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1、BRITISH STANDARD BS ISO 18395:2005 Animal and vegetable fats and oils Determination of monoacylglycerols, diacylglycerols, triacylglycerols and glycerol by high-performance size-exclusion chromatography (HPSEC) ICS 67.200.10 ? Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00
2、 2006, Uncontrolled Copy, (c) BSI BS ISO 18395:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 7 July 2005 BSI 7 July 2005 ISBN 0 580 45921 7 National foreword This British Standard reproduces verbatim ISO 18395:2005 and implements it as
3、 the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/307, Oil seeds animal present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related i
4、nternational and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iii, a blank page, pages 1 to 10, an inside back cover and a back cover. The BSI copyright notice displayed in this doc
5、ument indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference number ISO 18395:2005(E) INTERNATIONAL STANDARD ISO 18395 First edition 20
6、05-04-15 Animal and vegetable fats and oils Determination of monoacylglycerols, diacylglycerols, triacylglycerols and glycerol by high-performance size- exclusion chromatography (HPSEC) Corps gras dorigines animale et vgtale Dtermination de la teneur en monoacylglycrides, en diacylglycrides, en tria
7、cylglycrides et en glycrol par chromatographie liquide dexclusion (CLHP dexclusion) BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00
8、:00 2006, Uncontrolled Copy, (c) BSI iii 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 b
9、ody 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 Electro
10、technical Commission (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
11、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 document may be the subject of patent r
12、ights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 18395 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GM
13、T+00:00 2006, Uncontrolled Copy, (c) BSI blank BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 1 Animal and vegetable fats and oils Determination of monoacylglycerols, diacylglycerols, triacylglycerols and glycerol by high-
14、performance size-exclusion chromatography (HPSEC) 1 Scope This International Standard specifies a method for the determination of monoacylglycerols, diacylglycerols and triacylglycerols and also free glycerol by high-performance size-exclusion chromatography. It is applicable to products (e.g. emuls
15、ifiers) comprising monoacylglycerols and diacylglycerols as main constituents in concentrations ?10 %, and to triacylglycerols in a proportion of ? 20 %. The method is not applicable to dairy fats or fats and oils having a wide range of fatty acid chain lengths, since diacylglycerols of short fatty
16、acids have a lower molecular mass than monoacylglycerols of long-chain fatty acids. The method has restricted applicability to acylglycerol mixtures based on caprylic and capric acids. Here, only the monoacylglycerol content and the free glycerol content can be determined. NOTE References 1 to 4 giv
17、e background information. 2 Normative references The following referenced document is 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 amendments) applies. IS
18、O 661, Animal and vegetable fats and oils Preparation of test sample 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 contents of monoacylglycerols, diacylglycerols, triacylglycerols and glycerol proportion of monoacylglycerols, diacylglycerol
19、s, triacylglycerols and glycerol determined under the conditions of this International Standard NOTE The contents are expressed as a mass fraction (grams per 100 g) or as a percentage of all peaks. 4 Principle The sample is dissolved in tetrahydrofuran (THF). The solution obtained is analysed by gel
20、 permeation chromatography (GPC) using THF as the mobile phase. The acylglycerols and glycerol are separated according to their molecular size. Detection is by means of a refractive index detector. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Unco
21、ntrolled Copy, (c) BSI 2 5 Reagents WARNING Attention is drawn to regulations concerning the handling of hazardous substances. Observe all technical, organizational and personal protective measures. Use only reagents of recognized analytical grade, unless otherwise specified. 5.1 Tetrahydrofuran (TH
22、F), stabilized with 250 l/l of BHT. 5.2 Standard substances 5.2.1Glycerol (w W 99,5 %). 5.2.2Monoacylglycerols, diacylglycerols, triacylglycerols1). Standards used should be monoacylglycerols, diacylglycerols and triacylglycerols having a fatty acid distribution as similar as possible to those prese
23、nt in the sample. However, the acylglycerol composition does not have to be reproduced exactly. For most samples, monopalmitate and/or monostearate and/or monooleate, dipalmitate and/or distearate and/or dioleate, tripalmitate and/or tristearate and/or trioleate are sufficient. The response factors
24、for glycerol, monoacylglycerols, diacylglycerols and triacylglycerols are generally the same under the conditions indicated in this International Standard, so that in most cases the use of quantitative reference solutions is unnecessary and percentages by area may be used. However, it is necessary t
25、o determine a response factor for glycerol when the concentration in the sample is ? 3 %. No response factors at all are employed in the European Pharmacopoeia for acylglycerols. This shall be indicated in the test report. 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1
26、 Analytical balance, with a readability of 0,1 mg. 6.2 Volumetric flask, of 10 ml capacity. 6.3 Pipette, of 5 ml capacity. 6.4 Ultrasonic bath. 6.5 HPLC/GPC pump. 6.6 Injector, equipped with a sample loop of 20 l capacity. 6.7 Detector, differential refractometer (RI detector). 6.8 GPC column combin
27、ation, with an effective molecular mass up to 4 000 Da (e.g. three columns of 300 mm ? 7,5 mm Plgel2), 5 m, 100 ). It is also possible to use other columns (or column combinations) provided that the separation of the monoacylglycerols and diacylglycerols and also of the diacylglycerols and triacylgl
28、ycerols is ensured. The use of only two columns is also possible to achieve the required separation. 1) Available for example from Sigma-Aldrich (http:/ 2) Available for example from Polymer Laboratories (http:/ This information is given for the convenience of users of this International Standard an
29、d does not constitute an endorsement by ISO of these products. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 6.9 Column oven (column thermostat). 6.10 Data integration and evaluation system. 7 Sampling A representative
30、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 given in ISO 5555 5. 8 Preparation of test sample Prepare the test sam
31、ple in accordance with ISO 661. Before taking the test portion from the sample, mix the sample thoroughly to ensure homogeneity. For the same reason, melt solid samples completely for proper mixing. 9 Procedure 9.1 Preparation of the sample solution Weigh, to the nearest 0,1 mg, about 100 mg of the
32、sample into a 10 ml volumetric flask. The concentration is reported in milligrams of sample per 10 ml. After diluting to the mark with THF, place the flask in the ultrasonic bath for 5 min to 10 min. The clear solution obtained is directly injected into the GPC system. 9.2 Preparation of reference s
33、olutions Weigh, to the nearest 0,1 mg, amounts of glycerol, monoacylglycerols, diacylglycerols and triacylglycerols above and below the expected concentration in the sample into 10 ml volumetric flasks. After diluting to the mark with THF, place the flasks in the ultrasonic bath for 5 min to 10 min.
