Studies of Electroosmotic Flow Dynamics During Electrophoretic Separations

Instrumentation and techniques for monitoring electroosmotic flow (EOF) during capillary electrophoretic (CE) separations in both fused-silica capillaries and glass microfluidic devices are presented. These techniques were applied under conventional and sample stacking separation conditions. The instrumentation developed for monitoring EOF was also used to develop optically gated vacancy separations in microfluidic devices. A recently developed technique for monitoring EOF in capillary electrophoresis by periodic photobleaching of a neutral fluorophore added to the running buffer was further characterized and optimized and then applied to monitoring EOF during a typical capillary electrophoresis separation. The concentration of neutral fluorophore (rhodamine B) added to the running buffer for monitoring EOF was decreased by one order of magnitude to 40 nM. The rate at which EOF can be measured was increased from 0.2 to 1.0 Hz by decreasing the distance between the bleaching beam and the laser-induced fluorescence detector from 6.13 mm to 0.635 mm. The precision of the measured EOF ranged from 0.2 to 1.8%. Under typical experimental conditions, the dynamic range for flow measurements was 0.066 to 0.73 cm/s. Experimental factors affecting precision, signal-to-noise (S/N) ratio and dynamic range for EOF monitoring were studied. This technique was applied to measure EOF during a separation of phenolic acids with analyte detection by UV/VIS absorbance. The EOF monitoring method was shown not to interfere with UV/VIS absorbance detection of analytes. EOF was monitored in glass microfluidic devices at rates up to 2 Hz with a precision of 0.2 – 1.0% using the periodic photobleaching method. This EOF monitoring method was used to examine the performance of the current monitoring technique for measuring an average electroosmotic flow in a microfluidic device with a cross-T design. Flow measurements made with the current monitoring method gave a precision of 0.4 – 2.2%, but the periodic photobleaching method showed that the current monitoring technique caused changes in EOF as high as 41% during a single experiment. The periodic photobleaching method for EOF monitoring was also used to study EOF in channels on opposite sides of a cross-channel intersection. The opposite channels were shown to exhibit substantially different EOF dynamics during a current monitoring experiment as well as different steady state EOF rates during normal operating conditions. Electroosmotic flow dynamics during a field-amplified sample stacking experiment have been studied experimentally using the periodic photobleaching of a dilute, neutral fluorophore added to the separation buffer. Changes in electroosmotic flow during a separation of arsenic compounds with field-amplified sample stacking have been monitored at a rate of 1 Hz. The effects of hydrodynamically injecting different sample plug lengths of analyte dissolved in 0.125 mM (120, 240, and 600 s) and 41.7 mM (27, 45, and 74 s) phosphate buffer with a separation buffer concentration of 12.5 mM phosphate buffer were examined. The observed effects of increasing the sample plug length on electroosmotic flow and electrophoretic current agreed qualitatively with predictions by theoretical models presented in the literature. Electroosmotic flow changes greater than 100% (1.6 – 3.3 mm/s) have been observed. Broadening of the flow monitoring peaks has been used to examine laminar flow due to the discontinuous buffer systems used for sample stacking. Using the instrumentation and methods developed for monitoring EOF, electrophoretic vacancy separations with optically gated injections were developed as an alternative method of performing separations in microfluidic devices. Vacancy electrophoretic separations of FITC-labeled amino acids were performed over a separation distance of 0.9 cm (at a field strength of 353 V/cm,) with an injection every 8.0 s. For a series of 10 vacancy electrophoretic separations with 100-ms (177 pL) optically gated injections, the RSD for retention times was 0.17% and the RSD for peak height was 1.6%. The performance of optically gated vacancy electrophoresis was quantitatively compared to the performance of standard optically gated electrophoresis for the same separation using the same microfluidic device and identical experimental conditions. For these separations, the overall performance of the two optically gated injection methods was similar. The resolution obtained with the electrophoretic vacancy separations was slightly lower (6-14%) than with the standard electrophoretic separations due to the larger injection volume for optically gated vacancy injection, using the same injection time