I am using ABTS assay to investigate the antioxidant capacity on sorghum flour but I'm having issues with the calibration curve (trolox stock 0,025g in 100ml MetOH 50%) ranging from 5 to 900 µmol/L.
For the ABTS solution I am mixing
- 5mL of 2.6mM potassium persulphate
- 5mL of 8mM ABTS
and after letting them react for 16h at room temperature in the dark I adjust the absorbance at 743nm between 0.7 and 0.8 with 80% EtOH (HPLC grade).
The problem is that the curve is not linear with an R2 lower than 0.98 (the standards are fine and already checked) but I do not understand what I am doing wrong.
You have to prepare ABTS and Trolox solutions in 50 mM phosphate buffer at pH 7.2. Don't use alcohols. The sample could be in alcohol, but minimize its content.
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