1、February 2013 Translation by DIN-Sprachendienst.English price group 8No part of this translation 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).IC
2、S 65.080!$R7“1944720www.din.deDDIN EN 16328Fertilizers Determination of 3,4-dimethyl-1H-pyrazole phosphate (DMPP) Method using high-performance liquid chromatography (HPLC);English version EN 16328:2012,English translation of DIN EN 16328:2013-02Dngemittel Bestimmung von 3,4-Dimethyl-1H-pyrazolphosp
3、hat (DMPP) Verfahren mit Hochleistungs-Flssigchromatographie (HPLC);Englische Fassung EN 16328:2012,Englische bersetzung von DIN EN 16328:2013-02Engrais Dosage du 3,4-dimthyl-1H-pyrazole phosphate (DMPP) Mthode par chromatographie liquide haute performance (HPLC);Version anglaise EN 16328:2012,Tradu
4、ction anglaise de DIN EN 16328:2013-02www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 11 pages 02.13DIN EN 16328:2013-02 2 A comma is used as the decimal marker. National foreword This document (EN 16328:2012) has been prepared by Techn
5、ical Committee CEN/TC 260 “Fertilizers and liming materials” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-03-02
6、 AA Dngemittel. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and re
7、sults Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN
8、16328 December 2012 ICS 65.080 English Version Fertilizers - Determination of 3,4-dimethyl-1H-pyrazole phosphate (DMPP) - Method using high-performance liquid chromatography (HPLC) Engrais - Dosage du 3,4-dimthyl-1H-pyrazole phosphate (DMPP) - Mthode par chromatographie liquide haute performance (HP
9、LC) Dngemittel - Bestimmung von 3,4-Dimethyl-1H-pyrazolphosphat (DMPP) - Verfahren mit Hochleistungs-Flssigchromatographie (HPLC) This European Standard was approved by CEN on 27 October 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions fo
10、r 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 obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in thre
11、e 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 CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bod
12、ies of 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, S
13、pain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN
14、national Members. Ref. No. EN 16328:2012: EEN 16328:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Terms and definitions .44 Principle 45 Reagents .46 Apparatus .47 Sampling .58 Procedure .58.1 Preparation of the test solution 58.2 Preparation of calibration solution 58.3 HPLC
15、 conditions 58.4 HPLC determination 69 Calculation and expression of the results 610 Precision .610.1 Inter-laboratory test .610.2 Repeatability .610.3 Reproducibility .611 Test report 7Annex A (informative) Results of the inter-laboratory test .8A.1 Inter-laboratory tests .8A.2 Statistical results
16、.8Bibliography 9DIN EN 16328:2013-02 EN 16328:2012 (E) 3 Foreword This document (EN 16328:2012) 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, ei
17、ther by publication of an identical text or by endorsement, at the latest by June 2013, and conflicting national standards shall be withdrawn at the latest by June 2013. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or C
18、ENELEC shall not be held responsible for identifying any or all such 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 organisations
19、 of the following countries are bound to implement this European Standard: 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, N
20、etherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16328:2013-02 EN 16328:2012 (E) 4 1 Scope This European Standard specifies a method for the determination of 3,4-dimethyl-1H-pyrazole phosphate (DMPP, CAS-No: 202842-9
21、8-6) in mineral N containing 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 references, only the edition cited appli
22、es. 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 and soil improvers Vocabulary Part 1:
23、 General terms EN 12944-2:1999, Fertilizers and liming materials and soil improvers Vocabulary Part 2: Terms relating to fertilizers 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 The fertilizer sam
24、ple is dissolved in or extracted with water. The concentration of 3,4-dimethyl-1H-pyrazole phosphate is determined in diluted aqueous solution by high performance liquid chromatography (HPLC) at a reversed phase using a UV detector. 5 Reagents WARNING The described method includes the handling with
25、hazardous materials. Attention must be paid to technical, organizational and personal safety measures. Use only reagents of recognized analytical grade. 5.1 Water, de-ionized. 5.2 Acetonitrile, HPLC grade. 5.3 Sodiumdihydrogenphosphate-monohydrate, NaH2PO4H2O. 5.4 3,4-dimethyl-1H-pyrazole, 99 % puri
26、ty. 6 Apparatus 6.1 Ultrasonic bath and magnetic stirrer, respectively. DIN EN 16328:2013-02 EN 16328:2012 (E) 5 6.2 Filtration unit, manual or high-pressure filtration with membrane filter 0,45 m or comparable filter. NOTE High pressure filtration is advantageous for difficult samples. 6.3 HPLC uni
27、t, for isocratic operation with UV detector. 6.4 Evaluation unit. 6.5 Reverted phase-HPLC separation column, e.g. C18, 5 m, 150 mm 4,6 mm. NOTE With satisfactory separation performance, a shorter separation column can be used. 6.6 Analytical balance, capable of weighing to an accuracy of 0,001 g. 7
