Buffer salt effect on pH in the interior of an anion exchange resin.

The internal pH of Q Sepharose Fast Flow anion exchange resin in equilibrium with a bis-tris acetate buffer solution is investigated as a function of buffer salt concentration. Direct evidence of a resin phase pH shift is presented. At low buffer salt concentrations of 20 mM NaCl the resin phase pH is found to be as much as 1.1 pH units greater than that of the buffer phase, approaching to within 0.1 units of the buffer phase at salt concentrations greater than 250 mM. An ideal model with no adjustable parameters based on the Boltzmann distribution and the electroneutrality condition provides excellent agreement with experimental observations. The model assumes that small ions do not bind to the resin fixed charge sites and the agreement between the model predictions and observed resin internal pH suggests that strong electrolytes do not form ion pairs with the resin fixed charge sites.

Protein focusing in a conductivity gradient.

Conductivity gradient focusing (CGF) is one member of a family of gradient focusing techniques, characterized by two opposing forces which produce a dynamic equilibrium and which are able to simultaneously separate and concentrate proteins. In CGF, the two counteracting forces result from a constant convective flow of buffer opposed by an electric field gradient. This gradient in the electric field is formed by gradually decreasing buffer conductivity, i.e., when a slow-moving, relatively high conductivity buffer is dialyzed against a low conductivity purge buffer. This paper presents the design of an analytical-scale CGF device and the results of several experiments with colored proteins, both in free solution and with the use of a 45 micron size-exclusion (SEC) packing to decrease dispersion. Experimental results with hemoglobin suggest that CGF may one day be capable of resolving proteins with small charge differences. A linear computer model of conductivity gradient focusing is derived, and some suggestions are made for further development of this new electrophoretic method.

The development of recycle zone electrophoresis.

Milan Bier's contributions to preparative electrophoresis and, in particular, his work in "recycling", have had an enormous impact on the development of scaling strategies for continuous flow electrophoresis. This paper reviews my early work on the theory of recycle applied to zone electrophoresis and reports the results of several recent experiments in which proteins were purified from complex feed mixtures using recycle zone electrophoresis.

Electromagnetic stabilization of weakly conducting fluids.

Classical hydromagnetic theory predicts that the flow of dilute aqueous electrolyte in a slit can be stabilized by application of a strong, transverse magnetic field. However, recent experiments indicate that stabilization can be achieved with the use of a much weaker field in the presence of a small lateral current. A revised theory describes how the magnetic and electric fields interact to eliminate natural convection.

High resolution continuous flow electrophoresis.

Continuous free-flow electrophoresis (CFE) provides the biochemist with a powerful technique for preparative-scale purification of labile biomolecules. However, solute resolution in the classic CFE is limited by the deleterious effects of diffusion, electroosmotic dispersion, and connective dispersion. These problems can be overcome in the CFE by continuously recycling effluent through the chamber in a manner which allows complete recovery of undiluted product at arbitrarily high purities.Mathematical models of the continuous flow device with recycle (RCFE) are derived for the two limiting cases: rapid diffusion and slow diffusion. The analytical solutions of these models are used to predict separation factors under various operating conditions.