1、BRITISH STANDARDBS EN 15452:2008Fertilizers Determination of chelating agents Determination of iron chelated by o,p-EDDHA by reversed phase HPLCICS 65.080g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55
2、g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 15452:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 August 2008 BSI 2008ISBN 978 0 580 60412 6National forewordThis British Standard is the UK implementation of EN 15452:200
3、8. It supersedes DD CEN/TS 15452:2006 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee CII/37, Fertilisers and related chemicals.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not
4、purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORM
5、EN 15452July 2008ICS 65.080 Supersedes CEN/TS 15452:2006 English VersionFertilizers - 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 perfor
6、mance polarit de phase inverseDngemittel - Bestimmung von Chelatbildnern -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 conditio
7、ns for giving this EuropeanStandard the status of a national standard 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 offi
8、cial versions (English, French, German). A version in any other language 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, B
9、elgium, Bulgaria, 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
10、 STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR 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 Fo
11、reword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Interferences 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
12、of the inter-laboratory test 11 Annex C (informative) Complete names of chelating agents .12 Bibliography 13 BS EN 15452:2008EN 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
13、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 January 2009, and conflicting national standards shall be withdrawn at the latest by January 2009. Attention is drawn to the possibilit
14、y 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 supersedes CEN/TS 15452:2006. This document has been prepared under a mandate given to CEN by the European C
15、ommission 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,
16、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 15452:2008EN 15452:2008 (E) 4 1 Scope This European Standard specifies a method for th
17、e chromatographic determination of the amount of iron chelated 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 th
18、is chelating agent. The method is not applicable for the determination 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-EDD
19、HA) in fertilizers. NOTE 2 o,o-EDDHA and o,p-EDDHA are abbreviations 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 i
20、somers) exist, for o,p-EDDHA two different d/l pairs exist. 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 concent
21、ration of o,p-EDDHA. 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 latest edition of the referenced document (including any amendments) applies. EN 14
22、82-2, Fertilizers and liming materials Sampling and sample 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 ac
23、etonitrile as mobile phase. For both o,p-EDDHA and o,o-EDDHA, 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, DT
24、PA and EDDHMA do not interfere. 5 Reagents 5.1 General a) All reagents shall be of recognized analytical grade. BS EN 15452:2008EN 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
25、standard solutions, a correction should be made in order to obtain the required concentration in the solution. 5.2 Sodium hydroxide solution , c(NaOH) = 0,1 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 sol
26、ution, c(HCl) = 4 mol/l Dilute 395 g of concentrated hydrochloric 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) ni
27、trate nonahydrate Fe(NO3)3.9H2O in 80 ml of water. Add 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 solutio
28、n is stable for approximately one week. 5.6 o,p-Fe-EDDHA 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
29、 with a Fe(III)standard at constant pH = 6 (pH stat system), 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 t
30、he solution to 3,0 with NaOH (5.2) or HCl (5.4). Transfer 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
31、 to a 1 000 ml-volumetric flask. Dilute to the mark with 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 hav
32、e an influence on the retention times. To adjust the retention 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: BS EN 15452:2008EN 15452:2008 (E) 6 6.2 Magnetic stirrer Magnetic st
33、irrer with magnets. 6.3 Chromatograph equipped with: 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-det
34、ector with a 277-nm-filter; f) integrator. 6.4 Membrane 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
35、 preparation shall be carried out in accordance with 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 Procedu
36、re 8.1 Preparation of the sample solution Weigh to 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
37、homogenize. NOTE If the o,p-Fe concentration is lower 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 All
38、sphere ODS-2 from Alltech are examples of suitable 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. BS EN 15452:2008EN 15452:2008 (E) 7 8.2 Preparation of the standard solutions P
39、ipette a volume (V ml) (see Table 1) of the 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
40、chelated by o,p-EDDHA in milligrams per litre 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 pu
41、rity of the calibration standard in percent. 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
42、 15 15,0 15,0 5 20 20,0 20,0 6 25 25,0 25,0 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
43、two o,p-Fe-EDDHA isomers (d/l-1 and d/l-2) for 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)
44、. Identify the different isomers by the retention 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. BS EN 15452:2008EN 15452:2008 (E) 8 9 Express
45、ion of results The mass fraction, w, in 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 concent
46、ration of Fe chelated by the second set 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 diffe
47、rent 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 obtained with the same method on identical test m
48、aterial, in the same laboratory, by the same operator, using the same equipment within a shot interval of time, will in not more than 5 % of the cases exceed the values of r given in Table 2. 10.3 Reproducibility The absolute difference between two single test results obtained with the same method o
49、n identical test material, in different laboratories, by different operators, using different equipment, will in not more than 5 % of the cases exceed the values of R given in Table 2. BS EN 15452:2008EN 15452:2008 (E) 9 Table 2 Mean values, repeatability and reproducibility limits Sample o,p-Fe % p n r R Sample 1 2,17 10 2 0,037 0,25 Sample 2 1,33 10 2 0,051 0,29 Sample 3 0,47 9 2 0,047 0,16 Sample 4 0,15 8 2 0,0070 0,25 o,p-Fe: overall mean concentration in percent of Fe chelated by o,p-EDDHA p: number of parti
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