1、December 2010 Translation by DIN-Sprachendienst.English price group 11No 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).I
2、CS 65.080!$ldG“1736536www.din.deDDIN EN 15950Fertilizers Determination of N-(1,2-dicarboxyethyl)-D,L-aspartic acid(Iminodisuccinic acid, IDHA) using high-performance liquidchromatography (HPLC)English translation of DIN EN 15950:2010-12Dngemittel Bestimmung von N-(1,2-Dicarboxyethyl)-D,L-asparaginsu
3、re (Iminodibernsteinsure,IDHA) mit Hochleistungs-Flssigchromatographie (HPLC)Englische bersetzung von DIN EN 15950:2010-12Engrais Dosage de lacide N-(1,2-dicarboxythyl)-D,L aspartique (acide iminodisuccinique, IDHA)par chromatographique liquide haute performance (HPLC)Traduction anglaise de DIN EN 1
4、5950:2010-12www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.1712.10 DIN EN 15950:2010-12 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 260 “Fertilizers and li
5、ming materials” (Secretariat: DIN, Germany) under the mandate M/418. 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 AA Dngemittel. The D
6、IN Standards corresponding to the International Standards referred to in this document are as follows: EN ISO 3696 DIN ISO 3696 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-2, Accu
7、racy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15950 October 2010 ICS 65.080 English Version Fertilizers Determina
8、tion of N-(1,2-dicarboxyethyl)-D,L-aspartic acid (Iminodisuccinic acid, IDHA) using high-performance liquid chromatography (HPLC) Engrais Dosage de lacide N-(1,2-dicarboxythyl)-D,L aspartique (acide iminodisuccinique, IDHA) par chromatographique liquide haute performance (HPLC) Dngemittel Bestimmung
9、 von N-(1,2-Dicarboxyethyl)-D,L-asparaginsure (Iminodibernsteinsure, IDHA) mit Hochleistungs-Flssigchromatographie (HPLC) This European Standard was approved by CEN on 4 September 2010. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for givin
10、g 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 Management Centre or to any CEN member. This European Standard exists in three official vers
11、ions (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 Management Centre has the same status as theofficial versions. CEN members are the national standards bodies of Austria, Belgium,
12、 Bulgaria, Croatia, 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 United Kingdom. EUROPEAN COMMITTEE
13、 FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15950:2010: EEN 15950:2010 (E) 2 Contents
14、Page Foreword 31 Scope 42 Normative references 43 Terms and definitions .44 Principle 45 Interferences 46 Reagents .47 Apparatus .58 Sampling and sample preparation .59 Procedure .610 Calculation 811 Precision for IDHA .812 Test report 9Annex A (informative) Results of the inter-laboratory test 10An
15、nex B (informative) Stereo isomers of IDHA, chromatogram and calibration curve 12B.1 Stereo isomers of IDHA 12B.2 Chromatogram 13B.3 Calibration curve. 14Bibliography . 15DIN EN 15950:2010-12 EN 15950:2010 (E) 3 Foreword This document (EN 15950:2010) has been prepared by Technical Committee CEN/TC 2
16、60 “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 April 2011, and conflicting national standards shall be withdrawn at
17、 the latest by April 2011. 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 under a mandate given to CE
18、N 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, Croatia, Cyprus, Czech Republic, Denmark
19、, 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. DIN EN 15950:2010-12 EN 15950:2010 (E) 4 1 Scope This European S
20、tandard specifies a method for the determination of N-(1,2-dicarboxyethyl)-D,L-aspartic acid (Iminodisuccinic acid (IDHA) in fertilizers. The method is applicable to all fertilizers containing IDHA as chelating agent for contents 0,5 % (g/100 g). 2 Normative references The following referenced docum
21、ents 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 amendments) applies. EN 1482-2, Fertilizers and liming materials Sampling and sample preparation Par
