Antioxidant Activity I

We did a series of two experiments on antioxidants in the Introduction to Organic Chemistry laboratory this spring. I have seen antioxidant experiments in JCE and I performed one during the CASPiE Workshop a couple of years ago that has been reported in JCE by Burgess. So I have had this on my radar for a while. I had a senior in Biochemistry who wanted to do a course extension project so I suggested that he help me develop an antioxidant experiment for the intro class. There have to be close to ten distinctly different methods of determining antioxidant activity in the literature. He tried three different spectrophotometric antioxidant measurement methods. We chose DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and TPTZ (2,4,6-tripyridyl-s-triazine) to test. We also ordered some Folin-Ciocalteau reagent to test for total phenolics. We used aqueous solutions of gallic acid, ascorbic acid, and trolox as our standards. DPPH is really the most straightfoward assay. The bleaching of a dark blue-purple methanolic solution of DPPH is measured with a spectrophotometer. The problem with DPPH is that it tends to take awhile to stabilize after the antioxidant is added. Usually, readings are taken up to an hour after mixing. ABTS takes a little preparation because you have to generate the radical with something like K2S2O8 (potassium persulfate). The reagent works best if you mix it and let it sit overnight before using it. This is another bleaching assay – the ABTS radical is dark blue-green. This reagent reacts faster that DPPH and stabilizes within a minute. Student handout available at OChemOnline Wiki.


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