SIST EN ISO 9936:2006
(Main)Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents by high-performance liquid chromatography (ISO 9936:2006)
Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents by high-performance liquid chromatography (ISO 9936:2006)
This International Standard specifies a method for the determi nation of the contents of free , , , and -tocopherols and tocotrienols (referred to jointly as tocols) in animal and vege table fats and oils (referred to hereinafter as fats) by high- performance liquid chromatography (HPLC). For products contain ing tocopherol or tocotrienol esters, it is necessary to carry out a preliminary saponification.
Tierische und pflanzliche Fette und Öle - Bestimmung des Tocopherol- und Tocotrienol-Gehaltes mit Hochleistungsflüssigchromatographie (ISO 9936:2006)
Diese Internationale Norm legt ein Verfahren zur Bestimmung des Gehalts an freien a-, b-, g- und d Tocopherolen und Tocotrienolen (nachfolgend insgesamt als Tocole bezeichnet) in tierischen und pflanzlichen Fetten und Ölen (nachfolgend als Fette bezeichnet) durch Hochleistungsflüssigchromatographie (HPLC) fest.
Bei Produkten, die Ester von Tocopherolen oder Tocotrienolen enthalten, ist es erforderlich, eine vorausgehende Verseifung durchzuführen.
ANMERKUNG Ein geeignetes Verfahren mit einer Kaltverseifung wird in Anhang B nur zur Information beschrieben.
Corps gras d'origines animale et végétale - Détermination des teneurs en tocophérols et en tocotriénols par chromatographie en phase liquide a haute performance (ISO 9936:2006)
L'ISO 9936:2006 spécifie une méthode pour la détermination des teneurs en alpha-, bêta-, gamma- et delta-tocophérols et tocotriénols libres des graisses et des huiles d'origines animale et végétale, par chromatographie en phase liquide haute performance (CLHP).
Pour les produits contenant des esters de tocophérols ou de tocotriénols, il est nécessaire qu'ils subissent une saponification préalable.
Rastlinske in živalske maščobe in olja – Določevanje tokoferola in tokotrienola s tekočinsko kromatografijo visoke ločljivosti (ISO 9936:2006)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 9936:2006
01-september-2006
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD±'RORþHYDQMHWRNRIHURODLQWRNRWULHQRODV
WHNRþLQVNRNURPDWRJUDILMRYLVRNHORþOMLYRVWL ,62
Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents
by high-performance liquid chromatography (ISO 9936:2006)
Tierische und pflanzliche Fette und Öle - Bestimmung des Tocopherol- und Tocotrienol-
Gehaltes mit Hochleistungsflüssigchromatographie (ISO 9936:2006)
Corps gras d'origines animale et végétale - Détermination des teneurs en tocophérols et
en tocotriénols par chromatographie en phase liquide a haute performance (ISO
9936:2006)
Ta slovenski standard je istoveten z: EN ISO 9936:2006
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 9936:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 9936
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2006
ICS 67.200.10
English Version
Animal and vegetable fats and oils - Determination of tocopherol
and tocotrienol contents by high-performance liquid
chromatography (ISO 9936:2006)
Corps gras d'origines animale et végétale - Détermination Tierische und pflanzliche Fette und Öle - Bestimmung des
des teneurs en tocophérols et en tocotriénols par Tocopherol- und Tocotrienol-Gehaltes mit
chromatographie en phase liquide à haute performance Hochleistungsflüssigchromatographie (ISO 9936:2006)
(ISO 9936:2006)
This European Standard was approved by CEN on 13 April 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9936:2006: E
worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 9936:2006 (E)
Foreword
This document (EN ISO 9936:2006) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with 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 2006, and conflicting national
standards shall be withdrawn at the latest by October 2006.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 9936:2006 has been approved by CEN as EN ISO 9936:2006 without any
modifications.
