DIN EN 15688-2008 Fertilizers - Determination of urease inhibitor N-(n-butyl)thiophosphoric triamide (NBPT) using high-performance liquid chromatography (HPLC) English version of D.pdf

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1、November 2008DEUTSCHE NORM English price group 8No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.080!$TJF“1493935www

2、.din.deDDIN EN 15688Fertilizers Determination of urease inhibitor N-(n-butyl)thiophosphoric triamide(NBPT) using high-performance liquid chromatography (HPLC)English version of DIN EN 15688:2008-11Dngemittel Bestimmung von Urease-Hemmstoff N-(n-Butyl)-thiophosphortriamid (NBPT) mitHochleistungs-Flss

3、igchromatographie (HPLC)Englische Fassung DIN EN 15688:2008-11www.beuth.deDocument comprises 12 pagesDIN EN 15688:2008-11 2 National foreword This standard has been prepared by Technical Committee CEN/TC 260 “Fertilizers and liming materials” (Secretariat: DIN, Germany) under the mandate M/335. The

4、responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-02 AA Dngemittel. The DIN Standards corresponding to the International Standards referred to i

5、n this document are as follows, whereby EN ISO Standards are only listed below if these have not been published as DIN EN ISO Standards with the same number: EN ISO 3696 DIN ISO 3696 ISO 5725-1 DIN ISO 5725-1 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory

6、use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15688August 2008ICS 65.080English VersionFertilizers - Determination of urease inhibito

7、r N-(n-butyl)thiophosphoric triamide (NBPT) using high-performanceliquid 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)-thiophosp

8、hortriamid (NBPT) mit Hochleistungs-Flssigchromatographie (HPLC)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 withou

9、t any alteration. Up-to-date lists and bibliographical references 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 mad

10、e by translationunder the responsibility of a CEN member into its 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,

11、Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNG

12、Management Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15688:2008: EEN 15688:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Prin

13、ciple4 5 Reagents.4 6 Apparatus .5 7 Sampling and 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 DIN EN 15688:2008-11 EN 15688:2008 (E) 3 Foreword This docu

14、ment (EN 15688:2008) has been prepared by Technical 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 b

15、y February 2009, and conflicting national standards 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 suc

16、h patent rights. This document has been prepared under 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 St

17、andard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, 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 Kingdo

18、m. DIN EN 15688:2008-11 EN 15688:2008 (E) 4 1 Scope This 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)

19、. NOTE Pure NBPT reference material is required as a standard 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 e

20、dition of the referenced document (including any amendments) 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, Fertiliz

21、ers and liming materials and soil improvers Vocabulary Part 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 a

22、nd EN 12944-2:1999 apply. 4 Principle This analytical method 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

23、 water (grade 3 according to EN ISO 3696). 5.2 Reagents for liquid chromatography 5.2.1 Acetonitrile, HPLC-grade or spectroscopic grade. 5.2.2 Water, Milli-Q purified or equivalent quality. DIN EN 15688:2008-11 EN 15688:2008 (E) 5 5.2.3 N-(n-butyl)thiophosphoric triamide (NBPT), e. g. Sigma, B-3292,

24、 min 98 % n-Butyl ThioPhosphoric Triamide or TRC, catnr. B6940001. 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

25、 NBPT stock solution (5.3.1) to a volume of 100,00 ml in water. 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

26、in water. 5.3.5 Blank solution, water. 6 Apparatus 6.1 HPLC apparatus 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 c

27、onditions Two alternative systems may be used, as described in 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 Euro

28、pean Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 15688:2008-11 EN 15688:2008 (E) 6 Table 1 Alternative HPLC systems A and B Parameter A B Column C18 Nucleosil (250 mm4 mm) 5 m LiChroS

29、pher RP-8 (250 mm4 mm) 5 mMobile phase MeCN + H2O 10 + 90 MeCN + H2O 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 S

30、ampling is not part of the method specified in this document. A recommended 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

31、and dissolve by stirring to volume of 250 ml in water. 8.2 Calibration 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

32、be injected in duplicate. For calculations, see Clause 9. 8.3 Blank 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. DIN EN 15688:2008-11 EN 15688:2008 (E) 7 8.4 Control test The control test depends on validation linear

33、ity and precision of standard injections should be used as system suitability 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 di

34、viding by the fertilizer content (mass/volume) of the sample solution according 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 w

35、eighed into the sample solution (250 ml), in grams. The external standard 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+=+=

36、(2) where RC1, RC2, RC3are the response factors of the calibration standards; 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 calibrat

37、ion standard C2, in milligrams per millilitre; NBPTC3 is the mass concentration 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

38、has been carried out in 2006 with 7 participating laboratories and 3 different 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

39、 concentration ranges and matrices other than those given in Annex A. DIN EN 15688:2008-11 EN 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

40、using the same equipment within a short interval of time, will in not more than 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 b

41、y different operators using different equipment, will in not more than 5 % of 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 sampl

42、e 3 0,1119 0,0051 0,025411 Test report The test report shall contain at least 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

43、 procedure (if known); e) date when the analysis was finished; f) whether the 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

44、 might have influenced the test result(s). DIN EN 15688:2008-11 EN 15688:2008 (E) 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

45、of fertilizer. The statistical results are given in Table A.1. Table A.1 Statistical 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 valu

46、e, x (%) 0,0255 0,0553 0,1119 Repeatability standard deviation sr, (%) 0,0009 0,0010 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

47、0,0254 DIN EN 15688:2008-11 EN 15688:2008 (E) 10 Bibliography 1 EN 1482-1, Fertilizers 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 to fertilisers, Official Journal L 304, 21/11/2003, Pp. 1-194 DIN EN 15688:2008-11