34、 The clear solutions obtained in this way are directly injected into the GPC system. The concentration is reported in milligrams of component per 10 ml for each reference solution. If the expected content is not known, it is possible to make up a number of calibration solutions that cover a wider ra
35、nge. NOTE Refer to 5.2.2. 9.3 Gel permeation chromatography (GPC) Set up the GPC system as follows: Injection volume: 20 l Separation columns: Range: minimum 0 to 4 000 Da (6.8) Oven temperature: 35 C or 40 C ? 0,1 C Flow rate: 0,8 ml/min to 1,0 ml/min RI-detector: Temperature: 35 C or 40 C. Typical
36、 chromatograms obtained under these conditions are given in Annex A. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 4 Qualitative testing for the presence of monoacylglycerols, diacylglycerols and triacylglycerols and of g
37、lycerol is carried out by comparing the retention times with comparative substances (e.g. C18-monoacylglycerols/- C18-diacylglycerols/C18-triacylglycerols) or acylglycerol groups. For the quantitative determination of the contents, prepare at least two calibration solutions. Choose the concentration
38、s of the solutions so that they bracket the contents in the sample above and below. These solutions are injected in succession, twice in each case. The calibration curves produced make it possible to calculate the glycerol and acylglycerol concentrations in the sample. NOTE Refer to 5.2.2. 10 Calcul
39、ation The glycerol content in the sample, wG, expressed as a mass fraction in percent, is calculated as follows: GG G S 100 % mF w m ? ? where wGis the glycerol content of the sample; mGis the mass of the glycerol in the sample, in milligrams, calculated from the calibration curve; mSis the mass, in
40、 milligrams, of the test portion; FGis the response factor for glycerol. The mono-, di- and tri-acylglycerol contents are calculated in the same way. In the evaluation of the peak areas, a horizontal baseline is used as the integration boundary. The response factors for glycerol, monoacylglycerols,
41、diacylglycerols and triacylglycerols are generally the same under the conditions indicated here, so that in most cases the use of quantitative reference solutions is unnecessary and the response factors are assumed to be F = 1. However it is necessary to determine a response factor for glycerol when
42、 the concentration in the sample is ? 3 %. In most cases, free fatty acids are not separated from the monoacylglycerol peak group. Therefore in samples with acid values ? 1 the amount of fatty acids, calculated from the acid value, may be subtracted from the monoacylglycerol content. This shall be i
43、ndicated in the test report. Report the result of the determination, expressed as a mass fraction in percent, to one decimal place. 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 interla
44、boratory test may not be applicable to concentration ranges and matrices other than those given. 11.2 Repeatability The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the sam
45、e equipment within a short interval of time, will in not more than 5 % of cases exceed the value of r given in Tables B.1 to B.5. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 5 11.3 Reproducibility The absolute differenc
46、e between two single test results, obtained using the same method on identical test material in different laboratories by different operators using different equipment, will in not more than 5 % of cases exceed the value of R given in Tables B.1 to B.5. 12 Test report The test report shall specify:
47、? all information necessary for the complete identification of the sample; ? the sampling method used, if known; ? the test method used, with reference to this International Standard; ? all operating details not specified in this International Standard, or regarded as optional, together with details
48、 of any incidents which may have influenced the test result(s); ? the test result obtained; ? if the repeatability has been checked, the final quoted result obtained. BS ISO 18395:2005 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 6 Annex
49、A (informative) Examples of chromatograms Key X retention time, min Y peak intensity, mV 1 triacylglycerols (39,132) 2 diacylglycerols (40,407) 3 monoacylglycerols (42,552) 4 free glycerol (47,560) 5 diglycerol monostearate (41,719) a Partial separation of C16-/C18-monoacylglycerols. NOTE Five columns of 300 mm ? 7,5 mm Plgel, 5 m, 100 were used. Figure A.1 Chromatogram of a sample comprising mono-, di- and tri-acylglycerols