EN ISO 5509:2000
(Main)Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids (ISO 5509:2000)
Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids (ISO 5509:2000)
Tierische und pflanzliche Fette und Öle - Herstellung von Fettsäuremethylestern (ISO 5509:2000)
Diese Internationale Norm legt Verfahren zur Herstellung von Fettsäuremethylestern fest. Sie beinhaltet Verfahren zur Herstellung von Fettsäuremethylestern aus tierischen und pflanzlichen Fetten und Ölen, Fettsäuren und Seifen. Es werden die folgenden drei Methylierungsverfahren beschrieben, um unterschiedlichen Anforderungen gerecht zu werden: a) Bortrifluorid (BF3) Verfahren (siehe Abschnitt 3); b) Trimethylsulfoniumhydroxid (TMSH) Verfahren (siehe Abschnitt 4); c) Umesterungsverfahren (siehe Abschnitt 5).
Corps gras d'origines animale et végétale - Préparation des esters méthyliques d'acides gras (ISO 5509:2000)
La présente Norme internationale 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 des corps gras d'origines animale et végétale, des acides gras et des savons. Afin de répondre à différentes exigences, trois méthodes de méthylation sont spécifiées, à savoir:a) la méthode au trifluorure de bore (BF3) (voir l'article 3);b) la méthode à l'hydroxyde de triméthylsulfonium (TMSH) (voir l'article 4);c) la méthode de transestérification (voir l'article 5).Les esters méthyliques ainsi obtenus peuvent être utilisés dans diverses méthodes d'analyse exigeant des produits dérivés de ce type, telles que la chromatographie en phase gazeuse (CPG), la chromatographie en couche mince (CCM) ou la spectrométrie à infrarouge (IR).
Rastlinske in živalske maščobe in olja - Priprava metil estrov maščobnih kislin (ISO 5509:2000)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2001
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD3ULSUDYDPHWLOHVWURYPDãþREQLKNLVOLQ
,62
Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids (ISO
5509:2000)
Tierische und pflanzliche Fette und Öle - Herstellung von Fettsäuremethylestern (ISO
5509:2000)
Corps gras d'origines animale et végétale - Préparation des esters méthyliques d'acides
gras (ISO 5509:2000)
Ta slovenski standard je istoveten z: EN ISO 5509:2000
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.
INTERNATIONAL ISO
STANDARD 5509
Second edition
2000-04-01
Animal and vegetable fats and oils —
Preparation of methyl esters of fatty acids
Corps gras d'origines animale et végétale — Préparation des esters
méthyliques d'acides gras
Reference number
ISO 5509:2000(E)
©
ISO 2000
ISO 5509:2000(E)
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ii © ISO 2000 – All rights reserved
ISO 5509:2000(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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 5509 was prepared by Technical Committee ISO/TC 34, Agricultural food products,
Subcommittee SC 11, Animal and vegetable fats and oils.
This second edition cancels and replaces the first edition (ISO 5509:1978), which has been technically revised.
Annex A forms a normative part of this International Standard. Annex B is for information only.
ISO 5509:2000(E)
Introduction
ISO 5509 contains three different procedures to prepare methyl esters.
The general method is the BF method which is applicable to oils and fats and free fatty acids but which is less
applicable to caproic acid (C6) and not applicable to butyric acid (C4). The application field is GLC, TLC and IR.
Two alternative methods not involving BF are given using trimethylsulfonium hydroxide and potassium hydroxide
in methanol. Both methods are rapid methods for GLC analysis only.
The second method (trimethylsulfonium hydroxide method), which is for GLC analyses only, can be used for all fats
and oils including milk fat and milk fat containing blends. In the case of short fatty acids (C4 to C8) the use of an
internal standard is recommended.
The third method (trans-esterification method) can be used for neutral oils and fats, and can also be used for the
quantitative analysis of oils and fats with short-chain fatty acids down to butyric acid (C4). For the determination of
C4 and/or C6, only the internal standard method is maintained.
