Monitoring electroosmotic flow by periodic photobleaching of a dilute, neutral fluorophore

Electroosmotic now has been monitored in a capillary using a method based o n periodic photobleaching of a neutral, fluorescent buffer additive. Rhodam ine B was determined to be neutral between pH 6.0 and 10.8 and was added to the running buffer at a concentration of 400 nM. Rhodamine B was photoblea ched by opening a shutter under computer control for 250 ms every 5.00 s, t o expose the dye to a laser beam and create a photobleached zone. The time was measured for the photobleached zone to migrate 6.13 mm to a downstream laser-induced fluorescence detector, to determine the rate of electroosmoti c now in the entire capillary. The flow rate was sampled every 5.00 s, and the precision of the now measurements was 0.7% or better. Three fluorescent compounds were separated and detected by capillary electrophoresis with la ser-induced fluorescence detection, while simultaneously monitoring the ele ctroosmotic how rate.