DIN EN 15452-2008 Fertilizers - Determination of chelating agents - Determination of iron chelated by o p- EDDHA by reversed phase HPLC German version EN 15452 2008《肥料 螯合剂的测定 用反相高效.pdf

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1、November 2008DEUTSCHE NORM English price group 10No 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“1476897www

2、.din.deDDIN EN 15452Fertilizers Determination of chelating agents English version of DIN EN 15452:2008-11Dngemittel Bestimmung von Chelatbildnern Bestimmung von Eisen-chelatisiertem o,p-EDDHA mit Umkehrphasen HPLCEnglische Fassung DIN EN 15452:2008-11SupersedesDIN CEN/TS 15452:2007-01www.beuth.deDoc

3、ument comprises 15 pagesDetermination of iron chelated by o,p-EDDHA by reversed phase HPLCDIN EN 15452:2008-11 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 responsible

4、 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 EN is a

5、s follows: ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN CEN/TS 15452:2007-01 (German version only) as follows: a) In subclause 5.6, a note concerning the complexometric purity of the standard and its determination have been added. b) In clause 6, a new subclause 6.1 “General”

6、has been added and the subsequent subclauses have been renumbered. c) In Annex C (informative), the complete designation for HEDTA has been corrected. d) The standard has been editorially revised. Previous editions DIN CEN/TS 15452: 2007-01 National Annex NA (informative) Bibliography DIN ISO 5725-2

7、, 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 15452July 2008ICS 65.080 Supersedes CEN/TS 15452:2006 English Vers

8、ionFertilizers - Determination of chelating agents - Determination ofiron chelated by o,p-EDDHA by reversed phase HPLCEngrais - Dosage des agents chlatants - Dosage du ferchlat par o,p-EDDHA par chromatographie liquide haute performance polarit de phase inverseDngemittel - Bestimmung von Chelatbildn

9、ern -Bestimmung von Eisen-chelatisiertem o,p-EDDHA mitUmkehrphasen HPLCThis 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

10、 without any alteration. 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 langu

11、age made by translationunder 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,F

12、rance, 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 STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR

13、NORMUNGManagement Centre: 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 15452:2008: EEN 15452:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Interferenc

14、es 4 5 Reagents.4 6 Apparatus .5 7 Sampling and sample preparation.6 8 Procedure .6 9 Expression of results 8 10 Precision.8 11 Test report 9 Annex A (informative) Typical chromatogram .10 Annex B (informative) Statistical results of the inter-laboratory test 11 Annex C (informative) Complete names

15、of chelating agents .12 Bibliography 13 DIN EN 15452:2008-11 EN 15452:2008 (E) 3 Foreword This document (EN 15452:2008) 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

16、 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 some of the elements of this document may be the subject

17、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 15452:2006. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to

18、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, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithu

19、ania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15452:2008-11 EN 15452:2008 (E) 4 1 Scope This European Standard specifies a method for the chromatographic determination of the amount of iron chela

20、ted by each of the individual isomers of the chelating agent ortho-para EDDHA (o,p-EDDHA) in fertilizers. The method allows the identification of this chelating agent and the determination of the water soluble fraction of iron chelated by this chelating agent. The method is not applicable for the de

21、termination of the amount of free chelating agent. NOTE 1 This method has been shown to be also suitable for the determination of the amount of iron chelated by each of the individual isomers of the chelating agent ortho-ortho EDDHA (o,o-EDDHA) in fertilizers. NOTE 2 o,o-EDDHA and o,p-EDDHA are abbr

22、eviations used in this European Standard for the sake of simplicity. For complete names see Annex C. NOTE 3 The substances o,o-EDDHA and o,p-EDDHA both exist as different stereoisomers. For o,o-EDDHA a meso form and a d/l pair (the racemic isomers) exist, for o,p-EDDHA two different d/l pairs exist.

