I am trying to apply the Fenton system (h2o2/FeSO4) to the 20-hydroxyecdysone (20E) in order to study the formation of bioactive metabolites through ROS scavenging, but the reaction doesn't succeed, I mean the starting material is always present after many hours and I changed many parameters for my reaction with no results.
The experiment has to be carried out anaerobically. If you bring a iron(II) sulfate solution to pH 7 in air, then it will autoxidise rapidly and the Fenton reaction will not occur.
H2O2 is a strong oxidising reagent, together with Fe(II) it always results quickliy in Fe(III), a brownisch sometimes black-blue-green-grey mixture of Fe(II) +Fe(III) if pH is larger than 7..
Take a look at the reference below. It describes in sufficient detail how to perform Fenton-reaction experiments and proposes a control assay to account for the autoxidation of Fe(II) related by Dr. Koppenol.
Genaro-Mattos TC, Dalvi LT, Oliveira RG, et al (2009) Reevaluation of the 2-deoxyribose assay for determination of free radical formation. Biochim Biophys Acta 1790:1636–1642. doi: 10.1016/j.bbagen.2009.09.003
http://www.sciencedirect.com/science/article/pii/S0304416509002657
I am performing the reaction at pH3 in order to avoid the precipiration of Iron
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