Effect of ionic capacity on dynamic adsorption behavior of protein in ion-exchange electrochromatography

Four anion-exchangers of different ion-exchange capacities (21–129 μmol/mL) were prepared by coupling diethylaminoethyl to Sepharose 6FF. The adsorbents were used to study the dynamic protein adsorption in ion-exchange electrochromatography with an oscillatory transverse electric field perpendicular to the mobile-phase flow (IEEC). The static adsorption capacity of bovine serum albumin (BSA) increased from 67 to 186 mg/mL in the ionic capacity range, but the effective pore diffusion coefficient of the protein decreased from 10.9 × 10−12 to 1.85 × 10−12 m2/s with increasing the ionic capacity due to the hindrance effect of the bound protein molecules at the pore entrance. So, the dynamic binding capacity (DBC) of protein in ion-exchange chromatography decreased with increasing the ionic capacity. By applying an electric field of 30 mA, the DBC in IEEC packed with ion-exchangers of high ionic capacities (53 and 129 μmol/mL) increased significantly (over 30% and 100%, respectively). This was because the high surface charge density led to high electroosmotic flow that enhanced intraparticle mass transfer in IEEC. In comparison, the DBC in IEEC packed with ion-exchangers of low ionic capacities (21 and 35 μmol/mL) increased only slightly (ca. 10%) under the same condition. The results indicated the minor effect of electrophoretic mobility on the intraparticle mass transfer. Hence, it is beneficial to use ion-exchangers of high ionic capacity for high-capacity purification of proteins by IEEC.