Protein separation using a novel silica-based RPLC/IEC mixed-mode stationary phase modified with N-methylimidazolium ionic liquid.

Ionic liquids (ILs) immobilized on silica as a novel high-performance liquid chromatography (HPLC) stationary phase have attracted considerable attentions. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase with positive charges for protein separation. The results indicate that all of the basic proteins tested cannot be adsorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when being performed under reversed phase/ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial C4 column, the novel stationary phase can show good selectivity and resolution to acidic proteins. The effects of acetonitrile and salt concentration, pH as well as the ligand structure on protein separation were investigated in detail. In addition, the mix-mode retention mechanism of proteins on the SilprMim column was also discussed using stoichiometric displacement theory for retention in LC (SDT-R). The result shows that the protein retention can be controlled mainly by the electrostatic and hydrophobic interactions between the proteins and the stationary phase. As a result, with such characteristics of multi-interaction mechanism and multi-modal separation, not only the selectivity to the acidic proteins can be enhanced, but also a better resolution can be achieved. The result demonstrates that the SilprMim mixed-mode chromatography (MMC) column has a promising application in the separation and analysis of acidic proteins from the complex samples.