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This paper presents the integrated results of a series of new methods for preparing mesoporous silica spheres as high-performance liquid chromatography (HPLC) packing materials. The separation performance of the mesoporous spheres materials has also been determined. Micrometersized silica spheres with uniform spherical morphologies and ordered mesostructures were first successfully synthesized by the method employing a water-soluble polymer-assisted assembly. Then the templates for getting ordered mesoporous materials with high-density silanol groups were removed by using hydrothermal oxidation. Finally the silica spheres were functionalized with C8 alkyl groups by surface modification under hydrothermal conditions. The resultant functionalized silica spheres were demonstrated to be excellent packing materials for HPLC.