SIST EN 13368-2:2018
(Main)Fertilizers - Determination of chelating agents in fertilizers by chromatography - Part 2: Determination of Fe chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED, or the amount of chelating agents, by ion pair chromatography
Fertilizers - Determination of chelating agents in fertilizers by chromatography - Part 2: Determination of Fe chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED, or the amount of chelating agents, by ion pair chromatography
This European Standard specifies a method for the chromatographic determination of the iron chelated by each individual ortho(hydroxy)-ortho(hydroxy) isomer of the chelating agents [o,o] EDDHA, [o,o] EDDHMA and by HBED in fertilizers containing one or more of these substances, except for [o,o] EDDHMA and HBED mixes. The method allows the identification and the determination of the total concentration of water soluble iron chelates of these chelating agents. Also, after derivatization with Fe, the soluble amount of the chelating agents may be determined when other micro-nutrients, beside Fe are present in fertilizers containing [o,o] EDDHA, [o,o] EDDHMA or HBED.
This method is applicable to EC fertilizers covered by Regulation (EC) No 2003/2003 [4]. It is applicable to a mass fraction of the metal chelated of at least 0,625 %.
NOTE 1 The substances EDDHA (ethylenediamine-N,N'-di[(hydroxyphenyl)acetic acid] and EDDHMA (ethylenediamine-N,N'-di[(hydroxymethylphenyl)acetic acid] exist as several different isomeric forms. Positional isomers for the hydroxyl or methyl groups (in ortho, meta, and para positions) as well as stereo isomers (meso and dl-racemic forms) are known. Both meso and dl-racemic forms of the [ortho,ortho] EDDHA and [ortho,ortho] EDDHMA are positional isomers for the hydroxyl groups allowed by the Regulation (EC) No 2003/2003. Since para, meta and ortho methyl positional isomers of the EDDHMA present quite similar stability, they could be grouped: in the method here described the para, meta and ortho methyl positional isomers of the [o,o] EDDHMA are considered together. HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid) does not present isomeric forms.
NOTE 2 At present, analytically pure standards only exist for [ortho,ortho] EDDHA, [ortho,ortho] EDDHMA and HBED. All other substances being unavailable as a standard, the influence of their eventual presence in the samples (with respect to the sensitivity and the selectivity of this method) has not been studied.
NOTE 3 The meso and the dl-racemic forms of [o,o] EDDHA and [o,o] EDDHMA can be determined separately by this method.
Düngemittel - Bestimmung von Chelatbildnern in Düngemitteln mit Chromatographie - Teil 2: Bestimmung von Fe chelatisiertem [o,o] EDDHA, [o,o] EDDHMA und HBED, oder der Menge der Chelatbildner, mit Ionen-Paarchromatographie
Diese Europäische Norm legt ein Verfahren zur chromatographischen Bestimmung von Eisen, das mit jedem individuellen ortho(hydroxy)-ortho(hydroxy)-Isomer der Chelatbildner [o,o] EDDHA, [o,o] EDDHMA und mit HBED Chelate bildet, in Düngemitteln fest, die eine oder mehrere dieser Substanzen enthalten, mit Ausnahme von [o,o] EDDHMA und HBED-Gemischen. Das Verfahren erlaubt die Identifizierung und die Bestimmung der Gesamtkonzentration von wasserlöslichen Eisenchelaten dieser Chelatbildner. Nach der Derivatisierung mit Fe ist auch die Bestimmung des löslichen Anteils der Chelatbildner möglich, wenn andere Spurennährstoffe außer Fe in Düngemitteln vorhanden sind, die [o,o] EDDHA, [o,o] EDDHMA oder HBED enthalten.
Dieses Verfahren ist anwendbar für EG-Düngemittel, die der Verordnung (EG) Nr. 2003/2003 [4] unterliegen. Es gilt für einen Massenanteil an chelatisiertem Metall von mindestens 0,625 %.