23、 All four stereoisomers are observed separately in this method. NOTE 4 Currently, an analytically pure standard only exists for o,o-EDDHA. The method for o,p-EDDHA has been developed with an o,p-EDDHA standard containing an uncertain concentration of o,p-EDDHA. 2 Normative references The following r

24、eferenced documents 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

25、preparation Part 2: Sample preparation 3 Principle The technique used is reversed phase HPLC with UV detection at 277 nm. The sample is separated on a silica-based reversed phase column using sodium formate, c = 0,015 mol/l, pH = 3,0, and acetonitrile as mobile phase. For both o,p-EDDHA and o,o-EDDH

26、A, two stereoisomer peaks are observed. The concentration of iron chelated by o,p-EDDHA (o,p-Fe) is determined according to the external standard method. 4 Interferences No interferences have been detected. Iron chelates with EDTA, HEDTA, DTPA and EDDHMA do not interfere. 5 Reagents 5.1 General a) A

27、ll reagents shall be of recognized analytical grade. DIN EN 15452:2008-11 EN 15452:2008 (E) 5 b) All water should conform to EN ISO 3696:1995, grade 1. c) When products with a declared purity (P) of less than 99 % are used for the preparation of standard solutions, a correction should be made in ord

28、er to obtain the required concentration in the solution. 5.2 Sodium hydroxide solution , c(NaOH) = 0,1 mol/l Dissolve 4 g of NaOH in pellet form in water in a 1 000 ml-volumetric flask. Dilute to the mark and homogenize. 5.3 Hydrochloric acid solution, c(HCl) = 4 mol/l Dilute 395 g of concentrated h

29、ydrochloric acid (37 %) to 1 000 ml with water. 5.4 Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 25 ml of hydrochloric acid (5.3) to 1 000 ml with water. 5.5 Iron (III) nitrate solution, (Fe) = 4 200 mg/l Dissolve 3,04 g of iron (III) nitrate nonahydrate Fe(NO3)3.9H2O in 80 ml of water. Add

30、 1 ml of hydrochloric acid (5.3). Transfer to a 100 ml-volumetric flask. Dilute to the mark with water and homogenize. Check that the Fe concentration of this solution is 4 200 mg/ 100 mg/l for example by AAS or iodometric titration. This solution is stable for approximately one week. 5.6 o,p-Fe-EDD

31、HA standard solution, (o,p-Fe) = 100 mg/l Weigh to the nearest 0,1 mg (64,5 1) mg 100/P, where P is the complexometric purity of the standard in percentage of the acid form o,p-H4-EDDHA obtained by either manual or automatic photometric titration with a Fe(III)standard at constant pH = 6 (pH stat sy

32、stem), of o,p-H4-EDDHA (mco,p, see 5.1c) in a 100 ml beaker. Add 5 ml of NaOH (5.2) and carefully dissolve the sample by stirring. Add, after complete dissolution, 35 ml of water and 2,5 ml of iron (III) nitrate solution (5.5). Adjust the pH of the solution to 3,0 with NaOH (5.2) or HCl (5.4). Trans

33、fer the solution quantitatively to a 100 ml-volumetric flask. Dilute to the mark with water and homogenize. 5.7 Eluent solution Dissolve 1,0 g of sodium formate in 800 ml of water. Adjust the pH to 3,0 with HCl (5.3 or 5.4). Transfer the solution to a 1 000 ml-volumetric flask. Dilute to the mark wi

34、th water and homogenize. Filter the solution through a 0,45 m membrane filter. Mix 915 ml of the sodium formate solution, pH 3,0, with 85 ml of acetonitrile. Homogenize and degas the solution. NOTE The quality and the status of the column may have an influence on the retention times. To adjust the r

35、etention times, it may be necessary to slightly change the ratio of sodium formate solution and acetonitrile. 6 Apparatus 6.1 General Usual laboratory equipment, glassware, and: DIN EN 15452:2008-11 EN 15452:2008 (E) 6 6.2 Magnetic stirrer Magnetic stirrer with magnets. 6.3 Chromatograph equipped wi

36、th: a) isocratic pump delivering the eluent at a flow rate of 1,0 ml/min; b) injection valve with a 20 l injection loop; c) C18 modified silica column (ODS-2); 2504,6 mm ID; dp= 5 m1); d) use of a C18 guard column (ODS-2) is recommended; e) UV/VIS-detector with a 277-nm-filter; f) integrator. 6.4 Me

