Oscillatory transverse electric field enhances protein resolution and capacity of size-exclusion chromatography

Abstract

Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/ Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations.

High‐capacity purification of hen egg‐white proteins by ion‐exchange electrochromatography with an oscillatory transverse electric field

“…The experimental system for ion-exchange electrochromatography was set up as described earlier [22,23]. In this work, however, a smaller three-compartment rectangular column of 2-mL volume as the central compartment was used for the experiments (Fig.…”

“…Furthermore, the relatively short distance between the electrodes may create a strong electro-osmosis derived from a low-voltage power. Studies on the sizeexclusion chromatography of proteins with noncharged particles (Sephadex G-75) by this kind of electrochromatography have shown increased separation capability resulting from the electrophoresis migration and concentration polarization [22,23]. Moreover, the effect of electric field strength on the DBC of BSA was experimentally studied in this column packed with DEAE Sepharose Fast Flow as well [24].…”

“…They partly removed the Joule heat and electrolytic gases, but in principle, the electrochromatography in the five-compartment electrolyzer was still a typical axial-electric field electrochromatography. As described above, most preparative electrochromatography had been established by applying an axial electric field until Tan et al [22,23] designed a three-compartment rectangular plexiglass column with an oscillatory low-voltage electric field perpendicular to the mobile-phase flow driven by pressure.…”

Protein adsorption‐dependent electro‐kinetic pore flow: Modeling of ion‐exchange electrochromatography with an oscillatory transverse electric field

“…Three-compartment chromatographic column used in this paper had similar architecture with that described earlier [7][8][9][10]. In such a column, chromatographic bead was packed in central compartment with a dimension of 40 Â 7 Â 7 mm 3 (packed-bed volume, 2.0 mL) while cooled electrode solution (81C) flowed upward its neighboring electrode compartments.…”

“…The design was used for various adsorption electrochromatography and achieved high dynamic binding capacity (DBC) with increasing EF strength. Recently, Tan et al designed an electrochromatography with an oscillatory EF perpendicular to the mobilephase flow [7,8]. In this electrochromatographic column, electrolytic gases and the Joule heating can be readily removed by the cooled circulating electrode solutions.…”