Isothermal titration calorimetry in the single-injection mode with imperfect mixing.

Isothermal titration calorimetry (ITC) is now a method of choice to obtain thermodynamic information about the interaction between two molecular partners. Most often, the method in use is the so-called multiple-injection method (MIM) consisting in distinct short-time injections of the titrant separated by sufficient delay to reach equilibrium before each new injection. However, an alternative single-injection method (SIM) exists. It consists in a unique continuous injection and, despite the fact that it is quite simple and generally faster than MIM, it is very little used. The goal of this work is to reconsider its theoretical basis. A new equation taking into account the effect of dilution resulting from the continuous titration process is obtained. It allows to consider efficiently the continuum of possibilities from perfect to imperfect mixing of the cell content. It is shown that, to good approximation, imperfect mixing can be accounted for by considering the cell volume as an adjustable parameter. Most likely, this should lead to an artificial increase of it, although one cannot reject the possibility of a decrease. The processing of experimental data on the interaction of Ba++ with 18-crown-6 from led to an increase by 6.9%, which resulted in a much better fit of the titration curve and improved results on the association constant Ka and enthalpy variation ∆H. A criterion is also obtained on the maximum injection rate to be used for maintaining quasi-equilibrium during the whole titration for the association-dissociation mechanism [Formula: see text].