A native beta-cyclodextrin (beta-CD) stationary phase was prepared by covalently bonding beta-CD on silica particles via Huisgen [3+2] dipolar cycloaddition between the organic azide and terminal alkyne, the so-called Click chemistry. The resulting beta-CD bonded silica (Click beta-CD) was characterized by FT-IR, solid state (13)C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analyses, which proved the successful immobilization of beta-CD on the silica support with Click chemistry. The retentive properties of Click beta-CD were investigated under hydrophilic interaction liquid chromatography (HILIC) mode in different mobile phase conditions with a set of polar compounds including nucleosides, organic acids and alkaloids. The effects of water content, concentration of the salt and pH of the buffer solution on the retention time were studied and the results demonstrated the typical retention behavior of HILIC on Click beta-CD. Separation of very polar components, such as nucleosides and oligosaccharides, and chiral separation under HILIC mode were successfully achieved. In addition, Click beta-CD was chromatographically evaluated with a set of flavone glycosides. The retention curves depending on the mobile phase of acetonitrile content were "U" curves, which is an indication of HILIC/RPLC mixed-mode retention behavior. The difference of the separation selectivity between HILIC and RPLC was described as orthogonality by using geometric approach and the orthogonality reached 69.4%. The mixed-mode HPLC properties and excellent orthogonality demonstrated the flexibility in HPLC methods development and great potential in two-dimensional liquid chromatography separation.