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1、A Reference number ISO 6491:1998(E) INTERNATIONAL STANDARD ISO 6491 Second edition 1998-12-15 Animal feeding stuffs Determination of phosphorus content Spectrometric method Aliments des animaux Dtermination de la teneur en phosphore Mthode spectromtrique Copyright International Organization for Stan
2、dardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 6491:1998(E) ISO 1998 All rights reserved. Unless otherwise specified, no part of this p
3、ublication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from the publisher. International Organization for Standardization Case postale 56 CH-1211 Genve 20 Switzerland Internetisoiso.ch Printe
4、d in Switzerland ii 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 s
5、ubject 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 Electrotechnical Commission
6、(IEC) on all matters of electrotechnical standardization. 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 vote. International S
7、tandard ISO 6491 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 10, Animal feeding stuffs. This second edition replaces the first edition (ISO 6491:1980), which has been technically revised. Annexes A and B of this International Standard are for informatio
8、n only. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISOISO 6491:1998
9、(E) 1 Animal feeding stuffs Determination of phosphorus content Spectrometric method 1 Scope This International Standard specifies a spectrometric method for the determination of the phosphorus content of animal feeding stuffs. The method is applicable to animal feeding stuffs with a phosphorus cont
10、ent less than 50 g/kg. It is particularly appropriate for the analysis of products with low phosphorus content. For products with higher phosphorus content, application of a gravimetric method is advised, using for instance quinoline phosphomolybdate. 2 Normative references The following standards c
11、ontain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to invest
12、igate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 3696:1987, Water for analytical laboratory use Specification and test methods. ISO 6498:1998, Animal feeding stuffs P
13、reparation of test samples. 3 Principle A test portion of the sample is either ashed with lime and heated with acid (in the case of organic feeding stuffs) or wet oxidized with a mixture of sulfuric and nitric acids (in the case of mineral compounds and liquid feeding stuffs). An aliquot portion of
14、the acid solution is mixed with molybdovanadate reagent and the absorbance of the yellow solution obtained is measured at a wavelength of 430 nm. 4 Reagents Use only reagents of recognized analytical grade. 4.1 Water, complying to at least grade 3 in accordance with ISO 3696. 4.2 Calcium carbonate.
15、4.3 Hydrochloric acid, c(HCl) 6 mol/l. 4.4 Nitric acid, c(HNO3) 1 mol/l. 4.5 Nitric acid, c(HNO3) = 14 mol/l, r(HNO3) 1,40 g/ml. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 2
16、2:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 6491:1998(E) ISO 2 4.6 Sulfuric acid, c(H2SO4) = 18 mol/l, r(H2SO4) = 1,84 g/ml. 4.7 Ammonium heptamolybdate solution. Dissolve in hot water 100 g of ammonium heptamolybdate tetrahydrate (NH4)6Mo7O244H2O. Add 10 ml
17、 of ammonia c(NH4OH) = 14 mol/l, r(NH4OH) = 0,91 g/ml and dilute to 1 l with water. 4.8 Ammonium monovanadate solution. Dissolve 2,35 g of ammonium monovanadate (NH4VO3) in 400 ml of hot water. Stirring constantly, slowly add 7 ml of nitric acid (4.5) and dilute to 1 l with water. 4.9 Molybdovanadat
18、e reagent. In a 1 l volumetric flask, mix 200 ml of the ammonium heptamolybdate solution (4.7), 200 ml of the ammonium monovanadate solution (4.8) and 135 ml of nitric acid (4.5). Dilute to the mark with water. Filter if insoluble particles are present. 4.10 Reference solution. Dilute 10 ml of molyb