22、t 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 Part 2: Terms relating to fertilizers EN ISO 3696:1995, Water for analytical laboratory use Sp
23、ecification 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 IDHA is extracted with water, and measured and detected by reversed phase HPLC using UV detection at 260 nm
24、 after total conversion of the chelating agent into its iron-(III)-chelate by adding an excess of a solution of iron-(III)-nitrate (details see Clause 8 and the following). In the HPLC-chromatogram IDHA is represented by two dominant peaks, the first one representing the R,S-isomer (50 %), and the s
25、econd one representing the R,R- as well as the S,S-isomer (each 25 %). 5 Interferences Other chelating agents such as DTPA, o,o-EDDHA or o,p-EDDHA do not interfere the determination of IDHA. EDTA may interfere with the determination of IDHA with some equipment, especially with certain columns in the
26、 HPLC-equipment (see Table A.2). 6 Reagents Use only reagents of recognized analytical grade. 6.1 Water, distilled or demineralized (grade 1 according to EN ISO 3696:1995). 6.2 Tetra-n-butylammonium hydrogen sulfate (C16H37NO4S), for ion pair chromatography. Also tetra-n-butylammonium bromide or -ch
27、loride may be used. In that case the pH needs to be adjusted (see 6.9). DIN EN 15950:2010-12 EN 15950:2010 (E) 5 6.3 Tetra-n-butylammonium hydroxide, w (C16H37NO) = 40 % in water. 6.4 Iron (III) nitrate nonahydrate, p.a. 6.5 Nitric acid, w(HNO3) = 65 %, p.a. 6.6 Hydrochloric acid, c(HCl) = 1 mol/l a
28、nd c(HCl) = 0,1 mol/l. 6.7 Sodium hydroxide, c(NaOH) = 1 mol/l and c(NaOH) = 0,1 mol/l. 6.8 Buffer solution, pH = 8,0. Adjust 0,1 mol/l boron as boric/borate buffer at pH = 8,0 with hydrochloric acid or sodium hydroxide. NOTE Commercial buffers may be used. The buffer solution should not contain any
29、 phosphate or chelating agents like EDTA or others. 6.9 Eluent solution. In a 1 l volumetric flask dissolve 2,5 g tetra-n-butylammonium hydrogen sulfate (6.2) (= 7,36 mmol), 1,7 ml tetra-n-butylammonium hydroxide (6.3) and 0,04 ml nitric acid (6.5) in water and make up to volume. The pH of this solu
30、tion is approximately 2,5. In the case that tetra-n-butylammonium bromide or -chloride is used instead of tetra-n-butylammonium hydrogen sulfate, weigh 2,37 g or 2,05 g respectively and adjust pH to approximately 2,5 with additional nitric acid (6.5). 6.10 Derivatisation reagent. In a 100 ml volumet
31、ric flask dissolve 4,4 g tetra-n-butylammonium hydrogen sulfate (6.2) (= 12,95 mmol) and 1,5 g iron(III)nitrate nonahydrate (6.4) in water and make up to volume. The pH of this solution is approximately 1,5. In the case that tetra-n-butylammonium bromide or -chloride is used instead of tetra-n-butyl
32、ammonium hydrogen sulfate, weigh 4,17 g or 3,60 g respectively and adjust pH to approximately 1,5 with additional nitric acid (6.5). 7 Apparatus Laboratory equipment and glassware for preparation of solutions and dilutions. 7.1 Analytical balance, capable of weighing to an accuracy of 0,1 mg. 7.2 pH
33、-meter with electrode. 7.3 HPLC-system, with Diode Array Detector (DAD) or UV-detector. 7.4 Membrane filters, micro membrane filters resistant to aqueous solutions, with porosity of 0,45 m. 8 Sampling and sample preparation Sampling is not part of the method specified in this document. A recommended
34、 sampling method is given in EN 1482-1. Sample preparation shall be carried out in accordance with EN 1482-2. DIN EN 15950:2010-12 EN 15950:2010 (E) 6 For the size reduction of samples with a high amount of chelating agents, it is not recommended to use a high speed laboratory mill. It is more conve
35、nient to grind the sample to a particle size less than 1 mm with a mortar. Special care shall be taken with NPK samples due to their high hygroscopicity. 9 Procedure 9.1 System parameters of HPLC Analytical/separating column: silica column with C18 or C8 reverse phase, 5 m, 250 mm 4,6 mm 1)The use o
