Plant material extract was prepared in PCA. Then, it is treated with Potassium thiocynate and ferrous ammonium sulphate. OD at 480 nm. How to calculate the amount of H2O2 in the plant extract? What standard should I use?
Hach makes a kit to measure H2O2 that is fairly similar (link below). I used this a while ago when I needed to measure H2O2 concentration produced by a bench scale H2O2 reactor.
It does not state what absorbance peak to look at or a extinction coefficient though. This kit simply looks for a color change and then a calculation based on how much titrant was added is done to calculate H2O2 concentration. H2O2 does not absorb at 480 nm so you are trying to measure H2O2 based on some other species. Where did you get the protocol you are using from?
http://www.hach.com/hydrogen-peroxide-test-kit-model-hyp-1/product?id=7640218476&callback=qs
Thank you Michael.. I am using following article for protocol. Please check the URL
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC542013/
There is a little more description of the method in ref 13:
http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1972.tb01711.x/pdf
It says this method came from ref 16 but this is just a personal communication with Kreamer, M. which is not helpful. You really need the extinction coefficient for the ferrithiocyanate at 480 nm to directly calculate the concentration from an absorbance measurement. In the above reference they state that they compare the measured absorbance values with the results from standard H2O2 solutions. None of the papers I have found that reference this method give an extinction coefficient so I am guessing they all just compare to a standard curve. If you make a standard curve with solutions of known H2O2 concentration, you can then do a linear fit and get an equation y = mx + b where y is absorbance, x is H2O2 concentration, m is the slope and b is the intercept. Use this equation and the OD 480 nm value you measured to then calculate H2O2 of the plant sample. You should be able to do serial dilutions on store bought 3% H2O2 to make your standard curve. You can also also purchase 30% and 50% H2O2 but I would guess that the H2O2 concentration is plant material would be less than 3%.
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