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1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS ISO 12080-1:2000 ICS 6
2、7.100.10 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Dried skimmed milk Determination of vitamin A content Part 1: Colorimetric method Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI This British Standard, having b
3、een prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 July 2000 BSI 07-2000 ISBN 0 580 36172 1 BS ISO 12080-1:2000 Amendments issued since publication Amd. No.DateComments Nati
4、onal foreword This British Standard reproduces verbatim ISO 12080-1:2000 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products, which has the responsibility to: aid enquirers to un
5、derstand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented
6、on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled International Standards Correspondence Index, or by usi
7、ng the Find facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity f
8、rom legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 7 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Licensed Copy: sheffieldun sheffie
9、ldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference number ISO 12080-1:2000(E) INTERNATIONAL STANDARD ISO 12080-1 First edition 2000-06-01 Dried skimmed milk Determination of vitamin A content Part 1: Colorimetric method Lait crm en poudre Dtermination de la teneur en
10、vitamine A Partie 1: Mthode colorimtrique Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E) ii? Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E)
11、?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 body interested in a subject for whic
12、h 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 Electrotechnical Commission (IEC) on all ma
13、tters of 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 Stand
14、ard 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 part of ISO 12080 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International St
15、andard ISO 12080-1 was prepared by Technical Committee ISO/TC 34,Agricultural food products, Subcommittee SC 5,Milk and milk products,in collaboration with the International Dairy Federation (IDF) and AOAC International, and will also be published by these organizations. ISO 12080 consists of the fo
16、llowing parts, under the general titleDried skimmed milk Determination of vitamin A content: ?Part 1: Colorimetric method ?Part 2: Method using high-performance liquid chromatography Annex A of this part of ISO 12080 is for information only. Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:
17、35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E) iv? Introduction The methods specified in ISO 12080 have been selected after consideration and laboratory testing of a variety of alternative procedures. Their advantages include the absence of highly dangerous reagents as in, for
18、example, the Carr-Price method, and the avoidance of reagents that are not universally available. The decision to provide two separate methods was taken to meet the needs both of laboratories with sophisticated equipment (HPLC) and those without such apparatus. Although the International Standard fo
19、r vitamin A was discontinued in 1954, the International Unit for this substance has continued to be widely used and its use has been maintained in this International Standard. The International Unit for vitamin A was redefined in 1960 as the activity of 0,000 344 mg of pure all-trans-vitamin A aceta
20、te (see annex A). Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI INTERNATIONAL STANDARD?ISO 12080-1:2000(E) ?1 Dried skimmed milk Determination of vitamin A content Part 1: Colorimetric method WARNING The use of this International Standard
21、may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish safety and health practices and determine the applicability of regulatory limitatio
22、ns prior to use. 1?Scope This part of ISO 12080 specifies a colorimetric method for the determination of vitamin A in dried skimmed milk containing at least 10 IU (International Units) of vitamin A per gram. 2?Term and definition This part of ISO 12080 specifies a colorimetric method for the determi
23、nation of vitamin A in dried skimmed milk containing at least 10 IU (International Units) of vitamin A per gram. 2.1 vitamin A content of dried skimmed milk mass fraction of substances determined by the procedure specified in this part of ISO 12080 NOTE?It is expressed either in micrograms of retino
24、l per gram or in International Units of vitamin A activity per gram. 3Principle The test sample is saponified and extracted. The unsaponifiable matter is reacted with trifluoroacetic acid. The absorbance at 620 nm is measured. 4?Reagents Use only reagents of recognized analytical grade, unless other
25、wise specified, and distilled or demineralized water or water of equivalent purity. 4.1?Ethanol (CH3CH2OH), 95 % (by volume), free from aldehyde. 4.2?Sodium ascorbate solution, 200 g/l. If not available ready-made, prepare by dissolving 3,5 g of ascorbic acid (C6H8O6) in 20 ml of 1 mol/l sodium hydr
26、oxide (NaOH) solution and mix. Prepare this solution fresh daily. 4.3?Potassium hydroxide aqueous solution (KOH), 50 % (by mass) Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E) 2? Dissolve 50 g of potassium hydroxide in 5
27、0 ml of water. Mix and cool the solution. Prepare this solution freshly before use. 4.4?Potassium hydroxide aqueous alcoholic solution, 30 g/l. Dissolve 3 g of potassium hydroxide (KOH) in water and add 10 ml of ethanol (4.1) in a 100 ml one-mark volumetric flask. Dilute with water to the 100 ml mar
28、k and mix. Prepare this solution freshly before use. 4.5?Light petroleum, with a boiling range of between 40 C and 60 C, or of between 60 C and 80 C. 4.6?Chloroform (CHCl3) 4.7?Trifluoroacetic acid (CF3COOH) WARNING Chloroform and trifluoroacetic acid are carcinogenic. Take all necessary precautions
29、. 4.8?Colour reagent Mix by volume 1 part of pure trifluoroacetic acid (4.7) and 2 parts of chloroform (4.6). 4.9?Vitamin A standard solution Use US Pharmacopeia standard1)reference solution of vitamin A made from crystalline all-trans-retinyl acetate in cottonseed oil, equivalent to 30 mg of retino
