AbstractEnhanced separation of phosphoglucomutase (PGM1) isoenzymes has been achieved by addition of the separator N‐(2‐Hydroxyethyl)piperazine‐N′‐3‐propanesulphonic acid (EPPS) to pH 5–7 Ampholines in ultrathin and 1 mm isoelectric focusing gels. The resultant pH gradient ranges between pH 5.6 to pH 5.9 and effectively spreads PGM1isoenzymes across the whole width of the gel. The technique has been shown to be especially useful for separating some of the rare PGM1variants. The use of different electrolyte combinations has also been investigated and it has been shown that strong electrolytes,e. g.sodium hydroxide and orthophosphoric acid, result in a small but statistically significant increase in the gradient range compared with weaker electrolytes such as threonine/histidine. In addition, the effect of temperature on the rate of migration of PGM1isoenzymes has been