Using the ABTS method, I have made several analysis on the antioxidant activity of 4 different extracts of a food material where 4 different solvents were used (methanol, water, PBS [pH=7.4] and Tris-HCl). In order to calculate the Trolox equivalents of each extract and compare the 4 of them (to assess which solvent allowed the extractions of compounds with the higher antioxidant activity), I had to do 4 different curves where different percentages of Trolox were diluted on these 4 different solvents.
When the ABTS radical-cation is diluted in water, each curve is significantly different from each other and the absorbance values of the blanks of PBS (Abs=0.450) and Tris-HCL (Abs=0.300) presented a big difference compared to the absorbance of the blanks of methanol (Abs=0.680) and water (Abs=0.600).
To decrease this difference I had to dilute the ABTS radical on PBS. Nevertheless, the difference persists and I was wondering if anyone knows the reason that explains this high difference on the absorbance of these solutions, please?
First, I would not use methanol as a solvent. The antioxidant activity should be tested under physiological conditions, aqueous solutions at pH 7.4. ABTS is not a radical. The reactive species in the ABTS assay is the radical anion with the charge 3-, but not the radical cation. The abbreviation commonly used in the literature ABTS(+.) is not correct, but can be used. The stability of this radical in stock solutions depends on the ratio [ABTS(+.]/[ABTS]. The activity of such antioxidants, such as polyphenols, is strongly dependent on pH and ionic strength. Therefore, I would strongly advise to use the original protocol of ABTS assay.
Which type of the instrument and which method (protocol) is in your use for the determination of Trolox Eq. of food extracts? I think, there is problem in proper following of the method because such type of analysis needs several preparations and then skills.
I would try a different buffer. Also ABTS is normally used to pH 7.0.
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