The influence of β-cyclodextrin on acid–base and tautomeric equilibrium of fluorescein dyes in aqueous solution Original Research Article

The protolytic equilibrium of fluorescein in aqueous solutions was studied in the presence of cycloheptaamylose (β-cyclodextrin, or β-CD). The constants of stepwise ionization of the dye (image), Ka0, Ka1, and Ka2 were determined using vis-spectroscopy at ionic strength 0.05 M (NaCl + buffer) and 25 °C. In the presence of 0.0086 M β-CD, the indices of ionization constants are as follows: pKa0 = 1.21 ± 0.12, pKa1 = 5.08 ± 0.03, pKa2 = 6.35 ± 0.02. The changes in these pKas, as compared with the values determined without cyclodextrin, are unequal. Namely, the pKa0 value decreases by 1.0, while the pKa1 value increases by 0.7. Thus, the introduction of β-CD allows to govern the ratios Ka0/Ka1 and Ka1/Ka2, which are equal to, respectively, 141 and 151 in water, and 7.4 × 103 and 18.6 with cyclodextrin added. Rationalization of the observed phenomenon is possible taking into account the detailed scheme of protolytic equilibrium. Conclusions concerning tautomerism of dye molecules were deduced from absorption spectra; the fractions of tautomers, tautomerization constants, and microscopic ionization constants were evaluated. These data allow concluding that the main reason for the aforementioned pKa alterations is the binding of H2R by the cyclodextrin cavity accompanied by turning these neutral species into the colorless lactone. The host–guest interaction of neutral species of fluorescein isothiocyanate, 2,7-dichlorofluorescein, and 3′,4′,5′,6′-tetrachlorofluorescein also results in the cyclodextrin-assisted shift of tautomeric equilibrium. Such nature of interactions is proved by the addition of competing agents, camphor-4-carboxylic acid and sodium n-nonylsulfonate, which results in the removing of neutral dye species from the cycloheptaamylose cavity.