DERIVATIZATION OF DIPEPTIDES WITH 4-FLUORO-7-NITRO-2,1,3-BENZOXADIAZOLE FOR LASER-INDUCED FLUORESCENCE AND SEPARATION BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY

In capillary electrophoresis generally very small sample volumes are introduced, which often gives problems regarding determinations of low concentrations of the analytes. Frequently, therefore, they have to be transformed into products by suitable derivatization reagents. In this study 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used as a pre-capillary fluorigenic reagent for a series of dipeptides used as model compounds in order to study the characteristics of the derivatization procedure. Main emphasis was put on optimization of the reaction conditions using a chemometric approach involving a fractional factorial design for screening experiments and a central composite face-centred design for response surface modelling. The results showed that the reagent excess must be at least 70 times in order to get a linear response, the reaction mixture should consist of a phosphate buffer with low ionic strength (0.001) at pH 7 containing 15% of isopropanol. The presence of the micellar agent Brij 35 in the background electrolyte increased the fluorescence intensity of the analyte product at least 3 times, and the separation selectivity increased compared to using a neat buffer. Leu-Val, chosen as a model peptide for studies on quantitative determinations, could be determined at the level 10(-7) M (2 fmol injected) with a quantitative recovery and a relative standard deviation of 2.4%. The limit of detection was 4 . 10(-9) M (70 amol injected).