Abstract A method is presented for determining stepwise elution chromatographic conditions on the basis of the data obtained from linear gradient elution experiments. The separation of crude β-galactosidase by medium-performance (an)ion-exchange chromatography (MPIEC) was chosen as a model separation system. Some factors affecting the purification such as the column dimensions and the residence time were investigated. Large-scale MPIEC was carried out and its performance was compared with that of a small scale-column. The effect of sample loading was also examined.