ANMERKUNG 1 Die Substanzen EDDHA (Ethylendiamin-N,N'-di[(hydroxyphenyl)essigsäure)] und EDDHMA (Ethylendiamin-N,N'-di[(hydroxy-methylphenyl)essigsäure)] existieren in mehreren unterschiedlichen isomeren Formen. Es sind sowohl Stellungsisomere (in Ortho-, Meta- und Para-Stellungen) als auch Stereoisomere (Meso- und dl-Racemformen) der Hydroxyl- oder Methylgruppen bekannt. Die Meso- und dl-Racemformen von ortho,ortho-EDDHA und ortho,ortho-EDDHMA sind Stellungsisomere der Hydroxylgruppen, die nach der Verordnung (EG) Nr. 2003/2003 zugelassen sind. Da Para-, Meta- und Orthomethyl-Stellungsisomere von EDDHMA eine sehr ähnliche Stabilität aufweisen, könnten sie in einer Gruppe zusammengefasst werden: in dem hier beschriebenen Verfahren werden die Para-, Meta- und Orthomethyl-Stellungsisomere von [o,o] EDDHMA zusammen betrachtet. HBED (N,N'-Bis(2-hydroxybenzyl)ethylendiamin-N,N'-diessigsäure) bildet keine isomeren Formen aus.
ANMERKUNG 2 Gegenwärtig existieren analysenreine Standards nur für ortho,ortho-EDDHA, ortho,ortho-EDDHMA und HBED. Da alle anderen Substanzen als Standards nicht zur Verfügung stehen, ist deren Einfluss bei eventuellem Vorkommen in den Proben (in Bezug auf die Empfindlichkeit und die Trennschärfe dieses Verfahrens) noch nicht untersucht worden.
ANMERKUNG 3 Mit diesem Verfahren können die Meso- und dl-Racemformen von [o,o] EDDHA und [o,o] EDDHMA einzeln bestimmt werden.
Engrais - Détermination des agents chélatants dans les engrais par chromatographie - Partie 2: Détermination du fer chélaté [o,o] EDDHA, [o,o] EDDHMA et HBED, ou de la quantité d'agents chélatants, par chromatographie d'appariement d'ions
La présente norme européenne spécifie une méthode de détermination par chromatographie du fer chélaté par chacun des isomères ortho(hydroxy)-ortho(hydroxy) des agents chélatants [o,o] EDDHA et [o,o] EDDHMA et par l'HBED dans les engrais contenant l'une ou plusieurs de ces substances, à l'exception des mélanges de [o,o] EDDHMA et d'HBED. La méthode permet l'identification et la détermination de la concentration totale des chélates de fer solubles dans l’eau de ces agents chélatants. Par ailleurs, après dérivatisation avec le fer, la quantité d’agents chélatants soluble peut être déterminée, quand d’autres oligo-éléments que le fer sont présents, dans les engrais contenant [o,o] EDDHA, [o,o] EDDHMA ou HBED.
La méthode décrite concerne les engrais européens couverts par le Règlement (CE) n°2003/2003 [4]. Il est applicable à une teneur en métal chélaté d'au moins 0,625 %.
NOTE 1 Les substances EDDHA (acide éthylènediamine-N,N'-di[(hydroxyphényl)acétique] et EDDHMA (acide éthylènediamine-N,N'-di(hydroxy-méthylphényl)acétique] existent sous plusieurs formes isomères différentes. Des isomères de position des groupes hydroxyl et méthyl (en position ortho, méta et para) sont connus, aussi bien que des stéréo-isomères (formes méso et dl-racémiques). Les deux formes méso et dl-racémiques de [ortho,ortho] EDDHA et de [ortho,ortho] EDDHMA sont des isomères de position des groupes hydroxyl autorisés par la réglementation (CE) n° 2003/2003. Dans la mesure où les isomères de position ortho, méta et para des groupes méthyl de l'EDDHMA présentent une stabilité tout à fait similaire, ils ont pu être regroupés. Dans la méthode décrite ici, les isomères de position ortho, méta et para des groupes méthyl de l'[o,o]-EDDHMA sont donc considérés conjointement. L'HBED (acide N,N´-bis(2-hydroxybenzyl)-éthylènediamine-N,N´-diacétique) ne présente pas de formes isomères.