The principal new approach in this revision is the use of isooctane as solvent instead of hexane or pentane. This is
based on references [1] and [2], which showed better results especially for the BF method when using isooctane.
iv © ISO 2000 – All rights reserved
INTERNATIONAL STANDARD ISO 5509:2000(E)
Animal and vegetable fats and oils — Preparation of methyl esters
of fatty acids
1 Scope
This International Standard 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, three methylation methods are specified, as follows:
a) boron trifluoride (BF ) method (see clause 3);
b) trimethylsulfonium hydroxide (TMSH) method (see clause 4);
c) trans-esterification method (see clause 5).
Methyl esters so produced are used in various analytical procedures requiring such derivatives, for example gas-
liquid chromatography (GLC), thin-layer chromatography (TLC) and infrared spectrometry (IR).
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample.
ISO 3696, Water for analytical laboratory use — Specification and test methods.
3 General method using boron trifluoride
WARNING — The method described 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.
Boron trifluoride is poisonous. For this reason, it is not recommended that the analyst prepare the
methanolic solution of boron trifluoride from methanol and boron trifluoride. (See A.1 in annex A.)
3.1 Principle
The glycerides are saponified with methanolic sodium hydroxide. The soaps are converted into methyl esters by
reaction with a boron trifluoride/methanol complex.
For analysis of pure fatty acids and soaps, saponification with sodium hydroxide is not necessary and esters can be
prepared directly by reaction with boron trifluoride.
ISO 5509:2000(E)
3.2 Applicability
This method is to be preferred for most oils, fats and derivatives (fatty acids, soaps) with the exception of milk fats
and of fats containing fatty acids with specific groups.
During esterification, compounds containing the following configurations may be totally or partially decomposed:
� keto, epoxy, hydroxy, hydroperoxy groupings;
� cyclopropyl and cyclopropenyl groups;
� acetylenic fatty acids.
If the fatty matter contains such compounds in only very small amounts (e.g. cottonseed oil), the method can be
applied; otherwise the method described in clause 4 or 5 should be followed.
For gas chromatography, the optimum recovery of the methyl esters from the reaction mixture is obtained by using
isooctane. However, only about 75 % of the methyl caproate (C6) present will be recovered.
3.3 Reagents
Use only reagents of recognized analytical grade.
3.3.1 Water, complying with grade 3 of ISO 3696.
3.3.2 Sodium hydroxide, methanolic solution, approximately 0,5 mol/l.
Dissolve 2 g of sodium hydroxide in 100 ml of methanol containing not more than 0,5 % (mass fraction) of water. If
the solution has to be stored for a considerable time, a small amount of white precipitate of sodium carbonate may
be formed; this has no effect on the preparation of the methyl esters.
1�
3.3.3 Boron trifluoride (BF ), methanolic solution, 12 % to 15 % (mass fraction) .See A.1.
3.3.4 Isooctane (2,2,4-trimethylpentane), of chromatographic quality. See A.2.
WARNING — Isooctane is flammable and a fire risk. Explosive limits in air are 1,1 % to 6,0 % (volume
fraction). It is toxic by ingestion and inhalation. Use a properly operating fume hood when working with
this solvent.
3.3.5 Sodium chloride, saturated aqueous solution.
3.3.6 Sodium sulfate, anhydrous.
3.3.7 Nitrogen, having an oxygen content less than 5 mg/kg.
3.3.8 Hexane, of chromatographic quality, for dry methyl esters only. See A.2. Light petroleum, boiling range
40 °C to 60 °C, redistilled and residue-free, with a bromine value less than 1, may be used.
3.3.9 Methyl red, 1 g/l solution in 60 % (volume fraction) ethanol.
1� 14 %, 20 % (Merck No. 8.01663) and 50 % solutions 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.
2 © ISO 2000 – All rights reserved
ISO 5509:2000(E)
3.4 Apparatus
Usual laboratory equipment and, in particular, the following.
