SIST EN ISO 12966-2:2011

Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2011)

This part of ISO 12966 specifies methods of preparing the methyl esters of fatty acids. It includes methods for preparing fatty acid methyl esters from animal and vegetable fats and oils, fatty acids and soaps. To cover different requirements four methylation methods are specified, namely:
a) a “rapid” transmethylation procedure under alkaline conditions;
b) a “general” transmethylation/methylation procedure under sequential alkaline and acid conditions;
c) a BF3 transmethylation procedure;
d) an alternative procedure using acid-catalysed transmethylation of glycerides.
Methyl esters so produced are used in various analytical procedures requiring such derivatives, e.g. gas-liquid chromatography (GLC), thin-layer chromatography (TLC), and infrared spectrometry (IR).

Tierische und pflanzliche Fette und Öle - Gaschromatographie von Fettsäuremethylestern - Teil 2: Herstellung von Fettsäuremethylestern (ISO 12966-2:2011)

Diese Internationale Norm legt Verfahren zur Herstellung der Methylester von Fettsäuren fest.
Sie bezieht Verfahren für die Herstellung von Fettsäuremethylestern aus tierischen und pflanzlichen Fetten
und Ölen, Fettsäuren und Seifen mit ein. Um den verschiedenen Anforderungen gerecht zu werden, sind vier
Methylierungsverfahren festgelegt, insbesondere:
- ein „Schnellverfahren“ der Transmethylierung unter alkalischen Bedingungen (siehe 4.2);
- ein „allgemeines“ Transmethylierungs-/Methylierungsverfahren unter aufeinanderfolgenden alkalischen
und sauren Bedingungen (siehe 4.3);
- ein BF3-Transmethylierungsverfahren (siehe 4.4);
- ein alternatives Verfahren mit säurekatalysierter Transmethylierung der Glyceride (siehe 4.5).
Die so gewonnenen Methylester werden für verschiedene analytische Verfahren verwendet, bei denen diese
Derivate erforderlich sind, z. B. Gas-flüssig-Chromatographie (GC), Dünnschichtchromatographie (DC) und
Infrarotspektrometrie (IR).

Corps gras d'origines animale et végétale - Chromatographie en phase gazeuse des esters méthyliques d'acides gras - Partie 2: Préparation des esters méthyliques d'acides gras (ISO 12966-2:2011)

L'ISO 12966-2:2011 spécifie des méthodes de préparation des esters méthyliques d'acides gras.
Elle comprend des méthodes de préparation des esters méthyliques d'acides gras à partir de corps gras d'origines animale et végétale, d'acides gras et de savons. Afin de répondre à différentes exigences, quatre méthodes de méthylation sont spécifiées, à savoir: a) une méthode de transméthylation «rapide» en conditions alcalines; b) une méthode de transméthylation/méthylation «générale» en conditions alcalines et acides séquentielles; c) une méthode de transméthylation au BF3; d) une méthode alternative reposant sur la transméthylation par catalyse acide de glycérides.
Les esters méthyliques ainsi obtenus sont utilisés dans diverses méthodes d'analyse exigeant des produits dérivés de ce type, par exemple la chromatographie en phase gazeuse, la chromatographie sur couche mince et la spectrométrie infrarouge.

Rastlinske in živalske maščobe in olja - Plinska kromatografija metilnih estrov maščobnih kislin - 2. del: Priprava metilnih estrov maščobnih kislin (ISO 12966-2:2011)

Ta del ISO 12966 določa metode priprave metilnih estrov maščobnih kislin. Zajema metode za pripravo metilnih estrov maščobnih kislin iz živalskih in rastlinskih maščob in olj, maščobnih kislin in mil. Da bi zajeli različne zahteve, so določene štiri metode metilacije, in sicer:
a) »hiter« postopek transmetilacije v alkalnih okoliščinah;
a) »splošni« postopek transmetilacije/metilacije v zaporednih alkalnih in kislih okoliščinah;
c) postopek transmetilacije BF3;
d) alternativni postopek z uporabo transmetilacije gliceridov, kataliziranih s kislino.
Metlini estri, proizvedeni tako, se uporabljajo v različnih analitičnih postopkih, kjer so potrebni taki derivati, npr. plinska-tekočinska kromatografija (GLC), tankoplastna kromatografija (TLC) in infrardeča spektrometrija (IR).

