Kindly suggest how long should I incubate the sample mixture after adding FRAP reagent. The procedure I follow mentions to check absorbance after 4 min. But at 4 mins. The absorbance kept on increasing. The readings were stable only after an hour or so, but the OD readings where quite high > 1. Kindly suggest the proper way to do it
Hello Poovamma B C
FRAP values are strongly dependent upon incubation time. Therefore, the FRAP index evaluated at 4 min (as per the standard method) does not necessarily reflect the total antioxidant capacity of the sample. It may be possible that not all antioxidants are able to reduce the Fe+3-TPTZ complex rapidly enough to complete the reaction in a short time (4 mins). For this reason, when you increase the time of incubation of the sample with the FRAP reagent, the sample exhibiting lower reactivity will require longer time, and therefore, will be able to reduce the Fe+3-TPTZ complex into the ferrous form.
I feel it is important to standardize and apply adequate time for the reaction. If this is not taken into consideration, underestimation of the real antioxidant capacity will occur. I recommend that the kinetic FRAP profile should be realized for the sample which is to be analyzed to establish the most suitable incubation period to titrate all the reactive antioxidant species.
The below attached article will help!
https://www.alanrevista.org/ediciones/2011/3/art-13/#:~:text=The%20FRAP%20methodology%20has%20been,the%20ferrous%20complex%20(17).
Best.
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