Milan Bier

Computer simulation and experimental validation of isoelectric focusing in ampholine-free systems

Abstract An abbreviated version of a mathematical model of the steady state in isoelectric focusing is presente. Details of the mathematical trasformations leading to a model suitable for computer implementation and numerical solution are given in the Appendix. The model describes the structure of concentration, pH, conductivity and potential gradients arising from the focusing of electrochemically defined ampholytes. Some of the results of computer simulations of two and three component systems are of particular interest to the experimentalist and are presented together with experimental validation of this model.

Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor

A monoclonal antibody to phencyclidine was developed, produced in mouse ascites fluid, and purified. The purification used only preparative-scale isoelectric focusing in the Rotofor and dialysis. In 4 h, 25% (4 mg) of the antibody from 10 ml of ascites fluid was purified to homogeneity while 63% of the total antibody was recovered.

Experimental and theoretical dynamics of isoelectric focusing - Elucidation of a general separation mechanism

Abstract The transient states is isoelectric focusing were monitored using a potential gradient array detector. Electric field profiles are presented which show the formation of transient moving boundaries, as well as the approach of the steady state distribution, during the focusing of two and three component systems. The experimental results are completely consistent with corresponding computer simulation data. The focusing process is comprised of two sequential phases, a relatively rapid separation phase and a much slower stabilizing phase. A phenomenological separation mechanism is presented for the two and three component systems, based on distinct, transient moving boundaries, which describes the first phase. This mechanism is discussed with respect to n component diealized systems. The second phase provides insight into a primary cause of pH gradient instability. It was found that the time necessary for the adjustment to the steady state, the second phase, can be as much as twenty times longer than the time needed for the separation of the constituents.

Recycling isoelectric focusing and isotachophoresis

Of all electrophoretic methods, isoelectric focusing offers the highest resolution and is best suited for preparative applications. Over the years, several instruments were developed for this purpose, all operating in free fluids, in the absence of gels or other supporting matrices. In such systems, the avoidance of gravity or electrically driven convections is essential. Successful stratagems for fluid control included rapid recycling or rotation, in combination with either fine porosity screens or narrow gaps between parallel plates. The most successful apparatus so far is the Rotofor, in which fluid is stabilized by combining horizontal rotation with fine porosity screen partitioning. Recycling isotachophoresis offers the potential of separating proteins at high concentration. A new concept of tangential electrophoresis is described. To optimize the use of these devices for protein separation, low molecular weight, biologically acceptable buffers of known composition are essential. The buffering system developed for this purpose comprises a series of binary buffers that cover the pH range in steps of 1 pH unit or less. The pH gradient can be custom-designed and is of remarkable stability in operation.

Serum protein fractionation by isotachophoresis using amino acid spacers

Analytical and preparative isotachophoresis has been carried out using amino acids and peptides as discrete spacers in contrast to the usually employed continuous mobility spectrum Ampholine. Analytical isotachoresis in free solution, using the LKB Tachophor, demonstrated the separation of human serum into distinct mobility subgroups, n spacers giving rise to n + 1 protein subgroups. Preparative fractionation on polyacrylamide gel was carried out on the LKB Uniphor using threonine and glycine as spacers. Immunoelectrophoretic analysis showed that eight out of ten proteins assayed were clearly resolved in the three subgroups obtained, thus demonstrating the sharpness of isotachophoretic resolution.

Capillary isoelectric focusing: effects of capillary geometry, voltage gradient and addition of linear polymer

Abstract Free-fluid focusing of both simple buffer and Ampholine systems in various capillaries of rectangular cross-sections was investigated by following the temporal behavior of the current under constant voltage and the transient double peak approach to equilibrium of colored test proteins. For systems comprising two and three buffer constituents the ratio of initial to final focusing current compares well with data obtained by computer simulation. Experiments have also been performed in the presence of linear, non-crosslinked polyacrylamide to assess its fluid stabilizing potential in capillaries. Good focusing and resolution are commensurate with a high initial to final current ratio, no substantial drifts after separation (attainment of steady state) and proper boundary conditions at both column ends. The production of turbulent protein foci at high-voltage gradients is discussed with the aid of electric field profiles monitored along the focusing column.

Experimental and theoretical dynamics of isoelectric focusing. II: Elucidation of the impact of the electrode assembly

Abstract The effects of several different electrode assemblies on the establishment of pH gradients in isoelectric focusing (IEF) in free fluids were investigated. The term “assembly” refers to the nature and relative volume of the electrolytes and the types of membranes used to isolate those electrolytes from the separation space. Experiments were performed in capillary systems. Dialysis membranes, ion-exchange membranes and thin palladium foils were employed with electrolytes consisting of strong acids and bases, or ampholytes, in varying concentrations and volumes. The progress of focusing was monitored by recording the temporal behavior of the current, at constant voltage, or the voltage gradient profile across the capillary at constant current. Mixtures of simple ampholytes, as well as commercial carrier ampholytes were investigated. Computer simulation data were used to aid the interpretation of some of the experimental results. It was found that the different assemblies do not have an impact on the separation mechanism, but do affect separation speed, resolution, and the stability of the pH gradient. Specifically, palladium electrodes and ion-exchange membranes provide the greatest gradient stability and eliminate any impact of electrode solution concentration and volume. If dialysis membranes are used, small electrode volumes and high buffer concentrations constitute the best focusing conditions. In contrast, large electrolyte volumes of low concentration destabilize the separative pattern continuously and rapidly by an isotachophoretic mechanism. This destabilization can take the form of a cathodic, anodic or symmetrical drift. The arrangements discussed illustrate the similarities and differences of IEF and isotachophoresis and are relevant to free fluid and gel IEF.

An explanation for the plateau phenomenon in isoelectric focusing

Abstract Isoelectric focusing of mixtures of simple ampholytes occurs in two phases, an initial rapid separation phase and a second relatively slow stabilizing phase. Transient and steady-state computer simulation data are shown to predict the development of pH plateaus around neutrality during the stabilizing phase of the focusing of such mixtures. This occurs because a non-zero electrophoretic flux is present in a pure zone of focused ampholyte, which is a function of both its isoelectric point (pI) and its ΠpK vlaue. For an ampholyte with a pI > 9 or

Computer aided analysis of electric field gradients within isotachophoretic boundaries between weak electrolytes

Abstract Computer simulations are used to describe the temporal evolution of concentration and potential profiles in isotachophoresis. In contrast to other theoretical treatments of moving boundaries between weak electrolytes, diffusional effects are considered, which allows an exact prediction of boundary shape. The computer predicted shapes of three different electric field gradients between migrating protolytes are analyzed: (i) the commonly observed sigmoidal field increase from the leading to the terminating zone; (ii) the mirror image of such a gradient in the enforced migrational mode; (iii) the steady state “bump” gradient which can be established between a weak acid and its salt. The value of these predictions to the experimentalist is discussed.

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.