General Information

• Status: Withdrawn
• Publication Date: 16-Jun-2011
• Withdrawal Date: 05-Jun-2017
• ICS: 67.200.10 - Animal and vegetable fats and oils
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 9900 - Withdrawal (Adopted Project)
• Start Date: 06-Jun-2017
• Due Date: 29-Jun-2017
• Completion Date: 06-Jun-2017
• Ref Project: EN ISO 12966-2:2011 - Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2011)

Relations

• Replaced By: SIST EN ISO 12966-2:2017 - Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2017)
• Effective Date: 01-Jul-2017
• Replaced By: SIST EN ISO 12966-2:2017 - Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2017)
• Effective Date: 08-Jun-2022
• Replaces: SIST EN ISO 5509:2001 - Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids (ISO 5509:2000)
• Effective Date: 01-Jul-2011

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 12966-2:2011
01-julij-2011
1DGRPHãþD
SIST EN ISO 5509:2001
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD3OLQVNDNURPDWRJUDILMDPHWLOQLKHVWURY
PDãþREQLKNLVOLQGHO3ULSUDYDPHWLOQLKHVWURYPDãþREQLKNLVOLQ,62

Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters -
Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2011)
Tierische und pflanzliche Fette und Öle - Gaschromatographie von
Fettsäuremethylestern - Teil 2: Herstellung von Fettsäuremethylestern (ISO 12966-
2:2011)
Corps gras d'origines animale et végétale - Chromatographie en phase gazeuse des
esters méthyliques d'acides gras - Partie 2: Préparation des esters méthyliques d'acides
gras (ISO 12966-2:2011)
Ta slovenski standard je istoveten z: EN ISO 12966-2:2011
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 12966-2:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 12966-2:2011

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SIST EN ISO 12966-2:2011


EUROPEAN STANDARD
EN ISO 12966-2

NORME EUROPÉENNE

EUROPÄISCHE NORM
February 2011
ICS 67.200.10 Supersedes EN ISO 5509:2000
English Version
Animal and vegetable fats and oils - Gas chromatography of
fatty acid methyl esters - Part 2: Preparation of methyl esters of
fatty acids (ISO 12966-2:2011)
Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle -
Chromatographie en phase gazeuse des esters Gaschromatographie von Fettsäuremethylestern - Teil 2:
méthyliques d'acides gras - Partie 2: Préparation des Herstellung von Fettsäuremethylestern (ISO 12966-2:2011)
esters méthyliques d'acides gras (ISO 12966-2:2011)
This European Standard was approved by CEN on 14 February 2011.

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, 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: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12966-2:2011: E
worldwide for CEN national Members.

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SIST EN ISO 12966-2:2011
EN ISO 12966-2:2011 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 12966-2:2011
EN ISO 12966-2:2011 (E)
Foreword
This document (EN ISO 12966-2:2011) has been prepared by Technical Committee ISO/TC 34 "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 August 2011, and conflicting national standards shall be withdrawn at
the latest by August 2011.
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.
This document supersedes EN ISO 5509:2000.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 12966-2:2011 has been approved by CEN as a EN ISO 12966-2:2011 without any
modification.

3

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SIST EN ISO 12966-2:2011

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SIST EN ISO 12966-2:2011

INTERNATIONAL ISO
STANDARD 12966-2
First edition
2011-02-15


Animal and vegetable fats and oils — Gas
chromatography of fatty acid methyl
esters —
Part 2:
Preparation of methyl esters of fatty acids
Corps gras d'origines animale et végétale — Chromatographie en
phase gazeuse des esters méthyliques d'acides gras —
Partie 2: Préparation des esters méthyliques d'acides gras




Reference number
ISO 12966-2:2011(E)
©
ISO 2011

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
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©  ISO 2011
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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Published in Switzerland

ii © ISO 2011 – All rights reserved

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Reactions .1
4 Methodology .2
4.1 Preparation of test sample .2
4.2 Rapid method.2
4.3 General method .4
4.4 Transmethylation using boron trifluoride (BF ) catalyst .6
3
4.5 Acid-catalysed transmethylation of glycerides.9
Annex A (informative) Thin-layer chromatography method for testing the completeness of
derivatization .11
Bibliography.15

