OPTIMIZING FLUORESCENCE DETECTION IN CHEMICAL SEPARATIONS FOR ANALYTEBANDS TRAVELING AT DIFFERENT VELOCITIES

In many separation techniques, such as field flow fractionation, liqui d chromatography, and electrophoresis, chemical species form bands tha t migrate at distinct velocities. If these bands are to be quantified on-line using a shot-noise-limited detection system, then attention mu st be given to the data-digitization rate and to the removal rate of m olecules from the analyte pool as a result of the detection process. A theory is developed for calculating the signal-to-noise ratio under s uch conditions, and it is specialized to the case of fluorescence dete ction in capillary electrophoresis. Using standard detection procedure s in which the data-digitization rate and excitation intensity remain constant for the duration of a separation, detection sensitivity can v ary by more than a factor of five for bands that arrive at the detecti on zone between migration times tau(fast) and 10tau(fast), where tau(f ast) is the time after the start of the separation that the fastest mi grating band arrives at the detection zone. To compensate for differen t band velocities, both the data-digitization rate and the excitation intensity must be decreased as separation time (tau) increases by the factor tau(fast)/tau. Only when these corrections are made can uniform sensitivity with the highest possible signal-to-noise ratio be achiev ed for each peak. These predictions are experimentally tested and comp are favorably to observations.