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Three-dimensionally ordered macroporous microspheres (3DOMMs) with copolymer skeletons have been successfully fabricated using a colloidal crystal templating method. The spherical SiO2 colloidal crystal was firstly self-assembled as template via the evaporation-induced self-assembly process in a water/oil suspension system. The 3DOMMs were obtained by sealing the silica colloidal crystal template with polyethylene glycol 1500 (PEG 1500) after the infiltration of the precursor into the interstice of the template. The compositions of the precursor had an important effect on the structural order and integrity of the 3DOMMs. The structural strength of the microspheres was obviously improved by adding 2-hydroxyethyl methacrylate (HEMA) and 3-(trimethoxysilyl)propyl methacrylate (KH570) into the precursor. The chromatographic column packed with the 3DOMMs showed higher permeability and lower backpressure compared with the column packed with the traditional bulk materials and was suitable for the fast chromatographic separation. [ABSTRACT FROM AUTHOR]