19、dovanadate reagent (4.9) with 10 ml of water. 4.11 Phosphorus standard solution, r(P) = 1 mg/ml. In a 1 l volumetric flask, dissolve in water 4,394 g of potassium dihydrogen phosphate (KH2PO4), previously dried at 103 C for 1 h. Dilute to the mark with water. 5 Apparatus Usual laboratory apparatus a
20、nd, in particular, the following. 5.1 Ashing crucibles, of silica or porcelain. 5.2 Electric muffle-furnace, capable of being maintained at a temperature of 550 C 20 C. 5.3 Kjeldahl flask, of capacity 250 ml. 5.4 One-mark volumetric flasks, of capacities 500 ml and 1 000 ml. 5.5 Spectrometer, fitted
21、 with 10 mm cells, suitable for measurements at a wavelength of 430 nm. 5.6 Glass test tubes, of capacity 25 ml to 30 ml, fitted with ground glass stoppers. 5.7 Sand bath. 5.8 Beaker, of capacity 250 ml. 5.9 Graduated pipettes. 6 Sampling Sampling is not part of the method specified in this Internat
22、ional Standard. A recommended sampling method is given in ISO 6497 4. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Copyright International Organization for Standardization Provided by IHS under lic
23、ense with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- ISOISO 6491:1998(E) 3 7 Preparation of test sample Prepare the test sample in accordance with ISO 6498. If solid, grind the labor
24、atory sample (usually 500 g) so that it passes completely through a sieve with 1 mm apertures. Mix thoroughly. 8 Procedure 8.1 Selection of procedure If the test sample contains organic substances and if it is free from phosphates rendering insoluble products on ashing, proceed in accordance with 8.
25、2. If the test sample concerns a mineral compound or a liquid feeding stuff, proceed in accordance with 8.3. 8.2 Dry ashing Weigh about 2,5 g of the prepared test sample to the nearest 1 mg in an ashing crucible (5.1). Mix the test portion thoroughly with 1 g of the calcium carbonate (4.2). Ash in t
26、he furnace (5.2), set at a temperature of 550 C, until white or grey ash is obtained (a small quantity of carbon does not interfere). Transfer the ash to a 250 ml beaker (5.8) with 20 ml to 50 ml of water. Add hydrochloric acid (4.3) until effervescence ceases. Add a further 10 ml of the hydrochlori
27、c acid (4.3). Place the beaker on the sand bath (5.7) and evaporate to dryness to render the silica insoluble. Allow to cool. Add 10 ml of nitric acid (4.4) to the residue and boil on the sand bath for 5 min, without evaporating to dryness. Decant the liquid into a 500 ml volumetric flask (5.4), rin
28、sing the beaker several times with hot water. Leave to cool, dilute to the mark with water, mix and filter. Proceed in accordance with 8.4. 8.3 Wet destruction Weigh 1 g or more of the prepared test sample to the nearest 1 mg. Place the test portion in a Kjeldahl flask (5.3). Add 20 ml of sulfuric a
29、cid (4.6). Shake to impregnate the substance completely with acid and to prevent it from sticking to the wall of the flask. Heat and keep at boiling point for 10 min. Leave to cool slightly. Add 2 ml of nitric acid (4.5), heat gently then leave to cool slightly. Add a little more nitric acid (4.5) a
30、nd bring back to the boiling point. Repeat this procedure until a colourless solution is obtained. Cool, add a little water and decant the liquid into a 500 ml volumetric flask (5.5), rinsing the Kjeldahl flask with hot water. Leave to cool, dilute to the mark with water, mix and filter. 8.4 Develop
31、ment of the colour and measurement of absorbance Dilute an aliquot portion of the filtrate obtained (8.2 or 8.3) with water, to obtain a phosphorus content not exceeding 40 mg/ml. Transfer, by means of a pipette (5.9), 10 ml of this solution to a test tube (5.6). Add, by means of another pipette, 10
32、 ml of the molybdovanadate reagent (4.9). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 6
33、491:1998(E) ISO 4 Mix and leave to stand for at least 10 min at 20 C. Transfer a portion of the obtained solution to a measuring cell and measure the absorbance in the spectrometer (5.5) at a wavelength of 430 nm against the reference solution (4.10). 8.5 Preparation of the calibration curve 8.5.1 U
