1、 Reference number ISO/TS 17764-2:2002(E) ISO 2002TECHNICAL SPECIFICATION ISO/TS 17764-2 First edition 2002-11-01 Animal feeding stuffs Determination of the content of fatty acids Part 2: Gas chromatographic method Aliments des animaux Dtermination de la teneur en acides gras Partie 2: Mthode par chr
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5、entral Secretariat at the address given below. ISO 2002 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 IS
6、O at the address below or ISOs 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 copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2002 All rights reservedISO/TS 17764-2:2002(E) ISO 2
7、002 All rights reserved iiiForeword 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
8、 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 Comm
9、ission (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 technic
10、al 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. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may
11、 decide to publish other types of normative document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote;
12、 an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will
13、 be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is drawn
14、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/TS 17764-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs.
15、ISO/TS 17764 consists of the following parts, under the general title Animal feeding stuffs Determination of the content of fatty acids: Part 1: Preparation of methyl esters Part 2: Gas chromatographic method TECHNICAL SPECIFICATION ISO/TS 17764-2:2002(E) ISO 2002 All rights reserved 1Animal feeding
16、 stuffs Determination of the content of fatty acids Part 2: Gas chromatographic method 1 Scope ISO/TS 17764 specifies methods for the quantitative determination of individual fatty acids and of the sum of the fatty acids (elutable fatty acids). This part of ISO/TS 17764 specifies the application of
17、gas chromatography with capillary columns and flame ionization detection for the determination of the quantitative content of fatty acids in a fat by making use of the methyl esters of the fatty acids obtained in accordance with the method specified in ISO/TS 17764-1. This part of ISO/TS 17764 is ap
18、plicable to the investigation of animal and vegetable fats, oils and fatty acid mixtures for incorporation in animal feeding stuffs and fat extracts of animal feeding stuffs and raw materials for compound animal feeds, including fats and fatty acid mixtures containing butyric acid. This method is no
19、t applicable to polymerized fatty acids. 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 amendm
20、ents) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO/TS 17764-1, Animal feeding stuffs Determination of the content of fatty acids Part 1: Preparation of methyl esters 3 Terms and definitions For the purposes of this document, the following terms and d
21、efinitions apply. 3.1 fatty acid content mass fraction of the fatty acids in the test portion of oil, fat, fat extract, free fatty acids or soaps NOTE The fatty acid content is expressed in grams per kilogram. 3.2 content of elutable material mass fraction of the sum of all the fatty acids elutable
22、using a gas chromatographic column as described in this part of ISO/TS 17764 ISO/TS 17764-2:2002(E) 2 ISO 2002 All rights reserved4 Principle The methyl esters prepared from fatty acids in accordance with ISO/TS 17764-1 are separated with gas-liquid chromatography, making use of a capillary column.
23、The peaks in the chromatogram are defined with the help of a reference sample of known composition and are quantified by means of an internal standard. 5 Reagents Use only reagents and solvents of recognized analytical grade. 5.1 Water, complying with at least grade 3 in accordance with ISO 3696:108
24、7. 5.2 n-Hexane or n-heptane. 5.3 n-Pentane. 5.4 Reference sample: an oil or fat sample with exactly known fatty acid pattern, or a mixture of reference fatty acid methyl ester materials or reference fatty acids materials. NOTE If the BF 3method for esterification is used, a mixture of reference fat
25、ty acid methyl esters cannot be used for calibration or correction factors for fatty acids with a chain length of less than 10 carbon atoms, because of possible solubility of the methyl esters in the water phase. 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1 Gas chrom
26、atograph, comprising a capillary column and an injection system specially designed for use with such columns. It may be of the split or the split-less type or a cold on-column injector. However, a warm split-less injector is not suitable for the analysis of milk fats due to overlap of the solvent pe
27、ak with the butyric acid peak. 6.2 Column, constructed of inert material (fused silica or glass) with a stationary phase preferable chemically bonded to the wall of the column. Column dimensions and film thickness are important factors in determining the separation efficiency and capacity of the col
28、umn. A resolution of at least 1,25 for the fatty acids C16:0 and C16:1, and C18:0 and C18:1 should be accomplished. NOTE In most cases a moderately polar phase will suit. In special cases, for instance for the separation of cis-trans- isomers and/or positional isomers, or if one must be sure that no
29、 peaks coincide, a more polar phase is warranted. The desirable effectiveness and capacity of the column should also be considered for column dimensions and film thickness. Moderately polar phases are, for instance, various esters of poly(ethylene glycol). More polar phases are often of the cyano-pr
30、opyl-polysiloxane type. 6.3 Injection system, for manual injection, with a capacity of at most 10 l, and graduated in 0,1 l divisions, suited for the injector (6.2), or an automatic injection system. NOTE The use of an automatic system is preferable and can improve repeatability and reproducibility.