37、mbrane filters Micromembrane filters resistant to aqueous solutions with porosity of 0,45 m. 7 Sampling and sample preparation Sampling is not part of the method specified in this document. A recommended sampling method is given in EN 1482-1 1. Sample preparation shall be carried out in accordance w

38、ith EN 1482-2. NOTE 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 grind the sample in a mortar to a particle size less than 1 mm. 8 Procedure 8.1 Preparation of the sample solution Weigh t

39、o the nearest 0,1 mg approximately (250 10) mg of sample (ms) in a 250 ml-beaker. Add 200 ml of water and dissolve the sample by stirring for 30 min. Transfer the solution quantitatively to a 250 ml volumetric flask. Dilute to the mark with water and homogenize. NOTE If the o,p-Fe concentration is l

40、ower than 0,5 % or higher than 5 % the concentration of these iron chelates will be outside the calibration range. For a more accurate determination the intake should be accordingly either increased or decreased. 1) Spherisorb ODS-2 from Waters or Allsphere ODS-2 from Alltech are examples of suitabl

41、e products available 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 15452:2008-11 EN 15452:2008 (E) 7 8.2 Preparation of the standard solutions Pipette a volume (V ml) (see Table 1) of the

42、o,p-Fe-EDDHA standard solution (5.6) in six 50 ml-volumetric flasks. Dilute to the mark with water and homogenize. These solutions contain iron chelated by o,p-EDDHA in the approximated concentrations given in Table 1. The exact mass concentration of iron chelated by o,p-EDDHA in milligrams per litr

43、e is given by: 2d/l1d/lFepo, += (1) 35,360847,551001,0505,0po,c2d/l1d/l=PmV (2) where: V is the amount of standard solution used in millilitre; mco,p is the amount of o,p-H4-EDDHA used for the preparation of the standard solution in milligrams; P is the purity of the calibration standard in percent.

44、 NOTE It is assumed that the o,p-EDDHA acid standard contains equal amounts of the two stereoisomers. Table 1 Preparation of the standard solutions o,p-EDDHA d/l isomer-1 d/l isomer-2 Solution V ml Fe mg/l Fe mg/l 1 2,5 2,5 2,5 2 5 5,0 5,0 3 10 10,0 10,0 4 15 15,0 15,0 5 20 20,0 20,0 6 25 25,0 25,0

45、8.3 Chromatographic analysis Immediately before injection, all solutions should be filtered through a 0,45 m-membrane filter (6.4). Inject the standard solutions (8.2) into the chromatographic system (6.3). Measure the retention times and the areas of the two o,p-Fe-EDDHA isomers (d/l-1 and d/l-2) f

46、or all solutions. Draw a different calibration line for each of the two isomers with the value of the peak areas of the standard solutions versus the exact concentration of Fe (mg/l) chelated by each chelating agent isomer. Inject the sample solution (8.1). Identify the different isomers by the rete

47、ntion time of the obtained peaks (see Figure A.1). Measure the area of the peak for each isomer. Determine the concentration of the iron chelated (mg/l) for each isomer using the corresponding calibration line. DIN EN 15452:2008-11 EN 15452:2008 (E) 8 9 Expression of results The mass fraction, w, in

48、 percent of Fe chelated by o,p-EDDHA in the fertilizer is equal to: 25s2d/lpo,1d/lpo,Fepo,+=mw(3) where o,p-d/l-1is the mass concentration of Fe chelated by the first set of stereoisomers (d/l-1) of o,p-EDDHA, in milligram per litre o,p-d/l-2is the mass concentration of Fe chelated by the second set

49、 of stereoisomers (d/l-2) of o,p-EDDHA, in milligram per litre msis the mass of the sample taken for analysis, in milligrams. 10 Precision 10.1 Inter-laboratory test An inter-laboratory test was carried out in 2005 with 12 participating laboratories and 4 different commercial samples. The results of this inter-laboratory test are summarized in Annex B. Repeatability and reproducibility were calculated according to ISO 5725-2 2. 10.2 Repeatability The absolute difference between two independent test results o