Analytical- and Preparative-Scale Isoelectric Focusing Separation of Enantiomers

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Abstract
Isoelectric focusing has been used to achieve the analytical- and preparative-scale separation of the enantiomers of amphoteric analytes. By considering the simultaneous multiple equilibria involved in the chiral recognition process, a model has been developed to describe the magnitude of the ΔpI value that develops between the enantiomers in the presence of a noncharged chiral resolving agent, such as a noncharged cyclodextrin. Theoretical analysis of the model indicates that three kinds of IEF enantiomer separations are possible: aniono-selective and cationo-selective, when only the identically charged forms of the enantiomers bind selectively to the resolving agent, and duo-selective, when the differently charged forms of the enantiomers bind selectively to the resolving agent. The model predicts that the ΔpI vs cyclodextrin concentration curves approach limiting ΔpI values which can be as large as 0.1, even when the binding constants of the enantiomers differ only by 10%. The parameters of the model can be readily determined by free solution capillary electrophoretic or pressure-mediated capillary electrophoretic experiments. The validity of the proposed model has been tested with hydroxypropyl β-cyclodextrin as resolving agent and dansyl phenylalanine as probe. Capillary IEF enantiomer separations have been achieved using both ampholytes and binary propionic acid−serine buffers (Bier's buffers). Preparative-scale IEF enantiomer separations with production rates as high as 1.3 mg/h have been achieved in an Octopus continuous free-flow electrophoretic system.