Analytical and preparative-scale isoelectric focusing separation of enantiomers

Isoelectric focusing has been used to achieve the anatytical- and preparati ve-scale separation of the enantiomers of amphoteric analytes. By consideri ng the simultaneous multiple equilibria involved in the chiral recognition process, a model has been developed to describe the magnitude of the Delta pI value that develops between the enantiomers in the presence of a nonchar ged chiral resolving agent, such as a noncharged cyclodextrin. Theoretical analysis of the model indicates that three kinds of IEF enantiomer separati ons are possible: aniono-selective and cationo-selective, when only the ide ntically charged forms of the enantiomers bind selectively to the resolving agent, and due-selective, when the differently charged forms of the enanti omers bind selectively to the resolving agent. The model predicts that the Delta pI vs cyclodextrin concentration curves approach limiting Delta pI va lues which can be as large as 0.1, even when the binding constants of the e nantiomers differ only by 10%. The parameters of the model can be readily d etermined by free solution capillary electrophoretic or pressure-mediated c apillary electrophoretic experiments. The validity of the proposed model ha s been tested with hydroxypropyl beta-cyclodextrin as resolving agent and d ansyl phenylalanine as probe. Capillary IEF enantiomer separations have bee n achieved using both ampholytes and binary propionic acid-serine buffers ( Bier's buffers). Preparative-scale IEF enantiomer separations with producti on rates as high as 1.3 mg/h have been achieved in an Octopus continuous fr ee-now electrophoretic system.