EN ISO 29841:2014

Vegetable fats and oils - Determination of the degradation products of chlorophylls a and a' (pheophytins a, a' and pyropheophytins) (ISO 29841:2009)

ISO 29841:2009 specifies a procedure for the determination of the degradation products pheophytin a, a' and pyropheophytin a of chlorophylls. The method is applicable to vegetable fats and oils only.

Pflanzliche Fette und Öle - Bestimmung thermischer Abbauprodukte des Chlorophyll a und a' (Pheophytin a, a' und Pyropheophytin) (ISO 29841:2009)

Diese Internationale Norm legt ein Verfahren zur Bestimmung der Abbauprodukte Pheophytin a, a‘ und Pyropheophytin a des Chlorophylls fest. Das Verfahren gilt ausschließlich für pflanzliche Fette und Öle.

Corps gras d'origine végétale - Détermination des produits de décomposition des chlorophylles a et a' (phéophytines a, a' et pyrophéophytines) (ISO 29841:2009)

L'ISO 29841:2009 spécifie un mode opératoire permettant la détermination des produits de décomposition des chlorophylles, à savoir les phéophytines a et a′ et la pyrophéophytine a. La méthode s'applique uniquement aux corps gras d'origine végétale.

Rastlinske maščobe in olja - Določevanje razgradnih produktov klorofila a in a' (feofitini a, a' in pirofeofitini) (ISO 29841:2009)

Standard EN ISO 29841 določa postopek za določevanje razgradnih produktov feofitinov a, a' in pirofeofitinov a v klorofilih. Metoda je primerna le za rastlinske maščobe in olja.

General Information

• Status: Published
• Publication Date: 15-Apr-2014
• Withdrawal Date: 30-Oct-2014
• ICS: 67.200.10 - Animal and vegetable fats and oils
• Technical Committee: CEN/TC 307 - Oilseeds, vegetables and animal fats and oils and their by-products - Methods of sampling and analys
• Drafting Committee: CEN/TC 307 - Oilseeds, vegetables and animal fats and oils and their by-products - Methods of sampling and analys
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 16-Apr-2014
• Due Date: 22-Nov-2014
• Completion Date: 16-Apr-2014
• Ref Project: SIST EN ISO 29841:2014 - Vegetable fats and oils - Determination of the degradation products of chlorophylls a and a' (pheophytins a, a' and pyropheophytins) (ISO 29841:2009)

Relations

• Amended By: EN ISO 29841:2014/A1:2016 - Vegetable fats and oils - Determination of the degradation products of chlorophylls a and a' (pheophytins a, a' and pyropheophytins) (ISO 29841:2009/AMD 1:2016)
• Effective Date: 02-Jul-2014

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2014
5DVWOLQVNHPDãþREHLQROMD'RORþHYDQMHUD]JUDGQLKSURGXNWRYNORURILODDLQ
D
IHRILWLQLDD
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Vegetable fats and oils - Determination of the degradation products of chlorophylls a and
a' (pheophytins a, a' and pyropheophytins) (ISO 29841:2009)
Pflanzliche Fette und Öle - Bestimmung thermischer Abbauprodukte des Chlorophyll a
und a' (Pheophytin a, a' und Pyropheophytin) (ISO 29841:2009)
Corps gras d'origine végétale - Détermination des produits de décomposition des
chlorophylles a et a' (phéophytines a, a' et pyrophéophytines) (ISO 29841:2009)
Ta slovenski standard je istoveten z: EN ISO 29841:2014
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 29841
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2014
ICS 67.200.10
English Version
Vegetable fats and oils - Determination of the degradation
products of chlorophylls a and a' (pheophytins a, a' and
pyropheophytins) (ISO 29841:2009)
Corps gras d'origine végétale - Détermination des produits Pflanzliche Fette und Öle - Bestimmung thermischer
de décomposition des chlorophylles a et a' (phéophytines a, Abbauprodukte des Chlorophyll a und a' (Pheophytin a, a'
a' et pyrophéophytines) (ISO 29841:2009) und Pyropheophytin) (ISO 29841:2009)
This European Standard was approved by CEN on 11 April 2014.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 29841:2014 E
worldwide for CEN national Members.

Contents Page
Foreword .3
Foreword
The text of ISO 29841:2009 has been prepared by Technical Committee ISO/TC 34 “Food products” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 29841:2014 by
Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products -
Methods of sampling and analysis” the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by October 2014, and conflicting national standards shall be withdrawn at
the latest by October 2014.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 29841:2009 has been approved by CEN as EN ISO 29841:2014 without any modification.

