Improved calibration and use of stopped-flow instruments

The calibration procedure of stopped-flow instruments based on the dead-tim e concept has been improved. It is proved theoretically and shown experimen tally that the dead-time is not a simple instrumental parameter; it depends on the rate of the reaction. Two new parameters are introduced for improve d calibration: the filling time of the observation cell and the theoretical starting time, which is the time elapsed between the mixer and the end of the observation cell. Various experiments are presented for the determinati on of these parameters. The proposed calibration procedure is based on the evaluation of all experimental curves simultaneously, taking the concentrat ion constraints into account. The simultaneous evaluation is possible becau se the recently developed stopped-flow spectrophotometers give accurate mea sured absorbances. Therefore, the use of the pseudo-first-order approach is not necessary; measurements carried out under second-order conditions may also be evaluated without any simplification. The formation kinetics of the well-known FeSCN2+ complex has been reinvestigated in order to compare the results of the experiments carried out under pseudo-first-order and second -order conditions. The equilibrium and kinetic studies were carried out in an extended concentration and pH range. A slightly modified mechanism, invo lving the reaction between Fe-2(OH)(2)(4+) and SCN-, and the appropriate ra te constants are reported. The evaluation procedure developed can be extend ed to arbitrary reactions, and thus it increases the type of reactive chemi cal systems accessible for stopped-flow studies (e.g. accumulation of absor bing intermediates, autocatalytic reactions, etc.).