1、BRITISH STANDARD BS 5766-3:1999 ISO 6491:1998 Methods for analysis of animal feeding stuffs Part 3: Determination of phosphorus content (spectrometric method) ICS 65.120BS5766-3:1999 This British Standard, having been prepared under the directionof the Consumer Products and Services Sector Committee
2、, was published underthe authority of the Standards Committee and comesinto effect on 15May1999 BSI 03-2000 ISBN 0 580 32124 X National foreword This British Standard reproduces verbatim ISO6491:1998and implements it as the UK national standard. It supersedes BS5766-3:1981which is withdrawn. The UK
3、participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and kee
4、p the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publ
5、ications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary pro
6、visions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theISO title p
7、age, pages ii to iv, pages 1 to 4, an inside back cover and abackcover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date CommentsBS5
8、766-3:1999 BSI 03-2000 i Contents Page National foreword Inside front cover Foreword iii Text of ISO 6491 1ii blankBS5766-3:1999 ii BSI 03-2000 Contents Page Foreword iii 1 Scope 1 2 Normative references 1 3 Principle 1 4 Reagents 1 5 Apparatus 1 6 Sampling 1 7 Preparation of test sample 2 8 Procedu
9、re 2 9 Expression of results 3 10 Precision 3 11 Test report 3 Annex A (informative) Results of interlaboratory test 4 Annex B (informative) Bibliography Inside back cover Table A.1 Statistical results of interlaboratory test 4 Descriptors: Agricultural products, food, animal feeding products, chemi
10、cal analysis, determination of content, phosphorus, spectrometric method.BS5766-3:1999 BSI 03-2000 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 normal
11、ly 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 represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the
12、 work. ISO collaborates closely with the International Electrotechnical Commission (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 requ
13、ires approval by at least75% of the member bodies casting a vote. International Standard ISO6491was prepared by Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC10, Animal feeding stuffs. This second edition replaces the first edition (ISO6491:1980), which has been technicall
14、y revised. Annex A and Annex B of this International Standard are for information only.iv blankBS5766-3:1999 BSI 03-2000 1 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
15、 feeding stuffs with a phosphorus content less than50g/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
16、references The following standards contain 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 Internation
17、al Standard are encouraged to investigate 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. IS
18、O 6498:1998, Animal feeding stuffs Preparation 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 fee
19、ding stuffs). An aliquot portion of the acid solution is mixed with molybdovanadate reagent and the absorbance of the yellow solution obtained is measured at a wavelength of430nm. 4 Reagents Use only reagents of recognized analytical grade. 4.1 Water, complying to at least grade3in accordance with I
20、SO3696. 4.2 Calcium carbonate 4.3 Hydrochloric acid, c(HCl) . 6 mol/l. 4.4 Nitric acid, c(HNO 3 ) . 1 mol/l. 4.5 Nitric acid, c(HNO 3 ) = 14 mol/l, (HNO 3 ) . 1,40 g/ml. 4.6 Sulfuric acid, c(H 2 SO 4 ) = 18 mol/l, (H 2 SO 4 ) = 1,84 g/ml. 4.7 Ammonium heptamolybdate solution Dissolve in hot water 10
21、0 g of ammonium heptamolybdate tetrahydrate (NH 4 ) 6 Mo 7 O 24 4H 2 O. Add10ml of ammonia c(NH 4 OH) =14mol/l, (NH 4 OH) = 0,91g/ml and dilute to1l with water. 4.8 Ammonium monovanadate solution Dissolve 2,35 g of ammonium monovanadate(NH 4 VO 3 ) in400ml of hot water. Stirring constantly, slowly a
22、dd7ml of nitric acid(4.5) and dilute to1l with water. 4.9 Molybdovanadate reagent In a 1 l volumetric flask, mix 200ml of the ammonium heptamolybdate solution (4.7), 200ml ofthe ammonium monovanadate solution (4.8) and135ml of nitric acid (4.5). Dilute to the mark with water. Filter if insoluble par
23、ticles are present. 4.10 Reference solution Dilute 10 ml of molybdovanadate reagent (4.9) with10ml of water. 4.11 Phosphorus standard solution, (P) = 1 mg/ml In a 1 l volumetric flask, dissolve in water4,394g ofpotassium dihydrogen phosphate (KH 2 PO 4 ), previously dried at 103 C for1h. Dilute to t
24、he mark with water. 5 Apparatus Usual laboratory apparatus and, 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,
25、 of capacities 500 ml and 1 000 ml. 5.5 Spectrometer, fitted with 10 mm cells, suitable for measurements at a wavelength of 430nm. 5.6 Glass test tubes, of capacity25ml to30ml, fitted with ground glass stoppers. 5.7 Sand bath 5.8 Beaker, of capacity 250 ml. 5.9 Graduated pipettes 6 Sampling Sampling
26、 is not part of the method specified in this International Standard. A recommended sampling method is given in ISO64974. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage.BS5766-3:1999 2 BSI 03-2000 7 Pr
27、eparation of test sample Prepare the test sample in accordance with ISO6498. If solid, grind the laboratory sample (usually500g) so that it passes completely through a sieve with1mm apertures. Mix thoroughly. 8 Procedure 8.1 Selection of procedure If the test sample contains organic substances and i
28、f it is free from phosphates rendering insoluble products on ashing, proceed in accordance with8.2. If the test sample concerns a mineral compound or a liquid feeding stuff, proceed in accordance with8.3. 8.2 Dry ashing Weigh about 2,5 g of the prepared test sample to the nearest1mg in an ashing cru
29、cible (5.1). Mix the test portion thoroughly with1g of the calcium carbonate (4.2). Ash in the furnace (5.2), set at a temperature of550 C, until white or grey ash is obtained (a small quantity of carbon does not interfere). Transfer the ash to a250ml beaker (5.8) with 20 ml to50ml of water. Add hyd
30、rochloric acid (4.3) until effervescence ceases. Add a further 10 ml of the hydrochloric 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 for5min, withou
31、t evaporating to dryness. Decant the liquid into a500ml volumetric flask(5.4), rinsing the beaker several times with hot water. Leave to cool, dilute to the mark with water, mix and filter. Proceed in accordance with8.4. 8.3 Wet destruction Weigh 1 g or more of the prepared test sample to the neares
32、t 1 mg. Place the test portion in a Kjeldahl flask (5.3). Add20ml of sulfuric acid (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 for10min. Leave to cool slightly. Add2ml of nitric acid (4.5), hea
33、t gently then leave to cool slightly. Add a little more nitric acid (4.5) and 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 a500ml volumetric flask (5.5), rinsing the Kjeldahl flask with hot water.
