I was determining flavonoid concentration using AlCl3 method from natural product extract (based on Estimation of Total Phenols and Flavonoids in Extracts of
Actaea spicata Roots and Antioxidant Activity Studies; by Madan, Banshal, Kumar, Sharma; 2011) and used Sodium acetate instead of Potassium acetate (Evaluation of Antioxidant Activity, Total Flavonoids, Tannins
and Phenolic Compounds in Psychotria Leaf Extracts; Formagio et al, 2014).
EXPT-1.
I have prepared a calibration curve (With R^2=0.9918); by preparing different concentration of Quercetin (SRL chemicals) yellow powder in methanol. I did not notice any 'perceivable' colour change after I add reagents into Quercetin aliquot. However I prepared a conventional calibration curve.
It gave me a strange result. Very high flavonoid content in the test extracts; even greater than Total phenol content (by FCR method) of the test extracts
EXPT-2
Based on this ( https://chemistry.stackexchange.com/questions/35389/does-the-aluminium-chloride-assay-ordonez-et-al-2006-for-total-flavonoid-conte ) stackexchange post; I prepared a control set for the yellowish background correction (for each concentration of quercetin) where only water was added to quercetin aliquot (instead of same volume of AlCl3 reagent). This second experiment gave me weird result; the ODs increase with quercetin aliquot but OD(Reaction) - OD(Control) keeps fluctuating (positive and negative values at random).
Where I might be doing the mistake? Anybody else encountered similar problems?
Hello Rajarshi. First check again - Does quercetin dissolved in methanol?. Second can you try using Rutin in place of quercetin for the calibration curve? Just prepare concentration of 20-100microgram of rutin using serial dilution. That will give you a straight line graph with equation for the estimation of your flavonoid content.
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