Influence of pH on the intensity and stability of the fluorescence of p-aminobenzoic acid in aqueous solutions

It is known that the fluorescence of p-aminobenzoic acid (PABA) is pH-dependent. However, recent observations showed that the fluorescence of PABA dissolved in water and in buffered solutions at physiological pH (7.4) decreases with time. A study was therefore performed to investigate these changes and find solutions for the problem. The first part of this study was to investigate the interdependence of the fluorescence intensity and the pH of a PABA solution in various acid-base mixtures. It turned out that this dependence is sigmoid, i.e. the fluorescence intensity is very low below pH 4, and it maximizes at pH 6 and beyond. In the second part, the fluorescence intensity was studied as a function of time. PABA was dissolved at the concentrations of 10, 50, 100 ng/ml and 1 mu g/ml in various standard solutions: 0.1 N HCl, phosphate buffered saline (PBS) (pH 7.4), TRIS buffer (pH 7.4), berate buffer (pH 9.0) and 1 N NH4OH. PABA did not show any fluorescence at all in 0.1 N HCl, while showing stable fluorescence up to 7 days in berate buffer (pH 9.0) and in 1 N NH4OH. In water, PBS and TRIS buffer, the fluorescence of PABA decreased with time, with a higher decay rate in more diluted solutions. After 4 days, the fluorescence intensity of 10 and 50 ng/ml PABA in these solutions was practically zero. When concentrated NaOH was added to those solutions at time zero, the fluorescence intensity could be maintained. Apparently, at any pH below 9.0, a transformation of the fluorescent form of PABA to the unionized form takes place. Hence, pH should be taken into careful consideration when analysing PABA in aqueous solutions using fluorescence spectrometry.