Retention behavior of proteins in size‐exclusion electrochromatography with a low‐voltage electric field perpendicular to the liquid phase streamline

Abstract

A novel preparative size-exclusion electrochromatography with an oscillatory low-voltage electric field perpendicular to the liquid phase streamline (pSEEC) was proposed with a column design of rectangular cross-section. 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 on increasing the current strength as well as the difference between its pI and pH. The partition coefficient also increased on decreasing the mobile phase conductivity. For the gel-excluded protein like BSA, the concentration polarization (CP) on the gel surface induced by the protein electromigration was the main reason for the increased retention. For a gel-permeable protein like Myo, both the CP and electrophoretic migration in the solid phase contributed to its increased retention. Further results exhibited that the polarization would be offset by diffusion, because the accumulated protein would flux back to the bulk liquid phase. Therefore, when the electrically induced mass flux was equal to the diffusion flux, the partition coefficient did not change with a further increase of the oscillatory current cycle. Finally, pSEEC was compared with SEC in the separation of protein mixtures of BSA/Myo as well as BSA/Myo/lysozyme. The results showed much better resolutions of the protein mixtures in pSEEC with the column as short as 12 cm. The potential of pSEEC for preparative protein separation was therefore demonstrated.

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.…”

“…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.…”

“…In this newly designed column [22], the electrolytic gases formed at the electrodes are readily kept out from the packed-bed and are removed by circulating electrode solutions. As to the Joule heat generated in the packedbed column, it can be readily removed by the cooled mobile phase flowing through the column in a pressuredriven mode.…”

“…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].…”

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

“…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.…”