The simulation of a chromatofocusing column for the separation of a model mixture of four proteins, sperm-whale myoglobin, horse myoglobin, transferrin, and albumin, was performed under transient pH gradients and by computer calculation with a differential model including the buffering action. A simulated mixture of four proteins was eluted at the pH of their isoelectric points, and the internal elution pattern of horse myoglobin was visually determined. The focusing effect of protein by pH was illustrated with the elution of intermittent feeds into a single band. The smooth and linear pH gradients, having interaction with ion exchanger and mixed buffers, were produced by a differential model with a model buffer for the pH gradients and the protein elutions. The calculated linear pH gradients and elution profiles showed good agreements with experiments. The protein separation and focusing effect were calculated with a model buffer and the differential model.