34、Leave to cool, dilute to the mark with water, mix and filter. 8.4 Development of the colour and measurement of absorbance Dilute an aliquot portion of the filtrate obtained(8.2 or8.3) with water, to obtain a phosphorus content not exceeding404g/ml. Transfer, by means of a pipette (5.9), 10ml of this
35、 solution to a test tube (5.6). Add, by means of another pipette, 10ml of the molybdovanadate reagent (4.9). Mix and leave to stand for at least 10 min at20 C. Transfer a portion of the obtained solution to a measuring cell and measure the absorbance in the spectrometer (5.5) at a wavelength of430nm
36、 against the reference solution (4.10). 8.5 Preparation of the calibration curve 8.5.1 Using the phosphorus standard solution(4.11), and by means of graduated pipettes(5.9), prepare solutions with a phosphorus content of54g/ml, 104g/ml, 204g/ml, 304g/ml and404g/ml respectively. 8.5.2 Transfer, by me
37、ans of pipettes (5.9), 10 ml of each of these solutions to a series of five test tubes(5.6). Add, to each by means of another pipette, 10ml of the molybdovanadate reagent (4.9). Mix and leave to stand for at least10min at20 C. Measure the absorbance of each solution as specified in8.4. 8.5.3 Draw th
38、e calibration curve by plotting the absorbances against the corresponding phosphorus contents, in micrograms per millilitre, of the phosphorus standard solutions (8.5.1). For phosphorus contents between 04g/ml and404g/ml, the curve shall be linear. 8.6 Blank test Carry out a blank test in parallel w
39、ith the determination, using the same procedure and the same quantities of the reagents, but omitting the test portion.BS5766-3:1999 BSI 03-2000 3 9 Expression of results Calculate the phosphorus content of the test sample,w P , in grams per kilogram, using the following equation: where Report the r
40、esult 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 be applicable to concentration ranges and matrices other than those given. 10.2
41、 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, will in not more than5% of cases exceed1g/kg. 10.3 Repro
42、ducibility 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 exceed7g/kg. 11 Test report The test report shall specify: all i
43、nformation 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, or regarded as optional, together with details of any incid
44、ents 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. f 1 is the reciprocal dilution factor for the aliquot portion (see 8.4); f 2 is the units correction factor, in grams per milligram (
45、f 2= 10 3g/mg); w Pc is the phosphorus content, in micrograms per millilitre, of the diluted aliquot portion of the test solution, read from the calibration curve(8.5.3); V is the volume, in millilitres, of each of the calibration solutions taken in8.5.2 (V =10ml); m is the mass, in grams, of the te
46、st portion(8.2 or8.3).BS5766-3:1999 4 BSI 03-2000 Annex A (informative) Results of interlaboratory test An interlaboratory test was organized by ISO/TC34/SC10, Animal feeding stuffs, in1987and carried out in accordance with ISO5725 1 1) . The final statistical analysis was carried out in accordance
47、with ISO5725-2 3. In this test 24laboratories participated. Samples of corn gluten feed, finished mixed feed stuff, fish meal, mixed feed stuff concentrate (two types), premixed feed stuff and yeast were investigated. Table A.1 Statistical results of interlaboratory test 1) ISO5725:1986 (now withdra
48、wn) was used to obtain the precision data. Parameter Sample a 1 2 3 4 b 5 6 7 Number of laboratories retained after eliminating outliers 24 24 24 24 24 24 24 Mean phosphorus content, g/kg c 28 5,4 9,2 80,1 27,4 22,5 11,4 Repeatability standard deviation (s r ), g/kg 0,40 0,32 0,11 1,48 0,75 0,30 0,2
49、4 Repeatability relative standard deviation,% 1,4 5,9 1,2 1,9 2,7 1,3 2,1 Repeatability limit (r) r = 2,8 s r , g/kg 1,12 0,90 0,31 4,14 2,10 0,84 0,67 Reproducibility standard deviation (s R ), g/kg 2,6 3,0 1,9 14,5 4,1 2,0 1,4 Reproducibility relative standard deviation,% 9,4 55,3 21,1 18,1 14,9 8,7 12,3 Reproducibility limit (R) R = 2,8 s R , g/kg 7,28 8,40 5,32 40,60 11,48 5,60 3,92 a Sample 1: fish meal; Sample 2: corn gluten feed; Sample 3: yeast; Sample 4: premixed feed stuff; Sample 5: mixed feed stuff concent
