The high-performance liquid chromatography of soybean trypsin inhibitor is re-examined by using simultaneous optical activity and ultraviolet absorption detection. Ratio plots of the two detector responses allow easy identification of impurities that are not related to the protein. The specific rotations of each of the separated components can be derived. We find that one denatured form has a distinctly lower specific rotation while another form shows no change in specific rotation. The on-column denaturation rate here was found to be slower than that from previous work. Column pretreatment may have resulted in milder column conditions through the elimination of irreversibly adsorbing sites.