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(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 12966-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
This first edition of ISO 12966-2 cancels and replaces ISO 5509:2000, of which it constitutes a technical
revision.
ISO 12966 consists of the following parts, under the general title Animal and vegetable fats and oils — Gas
chromatography of fatty acid methyl esters:
⎯ Part 2: Preparation of methyl esters of fatty acids
⎯ Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)
The following part is under preparation:
⎯ Part 4: Determination of cis-, trans-, saturated, mono- and polyunsaturated fatty acids in vegetable or
non-ruminant oils and fats
The following part is planned:
⎯ Part 1: Guidelines on gas chromatography

iv © ISO 2011 – All rights reserved

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
Introduction
General
Oils and fats (i.e. liquid and solid lipids) are predominantly composed of fatty acid esters of glycerol
(triacylglycerols, TAGs), with smaller amounts of fatty acid esters of sterols and long chain aliphatic alcohols.
Due to the high molecular mass of the TAGs and their consequent low volatility, they are difficult to analyse
directly by gas chromatography (GC), especially if a detailed analysis of unsaturated fatty acids is required.
Fatty acids themselves do not chromatograph well (except for short-chain-length fatty acids, e.g. butanoic and
pentanoic acids). It is therefore better practice to form fatty acid esters, usually the fatty acid methyl esters
(FAMEs), prior to GC.
The analysis of oils and fats has been extensively reviewed in Reference [9].
The formation of FAMEs is a critical stage in the analysis of fatty acids. Non-quantitative conversion of fatty
acids to FAMEs, modification of the structure of fatty acids (e.g. changes in positional and geometric isomers
present) and formation of non-FAME artefacts may all affect the quantitative determination of fatty acid
composition.
Transesterification is one mechanism which can be employed to form FAMEs from fatty acid esters in fats (i.e.
triacylglycerol). Alkali- or acid-catalysed transesterification procedures can be used to form FAMEs in a
methanolic medium; the procedure can be termed transmethylation. Transmethylation is a reversible process
and a large excess of methanol is required to maintain an equilibrium position which favours formation of the
FAMEs. Water can prevent the reaction going to completion, and its presence should therefore be minimized.
Alkali-catalysed procedures do not produce FAMEs from free fatty acids, due to the formation of soaps.
Esterification is an acid-catalysed mechanism which can be employed to form FAMEs from fatty acids. It is
possible that the fatty acids are naturally present in the sample of fat under examination. Formation of FAMEs
by this mechanism is commonly termed methylation. Again, an excess of methanol and the absence of water
are preconditions for the quantitative formation of FAMEs.
This part of ISO 12966 provides guidelines for the preparation of fatty acid methyl esters. In support of these
guidelines, various procedures to prepare fatty acid methyl esters are specified. These include:
a) “rapid” transmethylation under alkaline conditions;
b) “general” transmethylation/methylation under sequential alkaline and acid conditions;
c) boron trifluoride (BF ) transmethylation/methylation.
3
“Rapid” transmethylation method under alkali-catalysed conditions
This method is applicable to the routine analysis of edible fats and oils containing fatty acids down to butanoic
acid (C4:0) and/or for the determination of butanoic acid or hexanoic acid (C6:0) by GC using an internal
standard.
Alkaline catalysts transesterify neutral lipids in the presence of anhydrous methanol (transmethylation) more
rapidly than acid catalysts. The disadvantages of such alkali-catalysed procedures are that free fatty acids are
not esterified, and the presence of water may prevent the transmethylation going to completion (hydrolysis of
the FAMEs to free fatty acids). The most commonly used reagents are potassium and sodium hydroxide and
sodium methoxide in the presence of anhydrous methanol.
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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
“General” transmethylation/methylation under sequential alkaline and acid conditions
This method under sequential alkali- and acid-catalysed conditions is applicable to all oils and fats including
