Dr Prieto's DPPH Microplate Protocol 02/07/12

Procedure: Preparation of DPPH Radical, and

antioxidant scavenging assay
Dr Jose M. Prieto
This is an assay for scavenging activity against free radicals. The scavenging activity of Natural
products can be assayed by measuring the decrease in absorbance at 517 nm of the stable free
radical DPPH. The purple-coloured free radical reacts with scavenger to yield the colourless
product 1,1-diphenyl-2-picrylhydrazine.

Apparatus:
• Multichannel pipette
• Flat transparent 96 well plate with lid (may be reused if washed)
• Spectrophotometer (plate reader)
• High precision balance
• Volumetric flask (500ml/50ml)
• Beakers

Reagents:
• DPPH powder
• Methanol (buffered or plain, HPLC grade)
• Ultrafiltered (0.22 µm), bidistilled water (MiliQ system)

Preparation of the DPPH radical at 0.2 mM in MeOH.

500ml of the DPPH are prepared by weighing 39.4mg of DPPH powder on a high precision balance
preferably using an eppendorff microcentrifuge tube, in which 1ml of MeOH is added, the tube

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Dr Prieto's DPPH Microplate Protocol 02/07/12

closed and vortexed until complete dissolution. Spin down the tube in a centrifuge and transfer
quantitatively to the 500ml Volumetric flask with the help of a micropipette and rinsing several
times with MEOH.. The volumetric flask is made up to the meniscus, with MeOH.

Storage and further use

This solution is kept in the fridge wrapped in foil when not in use, to reduce its degradation (light
induced). The solution degrades at a rate of 2-4% each week, and was remade weekly if necessary,
or if the absorbance at 515nm, was significantly changed.
Before use, it must be taken out from the fridge and allowed to reach room temperature, otherwise
the concentration will be higher due to volume contraction.

Microplate assay
A column with DPPH only, a column for each sample only and a column with sample plus DPH in
equal volumes.
First step: add to the 96-wells microplate the following reagents
1 2 3 4 5 6 7 8 9 10 11 12
Control Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 Cal line
Blank + DPPH Blank + DPPH Blank + DPPH Blank + DPPH Blank + DPPH
A MeOH Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 Sample 1 MeOH
100µL 200µL 200µL 200µL 200µL 200µL 200µL 200µL 200µL 200µL 200µL 100µL
B MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
C MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
D MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
E MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
F MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
G MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
H MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL

Note: MeOH can be replaced by Water if the sample is insoluble in alcohol. DPPH is insoluble in
water, but in our hands the reaction do not change significantly in up to 50% water in MeOH. If
neded, buffered MeOH or buffered water may be used (We use MeOH HPLC grade buffered with
Ammonium acetate which is commercially available and ready to use).
Second step: with a multichannel pipette, take 100 µL from the first row of columns 2 to 12 and
transfer them into the matching wells in the second row. Mix the contents with the pipette 4-5 times.
After this, transfer exactly 100 µL to the thrid row and repeat the process until the lat one,
discatrding the last 100 µL.
Every well has now only 100 µL and 8 double dilutions have been done across the columns 2 – 12.
Third step: add 100 µL of the DPPH 0.2 mM solution to each well in the plate.

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Dr Prieto's DPPH Microplate Protocol 02/07/12

1 2 3 4 5 6 7 8 9 10 11 12
Control Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 Cal line
Blank + DPPH Blank + DPPH Blank + DPPH Blank + DPPH Blank + DPPH
A DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 200 200 200 200 200 200 200 200 200 200 100 µM
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
B DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 100 100 100 100 100 100 100 100 100 100 50 µM
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
C DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 50 50 50 50 50 50 50 50 50 50 25 µM
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
D DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 25µg/mL 12.5 µM
E DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 6.25 µM
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
F DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 3.125
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µM
G DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 1.075
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µM
H DPPH Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5 DPPH
0.1 mM 1.075 1.075 1.075 1.075 1.075 1.075 1.075 1.075 1.075 1.075 100 µM
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL

Each compounds IC50 value was determined using the same method.
The plate is covered with the lid to minimise evaporation and then wrapped in foil or kept in a
drawer (or incubator if constant temperature different from room temperature is needed) in order
to protect the DPPH radical from degradation by light, and left for 30 minutes. For most samples
this is enough time to ensure the reaction is complete and a steady state had been achieved in each
well. However the plate could be read at later time points if needed. The absorbance (515nm) is
then read in a microplate reader.
For each sample, assays are run in duplicate or triplicate.

Statistics
• The percentage of radical scavenging can be obtained using the following expression:
% DPPH scavenging = 100 x [(Abs Sample+DPPH)-(Abs Sample Blank)]/[(Abs DPPH)-(Abs
Solvent)]
• The absolute moles of DPPH scavenged or left can be calculated by interpolating the
[(Abs of the sample)-(Abs Sample Blank)] into a calibration line generated by the Abs of
DPPH at diferent concentrations (Column 12). Be careful to eliminate the point deviating
from linearity, which depends on the spectrophotometer.
• The Efficient concentration (EC50) (concentration of sample scavenging 50% of the
initial DPPH radical). It can be calculated by interpolating the [(Abs of the sample)-(Abs
Sample Blank)] into a calibration line generated by the Abs of DPPH at diferent
concentrations (Column 12). Be careful to eliminate the point deviating from linearity,
which depends on the spectrophotometer.

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