Hagerman Butler assay

Figure 1. Parallel Determination of Tannins and Polymeric Pigments. The procedure shown in the rectangle (left) is a Somers assay performed at pH 4.9 and provides a measure of total pigments and total polymeric pigments (LPP + SPP) at that pH. The procedure shown in the polygon (right) is the Hagerman Butler assay for tannin. 2003 American Society for Enology and Viticulture.

The procedure in the polygon in Figure 1 is the Hagerman Butler assay for tannin. The only modification is that we retain the supernatant after centrifugation of the precipitation reaction mixture and bleach the monomeric anthocyanins with bisulfite. The residual absorbance represents pigments that do not precipitate with protein and do not bleach in the presence of bisulfite. We have designated this material small polymeric pigment solely to denote these two characteristics. The LPP removed from the sample by protein precipitation... 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

The contents of the ampule are diluted in water to a final volume of 50 mL. A 1-mL sample is then taken for the assay. To this sample 1.5 mL 0.667% (w/v) rhodanine in methanol is added. After exactly 5 min. 1 mL 0.5N KOH is added. After 2.5 min. water is added to a final volume of 25 mL. The absorbance is read at 520 nm after a 5-10 min. incubation. A standard curve is made by reaction of gallic acid in 0.2N sulfuric acid with the rhodanine solution. Hagerman and Butler (1989) argued that this assay is more suitable than the potassium iodate assay for the determination of hydrolysable tannins, although it has to be kept in mind that the rhodanine assay is sensitive to any gallic acid ester, including those in non-tannin compounds. 

Hagerman, A., And Butler, L. G., 1989, Choosing appropriate methods and standards for assaying tannin, J. Chem. Ecol. 15 1795-1810. 

As mentioned before, the amount of soluble tannin that causes astringency in persimmon fruits is usually estimated visually by the tannin print method and can be measured quantitatively by the Folin-Denis method. There is also a protein precipitation method for the measurement of soluble tannins (Hagerman and Butler 1978). In that method, the soluble tannin content is assayed by the addition of the sample to a standard solution of protein and the isolation of insoluble tannin-protein complexes. The complexes are dissolved in alkaline solution, to which ferric chloride is added. The absorbance of the solution at 510 nm is measured. 

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