2
---------------------- Page: 3 ----------------------
INTERNATIONAL ISO
STANDARD 9936
Second edition
2006-04-15
Animal and vegetable fats and oils —
Determination of tocopherol and
tocotrienol contents by high-performance
liquid chromatography
Corps gras d'origines animale et végétale — Détermination des teneurs
en tocophérols et en tocotriénols par chromatographie en phase liquide
à haute performance
Reference number
ISO 9936:2006(E)
©
ISO 2006
---------------------- Page: 4 ----------------------
ISO 9936:2006(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2006
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2006 – All rights reserved
---------------------- Page: 5 ----------------------
ISO 9936:2006(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 1
5 Reagents. 2
6 Apparatus . 2
7 Sampling. 3
8 Preparation of test sample. 3
9 Procedure . 4
9.1 Preparation of calibration solutions . 4
9.2 Optimization of working parameters . 5
9.3 Preparation of test solution . 5
9.4 Determination. 5
10 Expression of results . 6
11 Precision. 7
11.1 Interlaboratory test . 7
11.2 Repeatability. 7
11.3 Reproducibility. 7
12 Test report . 7
Annex A (informative) Examples of chromatograms. 8
Annex B (informative) Saponification . 10
Annex C (informative) Results of interlaboratory tests. 12
Bibliography . 17
© ISO 2006 – All rights reserved iii
---------------------- Page: 6 ----------------------
ISO 9936:2006(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 9936 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
This second edition cancels and replaces the first edition (ISO 9936:1997), which has been technically revised.
iv © ISO 2006 – All rights reserved
---------------------- Page: 7 ----------------------
INTERNATIONAL STANDARD ISO 9936:2006(E)
Animal and vegetable fats and oils — Determination of
tocopherol and tocotrienol contents by high-performance liquid
chromatography
1 Scope
This International Standard specifies a method for the determination of the contents of free α-, β-, γ-, and
δ-tocopherols and tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils (referred to
hereinafter as fats) by high-performance liquid chromatography (HPLC).
For products containing tocopherol or tocotrienol esters, it is necessary to carry out a preliminary
saponification.
NOTE A suitable method involving a cold saponification procedure is described in Annex B for information 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
tocol content
mass fraction of the individual tocols, determined using the method specified in this International Standard
NOTE The content is expressed in milligrams per kilogram as a whole number.
4 Principle
A test portion is dissolved in n-heptane and the individual tocols are separated by high-performance liquid
chromatography. The content of each tocol is calculated using calibration factors determined from calibration
solutions.
© ISO 2006 – All rights reserved 1
---------------------- Page: 8 ----------------------
ISO 9936:2006(E)
5 Reagents
Use only reagents of HPLC grade or equivalent.
5.1 α-, β-, γ- and δ-tocopherol and tocotrienol standards.
If tocopherol standards are not available, a blend of wheat germ and soya bean oil may be used to identify
α-, β-, γ- and δ-tocopherols.
If tocotrienol standards are not available, palm oil may be used to identify α- and γ-tocotrienols. The
chromatograms obtained can be used to assist peak identification in test sample chromatograms, in which
case the calibration factors for the corresponding tocopherols should be used.
1)
NOTE α-, β-, γ- and δ-tocopherol and tocotrienol standards can be obtained from Merck ; α-tocopherol can be
obtained from various suppliers. It has been reported that the purity of some commercially available tocopherol standards
may vary between 85 % and 100 %. Thus, it is important to determine the concentration of prepared calibration solutions
by UV spectrometry (see 9.1.1).
5.2 Tetrahydrofuran, filtered through an HPLC nylon filter (0,45 µm).
5.3 n-Heptane, filtered through an HPLC nylon filter (0,45 µm).
5.4 HPLC mobile phase: any suitable mixture of solvents that has been proved to reach a
chromatographic resolution of peaks as good as the one presented in Table 2 (relative retention time of
tocopherols and tocotrienols) and in Annex A (chromatograms of a mixture of vegetable oils), should be used
(see Table C.3).
The preparation of a suitable mobile phase, 3,85 % (volume fraction) tetrahydrofuran solution in n-heptane, is
as follows. Using a 1 000 ml graduated cylinder (6.5), introduce 1 000 ml of n-heptane (5.3) in a 2 litre bottle.
Add twice 20 ml of tetrahydrofuran (5.2) using a 20 ml volumetric pipette (6.6). Homogenize the mobile phase
by means of an ultrasonic bath (6.8) for 15 min.
5.5 Methanol.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 HPLC system, consisting of a high-pressure pump, a sample injection device, column thermostat
adjusted to 25 °C (optional), a fluorescence detector with the excitation wavelength set at 295 nm and
emission wavelength at 330 nm, and a recording integrator.
An ultraviolet (UV) detector may be used if a fluorescence detector is not available but it is not recommended.
However, if a UV detector is used, the wavelength should be set at 292 nm.
1) Merck Tocopherol set 613424 is available from Calbiochem (www.calbiochem.com). It contains one 50 mg vial each
of DL-α-tocopherol, D-β-tocopherol, D-γ-tocopherol, and D-δ-tocopherol with a purity of 95 % by HPLC (for each component).
