Ligand-substitution mode capillary electrophoretic reactor: Extending capillary electrophoretic reactor toward measurement of slow dissociation kinetics with a half-life of hours

A method employing capillary electrophoresis (CE) was developed to determine the rate constant of the very slow spontaneous dissociation of a complex species. The method uses a CE reactor (CER) to electrophoretically separate components from a complex zone and, thus, spontaneously dissociate a complex. The dissociation is accelerated by ligand substitution (LS) involving a competing ligand added to the electrophoretic buffer. The LSCER method is validated using the dissociation of a Ti(IV)-catechin complex and EDTA as a competing ligand. There is good agreement between the spontaneous dissociation rate constant ( k_d = (1.64 ± 0.63) x 10^-4 s ^-1) and the rate constant obtained by a conventional batchwise LS reaction ( k_d = (1.43 ± 0.04) x 10^-4 s ^-1). Furthermore, the usefulness of the method is demonstrated using a Ti(IV)-tiron complex, for which k_d = (0.51 ± 0.43) x 10 ^-4 s^-1, corresponding to a half-life ( t_1/2) of 3.8 h. Notably, a single run of LS-CER for the Ti(IV) complex is completed within 40 min, implying that LS-CER requires a considerably shorter measurement time (roughly equal to t_1/2) than conventional CER. LS-CER can be widely applied to determine the spontaneous dissociation rates of inorganic diagnostic and therapeutic reagents as well as of biomolecular complexes.