1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 15950:2010Fertilizers Determinationof N-(1,2-dicarboxyethyl)-D,L-aspartic acid (Iminodisuccinicacid, IDHA) using high-performance liquidchromatography (HPLC)BS EN 15950:201
2、0 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 15950:2010.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 beobtained on request to
3、its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 67488 4ICS 65.080Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was pu
4、blished under the authority of theStandards Policy and Strategy Committee on 31 October 2010.Amendments issued since publicationDate Text affectedBS EN 15950:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15950 October 2010 ICS 65.080 English Version Fertilizers - Determination of N-(1,2-d
5、icarboxyethyl)-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 von N-(1,2-
6、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 giving this Europ
7、ean 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 versions (Englis
8、h, 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 the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria,
9、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 FOR STAND
10、ARDIZATION 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: EBS EN 15950:2010EN 15950:2010 (E) 2 Con
11、tents 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 tes
12、t 10Annex B (informative) Stereo isomers of IDHA, chromatogram and calibration curve 12B.1 Stereo isomers of IDHA 12B.2 Chromatogram 13B.3 Calibration curve. 14Bibliography . 15BS EN 15950:2010EN 15950:2010 (E) 3 Foreword This document (EN 15950:2010) has been prepared by Technical Committee CEN/TC
13、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 April 2011, and conflicting national standards shall be withdrawn a
14、t 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 C
15、EN 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, Denmar
16、k, 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 15950:2010EN 15950:2010 (E) 4 1 Scope This European Stand
17、ard 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 documents
18、 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 Part 2:
19、 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 Specif
20、ication 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 af
21、ter 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 seco
22、nd 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 HP
23、LC-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. BS EN 15950:2010EN 15950:2010 (E) 5 Also tet
24、ra-n-butylammonium bromide or -chloride may be used. In that case the pH needs to be adjusted (see 6.9). 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 and c(HCl
25、) = 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 phospha
26、te 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 solution is
27、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 volumetric flas
28、k 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-butylammonium
29、 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-meter w
30、ith 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 samplin
31、g method is given in EN 1482-1. BS EN 15950:2010EN 15950:2010 (E) 6 Sample preparation shall be carried out in accordance with EN 1482-2. 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 convenient to grin
32、d 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 of a guard col
33、umn 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 of the tetra
34、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 down to room tem
35、perature 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 homogenize. This sol
36、ution 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 solution by
37、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) LiChrosorb RP-1
38、8 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. BS EN 15950:2010EN 15950:2010 (E) 7 and the areas of th
39、e two IDHA isomer peaks 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. Table 1 Preparation of calibration solutions Parameter ml of stock solution ml of water Content of IDHA mg/l Standar
40、d 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 sodium hydroxide (
41、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 solution shall be use
42、d 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 available, manually i
43、nject 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 graph (see Figure B
44、.3). The concentration of IDHA in the sample solutions shall be kept within the calibration limits to ensure sufficient reproducibility. BS EN 15950:2010EN 15950:2010 (E) 8 10 Calculation 10.1 Calculation of the content of IDHA in g/100 g of sample The calculation may be performed manually or by mea
45、ns 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 fra
46、ction of the sum of R,S- and R,R- and S,S-isomers of IDHA (free acid), RIDHA, in g/100 g according to Equation (1). EAR=40IDHA(1) where A is the IDHA concentration, in milligrams per litre, calculated by the system from the calibration graph; E is the mass of the test portion, in grams. 10.2 Calcula
47、tion of the content of IDHA in mmol/100 g of sample Calculate the content of IDHA, wIDHA, in mmol/100 g according to Equation (2). 249,11000IDHA=Rw (2) where R is the mass fraction of IDHA in grams per 100 g of the sample; 249,1 is the mole weight of the free acid of IDHA. 11 Precision for IDHA 11.1
48、 Inter-laboratory test Two inter-laboratory tests have been carried out in 2008 with eleven and ten participating laboratories and nine different samples of fertilizers. Repeatability and reproducibility were calculated according to ISO 5725-2. The values derived from this inter-laboratory test may
49、not be applicable to concentration ranges and matrices other than those given in Annex A. 11.2 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 than 5 % of the cases exceed the values of r given in Table 3. BS EN 15950:2010EN 15950:2010 (
