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Methods for Cider

Methods for Cider

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Published by: phamhoanglongduc on Mar 26, 2010
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Methods for Cider 'Tannin' Analysis
Two methods are given here - the Lowenthal Permanganate Titration and the Folin-CiocalteauColorimetric Reaction.
1. The Lowenthal 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 dilutedstock 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
1 g of indigo carmine in 1 litre of water to which 50 ml of concentrated sulphuric acid has been added(takegreat careand wear eye protectionwhen making up sulphuric acid solutions!! They can get very hot and bump violently. Always add the acid to the water andnever the other way around!).
 Samples are analysed by adding 1 ml sample and 5 ml indigo carmine to the 500 ml flask andadding
200 ml water (tap water is fine for this). Titrate this against the permanganate solutionuntil the royal blue fades to a light green. Then titrate dropwise until the lime green changes toyellow. 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
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 awhite tile or paper background in well lit conditions to see the endpoint more clearly. It is alsohelpful 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. Lowenthal "Uber die Bestimmung des Gerbstoffs" Z. Anal. Chem (1877)
33- 48Burroughs LF and and Whiting GC "Ann. Rept. Long Ashton Research Station for 1960" pp 140-143
2. The Folin-Ciocalteau Colorimetric Reaction
This method, although originally dating from 1912, was extensively upgraded and improved byProfessor Vernon Singleton of UC Davis during the 1960's and 70's, and is now pretty muchstandard in most of the wine industry. It is equally applicable to ciders, though rather morecomplicated to carry out than the permanganate titration.
 The Folin-Ciocalteau reagent is a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids. It oxidises phenolates, reducing theheteropoly acids to a blue Mo-W complex. The phenolates are only present in alkaline solution butthe reagent and products are alkali unstable. Hence a moderate alkalinity and a high reagentconcentration are used in the procedure below.
FC Reagent
- Dilute the concentrated commercially prepared reagent (Merck, Sigma etc) 1: 10with distilled water. Prepare fresh daily. The concentrate keeps well in closed dark conditions butthe 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) inwater. This is stable for several weeks.
Gallic Acid standard
- Make up a solution of 100 mg pure gallic acid (accurately measured) inone litre of water to give a stock standard of 100 ppm gallic acid. Crystalline gallic acidmonohydrate can be purchased as an ACS grade reagent which dissolves readily and is preferred tothe anhydrous form. Its concentration should be corrected for moisture content (
9.4%).Although gallic acid does not occur in apples, it is easier to use and more stable than the alternativeepicatechin 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.
- capable of reading at 740 nm. Disposable plastic cuvettes are recommended.
Normal laboratory glassware
 – including volumetric flasks and test tubes (to contain 10 ml withspace for vortex mixing). All glassware must be scrupulously clean. The use of disposable plastictubes has been found advantageous for the final dilution step. Reagent blanks should accompany allsamples.
Dispensing pipettes
– to deliver 1, 4 and 5 ml volumes. For assay of large numbers of samples, theFC 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 onesession, since time, temperature and reagent age can all affect the absolute values of the dataobtained.
For standards -
Use the 100 ppm gallic acid stock solution to prepare serial dilutions containing100, 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):
5 ml of diluted FC reagent
Wait 3 – 8 mins
4 ml of the 7.5% sodium carbonate solutionCover the tubes for 2 hours at room temperature and away from strong light. Then measure E
ina 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 dilutionfactor) to give the polyphenol concentration as ppm (parts per million or mg/l) gallic acidequivalents (GAE) in the original sample. To convert to percentage values, divide the ppm values by 10,000.

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