Multiple complex formation of fluorescent compounds with cyclodextrins: Efficient determination and evaluation of the binding constant with improved fluorometric studies

Equations are derived and allow the determination of the cyclodextrin compound complex primary and secondary binding constants by using fluorometric studies (by monitoring the fluorescence intensity of the compound in the presence of cyclodextrins). The fluorescence intensity is dependent on the concentration of cyclodextrins in the solution of the compound as well as on the stoichiometry of the cyclodextrin compound complex. It was found that riboflavin (fluorescent compound) forms 1:2 inclusion complex with cx-cyclodextrin and 1:1 with gamma-cyclodextrin. The complex stoichiometries were calculated using the continuous variation method, and the binding constants were calculated fluorometrically and were evaluated with kinetic studies in order to compare the values from the two methods. The fact that the same compound (riboflavin) can exhibit different binding behaviors with different cyclodextrins could result in new descriptive studies for each particular case.