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
absorbance is read at 530 nm. The concentration of proanthocyanidins is
expressed as cyanidin equivalents (used for the standard curve). The
molecular extinction coefficient εmol that can be used to convert the
absorbance values to a concentration is equal to 34700 L mol-1cm-1.