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1、Reference number ISO 3596:2000(E) ISO 2000 INTERNATIONAL STANDARD ISO 3596 First edition 2000-07-01 Animal and vegetable fats and oils Determination of unsaponifiable matter Method using diethyl ether extraction Corps gras dorigines animale et vgtale Dtermination de la teneur en matires insaponifiab
2、les Mthode par extraction loxyde dithylique Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO
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6、 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 ISOs member b
7、ody 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.ch Web www.iso.ch Printed in Switzerland ii ISO 2000 All rights reserved Copyright International Organization for Standardization Provided by
8、 IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 3596:2000(E) ISO 2000 All rights reservediii Foreword ISO (the International Organization for Standardization)
9、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 repres
10、ented 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
11、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 v
12、ote. 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 3596 was prepared by Technical Committee ISO/TC 34, Agr
13、icultural food products, Subcommittee SC 11, Animal and vegetable fats and oils. This first edition of ISO 3596 cancels and replaces ISO 3596-1:1988 and its Amendment 1:1997, of which it constitutes a minor revision. Annex A of this International Standard is for information only. Copyright Internati
14、onal Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided
15、by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 3596:2000(E) ISO 2000 All rights reserved1 Animal and vegetable fats and oils Determina
16、tion of unsaponifiable matter Method using diethyl ether extraction 1Scope This International Standard specifies a method using diethyl ether extraction for the determination of the unsaponifiable matter content of animal and vegetable fats and oils. This method is not applicable to waxes and, moreo
17、ver, gives approximate results with certain fats of high unsaponifiable matter content, for example with fats derived from marine animals. A method given in ISO 18609 may be used when climatic conditions, or regulations, do not permit the use of diethyl ether. 2Normative reference The following norm
18、ative document contains 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
19、 are encouraged to investigate the possibility of applying the most recent edition of the normative document 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
20、. ISO 661, Animal and vegetable fats and oils Preparation of test sample. 3Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 unsaponifiable matter all the substances present in the product which, after saponification of the latter by
21、potassium hydroxide and extraction by a specified solvent, are not volatile under the specified operating conditions 4Principle The fat or oil is saponified by boiling under reflux with an ethanolic potassium hydroxide solution. The unsaponifiable matter is extracted from the soap solution by diethy
22、l ether. The solvent is evaporated and the residue is weighed after drying. 5Reagents Use only reagents of recognized analytical grade and distilled or deionized water or water of equivalent purity. 5.1Diethyl ether, freshly distilled, free from peroxides and residue. Copyright International Organiz
23、ation for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 3596:2000(E) 2 ISO 2000 All rights reserved 5.2Acetone. 5.3Potassium hydro
24、xide, ethanolic solution, c(KOH) ? 1 mol/l. Dissolve 60 g of potassium hydroxide in 50 ml of water and dilute to 1000 ml with 95 % (by volume) ethanol. The solution should be colourless or straw-yellow. 5.4Potassium hydroxide, aqueous solution, c(KOH) ? 0,5 mol/l. 5.5Phenolphthalein, 10 g/l solution
25、 in 95 % (by volume) ethanol. 6Apparatus Usual laboratory apparatus and, in particular, the following. 6.1Round-bottomed flasks, of 250 ml capacity, with ground neck. 6.2Reflux condenser, with ground joint to fit the flasks (6.1). 6.3Separating funnels, of 500 ml capacity, with stopcock and stopper
26、made of polytetrafluoroethylene. 6.4Boiling water bath. 6.5Oven, capable of being maintained at 103 C ? 2 C. 7Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 5555 1. It is important the laboratory receive a sample wh
27、ich is truly representative and has not been damaged or changed during transport or storage. 8Preparation of test sample Prepare the test sample in accordance with ISO 661. 9Procedure 9.1Test portion Weigh, to the nearest 0,01 g, about 5 g of the test sample (clause 8) into a 250 ml flask (6.1). 9.2
28、Saponification Add 50 ml of the potassium hydroxide solution (5.3) and a few anti-bumping granules. Attach the reflux condenser (6.2) to the flask and boil the contents gently for 1 h. Stop heating. Add 100 ml of water through the top of the condenser and swirl. If the extraction of the unsaponifiab
29、le matter is carried out with a view to the determination of the composition of tocopherols, the addition of pyrogallol is necessary and the extraction should be completed quickly (say, within 30 min). Copyright International Organization for Standardization Provided by IHS under license with ISO Li
