pH dependent spectral properties of sodium fluorescein ophthalmic solutions revisited.

PURPOSE: To re-assess and further quantify the pH-related changes in the absorbance and fluorescence emission spectra of sodium fluorescein in buffered aqueous solutions.
METHODS: Analytical grade sodium fluorescein (NaF) or commercial NaF ophthalmic products (Minims 2% sodium fluorescein or Fluorets sodium fluorescein ophthalmic strips) were prepared over a range of dilutions in 1% NaCl with the pH set with phosphate, borate or acetate buffer mixtures between pH 4.0 and 10.0 at room temperature. Absorbance and fluorescence spectra were recorded in 10 mm pathlength cuvettes.
RESULTS: At pH 7.5-8.5, the main NaF absorbance in dilute solution (0.005% w/v, or c. 13 microM) is at 490 nm, but this peak shifts progressively to 460 nm as the pH is lowered below 7.5. The 490 nm absorption peak shows a 50% of maximum value at pH 6.75. For extremely diluted solutions of NaF (0.000002%, c. 50 nM), the fluorescence emission at 513 nm follows the same profile. At higher concentrations (>0.000005%), NaF solutions start to show marked fluorescence quenching at neutral pH with the emission wavelength progressively shifting to 530 nm and even to 560 nm at extremely high concentrations (0.125%) where the fluorescence is all but quenched.
CONCLUSIONS: The present studies confirm and extend some earlier reports that pH (over the range of 4.0-8.5), as well as concentration, predictably determines the fluorescence emission of dilute aqueous solutions of NaF.