1、BRITISH STANDARD BS 5766-12: 1986 ISO 6866:1985 Methods for Analysis of animal feeding stuffs Part 12: Determination of gossypol ISO title: Animal feeding stuffs Determination of free and total gossypol UDC 636.085:615.91.918:543.42.062BS5766-12:1986 This British Standard, having been prepared under
2、 the directionof the Food and Agriculture Standards Committee,was published underthe authority of the BoardofBSI and comes intoeffecton 28November1986 BSI 12-1999 The following BSI references relate to the work on this standard: Committee reference FAC/14 Draft for comment83/55938 DC ISBN 0 580 1544
3、4 0 Committees responsible for this British Standard The preparation of this British Standard was entrusted by the Food and Agriculture Standards Committee (FAC/-) to Technical Committee FAC/14, upon which the following bodies were represented: Association of Public Analysts Department of Trade and
4、Industry (Laboratory of the Government Chemist) Grain and Feed Trade Association Limited Ministry of Agriculture, Fisheries and Food National Farmers Union Overseas Development Administration (Tropical Development and Research Institute) Pet Food Manufacturers Association Royal Association of Britis
5、h Dairy Farmers United Kingdom Agricultural Supply Trade Association Ltd. United Kingdom Renderers Association Ltd. Amendments issued since publication Amd. No. Date of issue CommentsBS5766-12:1986 BSI 12-1999 i Contents Page Committees responsible Inside front cover National foreword ii 1 Scope 1 2
6、 Field of application 1 3 References 1 4 Principle 1 5 Reagents 1 6 Apparatus 1 7 Sampling 1 8 Procedure 1 9 Expression of results 2 10 Test report 3 Publications referred to Inside back coverBS5766-12:1986 ii BSI 12-1999 National foreword This Part of BS5766 has been prepared under the direction of
7、 the Food and Agriculture Standards Committee. It is identical with ISO6866:1985 “Animal feeding stuffs Determination of free and total gossypol” published by the International Organization for Standardization (ISO), in the preparation of which the United Kingdom played a full part. In the United Ki
8、ngdom, animal feeding stuffs are controlled by legislation. The method in this Part of BS5766 is technically equivalent to the method given in the Feeding Stuffs (Sampling and analysis) Regulations1982, SI1982 No. 1144 1) . Terminology and conventions. The text of the International Standard has been
9、 approved as suitable for publication as a British Standard without deviation. Some terminology and certain conventions are not identical with those used in British Standards; attention is drawn especially to the following. The comma has been used as a decimal marker. In British Standards it is curr
10、ent practice to use a full point on the baseline as the decimal marker. In British Standards it is current practice to use the symbol “L” for litre (and in its submultiples) rather than “l”. Wherever the words “International Standard” appear, referring to this standard, they should be read as “Briti
11、sh Standards”. Cross-references. When ISO6497, referred to in clause7, is published it is intended to implement it as a dual-numbered (identical) British Standard. In the meantime, parties concerned are recommended to reach agreement on sampling. The Technical Committee has reviewed the relevant pro
12、visions of ISO6498:1983, referred to in8.1 and has decided that they are acceptable for use in conjunction with this standard. The United Kingdom did not approve ISO6498 but, in the present context, test samples prepared in accordance with either ISO6498 or BS5766 “Methods for analysis of animal fee
13、dings stuffs” Part 10 “Preparation of test samples” are equally acceptable. 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 confe
14、r immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, pages1 to4, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in t
