ISO 14502-1:2005

INTERNATIONAL ISO
STANDARD 14502-1
First edition
2005-03-01


Determination of substances
characteristic of green and black tea —
Part 1:
Content of total polyphenols in tea —
Colorimetric method using Folin-
Ciocalteu reagent
Détermination des substances caractéristiques du thé vert et du thé
noir —
Partie 1: Dosage des polyphénols totaux dans le thé — Méthode
colorimétrique utilisant le réactif de Folin-Ciocalteu




Reference number
ISO 14502-1:2005(E)
©
ISO 2005

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ISO 14502-1:2005(E)
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ISO 14502-1:2005(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Principle. 1
4 Reagents. 1
5 Apparatus. 3
6 Sampling. 3
7 Preparation of test samples. 3
8 Procedure. 4
8.1 General. 4
8.2 Determination of dry matter content. 4
8.3 Test portion. 4
8.4 Extraction of polyphenols. 4
8.5 Dilution. 5
8.6 Determination. 5
9 Calculation. 5
10 Precision. 6
10.1 Interlaboratory test. 6
10.2 Repeatability. 6
10.3 Reproducibility. 6
11 Test report. 7
Annex A (informative) Gallic acid calibration graph . 8
Annex B (informative) Results of interlaboratory test . 9
Bibliography . 10

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ISO 14502-1:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 14502-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Tea.
ISO 14502 consists of the following parts, under the general title Determination of substances characteristic of
green and black tea:
 Part 1: Content of total polyphenols in tea — Colorimetric method using Folin-Ciocalteu reagent
 Part 2: Content of catechins in green tea — Method using high-performance liquid chromatography


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INTERNATIONAL STANDARD ISO 14502-1:2005(E)

Determination of substances characteristic of green and black
tea —
Part 1:
Content of total polyphenols in tea — Colorimetric method
using Folin-Ciocalteu reagent
1 Scope
This part of ISO 14502 specifies a method for the determination of the total polyphenol content of leaf tea and
[4]
instant tea by a colorimetric assay using Folin-Ciocalteu phenol reagent . It is applicable to both green and
black tea products.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 1572, Tea — Preparation of ground sample of known dry matter content
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 7513, Instant tea in solid form — Determination of moisture content (loss in mass at 103 °C)
3 Principle
Polyphenols are extracted with 70 % methanol from a test portion of finely ground leaf tea at 70 °C. Instant
teas are dissolved in hot water with 10 % acetonitrile (volume fraction) added to stabilize the extract. The
polyphenols in the extract are determined colorimetrically using Folin-Ciocalteu phenol reagent. The reagent
contains phospho-tungstic acids as oxidants, which on reduction by readily oxidized phenolic hydroxy groups
yield a blue colour with a broad maximum absorption at 765 nm. This is due to the formation of so-called
tungsten and molybdenum blues. The Folin-Ciocalteu phenol reagent reacts with a wide range of polyphenol
compounds and, although the response can vary with the individual components, selection of gallic acid as a
calibration standard enables useful total polyphenol data to be obtained.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
4.1 Water, conforming to grade 1 of ISO 3696:1987.
4.2 Acetonitrile.
4.3 Methanol.
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ISO 14502-1:2005(E)
4.4 Methanol/water extraction mixture, 70 % methanol (volume fraction).
Add 700 ml of the methanol (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and
mix.
4.5 Folin-Ciocalteu phenol reagent, commercially available ready prepared.
It is advisable to check the calibration linearity with respect to gallic acid in order to ascertain the suitability of
the supplied reagent.
4.6 Dilute Folin-Ciocalteu phenol reagent, 10 % (volume fraction).
Using a pipette, transfer 20 ml of Folin-Ciocalteu phenol reagent (4.5) to a 200 ml one-mark volumetric flask.
Dilute to the mark with water and mix.
Prepare fresh reagent solution daily.
To avoid contamination of the concentrated Folin-Ciocalteu reagent, discard any unused dispensed reagent.
4.7 Sodium carbonate solution, 7,5 % (mass concentration).
Weigh (37,50 ± 0,01) g of anhydrous sodium carbonate (Na CO ) into a 500 ml one-mark volumetric flask.
2 3
Add sufficient warm water to half-fill the flask. Swirl to dissolve the sodium carbonate, cool to room
temperature, dilute to the mark with water and mix.
NOTE This solution will remain stable at room temperature for up to 1 month.
4.8 Gallic acid stock standard solution, corresponding to approximately 1 000 µg/ml of anhydrous gallic
acid.
Weigh (0,110 ± 0,001) g of gallic acid monohydrate (M = 188,14) into a 100 ml one-mark volumetric flask.
Dissolve in water, dilute to the mark and mix.
Prepare a fresh standard solution daily.
NOTE Gallic acid monohydrate is preferred over anhydrous, due to its greater solubility, reduced hygroscopic
properties and availability of certified reagent grades, e.g. A.C.S., which is used to denote chemicals that meet
specifications outlined by the American Chemical Society. If not known, the moisture content (loss in mass at 103 °C) on a
portion of the standard material should be determined. The concentration of the stock standard solution on a gallic acid
anhydrous basis can then be calculated.
4.9 Gallic acid standard solutions A to E
Using pipettes, transfer the volumes of gallic acid stock standard solution (4.8) given in Table 1 to 100-ml one-
mark volumetric flasks. Dilute to the mark with water and mix. These dilute standard solutions should be
prepared on the day of use.
Table 1 — Gallic acid dilute standard solutions
Gallic acid standard solution Volume of gallic acid stock solution Nominal concentration of dilute standard
ml µg/ml
A 1,0 10
B 2,0 20
C 3,0 30
D 4,0 40
E 5,0 50
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