NOTE 2 À l'heure actuelle, il n'existe de produit de référence pur pour analyse que pour [ortho,ortho] EDDHA, [ortho,ortho] EDDHMA et l'HBED. Aucune autre substance n'étant disponible en tant que produit de référence, l'influence de leur présence éventuelle dans les échantillons n'a fait l'objet d'aucune étude (du point de vue de la sensibilité et de la sélectivité de la méthode).
NOTE 3 Les formes méso et dl-racémiques de [o,o] EDDHA et [o,o] EDDHMA peuvent être déterminées séparément par la présente méthode.
Gnojila - Določevanje sredstev za kelatiziranje v gnojilih s kromatografijo - 2. del: Določevanje železovih kelatov z [o,o] EDDHA, [o,o] EDDHMA in HBED ali količine sredstev za kelatiziranje s kromatografijo ionskih parov
Ta evropski standard določa metodo za kromatografsko določevanje železovih kelatov z vsakim posameznim orto(hidroksi)-orto(hidroksi) izomerom kelatov [o,o] EDDHA, [o,o] EDDHMA in HBED v gnojilih, ki vsebujejo eno ali več teh snovi, razen mešanic [o,o] EDDHMA in HBED. Metoda omogoča opredelitev in določevanje skupne koncentracije v vodi topnih železovih kelatov teh kelatov. Po derivatizaciji z železom se lahko količina topnih kelatov določi tudi, ko so v gnojilih, ki vsebujejo kelate [o,o] EDDHA, [o,o] EDDHMA ali HBED, prisotna druga mikrohranila razen železa.
Ta metoda se uporablja za gnojila ES, navedena v Uredbi (ES) št. 2003/2003 [4]. Uporablja se za masni delež kovinskih kelatov najmanj 0,625 %.
OPOMBA 1: Snovi EDDHA (etilendiamin-N,N'-di[(hidroksifenil)ocetna kislina] in EDDHMA (etilendiamin-N,N'-di[(hidroksimetilfenil)ocetna kislina] obstajajo v različnih izomernih oblikah. Znani so pozicijski izomeri hidroksilnih ali metilnih skupin (na pozicijah orto, meta in para) in stereo izomeri (oblike mezo in dl-racemične oblike). Oblike mezo ter dl-racemične oblike [orto,orto] EDDHA in [orto,orto] EDDHMA so pozicijski izomeri hidroksilnih skupin, ki jih dovoljuje Uredba (ES) št. 2003/2003. Ker je stabilnost para-, meta- in ortometil pozicijskih izomerov EDDHMA podobna, jih je mogoče razvrstiti v isto skupino: v metodi, opisani v tem dokumentu, so para-, meta- in ortometil pozicijski izomeri [o,o] EDDHMA obravnavani skupaj. HBED (N,N'-bis(2-hidroksibenzil)-etilendiamin-N,N'-diocetna kislina) nima izomernih oblik.
OPOMBA 2: Analitično neoporečni standardi trenutno obstajajo samo za [orto,orto] EDDHA, [orto,orto] EDDHMA in HBED. Ker druge snovi niso standardizirane, vpliv njihove možne prisotnosti v vzorcih (glede na občutljivost in primernost te metode) ni bil preučen.
OPOMBA 3: Oblike mezo in dl-racemične oblike [o,o] EDDHA in [o,o] EDDHMA je s to metodo mogoče določiti ločeno.
