Enantiomeric separation of clenbuterol by transient isotachophoresis–capillary zone electrophoresis–UV detection: New optimization technique for transient isotachophoresis

A method for the in-line preconcentration and enantioseparation of clenbuterol by transient isotachophoresis–capillary zone electrophoresis–UV absorbance detection (transient ITP–CZE–UV) has been developed. It implies the use of dimethyl-β-cyclodextrin as chiral selector and the application of a hydrodynamic counterflow during the ITP step. ITP is used to focus the sample constituents prior to CE whereas a counterpressure counterbalances the electrophoretic migration of the compounds. The sample is then focused and kept stationary in the proximity of the capillary inlet before CZE separation, leading to an extended-volume ITP–CZE system. A new strategy for the fast optimization of the counterpressure has been developed which implies the measurement of the hydrodynamic and electrophoretic velocities of the analyte during ITP. The in-line preconcentration and enantioseparation of clenbuterol selected as model compound was optimized using this method. Salbutamol was chosen as internal reference in order to check the reproducibility of the method. A 173-nl volume of aqueous sample solution was injected which implies an improvement of the injection volume of about 16 and a resolution of 4.8 was obtained for the clenbuterol enantiomers. A concentration detection limit of 10−6 mol/l was readily achieved for clenbuterol and salbutamol using only 3 min ITP preconcentration in in-line counterflow transient ITP–CZE–UV. Thanks to its fast optimization, the method is applicable to any enantioseparation by means of only five very short preliminary measurements.