36、f a guard column is recommended. Detection wavelength: 260 nm Eluent: according to 6.9 Flow rate: 0,5 ml/min Temperature: Constant between 20 C and 40 C Run time: 20 min Injection volume: 20 l 9.2 Calibration 9.2.1 Stock IDHA solution: (IDHA) = 1 000 mg/l Weigh 338,3 mg 100/R, where R is the purity
37、of the tetra sodium salt of IDHA in percent, equivalent to 250,0 mg of free acid, into a 250 ml beaker, add about 150 ml of water (6.1) and dissolve either by using an ultrasonic bath or stirring on a magnetic stirrer for about 15 min. When using an ultrasonic bath, the solution should be cooled dow
38、n to room temperature before the next step. Measure the pH of the solution. By the use of hydrochloric acid (6.6) or sodium hydroxide (6.7) and the pH-meter (7.2) adjust the pH to 8,0 0,1. Then add 20 ml of buffer solution (6.8). Transfer into a 250 ml volumetric flask, make up to volume and homogen
39、ize. This solution shall be used on the day of its preparation. 9.2.2 Calibration solution Into six beakers (e.g. 25 ml) take volumes from the stock solution (9.2.1) according to Table 1, make up to 10 ml with water (6.1) and add 3 ml of the derivatisation reagent (6.10). Homogenize and filtrate the
40、 solution by using the micro membrane filters (7.4) into the auto sampler vial. Inject the standard solutions into the chromatographic system. The evaluation of calibration is carried out manually or by means of a suitable PC-aided (computerized) calculation method. Measure the retention times 1) Li
41、Chrosorb RP-18 or RP-8 5 m 250/4,6 mm or equivalent is an example of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute any endorsement by CEN of these products. and the areas of the two IDHA isomer peaks
42、 for all solutions. Draw a calibration line with the sum of the peak areas of the standard solutions versus the IDHA concentration (mg/l), according to Table 1. DIN EN 15950:2010-12 EN 15950:2010 (E) 7 Table 1 Preparation of calibration solutions Parameter ml of stock solution ml of water Content of
43、 IDHA mg/l Standard 1 1,00 9,00 100 Standard 2 2,00 8,00 200 Standard 3 4,00 6,00 400 Standard 4 6,00 4,00 600 Standard 5 8,00 2,00 800 Standard 6 10,00 0,00 1 000 9.3 Preparation of the test solution Weigh an amount of the sample grounded to 5 0,25 1 to 5 1,0 1 5,0 Using hydrochloric acid (6.6) or
44、sodium hydroxide (6.7) and the pH-meter (7.2) adjust the pH to 8,0 0,1. Then add 20 ml of buffer solution (6.8). Transfer into a 250 ml volumetric flask, make up to volume with water (6.1) and homogenize. Filtrate the solution by using the micro membrane filters (7.4) (about 7 ml to 10 ml). This sol
45、ution shall be used on the day of its preparation. Transfer 5,00 ml into a beaker (e.g. 25 ml) and add 1,5 ml of the derivatisation reagent (6.10) and make homogeneous. Filtrate the solution by using the micro membrane filters (7.4) into the auto sampler vial. In the case that no auto sampler is ava
46、ilable, manually inject 20 l of the filtrated solution. 9.4 Measurement Run the chromatographic analysis and identify the IDHA isomers by the retention time of the obtained peaks (see Figure B.2). Measure the sum of the areas of both peaks. Determine the concentration of IDHA using the calibration g
47、raph (see Figure B.3). The concentration of IDHA in the sample solutions shall be kept within the calibration limits to ensure sufficient reproducibility. DIN EN 15950:2010-12 EN 15950:2010 (E) 8 10 Calculation 10.1 Calculation of the content of IDHA in g/100 g of sample The calculation may be perfo
48、rmed manually or by means of a PC using the calibration parameters in respect to the amount used. In the case of PC-aided (computerized) calculation and application of Table 1 regarding the amounts of stock solution, the concentration of IDHA in milligrams per litre will be calculated by the system. Calculate the mass fraction of the sum of R,S- and R,R- and S,S-isomers of IDHA (free acid), RIDHA, in g/10
