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1、Reference number ISO 13496:2000(E) ISO 2000 INTERNATIONAL STANDARD ISO 13496 First edition 2000-03-01 Meat and meat products Detection of colouring agents Method using thin-layer chromatography Viande et produits base de viande Dtection des agents colorants Mthode par chromatographie en couche mince
2、 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) PDF disclaimer This PDF file
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5、ation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2000 All rights reserved. Unle
6、ss 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 body in the country of the requester. ISO c
7、opyright office Case postale 56 ? CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 734 10 79 E-mail copyrightiso.ch Web www.iso.ch Printed in Switzerland ii ISO 2000 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA
8、Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) ISO 2000 All rights reservediii ContentsPage Foreword.iv 1Scope 1 2Normative references1 3Term and definition .1 4Principle2 5Reagents.2 6A
9、pparatus.3 7Sampling.4 8Preparation of test sample4 9Procedure.4 10Test report6 Annex A (informative) Synonyms and identity numbers of synthetic, water-soluble colouring agents .7 Annex B (normative) Possible interference by colours8 Annex C (informative) Absorbance spectra .9 Bibliography11 Copyrig
10、ht International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) iv ISO 2000 All rights reserved Fore
11、word 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 in a subject for which a techn
12、ical 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 Commission (IEC) on all matters of
13、electrotechnical standardization. International Standards are 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 requi
14、res 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 identifying any or all such patent rights. International Standa
15、rd ISO 13496 was prepared by Technical Committee ISO/TC 34,Agricultural food products, Subcommittee SC 6,Meat and meat products. Annex B forms a normative part of this International Standard. Annexes A and C are for information only. Copyright International Organization for Standardization Provided
16、by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 13496:2000(E) ISO 2000 All rights reserved1 Meat and meat products Detection of colouri
17、ng agents Method using thin-layer chromatography 1Scope This International Standard specifies a thin-layer chromatographic method for the detection of synthetic, water- soluble colouring agents in meat and meat products. The following colouring agents can be detected with the method: TartrazinePaten
18、t Blue V Quinoline YellowIndigotine Sunset Yellow FCFBrilliant Black PN AmaranthBlack 7984 Ponceau 4RFast Green FCF ErythrosineBlue VRS Synonyms and identity numbers of these colouring agents are listed in annex A. The plant colours and plant extracts which have been observed not to interfere with t
19、his method are listed in Table B.1. Natural colours which in some cases have been shown to interfere with this method are listed in Table B.2. 2Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International
20、 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 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indica
21、ted 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. ISO 3696:1987,Water for analytical laboratory use Specification and test methods. AOAC 46.1.08:1995,Official Meth
22、ods of Analysis(AOAC International). 3Term and definition For the purposes of this International Standard, the following term and definition apply. 3.1 detection of colouring agents detection of the presence or absence of colouring agents in accordance with the method specified in this International
23、 Standard Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) 2 ISO 2000 All righ
24、ts reserved 4Principle The colouring agents are extracted from a test portion with hot water and adsorbed onto polyamide powder. The extracted colouring agents are purified by column chromatography and the colours are eluted from the column. The colouring agents are identified by thin-layer chromato
25、graphy. 5Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 5.1Water, complying with at least grade 3 in accordance with ISO 3696. 5.2Petroleum ether, boiling range 40 ?C to 60 ?C. 5.3Methanol. 5.4Ammonia, 25 % aqueous solution, ?20= 0,910 g/ml. 5.5Acetic acid, 10
26、0 % mass fraction, ?20= 1,050 g/ml. 5.6Trisodium citrate dihydrate. 5.7Propan-1-ol. 5.8Ethyl acetate. 5.92-Methyl-2-propanol. 5.10Propionic acid. 5.11Eluent solution for column chromatography. Mix 95 volumes of methanol (5.3) with 5 volumes of ammonia solution (5.4). 5.12Acetic acid, 50 % solution i
27、n methanol. Mix 1 volume of acetic acid (5.5) with 1 volume of methanol (5.3). 5.13Polyamide powder, of particle size 0,05 mm to 0,16 mm. 5.14Sand, fine granular, hydrochloric acid-washed, neutralized and calcinated. 5.15Standard reference colours. The purities of the standard colours may vary so it
28、 is necessary to know the purity of the colours to be used as standards. The purity shall be determined by the method AOAC 46.1.08. NOTECertified food colours may also be used as standards. 5.16Standard reference solutions for thin-layer chromatography Separately make solutions in water of each of t
