1、November 2008DEUTSCHE NORM English price group 11No 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!$Rg“1476898www
2、.din.deDDIN EN 15451Fertilizers Determination of chelating agents Determination of iron chelated by EDDHSA by ion pair chromatographyEnglish version of DIN EN 15451:2008-11Dngemittel Bestimmung von Chelatbildnern Bestimmung von Eisen-chelatisiertem EDDHSA mit Ionen-PaarchromatographieEnglische Fassu
3、ng DIN EN 15451:2008-11SupersedesDIN CEN/TS 15451:2007-01www.beuth.deDocument comprises 17 pagesDIN EN 15451:2008-11 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 respo
4、nsible 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 Standard corresponding to the International Standard referred to in the E
5、N is as follows: ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN CEN/TS 15451:2007-01 (German version only) as follows: a) In subclause 5.7, it has been added that photometric titration may be manual or automatic. b) In clause 6, a new subclause 6.1 “General” has been added and t
6、he subsequent subclauses have been renumbered. c) In Annex D (informative), the complete designation for HEDTA and the abbreviation EDDHSA have been corrected. d) The standard has been editorially revised. Previous editions DIN CEN/TS 15451: 2007-01 National Annex NA (informative) Bibliography DIN I
7、SO 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 STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15451July 2008ICS 65.080 Supersedes CEN/TS 15451:2006 Eng
8、lish VersionFertilizers - Determination of chelating agents - Determination ofiron chelated by EDDHSA by ion pair chromatographyEngrais - Dosage des agents chlatants - Dosage du ferchlat par EDDHSA par chromatographie dappariementdionsDngemittel - Bestimmung von Chelatbildnern -Bestimmung von Eisen-
9、chelatisiertem EDDHSA mit Ionen-PaarchromatographieThis European Standard was approved by CEN on 30 May 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 without any alterat
10、ion. 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 made by translat
11、ionunder 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, Germany, Gree
12、ce, 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 NORMUNGManagement Ce
13、ntre: 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 15451:2008: EEN 15451:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Interferences 4 5 Reagents.4 6
14、Apparatus .5 7 Sampling and sample preparation.6 8 Procedure .7 9 Expression of the result7 10 Precision for Fe3+/EDDHSA.8 11 Test report 8 Annex A (informative) Chromatogram for Fe3+/EDDHSA 10 Annex B (informative) Spectra UV-visible for Fe3+/EDDHSA 11 Annex C (informative) Results of the inter-lab
15、oratory tests .12 Annex D (informative) Complete names of chelating agents .14 Bibliography 15 DIN EN 15451:2008-11 EN 15451:2008 (E) 3 Foreword This document (EN 15451:2008) has been prepared by Technical Committee CEN/TC 260 “Fertilizers and liming materials”, the secretariat of which is held by D
16、IN. 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 January 2009, and conflicting national standards shall be withdrawn at the latest by January 2009. Attention is drawn to the possibility that so
17、me 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 supersedes CEN/TS 15451:2006. This document has been prepared under a mandate given to CEN by the European Commission
18、 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany,
19、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 15451:2008-11 EN 15451:2008 (E) 4 1 Scope This European Standard specifies a method for the ch
20、romatographic determination of the total amount of iron chelated by EDDHSA in commercial products. 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 lates
21、t 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 ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle The iron ch
22、elates are determined by ion-pair high-performance liquid chromatography. When an iron chelate (anion) is added to a polar fluid (eluent), containing a large cation (ion-pair reagent), an ion pair is formed. This ion pair is retained by an apolar solid phase (stationary phase). The strength of the r
23、etention depends on the molecular size and its acidity. Each iron chelate presents a characteristic retention time and a characteristic spectrum depending on the chelating agent, and it is separated from the other substances present in the sample. The separation is carried out on a reverse phase sil
24、ica column and an aqueous solution of TBA+(tetrabutylammonium) and acetonitrile as eluent. The detection is based on photometry at 480 nm. For Fe3+/EDDHSA a gradient method is required. 4 Interferences No interferences have been detected. Iron chelates with o,o-EDDHA, o,p-EDDHA, o,o-EDDHMA, EDTA, DT
25、PA, CDTA, HEDTA, as well as the chelating agents do not interfere since they are separated from Fe3+/EDDHSA. 5 Reagents 5.1 General a) All reagents shall be of recognized analytical grade. b) All water used for the preparation of eluent, standards, and sample solutions should be water grade1 accordi
26、ng to EN ISO 3696:1995. c) When reagents with a declared purity of less than 99 % are used for the preparation of standard solutions, a correction should be made in order to obtain the required concentration in the solution. 5.2 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l Dissolve 20 g of NaOH in
27、 pellet form in a 1 litre-volumetric flask with water free of carbon dioxide. Dilute to the mark and homogenize. 5.3 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of NaOH in pellet form in a 1 litre volumetric flask with water free of carbon dioxide. Dilute to the mark and homogenize.