15、he amendment table on the inside front cover. 1) Published by HMSO.BS5766-12:1986 BSI 12-1999 1 1 Scope This International Standard specifies a method for the determination of the content of free and total gossypol and chemically related substances in animal feeding stuffs. 2 Field of application Th
16、e method is applicable to cottonseed and cottonseed meals and cakes, and to compound feeding stuffs containing these substances. The detection limit for free gossypol is20mg/kg and that for total gossypol is50mg/kg. 3 References ISO 6497, Animal feeding stuffs Sampling 2) . ISO 6498, Animal feeding
17、stuffs Preparation of test samples. 4 Principle Extraction of the gossypol in the presence of3-aminopropan-1-ol, either with a mixture of2-propanol and hexane for the determination of free gossypol or with dimethylformamide for the determination of total gossypol. Conversion of the gossypol into gos
18、sypol-dianiline using aniline. Measurement of the absorbance at the wavelength of maximum absorbance (between435 and445nm). 5 Reagents All reagents shall be of recognized analytical grade. The water used shall be distilled water or water of at least equivalent purity. 5.1 2-Propanol/n-hexane, 60+40
19、mixture by volume. 5.2 Solvent A Place in a1000ml volumetric flask approximately500ml of the2-propanol/hexane mixture (5.1), 2ml of3-aminopropan-1-ol, 8ml of glacial acetic acid and50ml of water. Make up to volume with the2-propanol/hexane mixture. This reagent is stable for one week only. 5.3 Solve
20、nt B Place in a100ml volumetric flask2ml of3-aminopropan-1-ol and10ml of glacial acetic acid. Cool to room temperature and make up to volume with N,N-dimethylformamide. This reagent is stable for one week only. 5.4 Aniline If the absorbance determined in the blank test exceeds0,022, distil the anili
21、ne over zinc dust, discarding the first and last10% fractions of distillate. Store in a stoppered brown glass bottle in a refrigerator (0to4 C). The reagent is stable for several months. 6 Apparatus Usual laboratory apparatus and in particular 6.1 Tumbler mixer, having a rotational frequency of appr
22、oximately35min 1 , or other shaker. 6.2 Spectrometer, suitable for making measurements at wavelengths between435 and445nm, with cells of optical path length10mm. 6.3 Volumetric flasks, of capacities25 and50ml. 6.4 Conical flasks, of capacities50, 100and250ml. 7 Sampling Take the laboratory sample in
23、 accordance withISO6497. 8 Procedure 8.1 Preparation of the test sample Prepare the test sample in accordance with ISO6498. 8.2 Test portion The mass of the test portion depends on the expected gossypol content. For the determination of free gossypol in cottonseed and cottonseed meals and cakes the
24、mass of the test portion should not exceed1g; for compound feeding stuffs, it may be as much as5g. For the determination of total gossypol, the mass of the test portion should be between0,5 and5g. NOTEThe mass of the test portion should be large enough to give the quantity of free gossypol indicated
25、 in8.3.1.2 or the quantity of total gossypol indicated in8.3.2.4 in each aliquot portion of the filtrate. 8.3 Determination 8.3.1 Determination of free gossypol 8.3.1.1 Transfer the test portion (8.2) to a250ml conical flask, the bottom of which has been covered with glass beads. Add, by means of a
26、pipette, 50ml of solvent A (5.2), stopper the flask and mix, using the tumbler mixer (6.1), for1h. Filter through a dry filter paper, collecting the filtrate in a100ml conical flask. During filtration, cover the funnel with a watch-glass. 2) At present at the stage of draft.BS5766-12:1986 2 BSI 12-1
27、999 8.3.1.2 Transfer, by means of a pipette, identical aliquot portions of the filtrate, containing50 to1004g of gossypol, to each of two25ml volumetric flasks (6.3) (C and D). If necessary, make up to10ml with solvent A (5.2). 8.3.1.3 Make up the contents of flask C to volume with the2-propanol/hex
28、ane mixture (5.1) and mix. This solution will be used as the reference solution against which the test solution will be measured. 8.3.1.4 Transfer, by means of a pipette, 10ml of solvent A (5.2) into each of two25ml volumetric flasks (E and F). 8.3.1.5 Make up the contents of flask E to volume with
