(533a) Fabrication, Optimization, and Application of a Micro-Free Flow Electrophoresis Microfluidic Chip

Free flow electrophoresis (FFE) is a continuous preparative separation technique that introduces a sample stream into a planar separation chamber pumped continuously with separation buffer. An electric field is applied laterally and analytes are separated based on electrophoretic mobility differences as illustrated in the attached figure. In addition, purified samples can be collected into fractions at the outlet of the separation channel. The main drawback of FFE is sample stream broadening due to many bulk phenomena, decreasing purified sample yields. Micro-FFE is the miniaturization of FFE for analytical applications which minimizes the effects of Joule heating and eliminates thermal convective mixing. From FFE to micro-FFE, the separation channel volume is decrease from ~25 mL to 0.005 mL. The microfluidic chip was fabricated using anodic bonding of two etched and electrode deposited wafers. To eliminate electrolysis product formation, four-fold deeper channels were etched for the electrodes to facilitate a 16 times greater linear velocity. Two-dimensional modeling was used to determine the best channel geometry for parallel buffer flow in the two-depth chip. The removal of electrolysis products increased resolution of fluorescent standards by a factor of 1.3 under similar separation conditions. Also, the new design allowed for a four-fold increase in applied electric field to 586 V/cm before Joule heating conditions persisted. Experimental variance data from the fluorescent analyte standards allowed for determination of a migration distance squared dependence on band broadening under non-diffusion-limited conditions. From this phenomenon, minimization of migration distance, possibly through suppression or modification of electroosmotic flow, allows for the highest resolution of analytes and efficiency of separations. Also, equations were derived to describe total peak variance, plate height and number, optimum linear velocity, peak capacity, and resolution. Linear velocity emerged as an important variable for optimization. Similarly, previously reported equations allow for precise positioning of analyte streams in the separation channel based on linear velocity and applied electric field. Separations of biomolecules have been performed to show the improved functionality of the micro-FFE through knowledge gained from the fundamental studies.