Channel electrophoresis for kinetic assays

A rectangular channel electrophoresis system and a cylindrical sampling capillary combination allows chemical changes in nanoliter-volume samples to be monitored as a function of time. The electrophoretic microseparation is carried out in a rectangular channel with a 7-cm-long, 40-μm x 2.5-cm geometry and is coupled to a 50-μm-i.d. cylindrical sample introduction capillary. The channel width dimension is used as a time axis by moving the outlet of the sampling capillary across the entrance of the separation channel. Detection of the separated analyte bands is achieved with laser-induced fluorescence and spatially resolved detection based on a charge-coupled device. The system is characterized with a series of fluorescein thiocarbamyl amino acid derivatives; limits of detection are <10^-8 M for amino acids and 10^-9 M (425 zmol) for fluorescein. The ability to achieve a time-based dynamic microseparation is demonstrated by monitoring fluorescent product formation during the enzyme-catalyzed hydrolysis of fluorescein di-β-D-galactopyranoside (FDG), a commonly used fluorescent substrate for enzymological studies.