Abstract Separation of enantiomers represents an extremely important task in the field of analytical chemistry. This paper contributes to the field of the on-line preconcentration of enantiomers by developing a novel setup based on the electrokinetic accumulation of ketoprofen enantiomers on the pH boundary followed by enantioselective mobilization by a mixture of SDS, sulfated-β-cyclodextrin (S-β-CD), and trimethyl-β-cyclodextrin (TM-β-CD). Under the best conditions, where the injection electrolyte was composed of 50 mmol L−1 borate/NaOH pH 9.5 with 60% (v/v) methanol, the background electrolyte contained 50 mmol L−1 phosphate/NaOH pH 2.5, and the mobilization electrolyte consisted of 50 mmol L−1 phosphate/NaOH pH 2.5 with 4.0% (w/v) S-β-CD, 0.5% (w/v) TM-β-CD, and 20 mmol L−1 SDS, the determination of nanomolar concentration levels of ketoprofen enantiomers was successful by using micellar electrokinetic chromatography with a common UV detection. LODs were 2.5 nmol L−1 and 3.4 nmol L−1, which represent enhancement factors of 9921 and 8529, respectively. The method was also applied to the determination of ketoprofen enantiomers in waste water samples by using simple filtration as a clean-up step. Here, the recovery of ketoprofen enantiomers was 91% at the concentration level of 5×10−9 mol L−1. Graphical abstract <alternatives> [...] </alternatives>
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