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DPPH Scavenging Assay Protocol- Detailed Procedure

DPPH Scavenging Assay Protocol- The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay is a widely used method to assess the ability of compounds or extracts to act as antioxidants. DPPH is a stable free radical that, when reduced, changes color from deep purple to yellow, allowing for spectrophotometric quantification of scavenging activity.

DPPH Scavenging Assay Protocol- Detailed Procedure
DPPH Scavenging Assay Protocol- Detailed Procedure

DPPH Scavenging Assay Protocol- Detailed Procedure

DPPH Scavenging Assay Principle

  • DPPH, a stable free radical, has an unpaired electron giving it a deep purple color and a strong absorbance around 517 nm.
  • Antioxidants can donate a hydrogen atom or an electron to DPPH, neutralizing it and forming the reduced, yellow-colored DPPH-H.
  • This color change is proportional to the antioxidant’s scavenging capacity. Spectrophotometric measurement at 517 nm allows quantification of the reaction.

Materials- DPPH Scavenging Assay Protocol

  • DPPH (2,2-diphenyl-1-picrylhydrazyl): Purchase from a reliable chemical supplier.
  • Methanol (or Ethanol): Spectrophotometric grade.
  • Test Samples: Extracts, compounds, or solutions to be tested for antioxidant activity.
  • Positive Control: Known antioxidant compound (e.g., ascorbic acid, Trolox).
  • Spectrophotometer
  • Cuvettes or Microplates: Suitable for spectrophotometer readings at 517 nm.
  • Pipettes: Adjustable micropipettes for accurate volume dispensing
  • Vortex Mixer (Optional): For thorough mixing of solutions.

Safety Precautions- DPPH Scavenging Assay Protocol

  • DPPH: Can be an irritant. Minimize skin exposure and inhalation. Wear gloves and potentially eye protection when handling.
  • Organic Solvents: Methanol and ethanol are flammable. Handle with appropriate precautions, away from open flames and with good ventilation.
  • Sample-Specific Hazards: Know the potential risks of your test samples and follow safe handling procedures.

DPPH Scavenging Assay Protocol Procedure

  1. DPPH Stock Solution Preparation:
    • Calculate the amount of DPPH needed based on the desired working solution volume and final concentration (typically 0.1 mM).
    • Weigh DPPH accurately using an analytical balance.
    • Dissolve DPPH in methanol or ethanol. 
    • Protect from light (wrap the container in aluminum foil) due to DPPH’s light sensitivity.
    • Store the DPPH stock solution in a refrigerator until use.
  2. Preparation of Working DPPH Solution:
    • Dilute the DPPH stock solution with methanol or ethanol to achieve the desired working concentration (usually 0.1 mM).
    • Check the absorbance of the working solution on a spectrophotometer at 517 nm. Ideal absorbance should be around 1.0 ± 0.1. Adjust dilution if needed.
    • Prepare fresh working solution daily.
  3. Preparation of Test Samples and Control:
    • Dissolve test samples in a suitable solvent (methanol, ethanol, DMSO, water, etc.). Ensure solubility and compatibility with the assay.
    • Prepare a series of dilutions to test a range of sample concentrations.
    • Positive Control: Dissolve ascorbic acid (or another chosen standard) in the same solvent. Prepare a serial dilution.
  4. Reaction Setup
    • Add Test Sample: Pipette a defined volume of each sample dilution into separate wells or cuvettes.
    • Add DPPH: Add an equal volume of DPPH working solution to each well/cuvette (this initiates the reaction).
    • Positive Control: Repeat with dilutions of ascorbic acid or your chosen standard.
    • Negative Control (Blank): Include a reaction containing only solvent and DPPH solution.
  5. Incubation
    • Mix solutions thoroughly (pipetting up/down or gentle vortexing).
    • Incubate in the dark at room temperature for a defined time (often 30 minutes). Incubation time optimization may be needed.
  6. Absorbance Measurement
    • After incubation, measure the absorbance of each reaction mixture at 517 nm on the spectrophotometer.
    • Use the blank (solvent + DPPH) to zero the instrument.

