1、BSI Standards Publication PD ISO/TS 17383:2014 Determination of the triacylglycerol composition of fats and oils Determination by capillary gas chromatographyPD ISO/TS 17383:2014 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of ISO/TS 17383:2014. The UK partic
2、ipation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and their by-products. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessar
3、y provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014 ISBN 978 0 580 76803 3 ICS 67.200.10 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Documen
4、t was published under the authority of the Standards Policy and Strategy Committee on 31 August 2014. Amendments issued since publication Date Text affectedPD ISO/TS 17383:2014 ISO 2014 Determination of the triacylglycerol composition of fats and oils Determination by capillary gas chromatography Dt
5、ermination de la composition des triacylglycrols des corps gras Dtermination par chromatographie en phase gazeuse sur colonne capillaire TECHNICAL SPECIFICATION ISO/TS 17383 First edition 2014-09-01 Reference number ISO/TS 17383:2014(E)PD ISO/TS 17383:2014ISO/TS 17383:2014(E)ii ISO 2014 All rights r
6、eserved COPYRIGHT PROTECTED DOCUMENT ISO 2014 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior w
7、ritten permission. Permission can be requested from either ISO 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 Switzerlan
8、dPD ISO/TS 17383:2014ISO/TS 17383:2014(E) ISO 2014 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Normative references 1 3 T erms and definitions . 1 4 Principle 1 5 Reagents 1 6 Apparatus . 2 7 Sampling 3 8 Preparation of test sample . 3 9 Procedure. 3 9.1 Sample purification (if n
9、ecessary) . 3 9.2 Separation of individual triglycerides by GC . 3 9.3 Identification 3 9.4 Verification of the response factors 3 10 Calculation 4 11 Precision . 4 11.1 Interlaboratory test. 4 11.2 Repeatability limit (r) . 4 11.3 Reproducibility limit (R) 5 12 Test report . 5 Annex A (informative)
10、 Chromatograms 6 Annex B (informative) Results of interlaboratory test .10PD ISO/TS 17383:2014ISO/TS 17383:2014(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standar
11、ds 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
12、 part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In par
13、ticular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives). Attention is drawn to the possibility that some of the elements of
14、 this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see
15、www.iso.org/patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence t
16、o the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils.iv ISO 2014 All rights reservedPD ISO/TS 17383:201
17、4TECHNICAL SPECIFICATION ISO/TS 17383:2014(E) Determination of the triacylglycerol composition of fats and oils Determination by capillary gas chromatography 1 Scope This Technical Specification describes the procedure for the capillary gas chromatographic determination of the qualitative and semi-q
18、uantitative composition of individual triglycerides of fats, oils, and fat mixtures. The separation of the triglycerides is based on their retention depending on the carbon number of the fatty acids in the triglycerides and their degree of unsaturation. This Technical Specification is applicable to
19、animal and vegetable fats, as well as to mixtures of natural and synthetic triglycerides, as long as the oil fatty acid composition does not contain high content of linolenic acid such as linseed oil and the total chain length does not exceed a total carbon number of C60. NOTE If quantitative result
20、s are expected, the response factors of several triglycerides have to be checked as the increase of the triglyceride unsaturation reduces the sensitivity. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the
21、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 T erms a nd definiti ons For the purposes of this document, the following terms and definitions ap
22、ply. 3.1 proportion of the triglyceride or triglyceride group composition of the mixture of triglycerides is expressed as a percentage of area assuming the total of the triglyceride peaks is normalized to 100 % 4 Principle Triglycerides of different polarities are separated by capillary gas chromato
23、graphy on a highly polar stationary phase without any further sample preparation. After normalization of all peak areas, the content of the relevant triglycerides of the same retention time is expressed as a percentage proportion of the sum of all peak areas in percent. 5 Reagents WARNING Attention
24、is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational, and personal safety measures shall be followed. Unless otherwise stated, analytically pure reagents are to be used. 5.1 n-Hexane, analytical grade. ISO 2014 All rights reserved 1PD ISO/TS 17383
25、:2014ISO/TS 17383:2014(E) 5.2 Diethyl ether, analytical grade. 5.3 Solvent mixture of hexane/diethyl ether, volume fraction hexane = 87 ml/100 ml, volume fraction diethyl ether = 13 ml/100 ml. 5.4 Isooctane, analytical grade. 5.5 Reference substances, triglycerides such as tripalmitin, tristearin, t
26、riolein, trilinolein, etc., as well as vegetable and animal fats of known composition. 5.6 Synthetic air, suitable for gas chromatography. 5.7 Hydrogen, for gas chromatography. 6 Apparatus 6.1 Injection vials for GC. 6.2 Gas chromatograph (GC), a chromatograph fitted with a cold on-column injection
27、system and a flame ionization detector (FID). NOTE Alternative injection systems, e.g. a split injector, a programmed-temperature vaporizer (PTV), or a moving-needle injector, can be used provided the same results are obtained as indicated in Annex A. 6.3 The separation and detection has been proven
28、 to be satisfactory if the following experimental conditions are followed: High temperature capillary GC column: fused silica coated with thermo stable 50 % to 65 % phenylmethyl-polysiloxane, 25 m to 30 m 0,25 i.d., film thickness of 0,1 m to 0,15 m. Temperature program: 100 C held 1 min (initial te
29、mperature), 100 C to 325 C at 30 C/min, 325 C to 345 C at 1 C/min, 345 C to 370 C at 5 C/min, 370 C held 5 min (final temperature). Carrier gas: hydrogen (purity 99,999 %). NOTE Operating conditions and type of GC column should be used provided the same results are obtained as indicated in Annex A.
