EN 15688-2008 en Fertilizers - Determination of urease inhibitor N-(nbutyl) thiophosphoric triamide (NBPT) using high-performance liquid chromatography (HPLC)《肥料 使用高效液相色谱法(HPLC)测定脲.pdf

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1、BS EN 15688:2008ICS 65.080NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFertilizers Determination ofurease inhibitor N-(n-butyl)thiophosphorictriamide (NBPT) usinghigh-performanceliquid chromatography(HPLC)This British Standardwas published under theauthority

2、of the StandardsPolicy and StrategyCommittee on 32008 BSI 2008ISBN 978 0 580 58650 7Amendments/corrigenda issued since publicationDate CommentsBS EN 15688:2008National forewordThis British Standard is the UK implementation of EN 15688:2008.The UK participation in its preparation was entrusted to Tec

3、hnicalCommittee CII/37, Fertilisers and related chemicals.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Complia

4、nce with a British Standard cannot confer immunityfrom legal obligations.0 SeptemberBS EN 15688:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15688August 2008ICS 65.080English VersionFertilizers - Determination of urease inhibitor N-(n-butyl)thiophosphoric triamide (NBPT) using high-performa

5、nceliquid chromatography (HPLC)Engrais - Dtermination de linhibiteur durase N-(n-butyl)triamine thiophosphorique (NBPT) par chromatographieliquide haute performance (HPLC)Dngemittel - Bestimmung von Urease-Hemmstoff N-(n-Butyl)-thiophosphortriamid (NBPT) mit Hochleistungs-Flssigchromatographie (HPLC

6、)This European Standard was approved by CEN on 4 July 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical referenc

7、es concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into i

8、ts own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lith

9、uania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN

10、 All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15688:2008: EBS EN 15688:2008EN 15688:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Principle4 5 Reagents.4 6 Apparatus .5 7 Sampling a

11、nd sample preparation.6 8 Procedure .6 9 Calculation and expression of the result 7 10 Precision.7 11 Test report 8 Annex A (informative) Results of the inter-laboratory test .9 Bibliography 10 BS EN 15688:2008EN 15688:2008 (E) 3 Foreword This document (EN 15688:2008) has been prepared by Technical

12、Committee CEN/TC 260 “Fertilizers and liming materials”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by February 2009, and conflicting national standards

13、shall be withdrawn at the latest by February 2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document has been prepared und

14、er a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Re

15、public, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15688:2008EN 15688:2008 (E) 4 1 Scope This

16、document specifies a method for the quantitative determination of the urease inhibitor N-(n-butyl)thiophosphoric triamide (NBPT) content in water-soluble matrices, i.e. urea based fertilizers using high performance liquid chromatography (HPLC). NOTE Pure NBPT reference material is required as a stan

17、dard for the quantitative calibration. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendmen

18、ts) applies. EN 1482-2, Fertilizers and liming materials Sampling and sample preparation Part 2: Sample preparation EN 12944-1:1999, Fertilizers and liming materials and soil improvers Vocabulary Part 1: General terms EN 12944-2:1999, Fertilizers and liming materials and soil improvers Vocabulary Pa

19、rt 2: Terms relating to fertilizers EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 12944-1:1999 and EN 12944-2:1999 apply. 4 Principle This analytical met

20、hod is based on the principles of liquid chromatography, with absorption in the ultraviolet region for detection of the separated compounds. 5 Reagents 5.1 General Use only reagents of recognized analytical grade and distilled or demineralized water (grade 3 according to EN ISO 3696). 5.2 Reagents f

21、or liquid chromatography 5.2.1 Acetonitrile, HPLC-grade or spectroscopic grade. 5.2.2 Water, Milli-Q purified or equivalent quality. BS EN 15688:2008EN 15688:2008 (E) 5 5.2.3 N-(n-butyl)thiophosphoric triamide (NBPT), e. g. Sigma, B-3292, min 98 % n-Butyl ThioPhosphoric Triamide or TRC, catnr. B6940