INTERNATIONAL ISO
STANDARD 29841
First edition
2009-03-15
Vegetable fats and oils — Determination
of the degradation products of
chlorophylls a and a′ (pheophytins a, a′
and pyropheophytins)
Corps gras d'origine végétale — Détermination des produits de
décomposition des chlorophylles a et a′ (phéophytines a, a′ et
pyrophéophytines)
Reference number
ISO 29841:2009(E)
©
ISO 2009
ISO 29841:2009(E)
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ii © ISO 2009 – All rights reserved

ISO 29841:2009(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 29841 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
INTERNATIONAL STANDARD ISO 29841:2009(E)

Vegetable fats and oils — Determination of the degradation
products of chlorophylls a and a′ (pheophytins a, a′ and
pyropheophytins)
1 Scope
This International Standard specifies a procedure for the determination of the degradation products
pheophytin a, a' and pyropheophytin a of chlorophylls. The method is applicable to vegetable fats and oils only.
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 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
pyropheophytin a (content in chlorophyll degradation products)
mass of pyropheophytin a divided by the sum of the masses of pheophytin a, a' and pyropheophytin a
4 Principle
Separation of the pigments (chlorophylls, pheophytins a and a', pyropheophytin a) using a miniaturized
column chromatography on a silica gel column from the major part of the lipids (triglycerides). The eluate is
analysed by RP18-HPLC, and the separated components are monitored at 410 nm using a photometric
detector.
The analysis of some seed oils (e.g. rapeseed oil) requires a higher sensitivity which can be achieved using
fluorescence detection with an excitation wavelength of 430 nm and an emission wavelength of 670 nm.
A direct HPLC determination with fluorescence detection is also possible and described in Annex C.
5 Reagents
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.
ISO 29841:2009(E)
5.1 Chlorophyll a.
NOTE Due to the lack of pheophytin and pyropheophytin reference materials, chlorophyll a is used to produce these
degradation products. Pheophytins are obtained from a chlorophyll a solution by acidification; pyropheophytins by
additional heat (> 100 °C) (Reference [4]).
5.2 Acetone.
5.3 Methanol.
5.4 Diethyl ether.
5.5 Petroleum ether, boiling point range 40 °C to 60 °C.
5.6 n-Heptane or, if not available, n-hexane.
CAUTION — Owing to its toxicity, avoid using n-hexane if n-heptane is available.
5.7 Petroleum ether and diethyl ether solvent mixture, volume fractions: ϕ = 90 ml/100 ml;
PetE
ϕ = 10 ml/100 ml.
Et O
6 Apparatus
Usual laboratory equipment, and in particular the following.
6.1 HPLC setup.
6.1.1 HPLC system, consisting of a pump, a sample-injecting device (20 µl loop) or auto sampler, either a
photometric detector for measurements at 410 nm or a fluorescence detector (excitation wavelength,
λ = 430 nm and emission wavelength, λ = 670 nm), and an integration system.
ex em
6.1.2 HPLC column, 250 mm length, 4,0 mm or 4,6 mm internal diameter, filled with reverse-phase type
RP18 particles, size 5 µm.
The following conditions have been found to be suitable:
1)
Stationary phase: Partisil ODS 3, 5 µm, 250 mm × 4,6 mm ;
Mobile phase: Water, methanol, and acetone (6.1.3);
Flow rate: 1,0 ml/min.
6.1.3 Mobile phase, water, methanol, and acetone mixture, volume fractions: ϕ = 4 ml/100 ml;
H O
ϕ = 36 ml/100 ml; ϕ = 60 ml/100 ml. All solvents shall be HPLC grade.
MeOH Me C=O
6.2 Taper-shaped flask, of capacity 10 ml or 25 ml.
6.3 Beakers, of different capacities.
6.4 Rotary evaporator, with water bath.
6.5 Auto sampler vials, of suitable capacity.

1) Example, available commercially, of a C18 reverse phase with a 10,5 % mass fraction carbon load, end-capped for
deactivation of silanols to minimize the need for ion suppression or ion pairing agents. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
2 © ISO 2009 – All rights reserved

ISO 29841:2009(E)
2)
6.6 Silica cartridge, 1 000 mg/6 ml, 55 µm, 700 nm or diol cartridge, 3 ml .
NOTE Home-made silica mini-columns can also be used for the separation.
7 Sample
7.1 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method
[1]
is given in ISO 5555 .
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
7.2 Preparation of the test sample
Prepare the test sample in accordance with ISO 661.
IMPORTANT — Test samples shall be stored cold and protected from light in glass vessels. After
clean-up, the test solutions shall be analysed immediately to prevent degradation and oxidation.
Avoid contact of test samples with plastics.
8 Procedure
8.1 Weigh about 300 mg of the test sample (7.2) into a small beaker (6.3), dissolve it in 1 ml of n-heptane
or n-hexane (5.6) and pour the solution on to the silica cartridge (silica gel column) (6.6). Rinse the beaker
twice with 1 ml portions of petroleum ether (5.5), pouring these washings on to the cartridge (column) also.
8.2 As soon as the solvent has drained to the top of the cartridge (column), elute the non-polar substances
twice with 5 ml petroleum ether/diethyl ether solvent mixture (5.7) each time.
8.3 Elute the pheophytin fraction twice with 5 ml acetone (5.2) and collect this fraction in a taper-shaped
flask (6.2) protected from light.
8.4 Evaporate the solvent to dryness on a rotary evaporator (6.4) at max 20 °C. Dissolve the residue in
200 µl acetone. Introduce this solution immediately into the HPLC setup (6.1).
IMPORTANT — Pheophytins are very unst
...