Abstract: A native β-cyclodextrin (β-CD) stationary phase was prepared by covalent bonding of β-CD on silica particles via thiol-ene click chemistry. The resulting β-CD bonded silica (Click TE-CD) was characterized by elemental analysis, which proved the successful immobilization of β-CD on the silica support with thiol-ene click chemistry. Click TE-CD was chromatographically evaluated with a set of flavone glycosides under hydrophilic interaction chromatography (HILIC) mode, reversed-phase chromatography (RPLC) mode and supercritical fluid chromatography (SFC) mode. The acetonitrile content dependent “U” retention curves indicated its HILIC/RPLC mixed-mode retention behavior. The difference of the separation selectivity between HILIC, RPLC and SFC was described as orthogonality by using geometric approach. The orthogonalities between HILIC/RPLC, HILIC/SFC, and RPLC/SFC reached 69.8%, 50.8% and 50.8%, respectively. The separation of Chinese traditional medicine Lignum Dalbergia Odorifera extract under HILIC, RPLC and SFC modes indicated the potential of Click TE-CD stationary phase in the analysis of complex samples. The mixed-mode HPLC properties and excellent orthogonality demonstrated its flexibility in HPLC method development and its great potential in two-dimensional liquid chromatography separation on one HPLC column by different separation modes.

Key words: flavone glycosides, orthogonality, thiol-ene click chemistry, β-cyclodextrin (β-CD) stationary phase