28、Sampling Sampling 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 1482-2. 8 Procedure 8.1 Preparation of the test solution Weigh (6.6) (30 0,001) g of sample (m) into a 1 000 ml v
29、olumetric flask. Add approximately 900 ml water (5.1) and dissolve the sample in the ultrasonic bath (6.1) for about 15 min. Fill up the flask with water (5.1) to capacity. Filter part of the sample solution through a membrane filter (6.2) to remove possible conditioners. If necessary, dilute the sa
30、mple for the calibration range. 8.2 Preparation of calibration solution Prepare from at least two weighted samples (stock solutions) made with the present 3,4-dimethyl-1H-pyrazole (5.4) at least four concentrations for calibration. The concentration range should be between 3 mg/l and 100 mg/l. 8.3 H
31、PLC conditions Separation column: filled for reversed phase-HPLC (6.5); column temperature: room temperature; elution agent: dissolve 1,38 g NaH2PO4H2O (5.3) in 1 l water and add 175 ml acetonitrile (5.2); the elution agent shall be de-gassed, e.g. in an ultrasonic bath (6.1); flow rate: 1,5 ml/min;
32、 injection volume: 10 l; wave length: 224 nm; retention time DMPP (at 1,5 ml/min flow rate): approximately 5,3 min. DIN EN 16328:2013-02 EN 16328:2012 (E) 6 8.4 HPLC determination To prepare a calibration curve, inject 10 I of each calibration solution three times consecutively. Calculate the compen
33、sation curve and correlation coefficient p by least-square method. The calibration curve can be used for concentration determination when p 0,99. Inject the test solution two times consecutively. 9 Calculation and expression of the results Carry out the evaluation on the basis of the calibration lin
34、e over the peak areas. Calculate the content of 3,4-dimethyl-1H-pyrazole phosphate, wDMPP, in mg/kg according to formula (1): 000101,962,194100)(dpkDMPP=maFbAw (1) where Apkis the peak height or peak area; b is the y-axis section of straight calibration line; a is the slope of straight calibration l
35、ine; Fdis the dilution factor; m is the sample weight, in milligrams. Calculate the arithmetic mean from both values obtained. Report the result to the nearest 0,001 mg/kg. 10 Precision 10.1 Inter-laboratory test Details of inter laboratory tests performed in 2010 on the precision of the method are
36、summarized in Annex A. The values derived from this test may not be applicable to concentration ranges and matrices other than those given. 10.2 Repeatability The absolute difference between two independent single test results, obtained using the same method on identical test material in the same la
37、boratory by the same operator using the same equipment within a short interval of time, will in no more than 5 % of the cases be greater than the repeatability limit r given in Table 1. 10.3 Reproducibility The absolute difference between two single test results, obtained using the same method on id
38、entical test material in different laboratories with different operators using different equipment, will in no more than 5 % of the cases be greater than the reproducibility limit R given in Table 1. DIN EN 16328:2013-02 EN 16328:2012 (E) 7 Table 1 Mean values, repeatability and reproducibility limi
39、ts for DMPP Sample x r R mg/kg mg/kg mg/kgSample 1 Ammonium sulfate nitrate (ASN) 1 700 90 170 Sample 2 NPK 24+8+7 1 270 70 410 Sample 3 NPK 13+10+20 706 56 210 11 Test report The test report shall contain at least the following information: a) all information necessary for the complete identificati
40、on of the sample; b) test method used with reference to this document; c) test results obtained; d) date of sampling and sampling 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 speci
41、fied 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). DIN EN 16328:2013-02 EN 16328:2012 (E) 8 Annex A (informative) Results of the inter-laboratory test A.1 Inter-laboratory tests A
42、n inter-laboratory test has been carried out in 2010 with nine participating laboratories and three different samples of fertilizers. Repeatability and reproducibility were calculated according to ISO 5725-1 and ISO 5725-2. A.2 Statistical results The statistical results are given in Table A.1. Tabl
43、e A.1 Statistical results for the determination of DMPP using HPLC Parameter Sample 1 Sample 2 Sample 3 Ammonium sulfate nitrate (ASN) NPK 24+8+7 NPK 13+10+20Year of the test 2010 2010 2010 Number of participating laboratories 9 9 9 Number of laboratories after eliminating outliers 7 8 8 Mean value,
44、 x , (mg/kg) 1 700 1 270 706 Repeatability standard deviation sr, (mg/kg) 30 20 20Relative standard deviation of repeatability, RSDr2 1,9 3 Repeatability limit, r, (mg/kg) 90 70 56 Reproducibility standard deviation, sR,(mg/kg) 60 150 75Relative standard deviation of reproducibility, RSDR4 11,6 11 R
45、eproducibility limit, R, (mg/kg) 170 410 210DIN EN 16328:2013-02 EN 16328:2012 (E) 9 Bibliography 1 EN 1482-1, Fertilizers and liming materials Sampling and sample preparation Part 1: Sampling 2 EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 IS
46、O 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions 4 ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 5 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 P. 0001-0194 DIN EN 16328:2013-02
