Alleviation of transport limitations in free-flow zone electrophoresis.

Abstract

With current free-flow zone electrophoresis (FFZE) technology, particles can be separated if their mobilities differ by more than 5%, at throughputs ranging between 10⁸ and 10⁹ cells/hr. These capabilities are inadequate for most biological applications. The objective of this research was to alleviate the limitations of FFZE through analysis of the underlying transport effects. A mathematical model was developed, which led to a chamber design that amplifies the peak-to-peak distance between sample components δ over five times; a mode of operation that virtually eliminates artifactual dispersion; and a new approach for scale-up. The model predicted the theoretical limit of resolution for symmetrically and asymmetrically cooled chambers to be 0.7% and 4.9% respectively. The latter compares well with published values of around 5%. The significantly higher resolution of symmetrically cooled chambers is explained in terms of the temperature dependence of mobility. The effects of electrosmosis, sedimentation, free convection, and the direction of the flow on resolution were also evaluated. The design feature that amplifies δ in the new chamber is a series of constrictions along the axis of separation. The amplification of δ at each constriction is shown to vary as the cube of the gap-width differential, and is explained in terms of selective increments in residence time and deflection rate of the faster component relative to that of the slower component. In the new mode of operation, called continuous-flow batch electrophoresis, the peaks are two to three times narrower and the distance between them 50% greater as compared to conventional FFZE. The higher resolution is accounted for in terms of uniformity of residence time and the absence of electroosmosis. In the new approach to scale-up, the ability to amplify δ is exploited to reduce the field strength required to obtain a given degree of separation. The disruptive effects of free convection and ohmic heating are thus suppressed by minimizing the heat generated by the field. Based on the findings of this study a new design for enhancing resolution and throughput is proposed. Simulation results indicate that under microgravity with the proposed design resolution would exceed 0.2%, while throughputs would be 10² times greater for cells and 10⁴ times greater for proteins.