Percy H. Rhodes

Dispersion effects in capillary zone electrophoresis

Abstract We qauntitatively analyzed possible causes of the observed spreading of sample components which degrades separation compared with theoretical limits, and identified four potentially significant causes, electrokinetic dispersion, wall adsorption, enhanced diffusion due to Poiseuille flows driven by pressure gradients, and enhanced diffusion due to mobility variations associated with transverse temperature differences. Electrokinetic dispersion is caused by changes in the conductivity and pH distributions, proportional to the concentration of the sample relative to the buffer components, and independent of the tube diameter. One-dimensional numerical results for the separation of seven species in a sodium acetate buffer are presented to illustrate the effect. A detailed discussion of methods to control it is also presented. It is suggested that both wall adsorption of sample species and most coating methods used to control it or to reduce electroosmosis can be understood as aspects fo the Debye double-layer theory. Large molecules, with a high degree of ionization with the opposite sign to the wall charge, are preferentially attracted to the layer, excluding smaller molecules, and decreasing the wall potential and mobility. We demonstrate the importance of choosing a combination of pH and tube material such that the wall and the large proteins in the sample have the same charge sign and repel each other. The diffusion enhancement analysis is based on the analytic approximation of balancing the flow and mobility distrubance terms in the concentration equation for a sample species with the transverse diffusion term. This determines the fluctuating part of the concentration, with zero transverse average. The interaction of this concentration distribution with the fluctuations modifies the equation for the transverse average of the concentration, by adding diffusivity a2Y2/48Di·. Here a is the radius, Di is the diffusivity of the species, and Y is a disturbance speed and is a sum of contributions from the pressure-gradient-driven Poiseuille flow, the transverse mobility variation due to the heating in the tube, and other effects which we believe are smaller. The numerical factor of 48 is exact for the Poiseuille flow and the transverse mobility variations effects, and presumably it improves the estimate for the other effects. The Poiseuille flow profile is driven by variations in the electroosmotic slip velocity, which are mostly caused by conductivity variations along the tube and by changes in the Debye layer structure associated with composition changes along the tube. Contributions from temperature or radius variations along the tube are estimated, and are apparently smaller. Relatively insignificant causes of dispersion include sample dispersion by the difference between the electroosmotic flow and the slower flow in the Debye layer (the layer is to thin), variations in the tube radius, and convection and electrohydrodynamics flows. Molecular diffusion is important (and theoretical plate numbers are achieved) if other dispersion effects are small; it is larger for sample species with small molecules.

Electrohydrodynamic distortion of sample streams in continuous flow electrophoresis

Abstract Prior theories involving electroosmotic, electrokinetics, and other effects have proved inadequate to explain the observed sample spreading and performance degradation in continuous flow electrophoresis (CFE). We suggest electrohydrodynamic flows as the main cause. These findings should contribute to efforts to improve the performance of CFE. It is shown theoretically that an electric field (AC or DC), perpendicular to a circular filament of conducting fluid surrounded by fluid of a different conductivity, produces an electrohydrodynamic flow, which distorts the filament into an ellipse. This distortion is similar to that described by G. I. Taylor for spherical drops. The major axis of the ellipse is either parallel to or normal to the field, depending on the conductivity and dielectric constant of the filament fluid relative to those of the surrounding fluid. For equal dielectric constants, the major axis is parallel to the field if the filament conductivity is greater than that of the surrounding fluid, and normal to the field otherwise. The flow and distortion rate is proportional to the square of the applied field. It is further shown theoretically that the flow associated with an elliptic cross section maintains the elliptic shape while it continues the distortion, provided the deviation from a circular cross section is small. As the ellipse stretches, small deviations from an elliptic cross section appear. It is shown, using an energy argument based on the assumption of an elliptic cross section, that the circular filament continues to flatten indefinitely, forming a ribbon, either parallel to the field or normal to it. The nature of the analysis, the physics of the flow, and the related experiments appear to confirm this behavior. For our experiments, we used an aqueous electrolyte (barbital buffer) and a sample of the same material with polystyrene latex added for visibility, in a CFE-type apparatus. The flow rate and configuration were typical of CFE. Electrokinetic and electrophoretic effects, and electroosmosis, were eliminated by using an AC field. Distinctive ribbons were formed, in both directions, at small fields of order 50 V/cm or less. All observations were consistent with the theories described above.