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Düngemittel - Bestimmung von Chelatbildnern in Düngemitteln mit Chromatographie - Teil 2: Bestimmung von Fe chelatisiertem [o,o] EDDHA, [o,o] EDDHMA und HBED, oder der Menge der Chelatbildner, mit Ionen-PaarchromatographieEngrais - Détermination des agents chélatants dans les engrais par chromatographie - Partie 2: Détermination du fer chélaté [o,o] EDDHA, [o,o] EDDHMA et HBED, ou de la quantité d'agents chélatants, par chromatographie d'appariement d'ionsFertilizers - Determination of chelating agents in fertilizers by chromatography - Part 2: Determination of Fe chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED, or the amount of chelating agents, by ion pair chromatography71.040.50Fizikalnokemijske analitske metodePhysicochemical methods of analysis65.080GnojilaFertilizersICS:Ta slovenski standard je istoveten z:EN 13368-2:2017SIST EN 13368-2:2018en,fr,de01-januar-2018SIST EN 13368-2:2018SLOVENSKI
STANDARDSIST EN 13368-2:20131DGRPHãþD
SIST EN 13368-2:2018
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 13368-2
November
t r s y ICS
x wä r z r Supersedes EN
s u u x zæ tã t r s tEnglish Version
Fertilizers æ Determination of chelating agents in fertilizers by chromatography æ Part
tã Determination of Fe chelated of chelating agentsá by ion pair chromatography Engrais æ Détermination des agents chélatants dans les engrais par chromatographie æ Partie
t ã Détermination de la quantité d 5agents chélatantsá par chromatographie d 5appariement d 5ions
Düngemittel æ Bestimmung von Chelatbildnern in Düngemitteln mit Chromatographie æ Teil
tã EDDHMA und HBEDá oder der Summe der Chelatbildnerá mit IonenæPaarchromatographie This European Standard was approved by CEN on
s s September
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
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CEN-CENELEC Management Centre:
Avenue Marnix 17,
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9
t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s u u x zæ tã t r s y ESIST EN 13368-2:2018
EN 13368-2:2017 (E) 2 Contents Page European foreword . 4 1 Scope . 6 2 Normative references . 6 3 Terms and definitions . 6 4 Principle . 7 5 Interferences . 7 6 Reagents . 7 7 Apparatus . 9 8 Sampling and sample preparation . 9 9 Procedure. 10 9.1 Preparation of the sample solution for iron chelates . 10 9.2 Preparation of the sample solution for other micronutrient chelates . 10 9.3 Preparation of the calibration solutions . 10 9.4 Chromatographic analysis . 11 10 Expression of the results . 14 10.1 Fe in Fe-chelates . 14 10.1.1 Fe chelated by [o,o] EDDHA or [o,o] EDDHMA in Fe-[o,o] EDDHA or Fe-[o,o] EDDHMA chelates . 14 10.1.2 Fe chelated by HBED in Fe-HBED chelates . 15 10.2 Chelating agent in fertilizers . 15 10.2.1 Mass fraction of the chelating agent in fertilizers containing [o,o] EDDHA or [o,o] EDDHMA . 15 10.2.2 Mass fraction of the chelating agent in fertilizers containing HBED . 16 11 Precision . 16 11.1 Inter-laboratory test . 16 11.2 Repeatability . 16 11.3 Reproducibility . 16 12 Test report . 18 Annex A (informative)
Statistical results of the inter-laboratory tests . 19 A.1 Statistical results of the inter-laboratory test performed in 2003 . 19 A.1.1 Test samples . 19 A.1.2 Inter-laboratory test procedure . 19 A.1.3 Results and statistical interpretation . 19 A.2 Statistical results of the inter-laboratory test performed in 2010 . 21 A.2.1 Test samples . 21 A.2.2 Inter-laboratory test procedure . 21 A.2.3 Results and statistical interpretation . 21 SIST EN 13368-2:2018
EN 13368-2:2017 (E) 3 A.3 Statistical results of the inter-laboratory test performed in 2014 — Part A . 22 A.3.1 Objective . 22 A.3.2 Test samples . 22 A.3.3 Inter-laboratory test procedure . 22 A.3.4 Results and statistical interpretation . 22 A.4 Statistical results of the inter-laboratory test performed in 2014 — Part B . 24 A.4.1 Objective . 24 A.4.2 Test samples . 24 A.4.3 Inter-laboratory test procedure . 25 A.4.4 Results and statistical interpretation . 25 Annex B (informative)
General procedure for the determination of the titrimetric purity of the chelating agents using a photometric automatic titrator . 26 Annex C (informative)
Complete names of chelating agents . 27 Bibliography . 28 SIST EN 13368-2:2018
