1、BRITISH STANDARDBS EN 15451:2008Fertilizers Determination of chelating agents Determination of iron chelated by EDDHSA by ion pair chromatographyICS 65.080g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g5
2、5g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 15451:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 August 2008 BSI 2008ISBN 978 0 580 60411 9National forewordThis British Standard is the UK implementation of EN 15451:20
3、08. It supersedes DD CEN/TS 15451:2006 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee CII/37, Fertilisers and related chemicals.A list of organizations represented on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 NOR
5、MEN 15451July 2008ICS 65.080 Supersedes CEN/TS 15451:2006 English 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 dappariementdionsDnge
6、mittel - Bestimmung von Chelatbildnern -Bestimmung von Eisen-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 EuropeanS
7、tandard 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 official versions (English, Fren
8、ch, 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, Belgium, Bulgaria, Cyprus, Cz
9、ech 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 STANDARDIZATIONCOMIT EUROPE
10、N 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 15451:2008: EEN 15451:2008 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normativ
11、e references 4 3 Principle4 4 Interferences 4 5 Reagents.4 6 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
12、Fe3+/EDDHSA 11 Annex C (informative) Results of the inter-laboratory tests .12 Annex D (informative) Complete names of chelating agents .14 Bibliography 15 BS EN 15451:2008EN 15451:2008 (E) 3 Foreword This document (EN 15451:2008) has been prepared by Technical Committee CEN/TC 260 “Fertilizers and
13、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 January 2009, and conflicting national standards shall be withdrawn at the latest by Jan
14、uary 2009. 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 supersedes CEN/TS 15451:2006. This document has been prepared
15、 under a mandate given to CEN 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, Cyprus, Czec
16、h 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 the United Kingdom. BS EN 15451:2008EN 15451:2008 (E) 4 1 Scope T
17、his European Standard specifies a method for the chromatographic 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 editi
18、on 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: Sample preparation EN ISO 3696:1995, Water for analytical laboratory use Specification and t
19、est methods (ISO 3696:1987) 3 Principle The iron chelates 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 so
20、lid phase (stationary phase). The strength of the retention 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. T
21、he separation is carried out on a reverse phase silica 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 chela
22、tes with o,o-EDDHA, o,p-EDDHA, o,o-EDDHMA, EDTA, DTPA, 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,
23、and sample solutions should be water grade1 according 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 sol
24、ution, c(NaOH) = 0,5 mol/l Dissolve 20 g of NaOH in 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
25、carbon dioxide. Dilute to the mark and homogenize. BS EN 15451:2008EN 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
26、 500 ml with water. 5.6 Iron (III) nitrate solution, (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 thi
27、s solution is (1 050 30) mg/l. As the Fe(NO3)39H2O 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 per
28、centage of the acid form H6EDDHSA obtained by 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). A
29、fter complete dissolution, add 25 ml of the Fe 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 ce
30、llulose filter and make up to volume (250 ml) 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
31、.4 and 5.5). Add 350 ml of acetonitrile (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). 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
32、6,0 with HCl (5.4 and 5.5). Add 750 ml of acetonitrile (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 bisulf
33、ate should be avoided because peak retention times and shape may significantly change. 6 Apparatus 6.1 General Usual laboratory equipment, glassware, and: 6.2 Magnetic stirrer Magnetic stirrer with magnets. BS EN 15451:2008EN 15451:2008 (E) 6 6.3 Chromatograph Chromatograph equipped with: a) gradien
34、t pump delivering the eluent at a flow rate 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 tab
35、le Time min Flow rate ml/min A % B % Curve 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
36、organic solutions, (e.g.: nylon micromembrane 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 E
37、N 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. 1) SYMMETRYTMC18, from WATERS cat.n: WAT054205, or equivalent a
38、re 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 15451:2008EN 15451:2008 (E) 7 8 Procedure 8.1 Preparation of the sample solution of iron chelates We
39、igh approximately 125 mg within 0,1 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
40、 water and homogenize. 8.2 Preparation 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
41、Solution V ml Concentration of Fe in 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
42、 the chromatographic system (6.3). Measure 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 va
43、lues of the two chromatographic peaks 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 us
44、ed, confirm with its UV-visible spectrum (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 chromatograp
45、hic peaks in Fe3+/EDDHSA appear overlapped. 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 fer
46、tiliser, wFe, is equal to: BS EN 15451:2008EN 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-l
47、aboratory 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 reproducibility were calculated accordin
48、g to ISO 5725-2 2. The values derived from the inter-laboratory test in 2005 are presented as the repeatability and reproducibility data of the method presented in 10.2 and 10.3. These results may not be applicable to concentration ranges and matrices other than those given in Annex C. 10.2 Repeatab
49、ility The absolute difference between two independent 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. 10.3 Reproducibility The absolute difference between two single test results obtained with the same method on identical test material, in different laboratories, by different operators, using different equipment, will, in no