Table-1: DPPH Scavenging Assay protocol

Methodology StepDescriptionImportant Considerations
DPPH PreparationPrepare a DPPH stock solution in methanol or ethanol. Protect from light.Use a spectrophotometric-grade solvent. DPPH is light-sensitive.
Working SolutionDilute the stock to the desired concentration (often 0.1 mM). Check absorbance at 517nm.Freshly prepare the working solution daily.
Sample & ControlPrepare samples and a positive control (e.g., ascorbic acid) at various dilutions in a suitable solvent.Ensure good sample solubility. Choose a solvent compatible with the assay.
Reaction SetupCombine samples and controls with an equal volume of DPPH working solution. Include a solvent-only blank.Mix thoroughly to initiate the reaction.
IncubationIncubate in the dark for a set time (e.g., 30 minutes).Choose incubation time based on the kinetics of your antioxidant.
MeasurementMeasure the absorbance of each reaction at 517 nm using a spectrophotometer.Zero the spectrophotometer with the blank.
Data AnalysisCalculate % scavenging activity for each sample. Determine IC50 values.Correct for any significant background absorbance of the samples.
DPPH Scavenging Assay protocol

Data Analysis and Calculations

  • % DPPH Radical Scavenging Activity: Calculate for each sample concentration using the following formula:

Scavenging Activity = [(Abs control – Abs sample) / Abs control] x 100

Where: * Abs control = Absorbance of the negative control (DPPH + solvent) * Abs sample = Absorbance of the sample reaction mixture

IC50 Calculation

  • Plot the % scavenging activity vs. sample concentration (or log concentration for a wider range).
  • Determine the sample concentration that causes 50% reduction of DPPH (the IC50 value). This represents the antioxidant’s potency. A lower IC50 indicates greater antioxidant activity.

Interpretation of Results

  • Comparison to Positive Control: Evaluate the test sample’s scavenging capacity relative to the known antioxidant standard.
  • Structure-Activity Relationships: If analyzing a series of related compounds, results can aid in investigating the impact of molecular features on antioxidant activity.

Additional Considerations

  • Solvent Choice: The solvent used for both DPPH and your samples can influence solubility and antioxidant activity. Methanol and ethanol are most common, but adjustments might be needed.
  • Kinetics: The DPPH reaction rate varies with different antioxidants. Ensure the chosen incubation time is sufficient, or consider time-course studies.
  • Interferences: Some compounds may absorb at 517 nm, interfering with the assay. Include sample-only controls (without DPPH) to correct for background absorbance.
  • Alternative Detection: While absorbance measurement is standard, methods like HPLC or electron paramagnetic resonance (EPR) can be used in more complex cases or for structural analysis of the products.


  • Low Scavenging Activity:
    • Increase sample concentrations
    • Extend the incubation time.
    • Ensure sample solubility and compatibility with the solvent.
  • Inconsistent Results:
    • Verify pipetting accuracy and calculations.
    • Check the stability and freshness of DPPH solution.
    • Control for temperature variations if they significantly impact your samples.
  • Unusual Absorbance Readings:
    • Check for precipitation in the reaction mixtures.
    • Exclude interference by sample or solvent by measuring their absorbance individually.

Advantages of DPPH Assay

  • Simple and Rapid: Easy to perform, with relatively quick results.
  • Versatile: Can be applied to various types of antioxidants and samples.
  • Cost-Effective: Requires basic laboratory equipment and reagents.

Limitations of DPPH Assay

  • Not Biologically Predictive: Antioxidant activity in vitro doesn’t always translate directly to activity within a living system.
  • Limited Specificity: DPPH is a synthetic radical; results shouldn’t be over-extrapolated to infer scavenging of all types of reactive oxygen species (ROS) found in biological systems.

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