Controllable preparation and application of mixed-mode stationary phases based on green peptide coupling reactions

Inspired by prebiotic reactions derived from wet-cool/dry-hot cycles, a green and efficient dry-wet cycling modification method was proposed for preparing peptide-modified polymer matrices, which are used as mixed-mode stationary phases for HPLC. Using polystyrene-divinylbenzene-glycidyl methacrylate (PS-GMA) as the matrices, alanine monomers were polymerized into peptide on the PS-GMA substrate surface through the dry-wet cycle reaction. By varying the number of dry-wet cycles, the length and density of peptides modified on PS-GMA were easily manipulated. The surface structures of the synthesized stationary phases were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, element analysis, Brunauer-Emmett-Teller test and liquid chromatography-mass spectrometry. Chromatographic separation results demonstrated that the stationary phase after six cycle reactions (PS-GMA-Ala-6C) exhibited the satisfactory selectivity for separating both hydrophilic and hydrophobic analytes. Using PS-GMA-Ala-6C column as the research object, the effects of chromatographic conditions on retention behavior were investigated. The retention mechanisms of the proposed mixed-mode chromatography were deeply analyzed through comparative studies with commercial reversed phase chromatography column, HILIC column and the unmodified PS-GMA column by Tanaka test. Finally, the proposed stationary phase was successfully applied to the qualitative and quantitative analysis of the dihydromyricetin in a real vine tea sample. This work not only presents a green and economically viable method for preparation but also achieves the controllable fabrication of the mixed-mode stationary phase, which is of great significance for the future development of chromatographic stationary phases.