Diode laser-induced fluorescence detection in capillary electrophoresis after pre-column derivatization of amino acids and small peptides

Abstract

The use of diode laser-induced fluorescence (DIO-LIF) detection in the field of capillary electrophoresis (CE) is examined. A simple but sensitive detection system was constructed. The performance of the system was evaluated with respect to design factors and its sensitivity was compared with the theoretically achievable sensitivity. To enhance the applicability of direct DIO-LIF detection in CE, a derivatization method for amines was developed. A red-absorbing label, consisting of a dicarbocyanine fluorophore with a succinimidyl ester functionality, was synthesized for this purpose. After derivatization of 1 × 10 −6 M glycine, a detection limit of 0.1 amol was observed for the labeled glycine. Similar detection limits were observed for other amino acids. To show that derivatization preserves the separation efficiency of CE for the analytes examined, 18 amino acids and tyramine were separated with micellar electrokinetic chromatography after labeling. In addition, even labeled peptides, including structurally related enkephalin-type compounds, were separated from each other with zone electrophoresis. To test the applicability of the derivatization method to biological samples, tyramine was determined in urine before and after the consumption of cheese.