Methods for Cider 'Tannin' Analysis

Two methods are given here - the L÷wenthal Permanganate Titration and the Folin-Ciocalteu Colorimetric Reaction.

1. The L÷wenthal Permanganate Titration

This was the standard method used at the Long Ashton Research Station from 1903 until the Cider Section's closure in the 1980's. The method relies on the oxidation of phenolics by potassium permanganate solution in the presence of indigo carmine as a 'redox indicator' to show the end point.

The following solutions are required. Both are sensitive to light and oxidation and should be prepared freshly on the day of use:

Potassium permanganate solution (N/40 or 0.005M). This may be made up from freshly diluted stock solution obtained from a laboratory supply house (generally as N/10 or 0.02M). Alternatively, it may be made up as required by accurately dissolving 0.79 g of analytical grade potassium permanganate in 1 litre of water to give the 0.005M solution.

Indigo carmine indicator. This is made up as a 0.1% working solution by dissolving ca 1 g of indigo carmine in 1 litre of water to which 50 ml of concentrated sulphuric acid has been added (take great care and wear eye protection when making up sulphuric acid solutions!!  They can get very hot and bump violently. Always add the acid to the water and never the other way around!).


Samples are analysed by adding 1 ml sample and 5 ml indigo carmine to the 500 ml flask and adding ca 200 ml water (tap water is fine for this). Titrate this against the permanganate solution until the royal blue fades to a light green. Then titrate dropwise until the lime green changes to yellow. Record this value as X ml.

A blank titration using 5 ml of indigo carmine alone in 200 ml water should also be carried out. The blank value should be ca 1 ml and should be recorded as Y ml.

With a little practice the endpoint is consistent to within 0.1 ml. It is advisable to work against a white tile or paper background in well lit conditions to see the endpoint more clearly. It is also helpful if the burette which contains the permanganate has a white background for easier reading of the scale, since this solution is intensely coloured.

Calculation of results

Total Tannin (%) = (X-Y)/10 expressed as 'tannic acid' equivalents.

To convert to ppm (parts per million or mg/l) multiply the tannin percentage figure by 10,000.


J. L÷wenthal  "Uber die Bestimmung des Gerbstoffs"   Z. Anal. Chem (1877) 16 33- 48

Burroughs LF and and Whiting GC  "Ann. Rept. Long Ashton Research Station for 1960" pp 140-143

2. The Folin-Ciocalteu 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.


The Folin-Ciocalteu 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.


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.


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.


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.


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 … means of Folin-Ciocalteu reagent  Methods in Enzymology, Oxidants and Antioxidants, Part A, Lester Packer (ed) (1999)  299  152-178 (ISBN 0121822001) Academic Press, San Diego.

Back to the "What is Tannin?" page

Back to my Contents Page