A study of photodecomposition of folic acid by ultraviolet radiation (254 nm) in the pH range 2.0-10.0 and in selected organic solvents and their aqueous solutions has been made. The role of riboflavin as a sensitizer in the photooxidation of folic acid in the pH range 4.0-9.0 has been evaluated.
A HPLC method has been developed for the separation, identification and assay of folic acid and its photodegradation products in the presence and absence of riboflavin. The method is specific and has a precision of ±3% p-Aminobenzoyl-L-glutamic acid and pterine-6- carboxylic acid have been identified as the major photoproducts of folic acid both in the presence and absence of riboflavin in the pH range studied.
Folic acid solution is photolysed by first order kinetics and the apparent first order rate constant for the reaction range from 0.155x10-3 min.-1 (pH 10.0) to 5.04x10-3 min.-1 (pH 2.5). The second order rate constants for the bimolecular interaction of folic acid ad riboflavin range from 0.500x103 M-1 (pH 9.0) to 3.00x10 3M-1 min.-1 (pH 6.2).
The pH-rate profiles for the photolysis of folic acid alone and in the presence of riboflavin have been prepared to observe the influence of pH on the rate of reaction. Folic acid photolysis shows a gradual decrease in rate in the pH range 2-10 and the profile indicates the appearance of three steps which may result from the participation of different species of folic acid (pka, 2.3, pka2 8.3) in the reaction. In the presence of riboflavin the pH rate profile (bell shaped curve) shows a gradual increase in the rate upto pH 6.2 (pH max) followed by a decrease upto pH 9.0. Thus the photolysis of folic acid depicts a different behavour in the presence of riboflavin and the rate may be influenced by te nature of interaction of the two species in a particular pH range.
An increase in HPO24 ion concentration (pH 7.0) in an equimkolar solution of folic acid and riboflavin leads to a gradual decrease in the rate of photolysis due to the formation of HPO24 riboflavin complex in the ground state. This has been confirmed by a decrease in the fluorescence intensity of riboflavin in the solutions. The HPO24 riboflavin complex is known to undergo cyclophotoaddition reaction thus inhibiting the mutual interaction of riboflavin and folic acid.
The influence of solvent parameter such as dielectric constant and viscosity on the photolysis of folic acid has been studied A plot of Kobs versus dielectric constant shows linear dependence of the rate on dielectric constant of the medium. Similarly a plot of k (organic solvents) and k (aqueous mixture of organic solvents) versus viscosity (cp-1) is also linear indicating a decrease in the rate as a function of reciprocal of solvent viscosity.
Reaction schemes for the photolysis of folic acid alone and in the presence of riboflavin have been presented and kinetic evidence for the participation of riboflavin as a photosensitizer in this reaction has been provided.