30、censee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 3596:2000(E) ISO 2000 All rights reserved3 9.3Extraction of unsaponifiable matter After cooling, transfer the solution to a 500 ml separating
31、 funnel (6.3). Rinse the flask and the anti-bumping granules several times with the diethyl ether (5.1), using 100 ml in all, and pour these rinsings into the separating funnel. Stopper and shake vigorously for 1 min, periodically releasing pressure by inverting the separating funnel and cautiously
32、opening the stopcock. Allow to stand until there is complete separation of the two phases. Then run off the lower layer as completely as possible into a second separating funnel. If an emulsion is formed, destroy it by adding small quantities of ethanol or concentrated potassium hydroxide or sodium
33、chloride solution. Extract the aqueous ethanolic soap solution twice more, each time in the same way with 100 ml of the diethyl ether. Collect the three ether extracts in one separating funnel containing 40 ml of water. 9.4Washing of ethereal extract Gently rotate the separating funnel containing th
34、e combined extracts and the 40 ml of water. CAUTION Violent shaking at this stage may result in emulsions. Allow the layers to separate completely and draw off the lower aqueous layer. Wash the ethereal solution twice more with 40 ml portions of water, shaking vigorously each time and discarding the
35、 lower aqueous layer after separation. Draw off each washing solution leaving 2 ml, then rotate the separating funnel around its axis. Wait some minutes to allow the remaining aqueous layer to collect. Draw this off, closing the stopcock when the ethereal solution reaches the bore of the stopcock. W
36、ash the ethereal solution successively with 40 ml of the potassium hydroxide solution (5.4), 40 ml of water, and again with 40 ml of potassium hydroxide solution, then at least twice more with 40 ml of water. Continue to wash with water until the washings no longer give a pink colour on the addition
37、 of a drop of the phenolphthalein solution (5.5). 9.5Evaporation of solvent Transfer the ethereal solution quantitatively, a little at a time, through the top of the separating funnel into a 250 ml flask (6.1), previously dried at 103 C in the oven (6.5), then cooled and weighed to the nearest 0,1 m
38、g. Evaporate the solvent on a boiling water bath (6.4). Add 5 ml of acetone (5.2) and evaporate the volatile solvent completely in a gentle current of air, holding the flask obliquely while turning it in a boiling water bath. 9.6Drying the residue and determination 9.6.1Dry the residue in the oven (
39、6.5) at 103 C for 15 min, with the flask in an almost horizontal position. Allow to cool in a desiccator and weigh to the nearest 0,1 mg. Repeat the drying for successive 15 min periods until the loss of mass between two successive weighings is less than 1,5 mg. If a constant mass is not obtained af
40、ter three periods of drying, the unsaponifiable matter is probably contaminated and the determination shall be repeated. NOTEIf available, a vacuum rotary evaporator may be used, particularly if the unsaponifiable matter is to be examined further. 9.6.2If a correction for free fatty acids is conside
41、red necessary, after weighing the residue dissolve it in 4 ml of the diethyl ether (5.1) and then add 20 ml of ethanol previously neutralized to a faint pink colour in the presence of the phenolphthalein (5.4) as indicator. Titrate with standard volumetric ethanolic potassium hydroxide solution, Cop
42、yright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 3596:2000(E) 4 ISO 2000 All rights reserved c(
43、KOH) = 0,1 mol/l, to the same final colour. Calculate the mass of free fatty acids as oleic acid and correct the mass of the residue accordingly (see clause 10). 9.7Number of determinations Carry out two determinations on the same test sample. 9.8Blank test Carry out a blank test, using the same pro
44、cedure and the same quantities of all the reagents, but omitting the test portion. If the residue exceeds 1,5 mg, investigate the technique and the reagents. 10 Expression of results The unsaponifiable matter content, expressed as a percentage by mass of the sample, is equal to 100 123 0 mmm m ?bg %
45、 where m0is the mass, in grams, of the test portion; m1is the mass, in grams, of the residue; m2is the mass, in grams, of the residue obtained with the blank; m3is the mass, in grams, of free fatty acids, if any (see 9.6.2), and equals 0,28 Vc where Vis the volume, in millilitres, of the standard vo
46、lumetric ethanolic potassium hydroxide solution used for the titration; cis the exact concentration, in moles per litre, of the standard volumetric ethanolic potassium hydroxide solution. Take as the result the arithmetic mean of the two determinations. 11 Precision Details of interlaboratory tests
47、on the precision of the method are summarized in annex A. The values derived from these interlaboratory tests may not be applicable to concentration ranges and matrices other than those given. 12 Test report The test report shall specify: ?all information necessary for the complete identification of
48、 the sample; ?the sampling method used, if known; ?the test method used, with reference to this International Standard; Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 09:24:37 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 3596:2000(E) ISO 2000 All rights reserved5 ?all operating details not specified in this International Stand