Determination Of Total Phenolics

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 and waste disposal (see

Alternate Protocol).

Materials

Sample, e.g., white wine or 10% (v/v) red wine in water

Gallic acid calibration standards (see recipe)

Folin-Ciocalteau (FC) reagent (Sigma; also Singleton and Rossi, 1965), stored in

the dark and discarded if reagent becomes visibly green

Sodium carbonate solution (see recipe)

100-ml volumetric flask

Spectrophotometer set to 765 nm, with 1-cm, 2-ml plastic or glass cuvettes

1. Place 1 ml sample, a gallic acid calibration standard, or blank (deionized or distilled

water) in a 100-ml volumetric flask.

Samples and standards should be analyzed in triplicate.

If any sample has an absorbance reading above that of the 500 mg/liter standard, it must

be diluted adequately and remeasured. White wine can typically be analyzed without

dilution. Red wine must be diluted with water (usually ten-fold) to fall into the range of the

standards.

2. Add ∼70 ml water, followed by 5 ml FC reagent. Swirl to mix and incubate 1 to 8

min at room temperature.

The incubation must not be >8 min (see Critical Parameters, discussion of reaction time

and temperature).

3. Add 15 ml sodium carbonate solution.

4. Add water to the 100-ml line, mix, and incubate 2 hr at room temperature.

5. Transfer 2 ml to a 1-cm, 2-ml plastic or glass cuvette and measure its absorbance at

765 nm in a spectrophotometer.

6. Subtract the absorbance of the blank from all readings and create a calibration curve

from the standards.

7. Use this curve to determine the corresponding gallic acid concentration of the

samples. Be sure to multiply by any dilution factor for the correct concentration (i.e.,

by ten for red wines). Report values in gallic acid equivalents (GAE) using units of

mg/liter (see Critical Parameters, discussion of standardization).

ALTERNATE

PROTOCOL

MICROSCALE PROTOCOL FOR FOLIN-CIOCALTEAU COLORIMETRY

This protocol is adapted for small sample volumes. The reaction is performed directly in

a 2-ml cuvette. For a list of materials needed, see Basic Protocol 1.

1. Put 20 μl sample, a gallic acid calibration standard, or blank (deionized or distilled

water) into a 1-cm, 2-ml plastic or glass cuvette.

Supplement 6 Current Protocols in Food Analytical Chemistry

I1.1.2

Determination of

Total Phenolics

2. Add 1.58 ml water, followed by 100 μl FC reagent. Mix thoroughly by pipetting or

inverting and incubate 1 to 8 min.

The incubation must not be >8 min (see Critical Parameters, discussion of reaction time

and temperature).

3. Add 300 μl sodium carbonate solution, mix, and incubate 2 hr at room temperature.

A final volume of 2 ml must fill the cell adequately for a reading.

4. Measure sample absorbance at 765 nm and analyze as described (see Basic Protocol

1, steps 6 to 7).

BASIC

PROTOCOL 2

DETERMINATION OF TOTAL PHENOLICS BY SPECTRAL ANALYSIS

Phenolic substances all absorb UV light, and all of them have some absorbance at 280

nm. This property can be used to determine phenolics by spectral analysis. One problem

with this method is that each class of phenolic substances has a different absorptivity

(extinction coefficient, e) at 280 nm. Thus, the results cannot be related to any specific

standard and are reported directly in absorbance units (AU). This also means that disparate

wines (or other disparate samples) are difficult to compare with this method, as they are

likely to have very different compositions.

The value of this method is that it is extremely simple and rapid, requiring only filtration

and, in some cases, dilution. It is very suitable for monitoring wines during various stages

of processing (e.g., fermentation) and for comparing similar wines (e.g., a single grape

variety from different vineyards, or wines from a particular vineyard over different

vintages).

Materials

Sample, e.g., red or white wine

Filter membrane, e.g., polytetrafluoroethylene (PTFE)

Cuvettes, transparent at 280 nm (e.g., quartz or methacrylate)

Spectrophotometer, set to 280 nm

1. Filter a sample or blank (deionized or distilled water) with a PTFE filter membrane

or other material to achieve clarity.

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