Here are the steps I'm taking:
- ABTS solution: 192 mg of ABTS in distilled water to 50 mL. Store this solution in an amber flask. - Potassium persulfate (140 mM): Mix 378.4 mg of the salt with 10 mL of distilled water. Store in the amber flask ABTS*: The day before the experiment mix in another amber flask 5 mL of the ABTS solution with 88 uL of the potassium persulfate and leave it at room temperature for 16 hours.
However, when I dilute the ABTS* with methanol or ethanol, I cannot achieve an absorbance of .700. Rather, the absorbance is 0.04.
I believe the issue might be coming from the potassium persulfate solution I mixed. Here is the potassium persulfate we purchased: https://www.laballey.com/products/potassium-persulfate-crystal-reagent
I think salt concentration and ABTS concentrations are not accurate. Check the below link. You will be able to get the absorbance of 0.700
https://www.ijpsonline.com/articles/estimation-of-phytochemical-content-and-antioxidant-activity-of-some-selected-traditional-indian-medicinal-plants.html?view=mobile#:~:text=ABTS%20radical%20scavenging%20assay&text=ABTS%C2%B7%2B%20cation%20radical%20was%20produced,of%200.700%20at%20734%20nm.
Evaluation of antioxidant activity using peroxynitrite as a source of radicals.
Geletii YV, Balavoine GG
Methods in Enzymology, 31 Dec 2001, 359:366-379 DOI: 10.1016/s0076-6879(02)59199-0
ABTS is available from Sigma and can be used without additional purification.
ABTS + is synthesized easily by oxidation of ABTS with solid PbO2. Dissolve
50 mg of ABTS in 50 ml of phosphate buffer solution (75 mM, pH 7.0) and then
under agitation add 150-220 mg of PbO2 (7- to 10-fold excess over ABTS). The
solution immediately becomes blue-green. After 2-3 min of agitation, thoroughly
remove all powder from the solution using a fine filter. Reaction time may vary
depending on the surface area of PbO2. ABTS conversion to ABTS + should be
~20-30%. If it is higher, ABTS + slowly decays and in several hours its concentration
reaches the value corresponding to 20-30% conversion. ABTS + can be quantified
by measuring its absorbency at 660 nm, e = 1.2 x 104 mo1-1 liter cm -1.
Its concentration should be checked before and after use. ABTS + stock solutions
(-0.5 mM) are rather stable at ambient temperature. The ABTS+/ABTS mixture
irreversibly precipitates at < 10 °C.
You can have a look Article Reduction of Oxidative Stress through Activating the Nrf2 me...
I think you are using a significant amount of persulfate compared to ABTS. Indeed with the same concentration of ABTS as you (7mM) I used around 2.5mM of persulfate or you use at a very high concentration. Also, the absorbance of 0.700 is linked to a wavelength of 734 nm (to be specified also if it is good at this level). Good luck.
Referenced used: //www.sciencedirect.com/science/article/pii/S0278691510006277
Take 140 mM Potassium persulfate (Dissolve 0.0378 g potassium persulfate in 1 ml of distilled water) and mix with 7 mM ABTS (0.072 g in 20 ml), and allowed to stand overnight in the dark at 25 degree Celsius.
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