Determining binding constants for 1:1 and 1:2 inclusion complexes of ester betulin derivatives with (2-hydroxypropyl)-β-cyclodextrin by affinity capillary electrophoresis

The complexation of ester betulin derivatives with (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) was studied by mobility shift affinity CE. Electrophoretic mobility for triangular peaks was calculated using the parameter a₁ of the Haarhoff–Van der Linde function instead of the peak top time. Dependences of the viscosity corrected electrophoretic mobility on HP-β-CD concentration were not described on the basis of only complexes with 1:1 stoichiometry due to the fact that these binding curves did not reach a plateau. However, the dependences were well described taking into account both 1:1 and 1:2 complexes. The presence of higher order equilibria was also revealed by x-reciprocal plots. The values of apparent binding constant logarithm, obtained for the first time, for 1:1 and 1:2 HP-β-CD complexes of betulin 3,28-diphthalate and betulin 3,28-disuccinate with 95% confidence interval limits in brackets are the same within error and are equal to 4.85 (4.73–4.95), 8.56 (7.75–8.82), 4.92 (4.86–4.97), and 8.54 (8.23–8.72) at 25°C, respectively. These values for 1:1 and 1:2 HP-β-CD complexes of betulin 3,28-disulfate at 25°C are 4.61 (4.57–4.64) and 7.11 (6.57–7.34), respectively. The binding constants for betulin 3,28-disulfate agree with the previously obtained results from the separation in the thermostatted capillary segment.