30、l (vitamin A alcohol, C20H30O) per gram of oil, or as stated when purchased. A secondary standard solution may be used if standardized against this primary standard reference solution or by UV measurement. Cut the tip from the capsule containing the vitamin A standard reference solution and express
31、the oil into a (tared) 100 ml amber-coloured one-mark volumetric flask. Weigh the contents to the nearest 0,1 mg. Dilute with chloroform (4.6) to the 100 ml mark. Use the vitamin A standard solution as soon as possible. Discard the solution after 8 h. 4.10Butylated hydroxytoluene (BHT) 5?Apparatus U
32、sual laboratory apparatus and, in particular, the following. 5.1?Photoelectric colorimeter or spectrometer, with an optical mechanism or filter for 620 nm wavelength (vitamin A derivative). Use matched absorption cells. An instrument providing linearity between absorbance and concentration is to be
33、preferred. 5.2?Beaker or conical flask, of capacity 250 ml. 5.3?Saponification flask, of capacity approximately 200 ml, fitted with a reflux condenser. 5.4?One-mark volumetric flasks, of capacities 100 ml and 200 ml. 5.5?One-mark pipettes, of capacities 2 ml, 10 ml, 25 ml and 50 ml. 1)The reference
34、vitamin A solution from United States Pharmacopeia Convention, Inc. 12601 Twinbrook Parkway, Rockville, Maryland 20852, USA, is an example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO 12080 and does not constitute an endorse
35、ment by ISO of this product. Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E) ?3 5.6?Automatic pipettes, suitable for organic solvents, or a pipette, to deliver 10 ml. 5.7?Steam bath, boiling water bath or electric heating
36、 mantle 5.8?Water bath, capable of operating at a temperature of up to 40 C. 5.9?Separating funnel, of capacity 500 ml, preferably with a polytetrafluoroethylene (PTFE) stopper. 5.10Ultrasonic bath 5.11Filter paper, of diameter 9 cm. 6?Sampling Sampling is not part of the method specified in this pa
37、rt of ISO 12080. A recommended sampling method is given in ISO 707 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 7?Preparation of test sample Thoroughly mix the test sample by repeatedly rotating
38、 and inverting the sample container. If necessary for this, transfer the complete test sample to an airtight container of sufficient capacity. 8?Procedure 8.1? General NOTE?If it is required to check whether the repeatability limit (10.2) is met, carry out two single determinations in accordance wit
39、h 8.2 to 8.5. For all operations, work in subdued light or use low-actinic glassware. 8.2? Test solution Weigh, to the nearest 0,001 g, about 20 g of the test sample into a beaker or conical flask (5.2) and dissolve in 50 ml of water at a temperature of least 80 C. Break down any lumps with a spatul
40、a or by using a ultrasonic bath (5.10). Cool to room temperature. Transfer quantitatively to a 100 ml one-mark volumetric flask (5.4). Dilute with water to the 100 ml mark. 8.3? Saponification and extraction 8.3.1?Transfer, by means of a pipette (5.5), 25 ml of the prepared test solution (8.2) to a
41、saponification flask (5.3). Add 20 ml of potassium hydroxide solution (4.3) and 10 ml of sodium ascorbate solution (4.2). Add 50 ml of ethanol (4.1) and mix well. 8.3.2?Reflux for 30 min on a steam bath (5.7), swirling from time to time. Cool immediately under running water. 8.3.3?Transfer the liqui
42、d to a separating funnel (5.9), using two 30 ml portions of water, two 10 ml portions of ethanol (4.1) and two 40 ml portions of light petroleum (4.5). Shake vigorously for 30 s and allow to stand until the two layers are clear. Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:
43、00 2006, Uncontrolled Copy, (c) BSI ISO 12080-1:2000(E) 4? Transfer the aqueous (lower) phase to a second separating funnel and shake with a mixture of 10 ml of ethanol (4.1) and 40 ml of light petroleum (4.5). Leave to separate. 8.3.4?Transfer the aqueous phase to a third separating funnel and the
44、light petroleum phase to the first separating funnel. Wash the second separating funnel with two 10 ml portions of light petroleum (4.5). Add the washings to the first separating funnel. 8.3.5?Shake the aqueous phase with 40 ml of light petroleum (4.5) and 10 ml of ethanol (4.1). Add the light petro
45、leum phase to the first separating funnel. Wash the combined light petroleum extracts with three 40 ml portions of freshly prepared potassium hydroxide alcoholic solution (4.4), shaking vigorously. Then wash with 40 ml portions of water until the last washing is neutral to phenolphthalein. Drain the
46、 last few drops of water, add two sheets of filter paper (5.11), cut into strips, to the separating funnel and shake. 8.3.6?Transfer the light petroleum extract, dried as described above, to a 200 ml one-mark volumetric flask (5.4). Rinse the separating funnel and paper with light petroleum (4.5), a
47、dd the rinsings to the volumetric flask, then add 10 mg to 20 mg of BHT (4.10). Dilute with light petroleum to the 200 ml mark and mix well. 8.4? Preparation of test colorimetric solution Pipette an aliquot part of the diluted extract (8.3.6) into a round-bottom flask. Evaporate to dryness under vac
48、uum by swirling in a water bath (5.8) at a temperature not exceeding 40 C. Cool under running water and restore atmospheric pressure, preferably with nitrogen. Dissolve the residue immediately in 10,0 ml of chloroform (4.6). NOTE?A typical concentration in the test solution is 10 IU of vitamin A per
49、 millilitre of chloroform. The amount of the aliquot may be adjusted depending on the size and sensitivity of the colorimetric cell. 8.5? Determination Designate two suitable matched colorimetric cells as 1 and 2. Pipette 2 ml of the test colorimetric solution (8.4) into cell 1. Pipette 2 ml of the diluted vitamin A standard solution (8.6) into cell 2. Rapidly add to each cell, preferably using an automatic pipette (5.6), 10,0 ml of the colour reagent (4.8) and mix. Monitor the absorbance of the solutions at 620 nm with the spectrometer or photo
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