ABSTRACT: To improve the chromatographic column’s separation efficiency, selectivity, and stability, small particle filler has become the new development direction of liquid chromatography packing. Since the enantiomers have the same chemical and physical properties, the separation of the enantiomers can only be carried out in a chiral environment. Therefore, the chromatographic technique using chiral stationary phases has become the preferred method for chiral separation. In this paper, a small particle size chiral stationary phase: p-methylphenyl urea-β-cyclodextrin chiral stationary phase (SiO2@PMPU-β-CD) was prepared for the rapid separation of chiral drugs. First, three kinds of 2 μm C18-modified silica microspheres with mesoporous structure, non-porous structure, and core-shell structure were prepared. Then applied to High Performance Liquid Chromatography to separate the four benzene homologues of uracil, benzene, fluorene, and naphthalene, the C18-modified mesoporous silica microspheres had the best separation effect.