Post-column reaction system for fluorescence detection in capillary electrophoresis

Abstract

A system for post-column derivatization in capillary electrophoresis has been developed and evaluated. In this system, reagent is added through a porous tube connecting the separation capillary with a reactor capillary. The reagent solution is driven by a regulated air pressure applied on the separation capillary inlet and the reagent vessel simultaneously. The system has been applied for the fluorescence detection of amino acids using o-phthalaldehyde as reagent. The reactor capillary geometry and flow-rates were optimized with respect to sensitivity and separation efficiency on the basis of measured reaction rate constants. A ten-fold increase in sensitivity could be obtained by using “bubble-cell” capillaries instead of normal fused-silica capillaries for detection. Amino acids were separated in a pH 9.7 borate buffer. Plate numbers in the order of 100 000 to 150 000 were obtained. Detection limits were typically between 2·10 −6 and 4·10 −6 mol l −1, or 0.05 to 0.1 pmol injected. The method was applied to the determination of free amino acids in urine samples.