The influence of beta-cyclodextrin on acid-base and tautomeric equilibrium of fluorescein dyes in aqueous solution.

The protolytic equilibrium of fluorescein in aqueous solutions was studied in the presence of cycloheptaamylose (beta-cyclodextrin, or beta-CD). The constants of stepwise ionization of the dye (H(3)R(+)left arrow over right arrowH(2)Rleft arrow over right arrow HR(-)left arrow over right arrowR(2-)), K(a0), K(a1), and K(a2) were determined using vis-spectroscopy at ionic strength 0.05 M (NaCl+buffer) and 25 degrees C. In the presence of 0.0086 M beta-CD, the indices of ionization constants are as follows: pK(a0)=1.21+/-0.12, pK(a1)=5.08+/-0.03, pK(a2)=6.35+/-0.02. The changes in these pK(a)s, as compared with the values determined without cyclodextrin, are unequal. Namely, the pK(a0) value decreases by 1.0, while the pK(a1) value increases by 0.7. Thus, the introduction of beta-CD allows to govern the ratios K(a0)/K(a1) and K(a1)/K(a2), which are equal to, respectively, 141 and 151 in water, and 7.4 x 10(3) 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 pK(a) alterations is the binding of H(2)R 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.