1、 Reference numbers ISO 15885:2002(E) IDF 184:2002(E) ISO and IDF 2002INTERNATIONAL STANDARD ISO 15885 IDF 184 First edition 2002-11-15 Milk fat Determination of the fatty acid composition by gas-liquid chromatography Matires grasses du lait Dtermination de la composition des acides gras par chromato
2、graphie en phases gazeuse et liquide ISO 15885:2002(E) IDF 184:2002(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and insta
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6、luding photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32
7、 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2002 All rights reservedISO 15885:2002(E) IDF 184:2002(E) ISO and IDF 2002 All rights reserved iiiForeword ISO (the Inter
8、national 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
9、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 st
10、andardization. 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
11、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 ident
12、ifying any or all such patent rights. ISO 15885IDF 184 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and sep
13、arately by AOAC International. ISO 15885:2002(E) IDF 184:2002(E) iv ISO and IDF 2002 All rights reservedForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be repr
14、esented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are c
15、irculated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. ISO 15885IDF 184 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the In
16、ternational Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Fat, of the Standing Committee on Main Components in Milk, under the aegis
17、of its project leader, Dr F. Ulberth (AT). INTERNATIONAL STANDARD ISO 15885:2002(E) IDF 184:2002(E) ISO and IDF 2002 All rights reserved 1Milk fat Determination of the fatty acid composition by gas- liquid chromatography 1 Scope This International Standard specifies a method for the determination of
18、 the fatty acid composition of milk fat and fat obtained from dairy products. 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 refe
19、renced document (including any amendments) applies. ISO14156IDF 172, Milk and milk products Extraction methods for lipids and liposoluble compounds ISO 15884IDF 182, Milk fat Preparation of fatty acid methyl esters 3 Terms and definitions 3.1 fatty acid composition of milk fat mass fraction of indiv
20、idual fatty acids determined by the procedure specified in this International Standard NOTE The fatty acid composition of milk fat (as grams of individual free acids) per 100 g of total fatty acids (free acids) is expressed as a mass fraction in percent. 4 Principle Fatty acid methyl esters (FAME) o
21、f milk fat are prepared by transesterification. They are separated and determined by capillary gas-liquid chromatography. Individual FAME are quantified by reference to a milk fat of known composition. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and disti
22、lled or demineralized water or water of equivalent purity. 5.1 Reference fatty acid methyl esters (FAME) for identification purposes, of high purity ( 90 %), consisting of at least the saturated, even-numbered, straight-chain FAME with C4 to C22 atoms in addition to oleic, linoleic and linolenic aci
23、d methyl esters. NOTE A set of reference fatty acid methyl esters can be obtained either as a commercially available mixture or as a laboratory-prepared mixture from single substances. ISO 15885:2002(E) IDF 184:2002(E) 2 ISO and IDF 2002 All rights reserved5.2 Reference milk fat, for quantification
24、purposes. Use a milk fat with known fatty acid composition (e.g. Reference material CRM 164 1) ). 5.3 Fat solvent: n-alkane (e.g. n-pentane, n-hexane or n-heptane), free of substances that appear in the region of interest in the chromatogram. 5.4 Carrier gas: hydrogen, helium or nitrogen, of purity
25、of at least 99,999 %, with an oxygen content of below 2 10 6 . 5.5 Other gases, free from organic impurities (C n H mcontent of below 1 10 6 ). Use nitrogen and hydrogen, of purity of at least 99,995 %, and synthetic air. 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1
26、Gas-liquid chromatograph, comprising the following. 6.1.1 Injector Maintain injectors of the vaporizing type (split injector or programmed temperature injector, PTV) at a temperature of at least 220 C (in the case of a PTV, the final injector temperature shall be at least 220 C). In the case of a co
27、ld on-column injector, maintain the injector at a temperature a few degrees below the boiling point of the solvent. 6.1.2 Column oven, capable of running temperature programmes from near ambient temperature up to 260 C. 6.1.3 Columns, narrow- or large-bore glass or silica capillary column, of suitab
28、le length and stationary phase thickness to attain the performance characteristics defined in Clause 7. NOTE 1 Commercial stationary phases containing nitroterephthalic-acid-modified or unmodified poly(ethylene glycol) or other polar phases have been found to be suitable. NOTE 2 The stationary phase
29、s may be substituted by other polar phases provided that a similar resolution of fatty acid methyl esters is obtained. 6.1.4 Flame ionization detector, capable of operating up to 20 C above the final temperature of the column oven (6.1.2). 6.1.5 Carrier gas pneumatics, oxygen-diffusion-proof type, c
30、apable of maintaining the column head pressure to give a linear carrier gas velocity, with suitable flow controls to provide the requisite flow rates. If using a vaporizing split injector, control the split vent flow to give a split ratio of 1:50 to 1:100. NOTE Examples of flow rates are: nitrogen,
