Concentration Polarization Effect In Micro Free-flow Electrophoresis And Its Preliminary Application Exploration

Proteins are very important contents of life science in the post-genomic era, the research focus has changed from all proteins to parts of proteins in one proteome, the innovation of proteins’ separation and purification technique is very significant. The free-flow electrophoresis as a pretreatment of method, it is superior in keeping biological activity and the characteristic of continuous samples injection and continuous separation mode are good at pretreatment of proteins and other biological samples. The concentration of charged component on micro-nano interface shows high concentration effect. In a self-build micro free-flow electrophoresis device, ion-exchange membranes are introduced to isolate separation chamber and electrode chamber, the concentration polarization would happen near the ion-exchange membrane in separation chamber, to realize the separation and concentration of proteins and other biological sample.In the self-build micro free-flow electrophoresis device, the concentration polarization near different ion-exchange membrane (anion and cation exchange membrane) was studied. Using the fluorescence imaging technique, obvious concentration phenomena was showed in the separation chamber, the ion concentration was decreased the carrier solution’s conductivity. The changes of collected solution showed water dissociation. The pH of the collected solutions by using different ion-exchange membrane showed that anion exchange membrane’s water dissociation was more violent than that of cation exchange membrane. Since the concentration near ion exchange membrane, the I-V curve of ion-exchange membrane free-flow electrophoresis system was divided to three regions.Using the concentration polarization and pH gradient caused by water dissociation, the bovine serum albumin (pI 6.9 Mw 66.4 KDa) and cytochrome C (pI 10.7 Mw 12.7 KDa) mixture was separated in anion exchange membrane free-flow electrophoresis device, the collected fractions’ SDS-polyacrylamide gel electrophoresis results showed the separation and concentration ability. The concentration of cytochrome C was completed at the edge of ion depletion zone near cation exchange membrane, the collected fractions through three outlets were characterized by SDS-polyacrylamide electrophoresis.This study showed that the FFE system could complete prefractionation and concentration of complex protein sample through introducing ion exchange membrane. It is expected that this prefractionation device may find application for online sample pretreatment of charged components and biological macromolecules.