It is generally accepted that the selective binding of enantiomers of the chiral analyte to a chiral selector is necessary for enantioseparations in CE, whereas the role of mobility differences between the temporary diastereomeric associates formed between the enantiomers and the chiral selector has been commonly neglected. One of the authors of this study suggested in 1997 that the mobility difference between the diastereomeric associates of two enantiomers with the chiral selector may be solely responsible for a separation of enantiomers in CE and enantioselective selector-selectand binding may be not necessarily required. Several indirect confirmations of this hypothesis have been described in the literature within the last few years but a dedicated study proving this concept has not been published yet. The present data obtained for the two chiral antimycotic drugs ketoconazole and terconazole by CE and NMR spectroscopy unequivocally support this concept.

Separations using CE employing non-aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with non-aqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non-aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non-aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non-aqueous methanolic BGEs with two single isomer sulfated derivatives of beta-CD, namely heptakis (2,3-diacetyl-6-sulfo)-beta-CD and heptakis (2,3-dimethyl-6-sulfo)-beta-CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non-aqueous BGE in the case of heptakis (2,3-dimethyl-6-sulfo)-beta-CD but remained the same in the case of heptakis (2,3-diacetyl-6-sulfo)-beta-CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non-aqueous BGEs.

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In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α-CD, β-CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), and heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD). EMO was reversed in the presence of α-CD and β-CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS-β-CD, and HDAS-β-CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS-β-CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.

In the present study the migration order of the propranolol enantiomers with various native CDs and neutral and charged CD derivatives was examined in capillary electrophoresis (CE). The reversal of the enantiomer migration order was observed due to sulfation of beta-CD on its primary hydroxy groups. The structures of intermolecular selector-select and temporary diastereomeric associates in solution were elucidated based on 1D rotating frame nuclear Overhauser effect spectroscopy (1D ROESY) experiments. Major structural differences were observed between the propranolol complexes with native beta-CD and heptakis(6-O-sulfo)-beta-CD.