Time-Course Reaction of Blue Dye after treatment with hydrogen peroxide

Objectives: To evaluate the time-course reaction of a blue food dye on exposure to hydrogen peroxide with the use of thin layer chromatography (TLC) and spectrophotometer and determine the relationship between the two types of measurements.
Methods: Time course reaction of 0.1ml of dye (Brilliant Blue FCF) and hydrogen peroxide (HP) with 0.5ml of pH 9 buffer was evaluated. Aliquots of reaction mixture at different interval times (0,5,10,15,20,30,45,60min) were transferred onto a TLC plate and intensity of dye was analyzed with computer program TNIMAGE. This procedure was repeated using a spectrophotometer (Power Wave X-I) on seven samples at peak wavelength of 630nm at respective time-intervals. Pearson correlation test was used to evaluate the relationship between the average absorbance level and the area under the curve for blue dye. Friedman test, followed by the post-hoc Bonferroni test, was used to detect the difference in absorbance levels among the eight different time points (alpha=0.05).
Results: Within one hour, the median reaction color absorbance dropped from 3.56(0min) to 0.24 (60min) and the area on the TLC plate decreased from 211 (0min) to 3 (60min). Significant differences in median absorbance levels were found among all eight time points (p<0.05 for all instances). The data showed that the absorbance levels significantly decreased as time increased (mean (SD): 3.57(0.05) for 0-min; 2.74(0.13) for 5-min; 2.14(0.10) for 10-min; 1.65(0.08) for 15-min; 1.28(0.07) for 20-min; 0.79 (0.07) for 30-min; 0.41(0.05) for 45-min; 0.23(0.03) for 60-min). A strong correlation between the absorbance level and the area under the curve was found (r=0.99, p<0.0001).
Conclusions: Time-course reaction of a blue dye on exposure to HP can be evaluated with TLC and spectrophotometer, which showed a strong correlation in this study.