- 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.
– 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).
- 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.