22、001. 5.2.4 Urea, p.a. quality. 5.3 Calibration standards 5.3.1 Stock solution, = 0,20 mg NBPT/ml. Weigh 50 mg NBPT into a 250-ml-measuring flask and dissolve to volume with water. 5.3.2 Standard solution, 0,01 mg/ml. Dilute 5,00 ml of the NBPT stock solution (5.3.1) to a volume of 100,00 ml in water

23、. 5.3.3 Standard solution, 0,05 mg/ml. Dilute 25,00 ml of the NBPT stock solution (5.3.1) to a volume of 100,00 ml in water. 5.3.4 Standard solution, 0,12 mg/ml. Dilute 60,00 ml of the NBPT stock solution (5.3.1) to a volume of 100,00 ml in water. 5.3.5 Blank solution, water. 6 Apparatus 6.1 HPLC ap

24、paratus 6.1.1 Automatic mixing of binary eluent systems 6.1.2 Automatic injector device, capable of injecting 30 l 6.1.3 UV absorbance detector, operable down to 200 nm in wavelength 6.1.4 Recording peak area integrating device 6.2 HPLC conditions Two alternative systems may be used, as described in

25、 Table 1. Columns equivalent to those mentioned under A and B, may be used. 1Sigma B-3292 and TCR, catnr B694000 are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of

26、 this product. Equivalent products may be used if they can be shown to lead to the same results. BS EN 15688:2008EN 15688:2008 (E) 6 Table 1 Alternative HPLC systems A and B Parameter A B Column C18 Nucleosil (250 mm4 mm) 5 m LiChroSpher RP-8 (250 mm4 mm) 5 mMobile phase MeCN + H2O 10 + 90 MeCN + H2

27、O 15 + 85 Flow rate 1,0 ml/min 1,0 ml/min Injection volume 30 l 30 l Detection UV absorption at 205 nm UV absorption at 203 nm Run time 30 min to 35 min 20 min to 25 min Expected tRNBPT 8 min 7 min 7 Sampling and sample preparation Sampling is not part of the method specified in this document. A rec

28、ommended sampling method is given in EN 1482-1 1. Sample preparation shall be carried out in accordance with EN 1482-2. 8 Procedure 8.1 Preparation of the test portion Weigh to the nearest 0,001 g an amount of 5 g of the test sample and dissolve by stirring to volume of 250 ml in water. 8.2 Calibrat

29、ion Use the standard solutions or stock solution to determine the retention time of NBPT in the HPLC system. Calculate the response factor of NBPT, R, by analysis of the calibration standards in the HPLC system. All standards should be injected in duplicate. For calculations, see Clause 9. 8.3 Blank

30、 test For each series of determinations, carry out a blank test using an urea sample according to 5.2.4, which is free from NBPT. BS EN 15688:2008EN 15688:2008 (E) 7 8.4 Control test The control test depends on validation linearity and precision of standard injections should be used as system suitab

31、ility test. 9 Calculation and expression of the result Determine the concentration of NBPT in the sample solution by the external standard principle. Calculate the mass fraction of NBPT, wNBPT, in percent of the dry sample by dividing by the fertilizer content (mass/volume) of the sample solution ac

32、cording to the following equation: 4100NBPT=mVRAw (1) where A is the peak area for NBPT; R is the response factor (see formula (2) (peak area/g NBPT); V is the injection volume in micro litre; m is the mass of the test portion weighed into the sample solution (250 ml), in grams. The external standar

33、d response factor, R, is calculated from the average of the peak areas and mass concentrations of NBPT of the 3 calibration standards according to the following equation: )NBPT()NBPT()NBPT(3C3C3C2C2C1C13C2C1C3C2C1CVVVAAARRRR+=+=(2) where RC1, RC2, RC3are the response factors of the calibration stand

34、ards; AC1, AC2, AC3 are the peak areas of the calibration standards; NBPTC1 is the mass concentration of NBPT of the calibration standard C1, in milligrams per millilitre; NBPTC2 is the mass concentration of NBPT of the calibration standard C2, in milligrams per millilitre; NBPTC3 is the mass concen

