A review of the zone broadening contributions in free-flow electrophoresis.

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.

On-chip pressure generation using a gel membrane fabricated outside of the microfluidic network.

On-chip generation of pressure gradients via electrokinetic means can offer several advantages to microfluidic assay design and operation in a variety of applications. In this article, we describe a simple approach to realizing this capability by employing a polyacrylamide-based gel structure fabricated within a fluid reservoir located at the terminating end of a microchannel. Application of an electric field across this membrane has been shown to block a majority of the electroosmotic flow generated within the open duct yielding a high pressure at the channel-membrane junction. Experiments show the realization of higher pressure-driven velocities in an electric field-free separation channel integrated to the micropump with this design compared to other similar micropumps described in the literature. In addition, the noted velocity was found to be less sensitive to the extent of Debye layer overlap in the channel network, and therefore more impressive when working with background electrolytes having higher ionic strengths. With the current system, pressure-driven velocities up to 3.6 mm/s were realized in a 300-nm-deep separation channel applying a maximum voltage of 3 kV at a channel terminal. To demonstrate the separative performance of our device, a nanofluidic pressure-driven ion-chromatographic analysis was subsequently implemented that relied on the slower migration of cationic analytes relative to the neutral and anionic ones in the separation channel likely due to their strong electrostatic interaction with the channel surface charges. A mixture of amino acids was thus separated with resolutions greater than those reported by our group for a similar analysis previously.

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.

Electrokinetic stacking of particle zones in confined channels enabling their UV absorbance detection on microchips.

In this article, we report a simple approach to stacking micro- and nanoparticle zones by electrokinetically migrating them through moderately confined channels of uniform cross-section. Experiments show the reported pre-concentration process to initiate at the tail end of the zone following its electrokinetic injection, with the stacked region migrating faster than the rest of the sample band. This effect causes the particles traveling in front to merge into the stacked region making it grow both in size and concentration. Because the stacked zone also gradually loses particles from its trailing edge, it eventually disintegrates upon running out of particles at its front end. Nevertheless, enhancements in peak height by over 100-fold were recorded using the reported approach for polystyrene beads with diameters comparable to the channel depth. This enhancement however, exhibited a temporal variation as the particle band migrated through the analysis column reaching a maximum value that depended on the particle diameter, particle concentration, channel depth, electric field strength, electroosmotic mobility, etc. Interestingly, the peak area recorded by the detector remained relatively constant during this particle migration period allowing reliable sample quantitation. Moreover, upon incubating antibody-coated particles against an antigen sample, the peak area for the particle zone was seen to scale linearly with the antigen concentration establishing the utility of the reported focusing phenomenon for chemical/biochemical analysis. The noted stacking technique was further applied to enabling UV absorbance detection of particle zones on microchips which then allowed us to determine the colloidal content in actual natural water samples. .