In this paper, a L-phenylalanine (L-Phe) imprinted polymer (MIP) based on monodisperse hybrid silica microspheres (MH-SiO2) with -CH=CH2 groups was synthesized by surface imprinting technique. The MH-SiO2 was synthesized by a sal-gel process in aqueous media using tetraethylorthosilicate (TEOS) and 3-methylacryloxypropyl trimethoxysilane (MATES) as the precursors. The morphologies of MH-SiO2 and MIP were characterized with scanning electron microscope (SEM) and the imprinted shell thickness on the surface of MH-SiO2 was calculated as 0.5 mu m. Compared with the imprinted polymer prepared only by beta-cyclodextrin (beta-CD) or methacrylic acid (MAA) as functional monomer, the imprinted polymer prepared with both beta-CD and MAA as binary functional monomers hold the highest adsorption capacity. Under the optimum chromatographic conditions, a complete baseline separation of phenylalanine racemates was observed only using the column packed with the imprinted polymer prepared with both beta-CD and MAA as binary functional monomers. And the separation factor and resolution of the imprinted polymer towards phenylalanine racemates were calculated as 1.41 and 1.46, respectively, which is higher than that of other imprinted polymers. (C) 2011 Elsevier B.V. All rights reserved.