Studies on fluorescein-V The absorbance of fluorescein in the ultraviolet, as a function of pH

The ultraviolet absorption spectum of an aqueous solution of highly-purified yellow fluorescein at ionic strength 0.10 has been measured at various pH values in the range from 0.15 to 8.70. The maxima at 227, 249 and 295 nm change little with pH, but the maximum found at 437 nm in acid medium changes greatly in absorbance and position on addition of alkali, resolving first into two maxima, at 455 and 475 nm, and finally becoming a single large maximum (at pH 8) at 490 nm. A unique feature of the absorption at 437 nm is that all four prototropic forms of fluorescein, H(3)Fl(+), H(2)Fl, HFl(-) and Fl(2-), absorb at this wavelength. The total absorbance at this wavelength first falls rapidly as the pH rises from 0.15, reaching a minimum at pH 3.63, then increases to a maximum at pH 5.3, and finally falls to steady value at pH > 8.0. The absorbance as a function of pH is defined by seven constants: three dissociation constants (K(H(3)Fl) = 6.61 x 10(-3), K(H(2)Fl) = 3.98 x 10(-5), K(HFl) = 4.36 x 10(-10)) and four molar absorptivities ((H(3)Fl) = 4.94 x 10(-4), (H(2)Fl) = 1.20(5) x 10(4), (HFl) = 2.16 x 10(4) and (Fl) = 7.61 x 10(3) 1.mole(-1).cm(-1)). Solutions of yellow fluorescein in water undergo rapid deterioration on exposure to daylight or fluorescent lighting but are stable in the dark.