distillate and acid oils, but is not recommended for lauric oils. Short-chain fatty acid methyl esters are easily
lost during reflux. For lauric acid oils, the “rapid” transmethylation method is recommended.
During methylation, substances containing the following configurations can be totally or partially decomposed:
a) keto, epoxy, hydroxyl, hydroperoxy groups;
b) cyclopropyl and cyclopropenyl groups;
c) acetylenic fatty acids.
Boron trifluoride (BF ) transmethylation/methylation
3
Owing to the toxicity of BF it is recommended that this method only be used in extremis.
3
The BF method is applicable for most oils, fats and derivatives (fatty acids, soaps) with the exception of milk
3
fats and fats containing fatty acids with specific groups.
During methylation, substances containing the following configurations can be totally or partially decomposed:
a) keto, epoxy, hydroxyl, hydroperoxy groups;
b) cyclopropyl and cyclopropenyl groups;
c) acetylenic fatty acids.
If the fatty matter contains such substances in only very small amounts (e.g. cottonseed oil), the method can
be applied, otherwise the “rapid” or “general” transmethylation/methylation methods should be followed.
For GC, the optimum recovery of the methyl esters from the reaction mixture is obtained by using isooctane
(2,2,4-trimethylpentane). However, only about 75 % of the methyl caproate present is recovered.
Boron trifluoride is a strong Lewis acid, and in the form of its coordination complex with methanol, under reflux
conditions, it can rapidly methylate fatty acids. Methanolic boron trifluoride does transmethylate fatty acid
esters (e.g. triglyceride), but the rate of reaction is slower than the methylation of fatty acids. Methanolic boron
trifluoride solution is commercially available, which enhances the attractiveness of this acid catalyst, but there
are potential disadvantages associated with the use of this reagent.
a) It has been reported that high concentrations of boron trifluoride (50 % mass fraction) produce methoxy
artefacts from unsaturated fatty acids.
b) The reagent has a limited shelf-life at ambient temperature and should be kept refrigerated.
c) Aged reagent may produce artefacts and therefore it is recommended that each new batch purchased be
tested before use and periodically during its lifetime.
d) Methanolic boron trifluoride is an acidic reagent and therefore may produce derivatives of fatty acids
containing labile groups which may give rise to spurious peaks on FAME chromatograms.
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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
Additional information
Much attention has been given to the preparation and analysis of esters of short-chain fatty acids by GC,
largely because of their occurrence in milk fats. Short-chain fatty acids, in the free state or esterified to
glycerol, can be converted completely to methyl esters by any of the reagents described in the preceding
paragraphs, but quantitative recovery from the reaction medium may not be achieved unless special
precautions are taken. Losses can occur at several stages in any procedure. Short-chain fatty acid esters
(methyl especially) are volatile and may be lost selectively on refluxing the esterification medium, they are
more soluble in water than longer-chain esters and can be lost in an aqueous extraction step or they may be
distilled off when the extracting solvent is evaporated. Selective losses can also occur if non-saponifiable
impurities have to be removed by sublimation or thin-layer chromatography (TLC) purification. The best
esterification procedures for short-chain fatty acids are those in which heating of the reagents is avoided and
in which stages involving aqueous extraction and solvent removal are absent.
Injection of reaction media containing basic and acidic esterification catalysts directly on to GC columns
shortens their working lives. The top few centimetres of packed columns can be replenished periodically, while
lengths of deactivated tubing or “retention gaps” ahead of capillary columns protect them. This can be a small
price to pay for the speed, simplicity, and accuracy of these procedures.
Additionally, this part of ISO 12966 gives a simple TLC procedure to check the effectiveness of the
transmethylation/methylation. This procedure may also be used to check the generic composition of an oil or
fat before transmethylation/methylation is undertaken.


