Preparation and chromatographic evaluation of a newly designed steviol glycoside modified-silica stationary phase in hydrophilic interaction liquid chromatography and reversed phase liquid chromatography

• A newly designed steviol glycoside modified-silica stationary phase-Click TE-RA.
• Thiol-ene click chemistry strategy was employed for immobilization of rebaudioside A.
• Click TE-RA with special structure could be used both in HILIC and HILIC/RPLC mixed-modes.
• Click TE-RA showed excellent selectivity and efficient retention for polar compounds in HILIC.

A diterpene glycoside compound, rebaudioside A (commonly abbreviated as RA), was immobilized onto porous silica surface through “thiol-ene” click chemistry strategy. The successful immobilization of the RA on the silica support was confirmed by FT-IR and elemental analysis. Chromatographic characteristics of the new stationary phase, named Click TE-RA, were evaluated by a set of diverse analytes such as carbohydrates, nucleosides, and organic acids in hydrophilic interaction liquid chromatography (HILIC) mode. The effects of water content, buffer pH and concentration were investigated and a typical HILIC retention feature of Click TE-RA was observed at high organic modifier content. The Click TE-RA stationary phase was further studied by a series of glycoside compounds. Tunable retention mechanisms from hydrophilic to hydrophobic interactions were observed. Separation of very polar compounds including oligosaccharides, nucleic acid bases and nucleosides using Click TE-RA in HILIC mode was successfully accomplished. In addition, separation of saponins both in HILIC and reversed-phase liquid chromatography (RPLC) modes was performed, demonstrating the presence of orthogonality between two different modes on Click TE-RA column. The multiple interactions induced by polar sugar group and hydrophobic aglycone group allowed this Click TE-RA to serve as a multi-mode stationary phase in two-dimensional liquid chromatography.