A successful enantioseparation of D,L-phenylalanine (Phe) was achieved for the first time by using D-Phe imprinted P(MAA-co-EGDMA) microbeads as HPLC stationary phase. The D-Phe imprinted microbeads were prepared by a novel modified suspension polymerization method without derivatization of the water-soluble template molecule. This preparation method was employed in order to capture template molecules in organic phase droplets during polymerization. The prepared D-Phe imprinted P(MAA-co-EGDMA) microbeads were packed into an empty stainless steel column, which was used for the separation of the Phe enantiomers. The selection of a suitable mobile phase, mobile phase composition, pH and flow rate were investigated for determining the best resolution of the Phe enantiomers. Baseline separation was achieved using an organic-aqueous buffer (EtOH-acetate buffer) solution as a mobile phase. Separation factors of more than 2.56, with a resolution of 1.38, were obtained with a mobile phase containing 9-18% (v/v) EtOH in a 0.030 M acetate buffer solution. D-Phe imprinted microbeads were superior to the majority of the reported molecularly imprinted polymers with respect to chiral separation abilities. The column backpressure was less than 300 psi. (C) 2008 Elsevier B.V. All rights reserved.