Application of an Electrokinetic Backflow for Enhancing Pressure-Driven Charge Based Separations in Sub-Micrometer Deep Channels

In this article, we report significant improvements in the resolving power of pressure-driven charge based separations performed in sub-micrometer deep glass channels upon introducing an electrokinetic backflow in the system. Such improvements are realized as axial electrophoresis aids the pressure-driven separation process in negatively charged glass conduits under these conditions. In addition, the electroosmotic backflow slows down the bulk transport of the background electrolyte subjecting the sample to the separation field for prolonged periods and yields a higher fluid shear across the channel depth further assisting the separation process. Although this increased shear also contributes to additional hydrodynamic dispersion, such contributions are usually small due to fast diffusion across the flow streamlines in sub-micrometer deep channels. In the present work, the pressure-driven flow was generated on-chip by fabricating a polyacrylamide based gel membrane within a chosen access hole upstream of the separation channel. Upon application of an electric field across this structure, the electroosmotic flow generated in the open channel interfacing the membrane was partially blocked producing the needed pressure-gradient. Optimization of the electrical voltage applied to the downstream end of the separation channel then yielded a suitable electrokinetic backflow that significantly improved the resolving power of our separations. For a sample comprising of three 5-TAMRA, SE-labeled amino acids, the noted strategy improved the separation resolution by over an order of magnitude compared to the case when no electrokinetic backflow was present. The band broadening in these separations was also assessed to understand its dependence on the operating conditions.