I was trying to measure antioxidant activity from bacterial crude extract. However, the absorbance of sample+DPPH is greater that absorbance of DPPH, therefore, I could not calculate the result. Is it unusual to get the result like this? since there was no problem with positive control (VitE).
My samples are colorless, very pale blue and very pale orange, but all the samples give similar trend of absorbance.
That should not be the case. The colour of the sample if too pale should not interfere with the absorbance. But since your positive control is doing fine, you should recheck your sample preparation
Nirjhar Dasgupta
I simply disrupted the cell in DMSO and did methanol extraction.
usually i face problems with DMSO. are you using DMSO analytical grade ?can you change it ..? maybe you can disrupted the cell in some other solvent .
It is assumed the DPPH is make up on methanol as usual. Increasing DPPH absorbance implies increased radical concentration? I would agree with Dr Khan and recommend doing a DMSO blank by estimating amount of DMSO in your sample and then preparing & analyzing a solvent blank (DMSO in DI water) accordingly . As discussed in another thread, DMSO use in extractions etc is problematic; this solvent is non-volatile and difficult to remove once added to a biological sample.
@muhammad I found that carotenoids in sample also interferes the abs value. I’m try another assay.
Carotenoid in the sample? Well that explains a great deal. Please see
Muller L. et al. (2011) Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP) , ABTS bleaching assay (aTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chemistry 129, 139-148.
[Crotenoids addition increasesd DPPH aborbance additively, showed no radical quenching + paper has aternative assays in which coretenoids show antioxidant activity (ABTS, FRAP). A choice of alternative assays depends whether you want to measure total antioxiant capacity with/out or with carotenoids.
@ Natthaporn Takpho yeah you can try that as well ..also prof Richard K Owusu-Apenten rise some valuable points . i am sure it will help you alot.
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