35、tration of NBPT of the calibration standard C3, in milligrams per millilitre; VC1, VC2, VC3 are the injection volumes of the calibration solutions in micro litre. 10 Precision 10.1 Inter-laboratory test An inter-laboratory test has been carried out in 2006 with 7 participating laboratories and 3 dif

36、ferent samples of fertilizers. This test yielded the data given in Annex A. Repeatability and reproducibility were calculated according to ISO 5725-1 2. The values derived from this inter-laboratory test may not be applicable to concentration ranges and matrices other than those given in Annex A. BS

37、 EN 15688:2008EN 15688:2008 (E) 8 10.2 Repeatability The absolute difference between two independent single test results, obtained with 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 th

38、an 5 % of the cases exceed the values of r given in Table 2. 10.3 Reproducibility The absolute difference between two single test results, obtained with the same method on identical test material in different laboratories by different operators using different equipment, will in not more than 5 % of

39、 the cases exceed the values of R given in Table 2. Table 2 Mean values, repeatability and reproducibility limits Sample x r R Urea based sample 1 0,0255 0,0026 0,0074Urea based sample 2 0,0553 0,0028 0,0119Urea based sample 3 0,1119 0,0051 0,025411 Test report The test report shall contain at least

40、 the following information: a) all information necessary for the complete identification of the sample; b) the test method used with reference to this document; c) the test results obtained; d) date of sampling and sampling procedure (if known); e) date when the analysis was finished; f) whether the

41、 requirement of the repeatability limit has been fulfilled; g) all operating details not specified in this document, or regarded as optional, together with details of any incidents occurred when performing the method, which might have influenced the test result(s). BS EN 15688:2008EN 15688:2008 (E)

42、9 Annex A (informative) Results of the inter-laboratory test The precision of the method has been determined in the year 2006 in an inter-laboratory trial with 7 laboratories participating and carried out on 3 samples of fertilizer. The statistical results are given in Table A.1. Table A.1 Statistic

43、al results of the inter-laboratory trial Parameter Sample 1 Sample 2 Sample 3 Year of the test 2006 2006 2006 Number of participating laboratories 7 7 7 Number of laboratories after eliminating outliers 7 7 7 mean value, x (%) 0,0255 0,0553 0,1119 Repeatability standard deviation sr, (%) 0,0009 0,00

44、10 0,0018 RSDr(%) 4 1,8 1,6 Repeatability limit r (2,83 sr) (%) 0,0026 0,0028 0,0051 Reproducibility standard deviation, sR(%) 0,0026 0,0043 0,0091 RSDR(%) 10 7,7 8,1 Reproducibility limit R (2,83 sR) (%) 0,074 0,0119 0,0254 BS EN 15688:2008EN 15688:2008 (E) 10 Bibliography 1 EN 1482-1, Fertilizers

45、and liming materials Sampling and sample preparation Part 1: Sampling 2 ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions 3 Regulation (EC) No 2003/2003 of the European Parliament and of the Council of 13 October 2003 relating

46、 to fertilisers, Official Journal L 304, 21/11/2003, Pp. 1-194 BS EN 15688:2008This page has been intentionally left blank BS EN15688:2008BSI GroupHeadquarters 389Chiswick High Road,London, W4 4AL, UKTel +44 (0)20 8996 9001Fax +44 (0)20 8996 - British Standards InstitutionBSI is the independent nat

47、ional body responsible for preparing BritishStandards. It presents the UK view on standards in Europe and at theinternational level. It is incorporated by Royal Charter.RevisionsBritish Standards are updated by amendment or revision. Users of BritishStandards should make sure that they possess the l

48、atest amendments oreditions.It is the constant aim of BSI to improve the quality of our products and services.We would be grateful if anyone finding an inaccuracy or ambiguity while usingthis British Standard would inform the Secretary of the technical committeeresponsible, the identity of which can be found on the inside front cover. Tel:+44 (0)20 8996 9000. Fax: +44 (0)20 8996 7400.BSI offers members an individual updating service called PLUS which ensurest