31、15 cm/s to 25 cm/s; helium, 25 cm/s to 35 cm/s; hydrogen, 35 cm/s to 55 cm/s at the initial oven temperature. 6.2 Injection syringe, of plunger-in-barrel type, manual with a maximum capacity of 10 l, or auto-injector as recommended for the equipment. 1) Reference material is available from Bureau of
32、 Reference, Commission of the European Communities, Brussels, Belgium. This information is given for the convenience of the users of this International Standard and does not constitute an endorsement by ISO or IDF of this product. ISO 15885:2002(E) IDF 184:2002(E) ISO and IDF 2002 All rights reserve
33、d 3Usually 10 l syringes are graduated to 0,2 l. If graduations of 0,1 l are required, it is recommended to use 5 l syringes. 6.3 Data system, capable of producing information required in Clauses 7, 10 and 11. 7 Performance specification Prepare a test mixture consisting of methyl butyrate, methyl s
34、tearate and methyl oleate at a concentration of 0,1 mg/ml of each in the fat solvent (5.3). Separate this mixture by gas chromatography using the same operating conditions as applied to the milk fat samples. Any test substance peak should be more than three- quarters full-scale of the data system (6
35、.3). The largest possible separation of methyl butyrate from the solvent peak and a resolution of 1,5 (baseline resolution) between methyl stearate and methyl oleate should be obtained. Calculate the resolution, R, by using the following equation: 12= 2 /( + ) R dWW where d is the numerical value of
36、 the distance between the respective peak maxima for methyl stearate and oleate, in millimetres; W 1is the numerical value of the width of the peak for methyl stearate, measured between the points of intersection of the tangents at the inflexion points of the curve with the baseline, in millimetres;
37、 W 2is the numerical value of the width of the peak for methyl oleate, measured between the points of intersection of the tangents at the inflexion points of the curve with the baseline, in millimetres. 8 Sampling It is important that the laboratory receive a sample which is truly representative and
38、 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 707. 9 Preparation of test sample and test portions For test samples of milk fat, see ISO 15884IDF 182. For those o
39、f milk and milk products, see ISO 14156IDF 172. 10 Procedure 10.1 Operating conditions Select operating conditions i.e. column length and inner diameter, stationary phase film thickness, initial oven temperature, temperature programme rate(s), final oven temperature, carrier gas flow rate to fulfil
40、the specifications of Clause 7. 10.2 Sample injection In general, follow the manufacturers instructions for the injector user. ISO 15885:2002(E) IDF 184:2002(E) 4 ISO and IDF 2002 All rights reservedIn case of a vaporizing split injector, draw up 0,5 l to 1,5 I of the test portion (Clause 9) with a
41、microsyringe (6.2). Withdraw the sample into the barrel of the syringe. Insert the needle into the heated injector and, after a dwell time of 3 s to 5 s, depress the plunger rapidly. Immediately thereafter, remove the syringe needle from the injector. If injection is carried out by the cold on-colum
42、n technique, dilute the test portion (Clause 9) sufficiently with a fat solvent (5.3) (e.g. a dilution ratio of 1:10). Inject the thus-prepared test portion at an oven temperature at or some degrees below the boiling point of the solvent used. 10.3 Qualitative analysis Analyse the reference fatty ac
43、id methyl esters (5.1) under the same working conditions as used for the test portion. Record the retention times of the reference substances. NOTE 1 Esters elute in order of increasing number of C atoms and in order of increasing number of double bonds for a given number (n) of C atoms (e.g. methyl
44、 palmitate elutes before methyl stearate). FAME with 18 C atoms elute in the order: methyl stearate, methyl oleate, methyl linoleate, methyl linolenate. Branched-chain FAME elute before the straight- chain esters with the same number of C atoms in the order: iso-branched (n 2) methyl esters before a
45、nteiso-branched (n 3) methyl esters. NOTE 2 The chromatogram shown in Figure 1 is an aid for the tentative identification of milk fat FAME. Identify the peaks of the test sample by comparison with the retention data obtained with the reference mixture and by comparison with the chromatogram shown in
46、 Figure 1. 10.4 Quantitative analysis 10.4.1 General Transmethylate the reference milk fat (5.2) as described in Clause 9. Analyse the obtained FAME mixture under the same working condition as used for the test portion. Terminate the recording of the chromatogram after complete elution of docosanoic
47、 acid (C22:0). To estimate the percentage of a component represented by a peak in the chromatogram, use the method of normalization, which assumes that all components of the sample are represented in the chromatogram. In that case, the sum of the area under the relevant peaks represents 100 % of the
48、 sample constituents (100 % elution). Determine the areas of peaks attributable to fatty acid methyl esters (i.e. all peaks in the chromatogram except the solvent peak, peaks due to addition of stabilizing agents and those which also appear in blank runs). 10.4.2 Calculation of the peak area Calcula
49、te the percentage, P Ai , of the total peak area represented by the peak of component i, by using the following equation: 100 % i Ai i A P A = where A iis the numerical value of the peak area corresponding to component i; A iis the numerical value of the sum of all peak areas corresponding to the component fatty acid methyl esters. ISO 15885:2002(E) IDF 184:2002(E) ISO and IDF 2002 All rights reserved 5Operating conditions Column dimensions: inner diameter 0,32 mm; length 30 m; Injector type manual
