1、BSI Standards PublicationBS EN 16651:2015Fertilizers Determinationof N-(n-Butyl)thiophosphoricacid triamide (NBPT) and N-(n-Propyl)thiophosphoric acidtriamide (NPPT) Methodusing high-performance liquidchromatography (HPLC)BS EN 16651:2015 BRITISH STANDARDNational forewordThis British Standard is the
2、 UK implementation of EN 16651:2015. The UK participation in its preparation was entrusted to TechnicalCommittee CII/37, Fertilisers and related chemicals.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include al
3、l the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 83533 9ICS 65.080Compliance with a British Standard cannot confer immunity from legal obligations.This British Sta
4、ndard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 16651:2015EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16651 May 2015 ICS 65.080 English Version Fertilizers -
5、 Determination of N-(n-Butyl)thiophosphoric acid triamide (NBPT) and N-(n-Propyl)thiophosphoric acid triamide (NPPT) - Method using high-performance liquid chromatography (HPLC) Engrais - Dosage du N-(n-butyl)triamide de lacide thiophosphorique (NBPT) et du N-(n-propyl)triamide de lacide thiophospho
6、rique (NPPT) - Mthode par chromatographie liquide haute performance (HPLC) Dngemittel - Bestimmung von N-(n-Butyl)thiophosphorsuretriamid (NBPT) und N-(n-Propyl)thiophosphorsuretriamid (NPPT) - Verfahren mit Hochleistungs-Flssigchromatographie (HPLC) This European Standard was approved by CEN on 16
7、April 2015. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be o
8、btained on application to the CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the
9、 CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Irel
10、and, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Cen
11、tre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16651:2015 EBS EN 16651:2015EN 16651:2015 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Pr
12、inciple 4 5 Reagents .4 6 Apparatus .5 7 Sampling .6 8 Procedure .6 8.1 Preparation of the test solution 6 8.2 Calibration 6 8.3 Blank test 6 8.4 Calculation and expression of the results 6 8.4.1 Concentration of NBPT .6 8.4.2 Concentration of NPPT .7 8.4.3 External response factors .7 8.4.4 Calcula
13、tion of the total content 8 8.5 Environmental aspects .8 9 Precision .8 9.1 Inter-laboratory test .8 9.2 Repeatability .8 9.3 Reproducibility .8 10 Test report 9 Annex A (informative) Results of the inter-laboratory test 10 Bibliography . 11 BS EN 16651:2015EN 16651:2015 (E) 3 Foreword This document
14、 (EN 16651:2015) 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 by No
15、vember 2015, and conflicting national standards shall be withdrawn at the latest by November 2015. 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 pa
16、tent 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 organisations of the following countries are bound to implement this European Standa
17、rd: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spai
18、n, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16651:2015EN 16651:2015 (E) 4 1 Scope This European Standard specifies a method for the quantitative determination of the urease inhibitors N-(n-Butyl)thiophosphoric acid triamide (NBPT, CAS-No. 94317-64-3) and N-(n-Propyl)thiophosphoric a
19、cid triamide (NPPT, CAS-No. 916809-14-8) content in urea based fertilizers using high-performance liquid chromatography (HPLC). 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated refere
20、nces, only the edition cited applies. For undated references, the latest edition 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
21、 and soil improvers - Vocabulary - Part 1: General terms EN 12944-2:1999, Fertilizers 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) 3 Terms and definitions For
22、the purposes of this document, the terms and definitions given in EN 12944-1:1999 and EN 12944-2:1999 apply. 4 Principle The fertilizer sample is diluted in water, grade 1. The concentration of N-(n-Butyl)thiophosphoric acid triamide (NBPT) and N-(n-Propyl)thiophosphoric acid triamide (NPPT) is dete
23、rmined in diluted aqueous solution by high-performance liquid chromatography (HPLC) using an UV detector at 205 nm. 5 Reagents Use only reagents of recognized analytical grade. 5.1 Water, grade 1 according to EN ISO 3696. 5.2 Acetonitrile, HPLC grade. 5.3 Urea, analytical pure. 5.4 NBPT, N-(n-Butyl)
24、thiophosphoric acid triamide, CAS-No. 94317-64-3, analytical pure. 5.5 NPPT, N-(n-Propyl)thiophosphoric acid triamide, CAS-No. 916809-14-8, analytical pure. 5.6 Eluent. Mix in a 1 000 ml volumetric flask (6.2), 750 ml of water (5.1) and 250 ml acetonitrile (5.2) and filter through a micro filter (6.
