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

Abstract

Scope and Method of Study: The aim of the study was, firstly, to introduce novel polar silica-based and acrylate-based monolithic columns for 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. 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.

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.