Protein Electrochromatography With An Oscillatory Transverse Electric Field

In this article, a novel preparative electrochromatography system with an oscillatory electric field perpendicular to liquid phase streamline was proposed with a column design of rectangle cross-section. This new system was applied in both size-exclusion electrochromatography (PSEEC) and ion-exchange electrochromatogra- phy (PIEEC) modes. The details in this work are summarized as follows.In order to resolve problems that often encountered in a conventional preparative electrochromatography with an axial electric field, such as removal of Joule heat and electrolysis gas, we have herein proposed a novel electrochromatography with an oscillatory transverse column electric field. That is, the electric field is perpendicular to liquid phase streamline in the electrochromatography. In this format, a perpendicular electro-kinetic mass transport was generated, and liquid phase flows through the column in the longitudinal direction driven by pressure.In PSEEC, the column of 12 cm length was packed with Sephadex G-75 and the retention behavior of bovine serum albumin (BSA) and myoglobin (Myo) was extensively investigated under various conditions. The results indicated that the partition coefficient of a charged protein increased significantly with increasing the electric field strength as well as the difference between its isoelectric point and pH. For the gel-excluded protein like BSA, the concentration polarization (CP) on gel surface induced by the protein electromigration was the main reason for the increased retention. For a gel-permeable protein like Myo, both CP and electrophoretic migration in the solid phase contributed to its increased retention. In addition, in PSEEC, charged proteins occurred both CP and electrophoretic migration on/in the solid phase, so mass transport characteristics was more complicated and influened by the electric field and character of the buffer used. H (height equivalent to a theoretical plate) at higher ionic strength was smaller than that at lower one under the same current strength, and its value increased with increasing the current.Retention experiments in PSEEC led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), Myo and lysozyme (Lys), BSA and Myo, as well as a ternary mixture of BSA, Myo and Lys. The separation results for the binary and ternary protein systems sufficiently exhibited the applicability of the PSEEC for various separations in terms of their molecular weights as well as isoelectric points. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar molecular weights proteins (Myo/Lys) by the PSEEC. Moreover, in the cases of BSA/Myo as well as BSA/Myo/Lys, which could be partially separated by SEC, the use of the PSEEC could greatly improve the resolution and the separation became possible at high sample loading. The results indicate that the PSEEC technology is promising forpreparative protein separations.In PIEEC, the effect of electric field strength on the dynamic binding capacity (DBC) was examined by frontal analysis of BSA adsorption to the packed beds of DEAE Sepharose FF in Tris-glycine buffer (pH 8.2). It was shown that the DBC at 10% breakthrough (q10) in the pIEEC increased linearly with increasing the electric field strength. For example, with a packed-bed height of 15 mm and electric potential of 38 V/cm, q10 increased four times over that in normal ion-exchange chromatography. So, the transverse electric field has created electro-kinetic mass transports (electroosmosis and electrophoresis) that intensified exterior liquid-film and intraparticle mass transfers, leading to the increased protein binding capacity. The results have revealed that an electric potential of 20 V/cm was enough to greatly enhance the DBC in the pIEEC, and when necessary, high electric field strength can be realized with a low applied voltage because the side distance of the column is usually an order of magnitude smaller than its height. The use of low voltage to carry out electrochromatography is a significant advantage of the pIEEC over those conventional axial electric field electrochromatography. Moreover, due to the increased capacity in the pIEEC, partial resolution of BSA and IgG under an overload condition was realized without any process optimization.