Column Electrophoresis on the Apollo-Soyuz Test Project

INTRODUCTION Electrokinetic separation techniques have been widely used for the analysis and characterization of charged materials of biological origin.3,4 Under terrestrial conditions preparative methods based on zone electrophoresis, isotachophoresis, and isoelectric focusing are prominent in the purification of charged macromolecules and small particles but have only been of limited usefulness in the separation of biological cells and larger particles. The major difficulties in the latter applications arise primarily from In the Apollo 16 experiment, a mixture of polystyrene latices was processed in an improved version of the apparatus used on Apollo 14. Whereas with such a device it is not possible to conduct the processing under terrestrial conditions without the appearance of extensive thermal convection which destroys the separation bound- aries, under microgravity conditions flight photographs showed no boundary deterioration, but did indicate that electroosmosis was a major factor in producing ov...

Thermal Considerations in Continuous Flow Electrophoresis

Abstract Two-dimensional heat flow in a continuous flow electrophoretic chamber is analyzed assuming Poiseuille flow and finite conductivity of the chamber walls. The thermal field can be characterized in terms of several dimensionless parameters which allow the solution to be applied to a wide variety of operating conditions. Since most electrophoretic chambers have a high aspect ratio, heat flow through the edge walls is not a major effect and the two-dimensional model is adequate. A major advantage of using this simplified approach is that analytical solutions can be obtained which provide insights that are difficult to get from three-dimensional numerical approaches. For example, a criterion is developed for determining the maximum power that can be used in machines operated in upflow or downflow configuration. Also it is shown that the actual structure of the flowfield has no effect on the fully developed thermal field. The model is compared with experimental measurements, and the implications of the...

Polystyrene latex separations by continuous flow electrophoresis on the Space Shuttle

Abstract The seventh mission of the Space Shuttle carried two NASA experiments in the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system. The objectives were to test the operation of continuous flow electrophoresis in a reduced gravity environment using stable particles with established electrokinetic properties and specifically to evaluate the influence of the electrical properties of the sample constituents on the resolution of the continuous flow electrophoretic device. Polystyrene latex microspheres dispersed in a solution with three times the electrical conductivity of the curtain buffer separated with a significantly larger band spread compared to the second experiment under matched conductivity conditions. It is proposed that the sample of higher electrical conductivity distorted the electric field near the sample stream so that the polystyrene latex particles migrated toward the chamber walls where electroosmosis retarded and spread the sample.

Stability of erythrocyte suspensions layered on stationary and flowing liquids

The apparent stability of erythrocyte suspensions layered on stationary and flowing Ficoll solutions was studied considering the effects of particle concentration, type and size, and the different flow rates of the particle suspensions and chamber liquid. The data from the flowing system were empirically fitted and, when extrapolated to zero chamber liquid flow rate, gave values comparable to the data from the stationary system, thus confirming the validity of the data and our approach to obtain that data.

The Effect of Small Temperature Gradients on Flow in a Continuous Flow Electrophoresis Chamber

Continuous flow electrophoresis employs an electric field to separate biological cells suspended in a flowing liquid buffer solution. Good separations based on differences in electrophoretic mobility are obtained only when a unidirectional flow is maintained. The desired flow has a parabolic structure in the narrow dimension of the chamber and is uniform acros the width, except near the edges where the no-slip condition prevails. However, because of buoyancy, very small laterall or axial temperature gradients deform the flow significantly. The results of experiments conducted with a specially instrumented chamber show the origin and structure of the buoyancy-driven perturbations. It is found that very small temperature gradients can disturb the flow significantly, as was predicted by earlier theoretical work.

Electrophoresis experiments for space

It has long been hoped that space could alleviate the problems of large-scale, high-capacity electrophoresis. Support media and reduced chamber dimensions of capillary electrophoresis have established the physical boundaries for Earth-based systems. Ideally, electrophoresis conducted in a virtual weightless environment in an unrestricted “free” fluid should have great potential. The electrophoresis and isoelectric focusing experiments done in the reduced gravity over the past twenty-five years have demonstrated the absence of thermal convection and sedimentation as well as the presence of electrohydrodynamics that requires careful control. One commercial venture produced gram amounts of an electrophoretically purified protein during seven Space Shuttle flights but the market disappeared in the six years between experiment conception and performance on the Space Shuttle. Our accumulated experience in microgravity plus theoretical models predict improvements that should be possible with electrophoresis if p...