Moving boundary electrophoretically mediated microanalysis

Moving boundary sample introduction is described as an alternative to zonal injection methods for the electrophoretically mediated microanalysis (EMMA) of leucine aminopeptidase (LAP). The capillary was initially filled with the analyte solution while the faster-migrating substrate, L-leucine-p-nitroanilide, was maintained in the inlet reservoir. Upon application of an electric field, electrophoretic merging of the reagents proceeded, and the detectable reaction product, p-nitroaniline, was transported to the detector. The area, maximum height, inclining slope, and declining slope of the resulting triangular product profile were each directly proportional to the activity of LAP, and the observed migration times of the product profile features defined the volume and time of the incubation. The moving boundary technique offered more than an order of magnitude greater concentration sensitivity than the zonal injection EMMA method. This heightened sensitivity facilitated rapid analysis as the use of elevated electric field strengths and short capillaries allowed for a 24-s kinetic determination of LAP.