28、DIN EN 15451:2008-11 EN 15451:2008 (E) 5 5.4 Hydrochloric acid solution, c(HCl) = 1,0 mol/l Dilute 88 ml of hydrochloric acid (35 % HCl) to 1 000 ml with water. 5.5 Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 50 ml of hydrochloric acid (5.4) to 500 ml with water. 5.6 Iron (III) nitrate sol
29、ution, (Fe) = 1 050 mg/l Dissolve 0,7594 g of iron (III) nitrate nonahydrate Fe(NO3)39H2O in 100 ml of water. Transfer to a 100 ml-volumetric flask. Dilute to the mark with water and homogenize. Check (for example by AAS) that the Fe concentration of this solution is (1 050 30) mg/l. As the Fe(NO3)3
30、9H2O is deliquescent, for preparation of the Fe3+/EDDHSA solution (5.7) it shall be added in solution of a known concentration. 5.7 Fe3+/EDDHSA solution, (Fe) = 100 mg/l Dissolve 0,233 0 g100/P, where P is the complexometric purity of the standard in percentage of the acid form H6EDDHSA obtained by
31、either manual or automatic photometric titration with a Fe(III)standard at constant pH = 6 (pH stat system), of ethylene diamine di(2-hydroxy-5-sulfophenyl) acetic acid and its condensation products (EDDHSA) in 150 ml of water and 2,7 ml of NaOH (5.2). After complete dissolution, add 25 ml of the Fe
32、 solution (5.6) to the chelating agent solution stirring about 5 min. The solution is adjusted to pH 7,0 with NaOH solution (5.3). Let the solution stand overnight in darkness to allow excess Fe to precipitate as oxide. Filter quantitatively through a cellulose filter and make up to volume (250 ml)
33、with water. 5.8 Eluent for the determination Two solutions are used to make a linear gradient eluent: a) Solution A: Add 3,33 ml of TBAOH 40 % (mass fraction) Tetrabutylammonium hydroxide solution in water to 500 ml of water. Adjust pH to 6,0 with HCl (5.4 and 5.5). Add 350 ml of acetonitrile (HPLC
34、grade) and make up to volume in a 1 litre-volumetric flask with water. Filter through 0,2 m membrane filter (6.4 b). b) Solution B: Add 3,33 ml of TBAOH 40 % (mass fraction) Tetrabutylammonium hydroxide solution in water to 150 ml of water. Adjust pH to 6,0 with HCl (5.4 and 5.5). Add 750 ml of acet
35、onitrile (HPLC grade) and make up to volume in a 1 litre-volumetric flask with water. Filter through 0,2 m-membrane filter (6.4 b). NOTE 1 TBACl or TBABr may be used, providing that pH is adjusted to 6,0 with NaOH or HCl. NOTE 2 Tetrabutylammonium bisulfate should be avoided because peak retention t
36、imes and shape may significantly change. 6 Apparatus 6.1 General Usual laboratory equipment, glassware, and: 6.2 Magnetic stirrer Magnetic stirrer with magnets. DIN EN 15451:2008-11 EN 15451:2008 (E) 6 6.3 Chromatograph Chromatograph equipped with: a) gradient pump delivering the eluent at a flow ra
37、te of 1,5 ml/min (see Table 1); b) injection valve with a 20 l injection loop; c) C18 column; 150 mm3,9 mm ID; dp = 5 m1); d) use of a C18 guard column is recommended; e) UV/VIS-detector with a 480 nm-filter or diode array; f) integrator. Table 1 Gradient table Time min Flow rate ml/min A % B % Curv
38、e type1,5 100 0 Linear 5 1,5 100 0 Linear6 1,5 0 100 Linear 11 1,5 0 100 Linear12 1,5 100 0 Linear 20 1,5 100 0 Linear6.4 Membrane filters a) Micromembrane filters resistant to aqueous solutions, with porosity of 0,45 m; b) Micromembrane filters resistant to organic solutions, (e.g.: nylon micromemb
