RESOLUTION OF STOPPED-FLOW KINETIC DATA FOR 2ND-ORDER REACTIONS WITH RATE CONSTANTS UP TO 10(8) M(-1) S(-1) INVOLVING LARGE CONCENTRATION GRADIENTS - EXPERIMENTAL COMPARISON USING 3 INDEPENDENT APPROACHES

A program has been developed for the implementation of a mathematical treatment which corrects for a concentration gradient within the stopp ed-flow observation cell for reversible second-order reaction kinetics studied by longitudinal absorbance measurements. This program has bee n tested using experimental kinetic data for three selected electron-t ransfer cross reactions with predicted rate constants of 1.1 x 10(6), 5.8 x 10(7), and 1.2 x 10(8) M(-1) s(-1), respectively. A second gradi ent-corrected approach has also been applied based on the steady-state absorbance which exists after the flow tube has been filled with the new reaction mixture just prior to the stopping of the flow (a permuta tion of the continuous-flow method). As a third comparison, the same d ata were also analyzed using a standard reversible second-order kineti c treatment, without corrections for the concentration gradient, by ap plying an appropriate time base correction. The experimental kinetic d ata were obtained using an unmodified commercial stopped-flow instrume nt with a 2.0 cm observation cell, a measured filling time of 3.8 ms, and a total dead time of 4.6 ms. For reactions with Delta epsilon grea ter than or equal to 10(4) M(-1) cm(-1) all three methods have been sh own to be capable of resolving second-order rate constants up to and e xceeding 10(8) M(-1) s(-1) under conditions where the initial half-lif e is as small as 600 mu s (i.e., about one-eighth the dead time). When the absorbance change becomes extremely small, the steady-state appro ach appears to generate the most reliable rate constant values. The mo st surprising observation is that the standard second-order treatment- which ignores the existence of a concentration gradient-yields rate co nstant values which are virtually identical to those obtained when the gradient correction is taken into account. The implications of this d iscovery are discussed. The demonstrated ability of a standard commerc ial stopped-flow instrument to yield accurate second-order rate consta nts up to 10(8) M(-1) s(-1) represents at least a 10-fold extension in the previously presumed limits for this method.