Analysis of Antioxidant Capacity using ABTS

UV-VIS Antioxidant Capacity (ABTS, 1 sample)

Analysis of Antioxidant Capacity using ABTS

Measuring antioxidant capacity using the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay with UV-Vis spectrophotometry is another widely used method for assessing the ability of compounds to scavenge free radicals. The ABTS assay is particularly versatile and can be adapted to measure the antioxidant capacity of various samples, including natural products, extracts, and compounds. Here's a general protocol for performing a UV-Vis antioxidant capacity assay using the ABTS reagent:

Materials and Reagents:

ABTS reagent (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))
Potassium persulfate (K2S2O8)
Antioxidant standard solution (eg, Trolox)
Sample extracts or solutions
Deionized water
Pipettes and pipette tips
Test tubes or microcentrifuge tubes
Vortex mixer or shaker
Timer or stopwatch
Lint-free tissue for cuvette cleaning

Procedure:

Prepare Antioxidant Standard Solutions:

Prepare a series of antioxidant standard solutions with known concentrations using the antioxidant of your choice (eg, Trolox). Typically, a concentration range of 0-100 µM is used for the standard solutions.

Prepare Sample Extracts:

If working with plant extracts or complex samples, prepare your sample extracts using an appropriate method (eg, solvent extraction). Ensure that your samples are appropriately diluted to fall within the linear range of the calibration curve.

Prepare ABTS Solution:

Dissolve the ABTS reagent in deionized water to prepare a 7-10 mM ABTS solution. Allow it to equilibrate in the dark for at least 12-16 hours before use.

Prepare Working ABTS Solution:

Mix the ABTS stock solution with potassium persulfate (K2S2O8) to initiate the radical formation. Typically, add a small volume of K2S2O8 solution (eg, 2.45 mM) to the ABTS solution to achieve a final concentration of approximately 2.5-10 mM ABTS radicals.
Allow this working ABTS solution to react in the dark for at least 6 hours before use. The solution will turn dark green.

Prepare Blank Solution:

Use a cuvette and add the working ABTS solution (without any antioxidant) as a blank reference for baseline correction.

Calibrate the Spectrophotometer:

Turn on the UV-Vis spectrophotometer and allow it to warm up.
Set the wavelength to the appropriate value for measuring the ABTS absorbance. The typical wavelength used is 734 nm.
Adjust the spectrophotometer's baseline using the blank solution, so that it reads zero absorbance at the chosen wavelength.

Prepare Reaction Mixtures:

In a test tube or microcentrifuge tube, mix the following components in the specified order:
0.2 mL of the antioxidant standard solution or sample extract
1.8 mL of the working ABTS solution
Mix well by vortexing or shaking the tube.

Incubate the Reaction Mixtures:

Allow the reaction mixtures to incubate in the dark at room temperature for an appropriate time, typically around 6-30 minutes, depending on the antioxidant's reactivity.

Measure Absorbance:

After the incubation, take a small volume (usually 1 mL) of each reaction mixture and transfer it to separate cuvettes.
Wipe the cuvettes with a lint-free tissue to remove any fingerprints or smudges.
Place each cuvette in the spectrophotometer and measure the absorbance at the chosen wavelength (734 nm).

Calculate Antioxidant Capacity:

Calculate the antioxidant capacity of the sample or standard by comparing the decrease in absorbance (scavenging of ABTS radicals) to that of the blank and using the calibration curve obtained from the standard solutions.