Hi im curious about how much ascorbic acid should i use to make a stock solution for stardard calibration curve (10,20,40,60,80,100ppm). Is it okay if I want to use gallic acid instead of ascorbic acid?
Abdullah S. Seddiek
Prepare a series of ascorbic acid standard solutions with known concentrations. It is recommended to prepare at least five or more solutions with a range of concentrations that cover the expected concentration range in your samples. For example, you can prepare solutions with concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM. Take aliquots of each ascorbic acid standard solution and transfer them to separate cuvettes or microplates. Each standard solution should be in duplicate or triplicate to ensure accuracy and reproducibility. Prepare a DPPH working solution by dissolving DPPH in a suitable solvent (e.g., ethanol or methanol) to a desired concentration. The exact concentration will depend on the sensitivity of your assay and the expected range of antioxidant activity in your samples. Typically, a concentration of 0.1-0.2 mM DPPH is used. Add a fixed volume of the DPPH working solution to each cuvette or well containing the ascorbic acid standard solutions. The volume should be the same for each sample. For example, you can add 200 μL of DPPH solution to each well. Incubate the cuvettes or microplates at a specific temperature (e.g., room temperature) for a fixed period (e.g., 30 minutes) to allow the reaction between DPPH and ascorbic acid to occur. During this time, the DPPH will be reduced by ascorbic acid, resulting in a decrease in the purple color of DPPH. Measure the absorbance of each well at a specific wavelength using a spectrophotometer. The wavelength used is typically 517 nm, which corresponds to the maximum absorption of the reduced form of DPPH. Plot a calibration curve by plotting the absorbance values (y-axis) against the known concentrations of ascorbic acid (x-axis). Use the absorbance of the DPPH solution without ascorbic acid (blank) as a reference and subtract it from all absorbance values before plotting the curve. Determine the equation of the calibration curve, usually a linear regression equation, using suitable data analysis software. This equation will allow you to calculate the concentration of ascorbic acid in your samples based on their absorbance values. Validate the calibration curve by assessing its linearity, accuracy, precision, and limit of detection. This step ensures the reliability of the curve for quantifying ascorbic acid in unknown samples.
To make a calibration curve using ascorbic acid for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which is commonly used to measure antioxidant activity, you can follow these steps:
By using the calibration curve, you can determine the concentration of ascorbic acid in your samples by measuring their absorbance values and using the equation of the curve.
0 votes
0 thanks
- Badges
- Science topic
More Siti Hawa Binti Yaman's questions See All
Similar questions and discussions