Silica- and organic polymer-based monolithic stationary phases for modern liquid phase separations

Scope and Method of Study. Scope and Method of Study: The aim of this study was, firstly, to introduce novel polar silica-based and acrylate-based monolithic columns for liquid normal phase chromatography (NPC) and electrochromatography of biomolecules and secondly, to develop silica-based and acrylate-based lectin affinity monolithic columns for the fractionation of glycoproteins and their glycan fragments by lectin affinity chromatography. The monolithic phases were obtained by in situ polymerization via sol-gel process in the case of silica monoliths and by vinyl co-polymerization of an acrylate functional monomer (e.g., glyceryl monomethacrylate) and a crosslinker (e.g., ethylene dimethacrylate) in the case of acrylate-based monoliths in the presence of porogens and initiators with gentle heating overnight. The silica monoliths were first functionalized with an epoxy silane and then with 1H-imidazole-4,5-dicarbonitrile to yield the so called 2CN-OH polar phases or with immobilized lectins via epoxy ring opening reactions. The acrylate-based monoliths had the diol functionalities on their surface which readily yielded polar diol monoliths or were conveniently converted to aldehyde monoliths prior to lectin immobilization. The polar and lectin affinity monolithic stationary phases were exploited in nano liquid chromatography and capillary electrochromatography using fused silica capillary columns of 100 mum I.D.;Findings and Conclusions. The investigation has yielded a simplified and time efficient method to fabricate acrylate-based monolithic stationary phases which can be effectively further modified into affinity stationary phases, and can also be applied readily as a polar stationary phase for NPC. The new acrylate-based lectin affinity monoliths exhibited strong interactions with target glycoproteins and 2-aminobenzamide (2-AB) derivatized glycans. The 2CN-OH silica-based monolith exhibited relatively high retention and selectivity toward a wide range of polar species, and proved useful in the separation of 2-AB derivatized glycans. The lectin silica-based monoliths were effective in the fractionation of selected acidic glycoproteins and the separation of some nitrophenyl derivatized monosaccharides.