Generation of stable pH gradients for preparative isoelectric focusing by electrolysis of two‐component buffer solutions in a multi‐compartment apparatus

A widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered, inter alia by the high cost of the carrier ampholytes used for creating the pH gradient. Efforts are therefore being made to replace the carrier ampholytes with simple two‐component buffers. To this end, a multimembrane electrolyzer, consisting of 22 compartments with attached cooling loops, has been constructed. The choice of membranes has proved to be one of the main problems. With permeable membranes, even small differences in hydrostatic pressure between the ends of the electrolyzer will cause internal liquid flows that prohibit the formulation of a useful pH gradient. Likewise, flow‐tight membranes generally give rise to a large internal flow due to electroosmosis. Among the types of membranes so far examined, polycrylamide membranes form an exception. With these membranes, useful and stable pH gradients have been created. There are drawbacks, however, relating to the low mechanical strength and to the difficulty of producing exactly equal membranes, thereby fulfilling the theoretical demand of constant transference numbers for the ions. The experimental observation that even small disturbances, such as variations of the transference numbers or a minute electroosmotic flow through the electrolyzer, may completely destroy the pH gradient, has been related to the mechanism operating during electrolysis of a simple buffer. While a possible local breakdown of a carrier‐ampholyte pH gradient is rapidly repaired by the current, a disturbance occurring in a buffer pH gradient can be repaired only by diffusion, which is a slow process.