Determination of the activity of an antioxidant by the DPPH° assay

Materials and reagents

• Glassware, pipettes…
• Visible spectrophotometer and cuvettes
• Ethanol 96° (spectrophotometric quality or HPLC grade)
• DPPH° solutions (M=394.3 g/mol) :
  • stock solution at 6.10^-3 mol/L, stable at -18°C for 1 week: weigh 0.0236 g of DPPH° in a 10 mL volumetric flask, make up with ethanol and place it in an ultrasonic bath for 15 minutes at room temperature.
  • daughter solution at 6.10^-5 mol/L, prepared extemporaneously by dilution at 1/100 in the ethanol 96° of the DPPH° stock solution. Make sure that the absorbance at 515 nm for this solution is between 0.6 and 0.7.
• Antioxidants: solutions to be prepared in ethanol 96° (make sure that they do not absorb at 515 nm).

Note: depending on the solubility of the antioxidants, the test can be performed in other solvents, provided that DPPH° is soluble in them.

PROTOCOL

• Zero the spectrophotometer with ethanol 96°.
• In the first spectrophotometric cuvette, place 3 mL of the daughter solution of DPPH° at 6.10^-5 mol/L. Add 77 μL of ethanol 96°. Mix by whirling. Start the stopwatch and measure the absorbance at 515 nm at regular intervals (e.g. every minute for 15 minutes, then every 15 minutes). This cuvette will be called the reference cuvette and will be kept in the dark between two measurements.
• In a second spectrophotometric cuvette, place 3 mL of the daughter solution of DPPH° at 6.10^-5 mol/L. Add 77 μL of the solution containing the antioxidant (pure molecule or extract). Start the stopwatch and measure the absorbance at 515 nm at regular intervals (e.g. every minute for 15 minutes, then every 15 minutes until reaching a constant value). This cuvette will be called a sample cuvette and will be kept in the dark between two measurements.
• Prepare as many spectrophotometric cuvettes as there are antioxidant samples to be tested (different concentrations, different antioxidants)