Mixtures of four proteins, sperm whale myoglobin, horse myoglobin, transferrin and albumin of isoelectric points, 8.3, 6.8-7.8, 5.9, 4.7 respectively, were successfully separated by chromato-focusing in a column packed with PBE 94 under transient pH gradients. Eluting the polybuffer, the smooth and linear pH gradient was developed for PBE 94 exchanger, and each of four proteins was recovered in the order of PI’s with high recovery yields, and within 0.25pH unit from the elution pH’s. A simulated mixture of four proteins was separated out at the corresponding pH. The internal elution pattern of myoglobin I was visually determined in the gradient column and the focusing effect of protein by pH was illustrated with the elution of intermittent feeds into a single band.