39、rane filters) with porosity of 0,2 m. 7 Sampling and sample preparation Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN 1482-1 1. Sample preparation shall be carried out in accordance with EN 1482-2. NOTE For the size reduction of
40、samples with a high amount of chelating agents, it is not recommended to use a high speed laboratory mill. It is more convenient to grind the sample in a mortar to a particle size less than 1 mm. 1) SYMMETRYTMC18, from WATERS cat.n: WAT054205, or equivalent are examples of suitable products availabl
41、e commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by CEN of these products. DIN EN 15451:2008-11 EN 15451:2008 (E) 7 8 Procedure 8.1 Preparation of the sample solution of iron chelates Weigh approximately 125 mg within 0,1
42、mg, into a 100 ml-beaker (for samples with more than 4 % of chelated iron weigh 80 mg and for samples with less than 2 % of chelated iron weigh 250 mg). Add 80 ml of water. Stir on a magnetic stirrer for 30 min. Dilute to the mark in a 100 ml-volumetric flask with water and homogenize. 8.2 Preparati
43、on of the calibration solutions Pipette a volume (V ml) (see Table 2) of the Fe3+/EDDHSA standard solution (5.7) in six 100 ml-volumetric flasks respectively. Make up to volume with water and homogenize. Table 2 Preparation of the calibration curve of Fe3+/EDDHSA Solution V ml Concentration of Fe in
44、 the form of Fe3+/EDDHSA mg Fe/l 1 1 1,0 2 5 5,0 3 10 10,0 4 25 25,0 5 50 50,0 6 100 100 8.3 Chromatographic analysis Immediately before injection, all solutions should be filtered through a 0.45 m-membrane filter (6.4 a). Inject the standard solutions (8.2.) into the chromatographic system (6.3). M
45、easure the retention times and the areas of the Fe3+/EDDHSA chromatographic peaks for all solutions. Two chromatographic peaks appear for Fe3+/EDDHSA, corresponding with the different forms of Fe3+/EDDHSA (see Annex A). Draw the calibration graph with the added values of the two chromatographic peak
46、s of the standard solutions of Fe3+/EDDHSA versus the chelated iron concentration (mg Fe/l) in the standards. Inject the sample solution (8.1). Identify the chelating agent by the retention time of the obtained peaks (see Annex A) and if diode array detector is used, confirm with its UV-visible spec
47、trum (see Annex B). Measure the areas of the peaks for the sample solution corresponding with the iron chelated by EDDHSA as chelating agent. Determine the concentration of the iron chelated (mg Fe/l) using the calibration graph. NOTE Normally the two chromatographic peaks in Fe3+/EDDHSA appear over
48、lapped. For integration baseline correction as indicated in Annex A, Figure A.1, can be used. 9 Expression of the result 9.1 Fe in Fe-chelates In case of the iron chelates prepared as in 8.1, the percentage by weight of the Fe in the form of Fe3+/EDDHSA in the fertiliser, wFe, is equal to: DIN EN 15
49、451:2008-11 EN 15451:2008 (E) 8 01,0Fe=mwwhere is the mass concentration of Fe in milligrams per litre of the Fe chelated by EDDHSA determined with the calibration graph; m is the mass of the sample taken for analysis in grams. 10 Precision for Fe3+/EDDHSA 10.1 Inter-laboratory test Two inter-laboratory tests have been carried out in 2004 and 2005 with 13 and 9 participating laboratories respectively. Three different samples were analyzed. The inter-laboratory tests yielded the data summarized in Annex C. Repeatability and r
