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The Folin-Ciocalteau Colorimetric Reaction

 

This method, although originally dating from 1912, was extensively upgraded and improved by Professor Vernon Singleton of UC Davis during the 1960's and 70's, and is now pretty much standard in most of the wine industry.  It is equally applicable to ciders, though rather more complicated to carry out than the permanganate titration.

Principle

The Folin-Ciocalteau reagent is a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids.  It oxidises phenolates, reducing the heteropoly acids to a blue Mo-W complex.  The phenolates are only present in alkaline solution but the reagent and products are alkali unstable. Hence a moderate alkalinity and a high reagent concentration are used in the procedure below.

Reagents

FC Reagent - Dilute the concentrated commercially prepared reagent (Merck, Sigma etc) 1: 10 with distilled water.  Prepare fresh daily. The concentrate keeps well in closed dark conditions but the diluted solution keeps only for a few days before high blanks are obtained.

Sodium Carbonate Reagent – Make up a 7.5% solution of sodium carbonate (anhydrous) in water.  This is stable for several weeks.

Gallic Acid standard  - Make up a solution of 100 mg pure gallic acid (accurately measured) in one litre of water to give a stock standard of 100 ppm gallic acid. Crystalline gallic acid monohydrate can be purchased as an ACS grade reagent which dissolves readily and is preferred to the anhydrous form.  Its concentration should be corrected for moisture content (ca 9.4%).  Although gallic acid does not occur in apples, it is easier to use and more stable than the alternative epicatechin standard, and the colour response per gram is very similar to that of apple polyphenols.  This solution is stable for a few days at 4° C.

Equipment

Spectrophotometer  - capable of reading at 740 nm.  Disposable plastic cuvettes are recommended.

Normal laboratory glassware – including volumetric flasks and test tubes (to contain 10 ml with space for vortex mixing).  All glassware must be scrupulously clean.  The use of disposable plastic tubes has been found advantageous for the final dilution step. Reagent blanks should accompany all samples.

Dispensing pipettes – to deliver 1, 4 and 5 ml volumes.  For assay of large numbers of samples, the FC and sodium carbonate reagents may be more conveniently dispensed from calibrated auto-dispensing bottles.

Procedure

Samples, standards and reagent blanks should be made up as one set for measurement all in one session, since time, temperature and reagent age can all affect the absolute values of the data obtained.

For standards -Use the 100 ppm gallic acid stock solution to prepare serial dilutions containing 100, 50, 25 and 10 ppm gallic acid (or as found appropriate).  Use 1 ml of each solution in the assay procedure (below) to construct a calibration graph or to calculate a ‘best-fit’ response factor (typically the 50 ppm standard will give ca 0.5 AU in the assay).

For samples - Filter the sample through paper or glass fibre and dilute with water 1:10.  Polymer filters (e.g. nylon) may remove polyphenols by adsorption and are not recommended. In the case of bittersweet ciders or red wines, an initial dilution 1:5 may be necessary.  Note that solutions cannot be diluted after the colorimetric reagents have been added.

Take a 1 ml aliquot of the diluted sample in a test tube and add in order, mixing well at each stage (e.g. using a vortex mixer):  

Cover the tubes for 2 hours at room temperature and away from strong light.  Then measure E740 in a 1 cm cell against a reagent blank carried through the same procedure.  A reagent blank should also be measured against a water blank – the background should be acceptably low.

Multiply the assay figure obtained from the calibration graph by 10 (or by the appropriate dilution factor) to give the polyphenol concentration as ppm (parts per million or mg/l) gallic acid equivalents (GAE) in the original sample.  To convert to percentage values, divide the ppm values by 10,000.

Note:  High levels of fructose, sulphite and ascorbic acid may interfere with the assay.  It is recommended that these factors be tested in model solutions if they are likely to be present in the samples in significant amounts.

References

Singleton and Rossi  -  Amer. J. Enol. Vitic.  (1965) 16  144
Kramling and Singleton -  Amer. J. Enol. Vitic.  (1969) 20  86
Somers and Ziemelis -  J. Sci. Fd. Agr.  (1980)  31  600

The procedure has been collated and reviewed by Singleton VL, Orthofer R and Lamuela-Raventos RM  in Analysis of total phenolics …..by means of Folin-Ciocalteau reagent  Methods in Enzymology, Oxidants and Antioxidants, Part A, Lester Packer (ed) (1999)  299  152-178 (ISBN 0121822001) Academic Press, San Diego.

 


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