25、3). 5.7 Stock solution, (NBPT) = 0,20 mg/ml and (NPPT) = 0,12 mg/ml. BS EN 16651:2015EN 16651:2015 (E) 5 Weigh in a 250 ml graduated flask (6.2), an amount of 50 mg NBPT and 30 mg NPPT. Add approximately 200 ml of water (5.1) and shake until NBPT and NPPT are diluted. Fill up to the mark with water
26、(5.1) and shake. This stock solution may be used within 24 h. 5.8 Calibration solution C1, (NBPT) = 0,01 mg/ml and (NPPT) = 0,006 mg/ml. Fill into a 100 ml graduated flask (6.2), an amount of 5,00 ml stock solution (5.7). Fill up to the mark with water (5.1). This calibration solution may be used wi
27、thin 24 h. 5.9 Calibration solution C2, (NBPT) = 0,05 mg/ml and (NPPT) = 0,030 mg/ml. Fill into a 100 ml graduated flask an amount of 25,00 ml stock solution (5.7). Fill up to the mark with water (5.1). This calibration solution may be used within 24 h. 5.10 Calibration solution C3, (NBPT) = 0,12 mg
28、/ml and (NPPT) = 0,072 mg/ml. Fill into a 100 ml graduated flask an amount of 60,00 ml stock solution (5.7). Fill up to the mark with water (5.1). This calibration solution may be used within 24 h. 6 Apparatus 6.1 Analytical balance, capable of weighing to an accuracy of 0,001 g. 6.2 Volumetric flas
29、ks, capacity 100 ml, 250 ml and 1 000 ml. 6.3 Micro filter, pore size 0,45 m. 6.4 One way shot, capacity 5 ml. 6.5 Vials, capacity 2 ml. 6.6 Appropriate shaking equipment. 6.7 HPLC apparatus, consisting of the following parts: 6.7.1 HPLC instrument with UV-Detector, and optional an auto sampler and
30、column oven. 6.7.2 HPLC separation column, e.g. LiChroSpher 100 RP-8 (250 mm 4 mm) 5 m or equivalent. The HPLC conditions based on this column are given in Table 1. BS EN 16651:2015EN 16651:2015 (E) 6 Table 1 Example of HPLC conditions aParameter Conditions Column LiChroSpher 100 RP-8 (250 mm 4 mm)
31、5 m Mobile phase Eluent (5.6) Flow rate 1,0 ml/min Injection volume 20 l UV detection at wavelength 205 nm Column temperature Room temperature Expected retention time NBPT approximately 7 min Expected retention time NPPT approximately 5 min aThe use of an equivalent column could lead to different pa
32、rameters. 7 Sampling Sampling from fertilizers, which are stored as bulk in storage buildings or warehouse or in packaged form is not part of the method specified in this document. A recommended sampling method is given in EN 1482-1. Sample preparation shall be carried out in accordance with EN 1482
33、-2. 8 Procedure 8.1 Preparation of the test solution Weigh to the nearest 0,001 g an amount of 7,500 g of the test sample into a 250 ml volumetric flask and dissolve in 200 ml water (5.1), shake the flask with shaking equipment (6.6) until the sample is completely dissolved. Fill up to the mark with
34、 water (5.1) and mix thoroughly. Fill the solution in a one way shot (6.4), filter through the micro filter (6.3) in a vial (6.5) and then inject the solution in HPLC apparatus (6.7). Solutions should be analysed within 24 h. 8.2 Calibration Determine the retention times of NBPT and NPPT by using th
35、e calibration solutions (5.8, 5.9 and 5.10). Activate the measure with the three different calibration solutions C1, C2 and C3 (manual handling or optional use of an auto sampler). 8.3 Blank test For each series of determination carry out a blank test using an urea solution containing 30 g urea in 1
36、 000 ml of water. Place the sample in the auto sampler and activate the measure. 8.4 Calculation and expression of the results1)8.4.1 Concentration of NBPT Calculate the concentration of NBPT in the sample solution by the external calibration. Calculate the mass fraction of NBPT, wNBPT, in percent o
37、f the sample by dividing by the fertilizer content (mass concentration) of the sample solution according to Formula (1): 1) Calibration curve e.g. calculated by the software of the HPLC apparatus can be used optionally. BS EN 16651:2015EN 16651:2015 (E) 7 1004NBPTAwRVm=(1) where A is the peak area f