34、sing the phosphorus standard solution (4.11), and by means of graduated pipettes (5.9), prepare solutions with a phosphorus content of 5 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml and 40 mg/ml respectively. 8.5.2 Transfer, by means of pipettes (5.9), 10 ml of each of these solutions to a series of five tes
35、t tubes (5.6). Add, to each by means of another pipette, 10 ml of the molybdovanadate reagent (4.9). Mix and leave to stand for at least 10 min at 20 C. Measure the absorbance of each solution as specified in 8.4. 8.5.3 Draw the calibration curve by plotting the absorbances against the corresponding
36、 phosporus contents, in micrograms per millilitre, of the phosphorus standard solutions (8.5.1). For phosphorus contents between 0 mg/ml and 40 mg/ml, the curve shall be linear. 8.6 Blank test Carry out a blank test in parallel with the determination, using the same procedure and the same quantities
37、 of the reagents, but omitting the test portion. 9 Expression of results Calculate the phosphorus content of the test sample, wP, in grams per kilogram, using the following equation: w = ffwV m P Pc 50 12 where f1is the reciprocal dilution factor for the aliquot portion (see 8.4); f2is the units cor
38、rection factor, in grams per milligram (f2 = 10-3 g/mg); wPcis the phosphorus content, in micrograms per millilitre, of the diluted aliquot portion of the test solution, read from the calibration curve (8.5.3); Vis the volume, in millilitres, of each of the calibration solutions taken in 8.5.2 (V =
39、10 ml); mis the mass, in grams, of the test portion (8.2 or 8.3). Report the result to the nearest 0,1 g/kg. 10 Precision 10.1 Interlaboratory test Details of an interlaboratory test on the precision of the method are summarized in annex A. The values derived from this interlaboratory test may not b
40、e applicable to concentration ranges and matrices other than those given. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted with
41、out license from IHS -,-,- ISOISO 6491:1998(E) 5 10.2 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 same equipment within a short interval of time, wil
42、l in not more than 5 % of cases exceed 1 g/kg. 10.3 Reproducibility The absolute difference 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
43、 7 g/kg. 11 Test report The test report shall specify: 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,
44、 or regarded as optional, together with details of any incidents that occurred which may have influenced the test result(s); the test result obtained; or if the repeatability has been checked, the final quoted result obtained. Copyright International Organization for Standardization Provided by IHS
45、under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 22:34:32 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 6491:1998(E) ISO 6 Annex A (informative) Results of interlaboratory test An interlaboratory test was organized by IS
46、O/TC 34/SC 10, Animal feeding stuffs, in 1987 and carried out in accordance with ISO 5725 11). The final statistical analysis was carried out in accordance with ISO 5725-2 3. In this test 24 laboratories participated. Samples of corn gluten feed, finished mixed feed stuff, fish meal, mixed feed stuf
47、f concentrate (two types), premixed feed stuff and yeast were investigated. Table A.1 Statistical results of interlaboratory test Parameter Sample1) 1234 2)567 Number of laboratories retained after eliminating outliers 24242424242424 Mean phosphorus content, g/kg3)285,49,280,127,422,511,4 Repeatabil
48、ity standard deviation (sr), g/kg0,400,320,111,480,750,300,24 Repeatability relative standard deviation, %1,45,91,21,92,71,32,1 Repeatability limit (r) r = 2,8 sr, g/kg1,120,900,314,142,100,840,67 Reproducibility standard deviation (sR), g/kg2,63,01,914,54,12,01,4 Reproducibility relative standard deviation, %9,455,321,118,114,98,712,3 Reproducibility limit (R) R = 2,8 sR, g/kg7,288,405,3240,6011,485,603,92 1)Sample 1: fish meal; Sample 2: corn gluten feed; Sample 3: yeast; Sample 4: premixed feed stuff;