Silica-based polypeptide-monolithic stationary phase for hydrophilic chromatography and chiral separation

Glutathione (GSH)-, somatostatin acetate (ST)- and ovomucoid (OV)-functionalized silica-monolithic stationary phases were designed and synthesized for HILIC and chiral separation using capillary electrochromatography (CEC). GSH, ST and OV were covalently incorporated into the silica skeleton via the epoxy ring-opening reaction between their amino groups and the glycidyl moiety in γ-glycidoxypropyltrimethoxysilane (GPTMS) together with polycondensation and copolymerization of tetramethyloxysilane and GPTMS. Not only could the direction and electroosmotic flow magnitude on the prepared GSH-, ST- and OV-silica hybrid monolithic stationary phases be controlled by the pH of the mobile phase, but also a typical HILIC behavior was observed so that the nucleotides and HPLC peptide standard mixture could be baseline separated using an aqueous mobile phase without any acetonitrile during CEC. Moreover, the prepared monolithic columns had a chiral separation ability to separate dl-amino acids. The OV-silica hybrid monolithic column was most effective in chiral separation and could separate dl-glutamic acid (Glu) (the resolution R=1.07), dl-tyrosine (Tyr) (1.57) and dl-histidine (His) (1.06). Importantly, the chiral separation ability of the GSH-silica hybrid monolithic column could be remarkably enhanced when using gold nanoparticles (AuNPs) to fabricate an AuNP-mediated GSH-AuNP-GSH-silica hybrid monolithic column. The R of dl-Glu, dl-Tyr and dl-His reached 1.19, 1.60 and 2.03. This monolithic column was thus applied to separate drug enantiomers, and quantitative separation of all four R/S drug enantiomers were achieved with R ranging from 4.36 to 5.64. These peptide- and protein-silica monolithic stationary phases with typical HILIC separation behavior and chiral separation ability implied their promise for the analysis of not only the future metabolic studies, but also drug enantiomers recognition.