Preparation And Chromatographic Characterization Of Two Ionic Liquid-Functionalized Monolithic Columns

As stationary phases of high performance liquid chromatography,organic polymer monolithic columns have led to high potential application for separation and purification of large bio-molecules such as proteins and peptides.However,its non-uniform internal structure and poor selectivity for proteins have caused problems in separation of proteins from complex bio-samples,which limited its further application.In this paper,two kinds of organic polymer monoliths were prepared by using ionic liquid and iron porphyrin,aiming to improve the uniformity and enrich the interactions,which can enhance the selectivity and performance of polymer monolith for bio-macromolecules.Firstly,an ionic liquid-based polymer monolithic column was proposed via in situfreeradicalpolymerization,inwhichprocesses,ionicliquid（1-allyl-3-methylimidazolium chloride）and stearyl methacrylate were used as dual-monomers,ethylene dimethacrylate as crosslinker,polyethylene glycol 200 and isopropanolasco-porogens.Scanningelectronmicroscopy,nitrogen adsorption-desorption instrument,and mercury intrusion porosimeter were used to investigate the morphology and pore size distribution of the prepared monoliths,respectively,which showed the homemade monolith column possessed relatively uniform macro-pore structure with the total macro-pore specific surface area of 44.72m² g^-1.Results obtained from chromatographic characterizations indicated that the present monolith exhibited excellent selectivity and high performance for separating proteins from complex bio-samples,such as egg white and snailase.It is should be emphasized that the present method is a promising fractionation separation method for human plasma in proteomic research.Secondly,in order to furtherly improve the selectivity of the homemade monolith for proteins,a double-functionalized polymer monolithic column was fabricated via redoxsysteminitiationbyusingironporphyrin,ionicliquid（1-allyl-3-methylimidazolium chloride）and 1,10-decanediol dimethacrylate as tri-monomers;ethylene dimethacrylate as crosslinker;polyethylene glycol 400 and N,N-dimethylformamide as co-porogens.Results obtained from scanning electron microscope,nitrogen adsorption-desorption instrument,and mercury intrusion porosimeter confirmed the uniform pore structure and pore size distribution of macro-pores.Comparedtotheworkmentionedabove,thepresent double-functionalized polymer monolithic column exhibited much improved selectivity for proteins,which obtained 21 baseline separated peaks.Furthermore,two chromatographic fractions from human plasma were identified by LC-MS,which results indicated that a number of unique peptides detected in fractions were seldom found in human plasma,confirming the present method was meaningful for fractionation separation of human plasma.