For the separation of enzyme, the characteristics of affinity chromatography was studied. The system employed Sepharose CL-4B as the gel matrix, 4-phenylbutylamine as a ligand and $\alpha$-chymotrypsin as a solute. Moment analysis was used to determine the retention time in a column, in which the longitudinal dispersion in the mobile phase, the radial dispersion inside the porous spherical gel beads and the sorption on the internal surface of the gel bead are simultaneously taking place. In the constant pattern assumption the averaged volumetric coefficient of mass transfer was obtained and the breakthrough curve could be calculated by the implicit scheme of finite difference method. To analyse the effect of pH on the retention time, an ionization model was incorporated into the chromatographic model. Dependence of adsorption equilibrium constant and the retention time on pH were investigated. From the experimental data the approximate pK values would be obtained. Also the dependence of retention time on the ligand concentration was investigated.