interface between the capillary wall and the electrolyte solution resulting in drastic variations of the positive charge density in the compact region of the electric double layer that, however, are is not suppressing completely the negative charges due to the ionization of silanol groups. Consequently, the net charge within the immobilized region of the electric double layer is negative as evidenced by the cathodic electroosmotic flow measured at any pH value within the range 3.0–8.0, indicative of negative zeta potential.