38、or NBPT; R is the response factor (see Formula (3), peak area/g NBPT); V is the injection volume in microlitres; m is the mass of the test portion weighed into the test solution (250 ml), in grams. 8.4.2 Concentration of NPPT Calculate the concentration of NPPT in the sample solution by the external
39、 calibration. Calculate the mass fraction of NPPT, wNPPT, in percent of the sample by dividing by the fertilizer content (mass concentration) of the sample solution according to Formula (2): 1004NPPTAwRVm=(2) where A is the peak area for NPPT; R is the response factor (see Formula (4), peak area/g N
40、PPT); V is the injection volume in microlitres; m is the mass of the test portion weighed into the test solution (250 ml), in grams. 8.4.3 External response factors 8.4.3.1 Calculate the external standard response factor, RNBPT, from the average of the peak areas and mass concentrations of NBPT of t
41、he three calibration standards according to Formula (3): 123 12311 2 2 333CC C CC CNBPTNBPTC C NBPTC C NBPTC C( )( )( )RRR AAARVVV+ += =+ + (3) where RC1,RC2,RC3are the response factors of the calibration standard; AC1,AC2,AC3 are the peak areas of the calibration standard; NBPTC1, NBPTC2,NBPTC3 are
42、 the mass concentrations of NBPT of the calibration standard C1, C2 and C3, in milligrams per millilitre; VC1,VC2,VC3 are the injection volumes of the calibration solution in microlitres. 8.4.3.2 Calculate the external standard response factor, RNPPT, from the average of the peak areas and mass conc
43、entrations of NPPT of the three calibration standards according to Formula (4): 123 12311 2 2 333CC C CC CNPPTNPPTC C NPPTC C NPPTC C( )( )( )RRR AAARVVV+ += =+ + (4) BS EN 16651:2015EN 16651:2015 (E) 8 where RC1,RC2,RC3are the response factors of the calibration standard; AC1,AC2,AC3 are the peak a
44、reas of the calibration standard; NPPTC1, NPPTC2,NPPTC3 are the mass concentrations of NPPT of the calibration standard C1, C2 and C3, in milligrams per millilitre; VC1,VC2,VC3 are the injection volumes of the calibration solution in microlitres. 8.4.4 Calculation of the total content Calculate the
45、total active-ingredient content, wTby addition of the calculated contents of NBPT and NPPT according to Formula (5). T NBPT NPPTww w= + (5) 8.5 Environmental aspects The substances, solutions, eluents and packaging materials should be disposed under consideration of safety data sheets and in accorda
46、nce with local, state and federal regulations. 9 Precision 9.1 Inter-laboratory test An inter-laboratory test has been carried out in 2013 with 11 participating laboratories and four different samples of fertilizers. This test yielded the data given in Annex A. The values derived from this inter-lab
47、oratory test may not be applicable to concentration ranges and matrices other than those given in Annex A. 9.2 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
48、 the same equipment within a short interval of time, will in no more than 5 % of the cases exceed the values of r given in Table 2 (NPPT) and Table 3 (NBPT). 9.3 Reproducibility The absolute difference between two single test results, obtained with the same method on identical test material in diffe
49、rent laboratories by different operators using different equipment, will in no more than 5 % of the cases exceed the values of R given in Table 2 (NPPT) and Table 3 (NBPT). BS EN 16651:2015EN 16651:2015 (E) 9 Table 2 NPPT: Mean values, repeatability and reproducibility limits Sample x r R LIMUS 1 a0,004 6 0,000 5 0,001 0 LIMUS 2 a0,010 7 0,000 8 0,001 7 LIMUS 3 0,023 7 0,001 7 0,003 4 LIMUS 4 0,036 3 0,002 5 0,004 2 aA more diluted calibration solution could be added if sample solutions are wanted to be between the calib