3.4.1 Flask, of capacity 50 ml or 100 ml, with ground neck and fitted with a ground glass stopper.
3.4.2 Reflux condenser, of 20 cm to 30 cm effective length, with a ground joint to fit the flask (3.4.1).
3.4.3 Boiling aid, fat-free.
3.4.4 Graduated or automatic pipette, of capacity at least 10 ml, and fitted with a rubber bulb.
3.4.5 Vial, of capacity 4 ml, with screw cap.
3.4.6 Separating funnels, of capacity 250 ml, for dry methyl esters only.
3.4.7 Rotary evaporator.
3.4.8 Analytical balance, capable of weighing to the nearest 0,001 g.
3.5 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.
3.6 Procedure
WARNING — Because of the toxic character of boron trifluoride, perform the methylation under a
ventilated hood. It is essential to wash all glassware with water immediately after use.
3.6.1 Test portion
Use Table 1 to select the appropriate size of flask and quantities of reagents and solvents required to methylate the
amount of test portion chosen.
Table 1
Test Flask NaOH solution BF solution Solvent
portion (3.4.1) (3.3.2) (3.3.4) or (3.3.8)
(3.3.3)
Purpose
mg ml ml ml
ml
100 to 250 50 4 5 1to3
GLC
250 to 500 50 6 7 2to5
500 to 750 100 8 9 4to8
IR/TLC
750 to 1 000 100 10 12 7to10
3.6.2 Saponification
3.6.2.1 For fats and oils, start the method at 3.6.2.2.
For fatty acids and soaps, start the method at 3.6.2.3.
3.6.2.2 Introduce the test portion into the appropriate flask. See Table 1 and annex A. Add the appropriate
amount (see Table 1) of the methanolic sodium hydroxide solution (3.3.2) and a boiling aid (3.4.3). Fit the
condenser (3.4.2) to the flask.
If the fatty acids contain more than two double bonds, remove the air from the flask by flushing the flask with dry
nitrogen (3.3.7) immediately prior to the reflux for a few minutes.
ISO 5509:2000(E)
Boil under reflux until the droplets of fat disappear, swirling the flask gently every 30 s to 1 min to prevent a solid
ring of sodium hydroxide forming around the walls of the flask. This usually takes 5 min to 10 min, but in certain
exceptional cases it may take longer. See A.3 and A.4. Add the appropriate amount (see Table 1) of the methanolic
boron trifluoride solution (3.3.3) through the top of the condenser.
Proceed in accordance with either 3.6.3 or 3.6.4.
3.6.2.3 Introduce the test portion into the appropriate flask (see Table 1). Add the appropriate amount (see
Table 1) of the methanolic boron trifluoride solution (3.3.3) into the flask. Fit the condenser (3.4.2) to the flask.
Proceed in accordance with either 3.6.3 or 3.6.4.
3.6.3 Preparation of the methyl esters in isooctane solution (mainly for gas liquid chromatography purposes)
3.6.3.1 Continue boiling for 3 min. In the case of oils with long-chain fatty acids, such as fish oils, continue
boiling for 30 min.
3.6.3.2 Add the appropriate amount (see Table 1) of isooctane (3.3.4) to the boiling mixture through the top of
the condenser.
3.6.3.3 Remove the flask from the heat source and remove the reflux condenser. IMMEDIATELY, without
allowing the flask to cool, add 20 ml of sodium chloride solution (3.3.5). Stopper the flask and shake it vigorously for
at least 15 s.
3.6.3.4 Add more of the saturated sodium chloride solution (3.3.5) to bring the liquid level of the mixture into
the neck of the flask. Allow the two phases to separate.
3.6.3.5 Transfer 1 ml to 2 ml of the upper isooctane layer into a 4 ml vial (3.4.5) and add a small amount of
anhydrous sodium sulfate (3.3.6) to remove any trace
...
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