Colorimetric assays involving complexation reactions

Analysis of antioxidant activity by performing a FRAP assay was proposed by Benzie and Strain [23]. It involves colorimetric determination of the reaction mixture in which the oxidants contained in the sample reduce Fe ions to Fe. At low pH, Fe(in)-TPTZ (ferric-tripyridltria-zine) complex is reduced to the ferrous (Fe ) form and intense blue colour at 593 nm can be observed. The FRAP reagent is prepared by mixing 2.5 ml of TPTZ (2,4,6-tris (l-pyridyl)-5-triazine) solution (10 mM in 40mM HCl), 25 ml acetate buffer, pH 3.6, and 2.5 ml FeCl3 H20 (20 mM). The colour of Fe(II)(TPTZ)2 which appears in the solution is measured colorimetri-cally after incubation at 37°C. The measurement results are compared to those of a blank sample, which contains deionised water instead of the analysed sample. The duration of the assay differs from one study to another 4 min [23, 24], 10 min [25] to 15 min [26]. The analysis results are converted and expressed with reference to a standard substance, which can be ascorbic acid [26], FeS04 [23, 25], Trolox [27,18]. 

The other major types of assay are all spectrophotometric (Alternate Protocols 1 and 2) and usually involve measuring the production of the highly colored end-product of the DPO reaction. They are used widely because they are rapid, can be carried out with the simplest of colorimeters/spectrophotometers, and can be adapted to use microtiter plates (Alternate Protocol 3). Thus they are ideal for simple routine comparative or monitoring studies. A major weakness of these colorimetric procedures is that they measure the end-product of a complex sequence of reactions, and different substrates yield different... 

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