30、6.4 Microliter syringe for the GC, injection volume 1 l to 2 l. 6.5 Pipettes, 1 ml. 6.6 Analytical balance, readability 0,1 mg. 6.7 V olum etric flasks, 10 ml. 6.8 Silica gel cartridges for solid phase extraction, 1 g (6 ml). 6.9 Rotary evaporator. 6.10 Micropipette.2 ISO 2014 All rights reservedPD
31、ISO/TS 17383:2014ISO/TS 17383:2014(E) 7 Sampling A representative sample should have been sent to the laboratory. It should not have 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 give
32、n in ISO 5555. 8 Preparation of test sample Prepare the test sample in accordance with ISO 661. Heat solid and semi-solid fats to temperatures slightly above their melting point to be completely clear, then mix well. Filter liquid or melted samples, or their solutions, which still contain visible co
33、ntaminations. 9 Procedure 9.1 S ample purificatio n (if nec essary) If a sample contains monoglycerides, diglycerides, free fatty acids, or polymerized fats in greater proportions, they should be separated in advance by preparative column chromatography according to the following procedure. Wash an
34、SPE silica gel cartridge ( 6.8) with 6 ml of hexane. Place a conical flask under the cartridge. Load a solution of the sample (0,12 g, approximately) in 0,5 ml of hexane (5.1) into the column and then elute the triglycerides fraction with 10 ml of the solvent mixture (5.3) of hexanediethyl ether (87
35、:13 v/v). Evaporate to dryness the eluted solvent in a rotary evaporator (6.9) under reduced pressure at room temperature. NOTE Oil purification can also be done using a silica gel column, as described in ISO 8420. 9.2 Separation of individual triglycerides by GC Prepare a solution of the sample at
36、approx. 0,50 mg/ml in isooctane (5.4). Weigh 50 mg of the sample in a 10 ml volumetric flask (6.7) and bring to volume with isooctane (5.4). Pipette 1 ml (6.5) of the resulting solution in another 10 ml volumetric flask (6.7) and bring to volume with the same solvent. Inject 1,0 l of the final test
37、solution into the GC system using the cold on-column injection system. Working (temperature) conditions shall be used to get a good separation C50-triglycerides (POP/PLP), C52-triglycerides (POS/POO/PLO), and C54-triglycerides (SOO/OOO/OLO) as shown in Annex A. 9.3 Identification For the identificat
38、ion of the peaks in the gas chromatogram, the relative retention times, e.g. relative to tripalmitin, shall be compared to those of the test substances. In general, triglycerides appear in order of increasing number of C atoms and of increasing unsaturation for the same number of C atoms. The elutio
39、n order of the triglycerides is given in the example chromatograms (Annex A). 9.4 V erification of the r esponse fact ors Prepare a solution of the reference substances (5.5) at approx. 0,1 mg/ml in isooctane (5.4). Weigh 50 mg of the sample in a 10 ml volumetric flask (6.7) and bring to volume with
40、 isooctane (5.4). Pipette 0,2 ml (6.10) of the resulting solution in another 10 ml volumetric flask (6.7) and bring to volume with the same solvent. ISO 2014 All rights reserved 3PD ISO/TS 17383:2014ISO/TS 17383:2014(E) Inject 1,0 l of the final test solution into the GC system using the cold on-col
41、umn injection system. Calculate the response factor as a ratio of the area of the tripalmitin, divided by the area of the triglyceride being checked: = (1) where F TGi is the response factor of the triglyceride i; A TGi is the peak area of the triglyceride i; A PPP is the peak area of the tripalmiti
42、n. 10 Calculation Calculate the composition of the mixture as a percentage of area by integrating all the peaks present in the chromatogram. The unknown peaks will be summed up in a category called “other TAGs”. A prerequisite for the calculation according to the 100 % method is the assumption that
43、all triglyceride groups contained in the sample are fully separated in the gas chromatogram with the same response. Calculation of the individual triglyceride groups according to the following Formula (2): (2) where C TGi is the proportion of the triglyceride or triglyceride group i, in %; A TGi is
44、the peak area of the triglyceride i; A T is the sum of the peak area of all triglycerides (A TGi ). NOTE For a quantitative analysis, a correction of the peak areas by means of the response factors is not necessary as long as the verification of the response factor leads to values lower or equal to
45、1,2 for the triglycerides present in the sample. As an example, the response factor for triolein was estimated at 1,18. 11 Precision 11.1 Interlaboratory test Details of an interlaboratory test on the precision of the method are summarized in Annex B. The values derived from this interlaboratory tes
46、t may not be applicable to concentration ranges and matrices other than those given. 11.2 Repeatability limit (r) The repeatability limit (r) is the value less than or equal to which the absolute difference between two test results obtained under repeatability conditions may be expected to be with a
47、 probability of 95 %. Repeatability conditions are conditions where independent test results are obtained with the same method on identical test items in the same laboratory by the same operator using the same equipment within short intervals of time.4 ISO 2014 All rights reservedPD ISO/TS 17383:201
48、4ISO/TS 17383:2014(E) 11.3 Reproducibility limit (R) The reproducibility limit (R) is the value less than or equal to which the absolute difference between two test results obtained under reproducibility conditions may be expected to be with a probability of 95 %. Reproducibility conditions are cond
49、itions where independent test results are obtained with the same method on identical test items in different laboratories with different operators using different equipment within short intervals of time. 12 Test report The test report shall include the following information: the method in accordance with which sampling was carried out (if known); the method used; the test result(s) obtained; if the repeatability has been checked, the final quoted result obtai
