I follow Ruch et al 1989 method for h202 scavenging assay but instead of decrease in absorbance I am getting increase in absorbance. I am using phosphate buffer as blank, h202(40mM)+phosphate buffer as control. the absorbance of control as also exceeding from range. please guide me?
Hi
I had also faced the same problem. I think the hydrogen peroxide which you are using might have degraded. H2O2 is usually degraded by autooxidation. Secondly you need to prepare fresh H2O2 at the time of experiment after all other reagent preparation. You should make H2O2 in amber coloured volumetric flask. If you follow all these you will get good results.
Good luck
We have faced the same problem. What we did was as Mr. Yadu said used fresh H2O2 and prepared tat the same time and stored in a amber bottle as well.
Good Luck!!
Hello Mr.Muhammad waqar Khan....you have to ensure the following in performing this radical scavenging assay..
1.Make use of PBS tablets in making the PBS..the other method of adding all the ingredients prescribed is tedious and often results in undesirable pH...if you don't have a pH meter just use the PBS tablets
2. Always prepare fresh Hydrogen-peroxide at the beginning of each experiment and store under refrigeration and away from light
3.Ensure you make the accurate concentration of hydrogenperoxide (40mM).Your calculations should be exact.
4.Ensure your UV-VIS spectrophotometer is well calibrated to record absorbance values accurately.
5.Perform all experiments in triplicate and express the results as mean+/-standard error of the mean.
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