ONPG disappearance and ONP appearance were synchronously measured during ONPG hydrolysis by beta-galactosidase using in situ on-line UV-vis spectroscopy. Intermediate formation was determined by the formula d[ONPG]/dt - d[ONP]/dt. The combined effects of temperature and time on nu(inst) and nu(inc) during the conversion of ONPG to ONP were expressed by the isogram method in which contour plots were used. Based on this approach, new insights were obtained into the irreversible-continuous conversion of ONPG to ONP during hydrolysis. The intermediate was a moving mass that flowed in three-dimensional space from the substrate to the product. The results of this study support the use of the isogram method for understanding the mechanisms of enzyme-catalyzed reactions via the dynamic resolution approach. (C) 2009 Elsevier Ltd. All rights reserved.