Automated micro stopped-flow/continuous-flow apparatus for serial measurement of enzyme reactions and its application as a real-time analyser for column chromatography

By functional combination of two modes of stopped-flow (SF) and flow-injection analysis (FIA), an automated micro apparatus is constructed for serial measurement of enzyme reactions. The complete operation of the apparatus, including kinetic data aquisition, is controlled by a personal computer. The apparatus has three specific features: (a) the volume required per measurement of a time course in an enzyme reaction is as small as 30 μl for each of enzyme and assay solution; (b) the time course can be traced by sampling ten observation points in a maximum of 1 s; and (c) the serial measurement of such a time course can be performed with automated cyclic operation (10 s per operating cycle as the lower limit). In addition to enzymatic activity, simultaneous monitoring of protein concentration, pH and conductivity in the enzyme solution can be sequentially conducted by the automated micro SF/FIA apparatus equipped with appropriate detectors. The successful application of such a four-channel apparatus to ion-exchange and affinity column chromatography of calf bone alkaline phosphatase proves that it is useful as a practical real-time analyser for enzyme activity together with other parameters to be monitored in liquid chromatography.