31、 6.4 Signal evaluation apparatus: an electronic system fitted with a recorder to transform the detector signal to a chromatogram (an integrator or a data station). ISO/TS 17764-2:2002(E) ISO 2002 All rights reserved 37 Procedure 7.1 Preparation of methyl esters Prepare the methyl esters of the fatty
32、 acids of the test portion and the reference sample (5.4) in accordance with ISO/TS 17764-1. 7.2 Selection of optimum operating conditions Optimize the equipment in accordance with the instructions given by the manufacturer. Optimize the flow of carrier gas in accordance with the recommendations of
33、the column manufacturer for the chosen column and the carrier gas. Maintain a detector temperature of 20 C to 50 C above the highest temperature of the column in a programmed heating, but at least at 150 C. The injector temperature depends on the type of the injector; follow the instructions given i
34、n the equipment manual. When using a split injector, set the split ratio between 1:30 and 1:100. 7.3 Analysis 7.3.1 Dissolve the fatty acid methyl esters of the test portion and the test portion with added internal standard in n-hexane (5.2) to a content of 1 % (mass fraction) when using a split inj
35、ector, or 0,05 % (mass fraction) in the case of a split-less injector or a cold on-column injector. Prepare a solution of the fatty acid methyl esters of the reference sample (7.1) in n-hexane with a comparable concentration. Inject separately 0,1 l to 1 l of the test sample, the test sample with in
36、ternal standard, and when necessary the reference sample. When using cold on-column injection, the use of n-pentane as solvent is necessary for a good separation of the fatty acid methyl esters with a chain length of less than 10 carbon atoms. Dissolve the methyl esters of the fatty acids in the tes
37、t portions and the reference sample in the same solvent. 7.3.2 Select a temperature programme depending on the fatty acid composition, allowing an effective resolution in the shortest possible time. Take into account the criteria mentioned in 6.3. Programme the oven temperature starting from 60 C if
38、 the sample contains fatty acids with a chain length shorter than 12 carbon atoms. If necessary, progress isothermally after the highest temperature in the programme has been reached until all components have been eluted. When using a cold on-column injector, start with an oven temperature of not mo
39、re than 10 C higher than the boiling point of the solvent at the prevailing pressure (50 C for n-pentane). Start the temperature programme immediately after the injection. Follow the manufacturers instructions. 8 Peak identification Identify the methyl ester peaks of the test portion according to th
40、e retention times in comparison with the retention times of the peaks of known fatty acid methyl esters in the reference sample. Peaks in the chromatogram of the test portion with the same retention time as peaks in the reference sample are considered to represent the same fatty acids. ISO/TS 17764-
41、2:2002(E) 4 ISO 2002 All rights reserved9 Calculation 9.1 Correction for heptadecanoic acid in the test portion Correct the peak area of heptadecanoic acid in the test portion with the added internal standard for heptadecanoic acid originating from the test sample, using the equation: ( ) () s17:0 s
42、r16:0 sr18:0 sr18:1 rsr sr17:0 s16:0 s18:0 s18:1 AAAA AA AAA + = + where A rsris the corrected area under the peak of the internal standard in the test portion with added internal standard, in area units; A sr16:0is the area under the peak of hexadecanoic acid (palmitic acid) in the test portion wit
43、h added internal standard, in area units; A sr17:0is the area under the peak of heptadecanoic acid (margarinic acid) in the test portion with added internal standard, in area units; A sr18:0is the area under the peak of octadecanoic acid (stearic acid) in the test portion with added internal standar
44、d, in area units; A sr18:1is the area under the peak of octadecenoic acid (oleic acid) in the test portion with added internal standard, in area units; A s16:0is the area under the peak of hexadecanoic acid (palmitic acid) in the test portion of the analysed sample without added internal standard, i
45、n area units; A s17:0is the area under the peak of heptadecanoic acid (margarinic acid) in the test portion of the analysed sample without added internal standard, in area units; A s18:0is the area under the peak of octadecanoic acid (stearic acid) in the test portion of the analysed sample without
46、added internal standard, in area units; A s18:1is the area under the peak of octadecenoic acid (oleic acid) in the test portion of the analysed sample without added internal standard, in area units. Correction for heptadecanoic acid in the test sample is not necessary if the relative quantity does n
47、ot exceed 0,5 % of total fatty acids. 9.2 Determination of relative calibration factors Determine the relative calibration factors for fatty acids with a chain length shorter than 10 carbon atoms. If another than cold on-column injection is used, it is necessary to account for selective evaporation
48、of fatty acid methyl esters. In this case, determine the relative calibration factors for the whole range of fatty acid methyl esters. Calibration factors are used to convert peak areas into mass fractions. Determine the calibration factors with the help of a chromatogram derived from the analysis o
49、f the reference mixture (5.4) carried out under operating conditions identical to those used for the test sample. Calculate the calibration factor for the fatty acid i by the equation: i i i m k A = ISO/TS 17764-2:2002(E) ISO 2002 All rights reserved 5where k iis the calibration factor for fatty acid i, in mass units per area unit; m iis the mass of fatty acid i in the reference sample, in mass units; A iis the peak area of fatty acid i in the reference sample, in area units. If it is not possible to det
