In the present paper, a sensitive, fast and suitable method for the calculation of pK(a) values of fluorescein is proposed. The effects of sodium dodecyl sulfate (SDS) and Triton X-100 (TX-100) as a surface-active agent on the acidic and basic forms, and the spectral properties of fluorescein were studied by the spectrophotometric method. The study was performed in sub-micelle surfactant concentration, and absorption spectra at 300-550 nm intervals were recorded. Stepwise acidity constants of fluorescein at pH range 1.50-10.00 and at constant ionic strength 0.1M and 25 degrees C were determined using DATAN program using pH-spectrophotometric titration data. The method is efficient, however the component spectra showed intensive overlapping. The calculated acidity constants of fluorescein in water at ionic strength 0.1 are pK(a1)=2.20, pK(a2)=4.30 and pK(a3)=6.43. The acidity constants of the dye in ethanol-water solution were studied by the same procedure. Effect of surfactants and ethanol on acidity constants and pure spectrum of each component are also discussed.
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