Binding constants determination of cyclodextrin inclusion complexes by affinity capillary electrophoresis. How to overcome the limitations induced by the UV-detector?

In most cases, determination of binding constant for analyte-cyclodextrin complexes in capillary electrophoresis is investigated by affinity capillary electrophoresis using a UV detector (ACE-UV). The limitations induced by the UV-detector include : (i) the difficulty of dealing with poor chromogenic analytes and more generally with any analyte presenting strong affinity towards the cyclodextrin (CD), i.e. for which the prerequisite to work with analyte concentration much smaller than those of the CD is difficult to fulfill (ii) the impossibility of studying non-chromogenic analyte. In this paper, two simple methodologies were developed to overcome these limitations. Regarding the analytes which present poor UV-absorbance and/or very high CD-affinity, a methodology using an algorithmic data treatment and taking into account the real analyte concentration in the capillary at the determined migration times allows to correctly estimate the binding constants, even if the experimental prerequisite ([analyte]<<[CD]) is not complied. Moreover, it is proved that classical linearization treatment by picking the migration time of the infinite diluted analytes (at the start of the peak) also provide satisfactory results. Regarding UV-transparent analyte, a competitive methodology combined with algorithmic data treatment allows the determination of their affinity towards cyclodextrins. Last, the applicability of the described competitive method is extended to the study of interaction between two neutral partners, which is another well-known limitation of ACE.

Improving ITC studies of cyclodextrin inclusion compounds by global analysis of conventional and non-conventional experiments.

The study of 1:1 cyclodextrin inclusion compounds by isothermal titration calorimetry was explored in a theoretical and experimental point of view to compare the efficiency of conventional and non-conventional experiments. All direct and competitive protocols were described and evaluated in terms of accuracy on both binding constant and inclusion enthalpy. Significant improvement in the calorimetric characterization may be obtained by means of the global analysis of non-conventional experiments coupled to the standard titration protocol. While the titration-release approach proved to be the most accurate strategy for classical complexations, the valuable contribution of other non-conventional experiments was demonstrated for issues concerning weak stability, enthalpy, or solubility.