Determination Of Total Phenolics
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Determination of Total Phenolics UNIT I1.1
The phenols or phenolics in wine are important to both red and white wines. In red wines,
this class of substances contributes to the astringency, bitterness, and other tactile
sensations defined as structure or body, as well as to the wine’s red color. In white wines,
higher levels of phenolics are generally undesirable, as they contribute to excessive
bitterness and to the tendency of the wine to brown when it is exposed to air. Phenolics
in grapes and wines include many different substances: phenolic acids (e.g., hydroxybenzoic
acids such as gallic acid, hydroxycinnamic acids found in grape juice), three classes
of flavonoids found in the skins and seeds (the red anthocyanins, the flavonols, and the
abundant flavan-3-ols, which comprise the monomeric catechins), oligomeric proanthocyanidins,
and polymeric condensed tannins. (For details on phenolics classes and
compound structures, refer to UNITS I1.2 & I1.3.) White wine is made by immediately pressing
off the skins and seeds after harvesting, and thus contains only small quantities of
flavonoids. In contrast, red wine is a whole-fruit extract made by fermenting with the
skins and seeds, and the alcohol thus produced is an excellent solvent for these substances.
Measuring these different substances and reporting meaningful values in a single number
is an analytical challenge. There are many different procedures for analyzing different
classes of phenolic substances, but few are used in wine analysis except for anthocyanin
or color measures. HPLC methods (UNIT I1.3) that give specific information on individual
substances are not widely used in wineries, but are becoming more common as the
significance of particular phenolic substances becomes better understood.
There are two widely used methods for the analysis of total phenolics in wine. The
Folin-Ciocalteau method (Basic Protocol 1 and the Alternate Protocol) has the advantage
of a fairly equivalent response to different phenols, with the disadvantage of responding
to sulfur dioxide and sugar. The direct spectral absorbance analysis (Basic Protocol 2) is
quick and simple, making it suitable for process monitoring. This method, however,
responds differently to the various phenolic classes, making comparisons between different
wine types problematic, and also gives significant interference for sorbate.
Wine, of course, is not the only food that contains phenolics. Phenolics are found in all
foods, though at low levels in most. Notable foods that are high in phenolics include coffee
and tea, chocolate, fruits and derived products, some oils, spices, and some whole grains.
Although the following methods were developed for—and first applied to—analysis of
wines and grapes, they can be adapted for other foodstuffs (also see Commentary).
BASIC
PROTOCOL 1
DETERMINATION OF TOTAL PHENOLICS BY FOLIN-CIOCALTEAU
COLORIMETRY
Folin-Ciocalteau (FC) colorimetry is based on a chemical reduction of the reagent, a
mixture of tungsten and molybdenum oxides. Singleton adapted this method to wine
analysis (Singleton and Rossi, 1965) and has written two major reviews on its use
(Singleton, 1974; Singleton et al., 1999). The products of the metal oxide reduction have
a blue color that exhibits a broad light absorption with a maximum at 765 nm. The intensity
of light absorption at that wavelength is proportional to the concentration of phenols. The
FC method has been adopted as the official procedure for total phenolic levels in wine;
the Office International de la Vigne et du Vin (OIV), the one international body that
certifies specific procedures for wine analysis, accepts the FC method as the standard
procedure for total phenolic analysis (OIV, 1990). An earlier variation was the Folin-Denis
procedure, but the FC method has displaced it except in a few historical cases of official
procedures that have not been updated (AOAC International, 1995).
Supplement 6
Contributed by Andrew L. Waterhouse
Current Protocols in Food Analytical Chemistry (2002) I1.1.1-I1.1.8
Copyright © 2002 by John Wiley & Sons, Inc.
I1.1.1
Polyphenolics
Color development is slow but can be accelerated by warming the sample. With excessive
heating, however, subsequent color loss is quite rapid, and timing the colorimetric
measurement becomes difficult to reproduce. The reagent is commercially available, but
can be prepared (Singleton and Rossi, 1965). The resulting solutions are treated as
hazardous waste, and the scale of the original procedure creates a lot of waste. Fortunately,
modern liquid-measuring equipment now allows for microscaling the reaction to the
volume of a UV-Vis cuvette, reducing the cost of the reagent
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