ISO-5509-2000.pdf

Reference number ISO 5509:2000(E) ©ISO 2000 INTERNATIONAL STANDARD ISO 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 ISO 5509:2000(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 2000 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 22 734 10 79 E-mail copyrightiso.ch Web www.iso.ch Printed in Switzerland ii© ISO 2000 All rights reserved -,-,- ISO 5509:2000(E) © ISO 2000 All rights reservediii 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) iv© ISO 2000 All rights reserved Introduction ISO 5509 contains three different procedures to prepare methyl esters. The general method is the BF3method 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 BF3are 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 BF3method when using isooctane. -,-,- INTERNATIONAL STANDARDISO 5509:2000(E) © ISO 2000 All rights reserved1 Animal and vegetable fats and oils Preparation of methyl esters of fatty acids 1Scope 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 (BF3) 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). 2Normative 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. 3General 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.) 3.1Principle 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) 2© ISO 2000 All rights reserved 3.2Applicability 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. 3.3Reagents Use only reagents of recognized analytical grade. 3.3.1Water, complying with grade 3 of ISO 3696. 3.3.2Sodium 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. 3.3.3Boron trifluoride (BF3), methanolic solution, 12 % to 15 % (mass fraction)1?. See A.1. 3.3.4Isooctane (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. 3.3.5Sodium chloride, saturated aqueous solution. 3.3.6Sodium sulfate, anhydrous. 3.3.7Nitrogen, having an oxygen content less than 5 mg/kg. 3.3.8Hexane, 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. 3.3.9Methyl 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. -,-,- ISO 5509:2000(E) © ISO 2000 All rights reserved3 3.4Apparatus Usual laboratory equipment and, in particular, the following. 3.4.1Flask, of capacity 50 ml or 100 ml, with ground neck and fitted with a ground glass stopper. 3.4.2Reflux condenser, of 20 cm to 30 cm effective length, with a ground joint to fit the flask (3.4.1). 3.4.3Boiling aid, fat-free. 3.4.4Graduated or automatic pipette, of capacity at least 10 ml, and fitted with a rubber bulb. 3.4.5Vial, of capacity 4 ml, with screw cap. 3.4.6Separating funnels, of capacity 250 ml, for dry methyl esters only. 3.4.7Rotary evaporator. 3.4.8Analytical balance, capable of weighing to the nearest 0,001 g. 3.5Preparation 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. 3.6Procedure 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. 3.6.1Test 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 Purpose Test portion mg Flask (3.4.1) ml NaOH solution (3.3.2) ml BF3solution (3.3.3) ml Solvent (3.3.4) or (3.3.8) ml GLC 100 to 250 250 to 500 50 50 4 6 5 7 1 to 3 2 to 5 IR/TLC 500 to 750 750 to 1 000 100 100 8 10 9 12 4 to 8 7 to 10 3.6.2Saponification 3.6.2.1For fats and oils, start the method at 3.6.2.2. For fatty acids and soaps, start the method at 3.6.2.3. 3.6.2.2Introduce 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) 4© ISO 2000 All rights reserved 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. 3.6.2.3Introduce 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. 3.6.3Preparation of the methyl esters in isooctane solution (mainly for gas liquid chromatography purposes) 3.6.3.1Continue boiling for 3 min. In the case of oils with long-chain fatty acids, such as fish oils, continue boiling for 30 min. 3.6.3.2Add the appropriate amount (see Table 1) of isooctane (3.3.4) to the boiling mixture through the top of the condenser. 3.6.3.3Remove 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. 3.6.3.4Add 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. 3.6.3.5Transfer 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 traces of water. The isooctane solution thus obtained may be injected as follows: a)directly onto a packed column for gas-liquid chromatography (see A.5); b)after appropriate dilution with isooctane for capillary column systems prior to the injection (see A.6); c)after dilution with a lower boiling solvent such as heptane for the special case of capillary on-column injection. 3.6.4Preparation of dry methyl esters (for TLC or IR spectroscopy) 3.6.4.1Continue boiling for 3 min. 3.6.4.2Add the appropriate amount (see Table 1) of hexane (3.3.8) to the boiling mixture through the top of the condenser. 3.6.4.3Remove the flask from the heat source and remove the reflux condenser. IMMEDIATELY, without allowing the flask to cool, add 20 ml sodium chloride solution (3.3.5). Stopper the flask and shake it vigorously for at least 15 s. 3.6.4.4Transfer the saline solution and hexane layer to a 250 ml separation funnel (3.4.6). Add about 30 ml of the saturated sodium chloride solution. Allow the two phases to separate. Retain the hexane solution. Extract the saline solution twice with 50 ml portions of hexane (3.3.8). 3.6.4.5Combine the hexane solution and the two extracts and wash them with 20 ml portions of water (3.3.1) until no free acid is obtained, using the methyl red solution (3.3.9) as indicator. Dry over anhydrous sodium sulfate (3.3.6). Filter the solution and evaporate the solvent cautiously on a water bath under a stream of nitrogen (3.3.7) or use a rotary evaporator (3.4.7). If the remaining portion contains a considerable amount of short-chain methyl esters (C6 to C10), a substantial loss of these can hardly be