29、the2-propanol/hexane mixture (5.1) and mix. This solution will be used as the reference solution against which the blank test solution will be measured. 8.3.1.6 Add2,0ml of the aniline (5.4) to flasks D andF. Heat for30min on a boiling water-bath to develop the colour. 8.3.1.7 Cool to room temperatu
30、re, make up to volume with the2-propanol/hexane mixture (5.1), mix and leave for1h. 8.3.1.8 Measure the absorbance of the blank test solution (flask F; 8.3.1.7) against the reference solution (flask E; 8.3.1.5) and the absorbance of the test solution (flask D; 8.3.1.7) against the reference solution
31、 (flask C; 8.3.1.3) using the spectrometer(6.2) at the wavelength of maximum absorbance (between435 and445nm). 8.3.1.9 Subtract the absorbance of the blank test solution from that of the test solution to obtain the corrected absorbance. 8.3.2 Determination of total gossypol 8.3.2.1 Transfer the test
32、 portion (8.2) into a50ml volumetric flask and add10ml of solvent B (5.3). 8.3.2.2 Transfer10ml of solvent B (5.3) to a second50ml volumetric flask (6.3). 8.3.2.3 Heat the two flasks (8.3.2.1 and8.3.2.2) for30min on a boiling water-bath. Cool to room temperature and make up to volume with the2-propa
33、nol/hexane mixture (5.1). Mix and leave to settle for10 to15min, then filter and collect the filtrates in50ml conical flasks (6.4). 8.3.2.4 Transfer, by means of a pipette, 2ml of the filtrate obtained from the test portion, containing40 to2004g of gossypol, into each of two25ml volumetric flasks an
34、d2ml of the filtrate obtained from the second flask into each of two other25ml volumetric flasks. Make up the contents of one of the flasks containing the test solution and of one flask from the blank test to volume with the2-propanol/hexane mixture (5.1). These solutions will be used as reference s
35、olutions. 8.3.2.5 Add2,0ml of the aniline (5.4) to the remaining test and blank test solutions. Heat for30min on a boiling water-bath to develop the colour. Cool to room temperature, make up to volume with the2-propanol/hexane mixture (5.1), mix and leave for1h. 8.3.2.6 Measure the absorbances of th
36、e solutions as specified in8.3.1.8 and calculate the corrected absorbance as specified in8.3.1.9. 8.4 Number of determinations Carry out two determinations on the same test sample. 9 Expression of results 9.1 Method of calculation and formula The free or total gossypol content, expressed in milligra
37、ms per kilogram of the product as received, is equal to where A is the corrected absorbance (8.3.1.9 or8.3.2.6); m is the mass, in grams, of the test portion(8.2); V is the volume, in millilitres, of the aliquot portion of filtrate taken in8.3.1.2 or8.3.2.4; a is the specific mass absorbance coeffic
38、ient(62,5cm 1 g 1 l in the case of free gossypol; 60,0cm 1 g 1 l in the case of total gossypol). Take as the result the arithmetic mean of the two determinations (see8.4) if the condition for repeatability (see9.2) is met. 9.2 Repeatability The difference between the results of two determinations, c
39、arried out simultaneously or in rapid succession on the same sample, shall not exceed 15% (relative) of the mean for gossypol contents less than500mg/kg; 75mg/kg (absolute) for gossypol contents not less than500mg/kg and not more than750mg/kg; 10% (relative) of the mean for gossypol contents more th
40、an750mg/kg.BS5766-12:1986 BSI 12-1999 3 10 Test report The test report shall show the method used and the result obtained. It shall also mention any operating details not specified in this International Standard, or regarded as optional, together with details of any incidents likely to have influenc
41、ed the result. The test report shall include all the information necessary for the complete identification of the sample.4 blankBS5766-12:1986 BSI 12-1999 Publications referred to See national foreword.BS 5766-12: 1986 ISO 6866:1985 BSI 389 Chiswick High Road London W4 4AL BSIBritishStandardsInstitu
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