Enantioseparation of Phenylglycine by Ligand Exchange Chromatography
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Abstract
Introduction: Since phenylglycine in IDJ configuration is pharmaceutically used for the synthesis of ampicillin and cephalexin, its enantiomeric purity needs to be confirmed prior to use. This study aimed to develop a chiral separation method for phenylglycine instead of using expensive chiral chromatographic columns. It was based on the use of a chiral mobile phase consisting of copper(II) and L-proline with a typical C18 column as a stationary phase. Methods: The optimal conditions for the separation of D- and L-phenylglycine, i.e. the pH of the mobile phase, concentration of copper(II) and L-proline, and percentage of methanol as an organic modifier in the mobile phase, were studied. The performance of the separation was evaluated by the resolution between the peaks of D- and L-isomers as well as the characteristics of the chromatograms. A standard curve was created to investigate the range and linearity. Results: D- and L-phenylglycine best separated from each other with the retention time of about 5 and 11 min, respectively, when a mixture containing 2.5:5 mM copper (II):L-proline, 10% methanol, and with a pH of 6.0 was used as the mobile phase. Under these optimal conditions, satisfactory linear relationships between the concentration and peak area were obtained for both D- and L-phenylglycine with correlation coefficients of 0.9997 and 0.9998 respectively. Conclusion: A copper(II)/L-proline mixture at a suitable concentration and pH in the presence of an appropriate amount of methanol could be effectively used as a mobile phase together with a C18 column for the separation of phenylglycine enantiomers. Thus, these optimal conditions can be used for the further development and validation of a simple and inexpensive method for the quantitation of D- and L-phenylglycine.
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