Good day! I am currently testing for the antioxidant activity of my extract via the hydrogen peroxide scavenging assay. I have already done the experiment in six trials using 30% hydrogen peroxide. Unfortunately the absorbances I got were almost all the same even with my positive control ascorbic acid. After checking the reagents, I realized that the hydrogen peroxide I used was already expired last 2015. I would like to ask if it is possible to use the commercially available 3% or 6% hydrogen peroxide solution for the working solution instead of the 30% since it is regulated in our country and will be hard to procure easily. Thank you very much and have a great day!
Good day! To address your query regarding the use of commercially available 3% or 6% hydrogen peroxide (H₂O₂) for the hydrogen peroxide scavenging assay, it is essential to consider both the concentration and the potential impact on your experimental results.
Feasibility and Considerations for Using Lower Concentration Hydrogen Peroxide:
1. Concentration Adjustment:
- 3% and 6% H₂O₂ Solutions: These concentrations are significantly lower than the 30% solution you initially used. Therefore, if you choose to use 3% or 6% H₂O₂, you will need to adjust the volume used in your assay to ensure the final concentration in your reaction mixture is comparable to that achieved with the 30% solution. This involves calculating the appropriate dilution factor.
- Dilution Calculation Example:
If your assay requires a specific final concentration of H₂O₂ (e.g., 0.1 mM in the reaction mixture), you can use the following formula to determine the volume needed from a lower concentration stock solution:
\[
C_1 \times V_1 = C_2 \times V_2
\]
Where:
- \(C_1\) = Initial concentration of the H₂O₂ stock solution (3% or 6%)
- \(V_1\) = Volume of the stock solution required
- \(C_2\) = Desired final concentration in the assay
- \(V_2\) = Final volume of the reaction mixture
For instance, if using a 3% H₂O₂ solution to achieve a final concentration of 0.1 mM in a 1 mL reaction mixture, you need to determine \(V_1\) by rearranging the formula.
2. Impact on Assay Sensitivity and Accuracy:
- Expired Reagents: The expired 30% H₂O₂ you used likely led to inconsistent results due to degradation. Fresh reagents are crucial for reliable results.
- Scavenging Assay Sensitivity: Lower concentrations of H₂O₂ may affect the sensitivity and dynamic range of your assay. Ensure that the final concentration used falls within the optimal detection range of your spectrophotometer and that it is sufficient to observe a measurable scavenging effect by your extract and control substances (e.g., ascorbic acid).
3. Practical Implementation:
- Preparation of Working Solution: Dilute the 3% or 6% H₂O₂ to the required concentration immediately before use to minimize degradation. Store the diluted solution in a dark, cool place if not used immediately.
- Control Comparisons:
Conduct preliminary tests to compare the scavenging activity results using the 3% or 6% H₂O₂ with those obtained previously using the 30% solution (prior to expiry). This will help validate the reliability of your adjusted protocol.
Conclusion:
Using commercially available 3% or 6% hydrogen peroxide for the hydrogen peroxide scavenging assay is feasible, provided you accurately adjust the volumes to achieve the desired final concentration in your assay. This approach ensures regulatory compliance and mitigates issues related to the procurement of higher concentration H₂O₂. By conducting preliminary validation experiments, you can confirm the reliability and sensitivity of your assay with the adjusted concentrations.
Should you have further inquiries or require additional guidance on specific calculations or assay adjustments, please feel free to ask. Have a great day!
For the hydrogen peroxide scavenging assay, use commercially available 3% or 6% hydrogen peroxide solutions instead of the 30% solution. Adjust the concentration to ensure the final concentration is 0.01 mM. Prepare the working solution by diluting it with phosphate buffer (pH 7.4) to reach the target concentration. Always prepare the working solution fresh before each experiment to avoid inconsistent results due to hydrogen peroxide decomposition.
Thank you very much for the advice! I’ll try to redo the experiment and update you soon. Have a great day!
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Agronomy
- Crop Science