The solution was continuously bubbled with a gentle stream of air. Samples of irradiated solutions learn more were examined for degradation using thin-layer chromatography and RF and photoproducts were assayed spectrophotometrically at appropriate intervals. The photoproducts formed during the degradation

of RF at pH 4.0–7.0 in the presence of citrate buffer were detected by TLC on cellulose plates (Whatman CC 40) using (a) 1-butanol–acetic acid–water (40:10:50, v/v, organic phase) and (b) 1-butanol–1-propanol–acetic acid–water (50:30:2:18, v/v) as solvent systems [8]. All spectral determinations on RF and its photodegraded solutions were carried out on a Shimadzu UV-1601 recording spectrophotometer using silica cells of 10 mm path length. Fluorescence measurements of RF solutions were MK8776 carried out at room temperature (∼25 °C) using a Spectramax 5 fluorimeter (Molecular Devices, USA) in the end point mode using λex=374 nm and λem=520 nm [33]. The fluorescence was recorded in relative fluorescence

unit using a pure 0.05 mM RF solution as standard. RF and photoproducts in degraded solutions were assayed using a specific multicomponent spectrophotometric method previously developed by Ahmad and Rapson [1]. The method is based on preadjustment of photolysed solutions to pH 2.0 (HCl–KCl buffer), chloroform extraction to remove the photoproducts, LC and LF, and their determination, after chloroform evaporation, at pH 4.5 (acetate buffer) by a two-component assay at 445 and 356 nm. The aqueous phase was assayed for RF and FMF by a two-component assay at 445 and 385 nm. The intensity of the radiation source (125 W Phillips HPLN lamp) was determined by the method of Hatchard and Parker [21] using potassium ferrioxalate actinometry as 1.15±0.10×1017 quanta s–1. It is important to ascertain the nature of RF photoproducts under the reaction conditions employed before the assay procedure could be applied to determine these compounds in degraded solutions.

The photoproducts formed during the degradation of RF at pH 4.0–7.0 in the presence of citrate buffer were detected by TLC and identified as formylmethylflavin (FMF), carboxymethylflavin (CMF), lumichrome (LC) and lumiflavin (LF) crotamiton by their characteristic fluorescence under UV excitation (RF, FMF, CMF, LF, yellow green; LC, sky blue) and comparison of Rf values with those of the authentic compounds. FMF, LC (major) and CMF (minor) are produced throughout the pH range whereas LF is produced only at pH 7.0 as observed in a previous study [10]. The formation of these products has been found to decrease with an increase in buffer concentration indicating the inhibitory role of citrate buffer on the photolysis reaction. The absorption spectra of the aqueous phase (pH 2.0) of photolysed solutions of RF show a gradual decrease in absorption at 445 nm with a concomitant increase at around 385 nm indicating the loss of RF.