Separation of phenylalanine racemates using d-phenylalanine imprinted microbeads as HPLC stationary phase

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.