Abstract: An improved method of Kohlrausch-regulated electrophoresis (isotachophoresis) is disclosed for the preparative separation of respective macromolecular components such as proteins in a crude sample. The preferred method involves use of a substantially linear, polyethylene glycol derivatized trailing ion having a relatively high mass to charge ratio and a low mobility which is substantially independent of pH changes over a relatively broad pH range. The most preferred trailing ion is a polyethylene glycol dicarboxylic acid having a molecular weight of about 6,000-7,500, a pKa value of about 3.7, and a fully ionized mobility of about -1.3.times.10.sup.-5 cm..sup.2 /volt/sec. The method hereof permits large scale protein separations and subsequent collection of purified protein fractions over a wide range of pH conditions and without interfering reactions between the proteins and trailing ion.