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SIST EN ISO 12966-2:2011

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SIST EN ISO 12966-2:2011
INTERNATIONAL STANDARD ISO 12966-2:2011(E)

Animal and vegetable fats and oils — Gas chromatography of
fatty acid methyl esters —
Part 2:
Preparation of methyl esters of fatty acids
1 Scope
This part of ISO 12966 specifies methods of preparing the methyl esters of fatty acids.
It includes methods for preparing fatty acid methyl esters from animal and vegetable fats and oils, fatty acids
and soaps. To cover different requirements four methylation methods are specified, namely:
a) a “rapid” transmethylation procedure under alkaline conditions;
b) a “general” transmethylation/methylation procedure under sequential alkaline and acid conditions;
c) a BF transmethylation procedure;
3
d) an alternative procedure using acid-catalysed transmethylation of glycerides.
Methyl esters so produced are used in various analytical procedures requiring such derivatives, e.g. gas-liquid
chromatography (GLC), thin-layer chromatography (TLC), and infrared spectrometry (IR).
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 Reactions
The determination of the fatty acid composition of oils and fats is one of the fundamental analyses within the
fats and oils sector and has been extensively reviewed in Reference [9]. For this purpose, the fatty acid
components of lipids are usually converted to methyl esters followed by GC analysis.
The “rapid” method (4.2) does not derivatize free fatty acids (FFAs) present in oil to fatty acid methyl esters
(FAMEs). If FFAs are present, the assumption is usually made that the FFAs have the same fatty acid
distribution as the triglycerides. This is usually true for crude oils, but less so for fractionated or refined oils.
Except for some cold-pressed oils, as a general rule oils with <0,5 % mass fraction FFAs have probably been
refined; oils above can be assumed to be crude. The tolerable concentration of FFAs in oil depends on the
particular oil being analysed and also the intended use of the FAME data generated. The presence of FFAs in
© ISO 2011 – All rights reserved 1

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
oil might introduce additional peaks on the final gas chromatogram and make the identification of FAMEs
synthesized using the “rapid” transmethylation procedure problematic.
The “general” (4.3) procedure derivatizes both FFAs and glyceryl esters to FAMEs (see 4.3.1).
It is up to the analyst to decide whether the use of either the “rapid” or “general” procedure is appropriate
based on the nature of the oil being analysed. This notwithstanding, as a general rule, use of the “rapid”
method is suggested only if the FFA content is u0,5 % mass fraction. The “general” method (4.3) is suggested
for oils with an FFA content >0,5 % mass fraction. Alternatively, if a partially hydrolysed fat is to be converted
to FAMEs, the acid-catalysed transmethylation procedure specified in 4.5 may be used.
Owing to the toxicity of BF , it is recommended that the BF method (4.4) be used only in extremis.
3 3
4 Methodology
WARNING — The method specified involves the use of potentially hazardous reagents. Normal
precautions shall be taken for eye protection and for protection from the dangers of corrosive
chemical burns. Methanolic potassium hydroxide solution is poisonous.
4.1 Preparation of test sample
The test sample shall be liquid, dry, and clear. Proceed in accordance with ISO 661, but heat the sample to
just above the melting point.
4.2 Rapid method
4.2.1 Applicability
This rapid transmethylation method, under alkali-catalysed conditions, is applicable to the routine analysis of
edible fats and oils containing fatty acids down to butanoic acid (C4:0) and/or for the determination of butanoic
acid or hexanoic acid (C6:0) by GC using an internal standard.
NOTE 1 This procedure does not derivatize FFAs to FAMEs. The analyst should note that the presence of FFAs in the
final solution can affect the quality of subsequent gas chromatography.
[8]
NOTE 2 According to COI/T.20/Doc. No. 24:2001 , a similar procedure can be applied directly to samples of the
following oil categories:
a) virgin olive oil with an acidity less than 3,3 %;
b) refined olive oil;
c) olive oil (blend of virgin and refined olive oil);
d) refined olive-pomace oil;
e) olive-pomace oil (blend of virgin olive oil and refined olive-pomace oil).
4.2.2 Principle
Methyl esters are formed by transmethylation with methanolic potassium hydroxide. Free fatty acids are not
esterified by this procedure.
4.2.3 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
The reagents shall not produce peaks which interfere with those of the fatty acid methyl esters during GC. Any
new batch of reagent or solvent should be checked by using it to prepare the methyl ester of pure oleic acid. If
any extra, unexpected, peaks appear during the final GC analysis the reagent should be rejected.
4.2.3.1 Methanol, containing not more than 0,5 % mass fraction water.
[3]
4.2.3.2 Water, complying with ISO 3696 , grade 3.
4.2.3.3 Sodium hydrogensulfate, anhydrous.
4.2.3.4 Isooctane (2,2,4-trimethylpentane), chromatographic quality.
WARNING — Isooctane is flammable and a fire risk. Explosive volume fraction limits in air are 1,1 % to
6,0 %. It is toxic by ingestion and inhalation. Use a properly operating ventilated hood when working
with this solvent.
4.2.3.5 Potassium hydroxide, methanolic solution, amount of substance concentration c ≈ 2 mol/l.
Dissolve, with gentle heating, 13,1 g of potassium hydroxide (mass fraction w = 85 g/100 g) in 100 ml of
absolute methanol.
4.2.3.6 Internal standard stock solution, for butanoic and/or hexanoic acid determination only.
Weigh 250 mg (to the nearest 0,1 mg) of valeric acid methyl ester (methyl pentanoate) into a 50 ml one-mark
volumetric flask (4.2.4.4). Use isooctane to dissolve the sample and make up to the mark with the same
solvent.
4.2.3.7 Internal standard reference solution, for butanoic and/or hexanoic acid determination only.
Add (4.2.4.2) 10 ml of stock solution to a 100 ml volumetric flask (4.2.4.4) and make up to the mark with
isooctane. Calculate the concentration of this reference solution.
4.2.3.8 Sodium chloride solution. Dissolve 40 g of sodium chloride in 100 ml of water.
4.2.4 Apparatus
Usual laboratory apparatus and, in particular, the following.
4.2.4.1 Screw-top test tubes, 10 ml, with cap fitted with a PTFE-joint.
[6]
4.2.4.2 Pipettes, capacities 0,1 ml, 2 ml and 10 ml, ISO 8655-2 .
4.2.4.3 Glass sample vials, 3 ml.
[2]
4.2.4.4 One-mark volumetric flasks, capacities 50 ml and 100 ml, ISO 1042 class A.
4.2.5 Procedure
In a 10 ml screw-top test tube (4.2.4.1), weigh approximately 0,1 g of the test sample (4.1). If fatty acids are to
be determined quantitatively by GC using internal standard(s), it is essential to weigh the test portion
accurately; i.e. to the nearest 0,1 mg. The results are then expressed as percentage mass fractions of the
fatty acid in the fat or oil. These results do not necessarily agree with results obtained by internal
normalization.
Add (4.2.4.2) 2 ml of isooctane (4.2.3.4), and shake. In certain instances, it may not be possible to use the
specified 0,1 g sample size, in which case the amount of isooctane added to the test portion should be
changed proportionately.
© ISO 2011 – All rights reserved 3