Merck Tocotrienol set 613432 is available from Calbiochem also. It contains one 50 mg vial each of α-tocotrienol,
β-tocotrienol, γ-tocotrienol, and δ-tocotrienol with a purity of 95 % by HPLC (75 % for γ-tocotrienol).
This information is given for the convenience of users of this International Standard and does not constitute an
endorsement by ISO of these products.
2 © ISO 2006 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 9936:2006(E)
6.2 HPLC analytical column, two types are possible:
⎯ 250 mm × 4 mm, packed with microparticulate diol having a mean particle size of about 5 µm, or
⎯ 250 mm × 4,6 mm, packed with microparticulate silica having a mean particle size of about 5 µm.
NOTE 1 Suitable diol silica column packing material available commercially is 5 µm LiChrospher 100 Diol; suitable
2)
silica column packing materials available commercially are 5 µm LiChrosob SI 60 and Kromasil 100 . When β-tocotrienol
is expected in the sample, the diol silica column is preferred as γ-tocopherol and β-tocotrienol are co-eluted when using
the silica column.
NOTE 2 The length and the diameter of the column can be varied according to the HPLC technique used.
6.3 UV spectrometer, capable of absolute measurement of absorbance at precisely defined wavelengths,
with a 10-mm path length cell.
6.4 Rotary evaporator.
6.5 Graduated cylinder, of 1 000 ml capacity.
6.6 Volumetric pipettes, of 5 ml, 10 ml and 20 ml capacities.
6.7 Volumetric flasks, 50 ml and 25 ml capacities.
6.8 Ultrasonic bath.
7 Sampling
A representative sample should be sent to the laboratory. It is important that the sample has not been
damaged or changed during transport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling method
is given in ISO 5555.
8 Preparation of test sample
In the case of liquid laboratory samples, prepare the test sample by homogenization as described in ISO 661,
except that filtration should be avoided.
In the case of solid samples, transfer a representative portion (i.e. not less than 10 % by mass of the
laboratory sample) to a glass beaker and carefully homogenize by melting, with gentle mixing, in a water bath
at a temperature not exceeding 40 °C.
Preparation of the test samples should be carried out, as far as is practicable, in subdued light and in all cases
out of direct sunlight.
2) These types of columns are examples of suitable products which are available commercially.
This information is given for the convenience of users of this International Standard and does not constitute an
endorsement by ISO of these products.
© ISO 2006 – All rights reserved 3
---------------------- Page: 10 ----------------------
ISO 9936:2006(E)
9 Procedure
IMPORTANT — In general, the oxidation of tocols during the analysis may lead to low results. The rate
of oxidation is increased in the presence of alkalis, or under the influence of heat or light, and
measures should be taken to guard against these influences.
9.1 Preparation of calibration solutions
9.1.1 Stock calibration solutions
Prepare a stock solution of each tocol by weighing 10 mg ± 1 mg of the standard (5.1) into a 50 ml volumetric
flask and diluting to the mark with n-heptane (5.3).
Pipette 5 ml of this solution into an amber glass round-bottomed flask and remove all n-heptane on a rotary
evaporator (6.4) under vacuum at a temperature not greater than 40 °C. Restore atmospheric pressure with
nitrogen and remove the flask from the evaporator as soon as all the solvent has been removed. Pipette into
the flask 10 ml of methanol (5.5) and swirl to dissolve the residue. Measure the maximum absorbance of this
solution in a wavelength range between 270 nm and 310 nm (see appropriate wavelength in Table 1) using
the UV spectrometer (6.3) with a 10-mm path length cell. The measured absorbance should be between 0,2
and 0,8. Calculate the concentration (in micrograms per millilitre) by dividing the absorbance value by the
appropriate factor given in Table 1.
Table 1 — Division factors
Wavelength
Tocopherol Division factor
nm
292 0,007 6
α-tocopherol
296 β-tocopherol 0,008 9
298 γ-tocopherol 0,009 1
298 0,008 7
δ-tocopherol
NOTE The factors quoted are derived from the E values (1 %/1 cm) of the tocopherols. For
example, the E value (1 %/1 cm) of α-tocopherol is 76 at 292 nm (in methanol); therefore a 1 µg/ml
solution of α-tocopherol will have an absorbance of 0,007 6 at 292 nm.
9.1.2 Standard solution
A suitable standard solution should be prepared, according to the sensitivity of the fluorescence detector used.
T
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.