Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with...

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20mM sodium tetraborate, 20mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.