I’m performing a DPPH radical scavenging assay using Trolox as a standard antioxidant, but my inhibition curve plateaus just below 50%, even at higher Trolox concentrations. I’ve run the assay in duplicate with the following Trolox concentrations (in µg/mL): 0, 12.5, 25, 50, 100, 125, and 250.
Details of my protocol:
- DPPH concentration: 0.2 mM
- Solvent: 95% ethanol (for both DPPH and Trolox solutions)
- Absorbance measured at 517 nm at 1-minute intervals.
- Solutions mixed at 1-minute intervals.
- Incubation: Samples were incubated in the dark for 30 minutes before final readings.
Despite increasing the Trolox concentration up to 250 µg/mL, inhibition does not exceed 50%. I’ve ensured my reagents are fresh, and my 0 µg/mL control gives the expected DPPH absorbance.
Could this plateau be due to:
Has anyone experienced this, or can you suggest adjustments? I’d appreciate any insights!
Play with different concentrations of DPPH. 0.2 mM (200 microM) DPPH is very high to be neutralized. The availability and reactivity of Trolox may not be ideal under these conditions.
Trolox as an antioxidant for the DPPH-generated radicals may not be ideal.
DPPH radicals react with phenols, which are the most reactive and important ones that react easily with DPPH.
Try other phenols (natural polyphenols) along with Trolox.
Great project.
Parinandi
The Ohio State University Wexner Medical Center
Columbus, OH
One possible reason for the inhibition plateauing below 50% could be the DPPH concentration being too high (0.2 mM), leading to an excess of radicals that even higher Trolox concentrations cannot fully neutralize. You may try reducing the DPPH concentration to 0.1 mM or lower to see if it improves inhibition.
Another factor could be solubility or reaction time ensuring thorough mixing and extending incubation slightly might help. Also, check if Trolox degrades in ethanol over time, as this could affect its antioxidant activity.
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