29、he standard reference colours (5.15) with a standard colour content of about 1 g/l. Prepare solutions of indigotine on the day of use. Other solutions will keep for at least 3 months (solutions of erythrosine for 1 month) when stored in the dark. Copyright International Organization for Standardizat
30、ion Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) ISO 2000 All rights reserved3 5.17Eluent for thin-layer chromatography: solution I
31、 Weigh, to the nearest 0,1 g, 25 g of trisodium citrate dihydrate (5.6) into a 1 000 ml one-mark volumetric flask. Dissolve in water, dilute to the mark with water and mix. Mix 80 volumes of this citrate solution with 20 volumes of ammonia solution (5.4) and 12 volumes of methanol (5.3). To avoid or
32、 reduce interference from safflor or saffran, it is advisable to use chromatography solution II (5.18). 5.18Eluent for thin-layer chromatography: solution II Mix 6 volumes of propan-1-ol (5.7) with 1 volume of ethyl acetate (5.8) and 3 volumes of water. 5.19Eluent for thin-layer chromatography: solu
33、tion III Mix 50 volumes of 2-methyl-2-propanol (5.9) with 12 volumes of propionic acid (5.10) and 38 volumes of water. 6Apparatus Usual laboratory apparatus and, in particular, the following. 6.1Mechanical or electrical homogenizing equipment, capable of homogenizing the laboratory sample. Use a hig
34、h-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding 4,0 mm in diameter. 6.2Centrifuge tubes, of capacity 75 ml, made of glass. 6.3Flat-bottomed flasks, of capacity 250 ml, with ground glass stoppers. 6.4Round-bottomed flasks, of capacity 100 ml, with ground glass
35、joint. 6.5Centrifuge, operating at a radial acceleration of about 2 000gn. 6.6Rotary evaporator. 6.7Chromatographic column, of glass, with fritted filter and tap, of length about 20 cm, diameter about 30 mm, filter pore size 40 ?m to 100 ?m (porosity grade P 100 according to ISO 4793 2). Put some gl
36、ass wool in the column and add 1 g to 2 g of sand (5.14). 6.8Plastics container, of volume about 10 ml, with lid. 6.9Thin-layer plates, coated with a layer of cellulose powder of 0,10 mm thickness, or equivalent. Ready-to-use plates are suitable. 6.10Micropipettes, of capacity approximately 5 ?l. 6.
37、11pH-meter, accurate to within 0,1 pH unit. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO
38、 13496:2000(E) 4 ISO 2000 All rights reserved 7Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 3100-1 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or
39、changed during transport or storage. Proceed from a representative sample of at least 200 g. Store the sample in such a way that deterioration and change in composition are prevented. 8Preparation of test sample Homogenize the laboratory sample with the appropriate equipment (6.1). Take care that th
40、e temperature of the sample material does not rise above 25 C. If a mincer is used, pass the sample at least twice through the equipment. Fill a suitable airtight container with the prepared sample. Close the container and store in such a way that deterioration and change in composition of the sampl
41、e are prevented. Analyse the sample as soon as practicable, but always within 24 h after homogenization. 9Procedure WARNINGS If the sample contains indigotine, the temperature shall not at any time during the analysis exceed 35 ? ? ? ?C. Indigotine partially decomposes in chromatography solution I,
42、so chromatography solution II shall be used. Erythrosine is sensitive to light. When pausing in the course of the analysis, solutions and plates shall be stored in the dark. The same also holds for indigotine. 9.1Test portion Weigh, to the nearest 0,1 g, 5 g of the prepared test sample (see clause 8
43、) into a centrifuge tube (6.2). For fatty samples, proceed in accordance with 9.2. For non-fatty samples, proceed in accordance with 9.3. 9.2Fatty samples Add about 20 ml of petroleum ether (5.2) to the centrifuge tube and mix with a glass rod. Decant the petroleum ether. Repeat this procedure three
44、 times. 9.3Non-fatty samples Add 25 ml of boiling water (see warning above) and mix. Add 25 ml of the eluent solution(5.11). Check that the pH is 9 ? 0,5 using the pH-meter (6.11). If not, adjust the pH with acetic acid (5.5) or ammonia solution (5.4). Mix well. Chill the sample in a freezer for 15
45、min (to prevent turbidity). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/21/2007 01:58:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13496:2000(E) I
46、SO 2000 All rights reserved5 Centrifuge (6.5) for 10 min at a radial acceleration of about 2000gn. Decant the clear solution into a flat-bottomed flask (6.3). In the case of indigotine, use a round-bottomed flask (6.4). Add 5 ml of water to the centrifuge tube containing the residue. Mix and add 10
47、ml of the eluent solution (5.11). Mix and centrifuge as above. Repeat the procedure until all colour has been extracted from the sample then combine all extracts. Evaporate the combined extract on a water bath to about 25 ml in order to remove methanol. In the case of indigotine, use a round-bottome
48、d flask (6.4) and the rotary evaporator (6.6) at 35 ?C. Add 25 ml of boiling water (see warnings) and mix. 9.4Transfer of the colours to polyamide powder Using acetic acid (5.5) or ammonia solution (5.4).adjust the pH to between 4 and 5. Add 1 g of polyamide powder (5.13) to the warm solution (see warnings). Shake vigorously for 1 min. Allow the powder to form a sediment. Check that no colour remains in the solution. If the solution is coloured, add some more polyamide powder and shake