Electrophoresis in one buffer at two pH values

A theory for discontinuous electrophoresis in a polyacrylamide gel is presented for one buffer at two pH values. It is shown that polyions stack between identical leading and trailing ions, and resolve in a gel of constant polyacrylamide concentration. The theory is illustrated by the separation of serum protein polyions in a Tris‐glycinate buffer of pH 8.19 in the well‐forming gel, and pH 9.16 in the resolving gel. The selected concentrations and electrolyte ionization degrees of Tris and glycine have values at which the serum protein polyions stack between the resolving and electrode buffers, followed by separation in the resolving gel.