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SIST EN ISO 12966-2:2011
ISO 12966-2:2011(E)
For butanoic and/or hexanoic acid determination, pipette (4.2.4.2) 2 ml of reference solution (4.2.3.7) instead
of isooctane. In certain instances, it may be necessary to analyse fats and oils that contain low amounts of
these fatty acids. In this instance, the volume of reference solution added to the test sample may be reduced
proportionately.
Add (4.2.4.2) 0,1 ml of 2 mol/l methanolic potassium hydroxide solution (4.2.3.5), immediately put on the cap
fitted with a PTFE-joint, tighten the cap, and shake vigorously for 1 min. The solution becomes clear and then
shortly afterwards becomes cloudy again as glycerol separates. Allow to stand for approximately 2 min. Add
approximately 2 ml of sodium chloride solution and shake briefly. Draw off the isooctane layer and transfer to
a sample vial (4.2.4.3). Add approximately 1 g of sodium hydrogensulfate (4.2.3.3) and shake the solution.
The isooctane solution is suitable for analysis using GC in accordance with ISO 12966-4.
NOTE The effectiveness of derivatization using the “rapid” procedure can be determined by TLC as described in
Annex A.
4.3 General method
4.3.1 Applicability
This general transmethylation/methylation method, under sequential alkali- and acid-catalysed conditions, is
applicable to all oils and fats including distillate and acid oils, but not recommended for lauric oils. Short-chain
fatty acid methyl esters are easily lost during reflux. For lauric oils, the method specified in 4.2 is
recommended.
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