Dua metode PK tanin yang sering dipakai adalah metode titrasi permanganometri dan spektrofotometri

1. Permanganometri

Prinsip PK: Tanin termasuk golongan senyawa bergugus fenol. Jumlah gugus fenol ini diasumsikan
mewakili jumlah tanin secara keseluruhan. Dengan larutan kalium permanganat, gugus fenol ini
dioksidasi. Jumlah gugus fenol berbanding lurus dengan jumlah kalium permanganat yang diperlukan
untuk titrasi. Sebagai indikator redoks digunakan larutan indigo carmine.

2. Spektrofotometri UV-Vis

Prinsip PK: Gugus fenolat (dalam suasana alkali) direaksikan dengan reagen Folin-Ciocalteau (…saya
juga baru tahu kali ini), membentuk senyawa kompleks yang berwarna biru. Kadar senyawa kompleks
ini diukur dengan spektrofotometer UV-Vis pada panjang gelombang 740 nm. Sebagai standar,
digunakan larutan asam galat.

Methods for Cider 'Tannin' Analysis

Two methods are given here - the Lowenthal Permanganate Titration and the Folin-Ciocalteau
Colorimetric 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 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!).
 
Procedure
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.
 
References
J. Lowenthal  "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-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):
 
  5 ml of diluted FC reagent
  Wait 3 – 8 mins
  4 ml of the 7.5% sodium carbonate solution

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.