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Reagents preparation: 1. For standard solution add exactly 1 mL of deionized water in standard vial. Keep it at -20 °C. Then, dilute it 100 times to perform the calibration curve. 2. Dissolve 10 μL of standard solution in 1000 μL of deionized water. 3. Maintain the rest of the standard solution at -20 °C. 4. For DMPD•+ solution formation, mix 25 mL of DPMD solution in a 25 mL tube (not provided) with one vial of Reagent A (this mixture is stable for 12h at room temperature).
Sample preparation: If your sample is coloured, dilute it to an absorbance value lower than A0.
Standard Preparation Antioxidant activity is expressed as CEAC (Vitamin C equivalents antioxidant capacity). For this purpose, use the 1:100 diluted standard previously prepared (see Reagents preparation). Prepare calibration curves in 1 mL tubes as shown in Table 1 (see kit book let).Assay: 1. Add 20 μL of the sample or standard in each well. 2.Add 280 μL of DMPD•+ solution (see Reagents preparation) in each well. 3.Mix the mixture at 25 °C for 10 minutes under continuous stirring. 4.Read the absorbance at 553 nm.
1.Calculate the absorbance at 553 nm as percentage of the absorbance of the uninhibited radical cation solution (Blank) according to the equation: Inhibition of A553 (%) = (1-Af/A0) x 100 Where A0 is the absorbance of uninhibited radical cation and Af is the absorbance measured 10 min after the addition of antioxidant samples. 2. Plot the inhibition of standards as a function of their final concentrations (Table 1 in book let). See Figure 2 for a typical standard curve ( see images). 3.Calculate the CEAC value of the samples using the equation obtained from the linear regression of the standard curve substituted inhibition percentage values for each sample.CEAC (μg/ ml) = (sample inhibition A553- intercept)/slope