The classical isoelectric trapping (IET) technique, using the multicompartment electrolyzer (MCE), has been one of the most successful electrophoretic techniques in preparative-scale protein separations. IET is capable of achieving high resolution discrimination of proteins, by isolating proteins in between buffering membranes, in their isoelectric state. However, due to the inherent nature of the IET process, IET has suffered several shortcomings which have limited its applicability. During a classical IET separation, a protein gets closer and closer to its pI value, thus the charge of the protein gets closer and closer to zero. This increases the likelihood of protein precipitation and decreases the electrophoretic velocity of the protein, thus making the separation very long. Furthermore, the problems are aggravated by the fact that the instrumentation currently used for IET is not designed to maximize the efficiency of electrophoretic separations. To address these problems, a new approach to IET has been developed, pH-biased IET. By controlling the solution pH throughout the separation, such that it is not the same as the protein’s pI values, the problems of reduced solubility and low electrophoretic migration velocity are alleviated. The pH control comes from a novel use of isoelectric buffers (also called auxiliary isoelectric agents or pH-biasers). The isoelectric buffers are added to the sample solution during IET and are chosen so that they maintain the pH at a value that is different from the pI value of the proteins of interest. Two new pieces of IET instrumentation have been developed, resulting in major improvements in protein separation rates and energy efficiency. A variety of separations, of both small molecules and proteins, have been successfully performed using the pH-biased IET principle together with the new instrumentation.

Isoelectric trapping (IET) in multicompartment electrolyzers (MCE) has been widely used for the electrophoretic separation of ampholytic compounds such as proteins. In IET, the separation occurs in the buffering membranes that form a step-wise pH gradient in the MCE. Typically, buffering membranes have been made by copolymerizing acrylamide with Immobiline compounds, which are acidic and basic acylamido buffers. One major problem, however, is that these buffering membranes are not stable when exposed to high concentrations of acid and base due to hydrolysis of the amide bonds. Poly(vinyl alcohol)-based, or PVA-based, membranes were made as an alternative to the polyacrylamide-based membranes since they provide more hydrolytic and mechanical stability. Four mid-pH, PVA-based buffering membranes that contain single ampholytes were synthesized. These buffering membranes were used to trap small molecular weight pI markers for up to three hours, and were also used in desalting experiments to remove strong electrolytes from a solution of ampholytes. Additionally, the membranes were used in IET experiments to separate mixtures of pI markers, and to fractionate the major proteins in chicken egg white. The membranes did not show any degradation when stored in 3 M NaOH for up to 6 months and were shown to tolerate current densities as high as 16 mA/cm2. In addition, six series of PVA-based membranes, whose pH values can be tuned over the 3 < pH < 10 range, were synthesized by covalently binding aminodicarboxylic acids, and monoamines or diamines to the PVA matrix. These tunable buffering membranes were used in trapping experiments to trap ampholytes for up to three hours, and in desalting experiments to remove strong electrolytes from a solution of ampholytes. These tunable buffering membranes were also used in IET experiments to separate proteins, some with pI values that differ by only 0.1 pH unit. The tunable buffering membranes did not show any signs of degradation when exposed to 3 M NaOH for up to 3 months, and could be used in IET experiments with current densities as high as 20 mA/cm2. These tunable buffering membranes are expected to broaden the application areas of isoelectric trapping separations.

Isoelectric trapping (IET) in multicompartment electrolyzers (MCE) has been widely used for the electrophoretic separation of ampholytic compounds such as proteins. In IET, the separation occurs in the buffering membranes that form a step-wise pH gradient in the MCE. Typically, buffering membranes have been made by copolymerizing acrylamide with Immobiline compounds, which are acidic and basic acylamido buffers. One major problem, however, is that these buffering membranes are not stable when exposed to high concentrations of acid and base due to hydrolysis of the amide bonds. Poly(vinyl alcohol)-based, or PVA-based, membranes were made as an alternative to the polyacrylamide-based membranes since they provide more hydrolytic and mechanical stability. Four mid-pH, PVA-based buffering membranes that contain single ampholytes were synthesized. These buffering membranes were used to trap small molecular weight pI markers for up to three hours, and were also used in desalting experiments to remove strong electrolytes from a solution of ampholytes. Additionally, the membranes were used in IET experiments to separate mixtures of pI markers, and to fractionate the major proteins in chicken egg white. The membranes did not show any degradation when stored in 3 M NaOH for up to 6 months and were shown to tolerate current densities as high as 16 mA/cm2. In addition, six series of PVA-based membranes, whose pH values can be tuned over the 3 < pH < 10 range, were synthesized by covalently binding aminodicarboxylic acids, and monoamines or diamines to the PVA matrix. These tunable buffering membranes were used in trapping experiments to trap ampholytes for up to three hours, and in desalting experiments to remove strong electrolytes from a solution of ampholytes. These tunable buffering membranes were also used in IET experiments to separate proteins, some with pI values that differ by only 0.1 pH unit. The tunable buffering membranes did not show any signs of degradation when exposed to 3 M NaOH for up to 3 months, and could be used in IET experiments with current densities as high as 20 mA/cm2. These tunable buffering membranes are expected to broaden the application areas of isoelectric trapping separations.