EN 13368-2:2017 (E) 4 European foreword This document (EN 13368-2:2017) 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 a national standard, either by publication of an identical text or by endorsement, at the latest by May 2018 and conflicting national standards shall be withdrawn at the latest by May 2018. 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 EN 13368-2:2012. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. In comparison with EN 13368-2:2012, the following changes have been made: a) determination of the chelating agent added to the scope and to the title; b) a derivatization method for the determination of the chelating agent explained in Clause 4, Principle; c) a new option added for the preparation of the Fe-[o,o] EDDHA solution, starting from a Fe-[o,o] EDDHA standard, in 6.6; d) calculation of the mass fraction of the chelating agents included in Clause 10 including Formulae (3) and (4); e) Table 2 enlarged by the precision data concerning the 2014 inter-laboratory test; f) information on the type of standard used for Fe-[o,o] EDDHA samples and the possibility to report on the chelating agent contents included in Clause 12; g) results of the inter-laboratory test performed in 2014, part A and B respectively, added (A.3 and A.4); h) complete names of chelating agents in Annex C technically revised; i) editorially revised. EN 13368, Fertilizers — Determination of chelating agents in fertilizers by chromatography consists of the following parts: — Part 1: Determination of EDTA, HEEDTA and DTPA by ion chromatography — Part 2: Determination of Fe chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED, or the amount of chelating agents, by ion pair chromatography — Part 3: Determination of [S,S]-EDDS by ion pair chromatography SIST EN 13368-2:2018
EN 13368-2:2017 (E) 5 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, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 6 1 Scope This European Standard specifies a method for the chromatographic determination of the iron chelated by each individual ortho(hydroxy)-ortho(hydroxy) isomer of the chelating agents [o,o] EDDHA, [o,o] EDDHMA and by HBED in fertilizers containing one or more of these substances, except for [o,o] EDDHMA and HBED mixes. The method allows the identification and the determination of the total concentration of water soluble iron chelates of these chelating agents. Also, after derivatization with Fe, the soluble amount of the chelating agents can be determined when other micro-nutrients, beside Fe are present in fertilizers containing [o,o] EDDHA, [o,o] EDDHMA or HBED. This method is applicable to EC fertilizers covered by Regulation (EC) No 2003/2003 [4]. It is applicable to a mass fraction of the metal chelated of at least 0,625 %. NOTE 1 The substances EDDHA (ethylenediamine-N,N'-di[(hydroxyphenyl)acetic acid] and EDDHMA (ethylenediamine-N,N'-di[(hydroxymethylphenyl)acetic acid] exist as several different isomeric forms. Positional isomers for the hydroxyl or methyl groups (in ortho, meta, and para positions) as well as stereo isomers (meso and dl-racemic forms) are known. Both meso and dl-racemic forms of the [ortho,ortho] EDDHA and [ortho,ortho] EDDHMA are positional isomers for the hydroxyl groups allowed by the Regulation (EC) No 2003/2003. Since para, meta and ortho methyl positional isomers of the EDDHMA present quite similar stability, they could be grouped: in the method here described the para, meta and ortho methyl positional isomers of the [o,o] EDDHMA are considered together. HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid) does not present isomeric forms. NOTE 2 At present, analytically pure standards only exist for [ortho,ortho] EDDHA, [ortho,ortho] EDDHMA and HBED. All other substances being unavailable as a standard, the influence of their eventual presence in the samples (with respect to the sensitivity and the selectivity of this method) has not been studied. NOTE 3 The meso and the dl-racemic forms of [o,o] EDDHA and [o,o] EDDHMA can be determined separately by this method. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. 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: 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, Water for analytical laboratory use - Specification and test methods (ISO 3696) 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. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 7 4 Principle The iron chelates are separated and determined by isocratic ion-pair high-performance liquid chromatography. When an iron chelate (anion) is added to a polar fluid (eluent), containing a large cation, an ion pair is formed. This ion pair is retained by a non-polar solid phase (stationary phase). The strength of the retention depends on the molecular size and its acidity. Then, each iron chelate presents a characteristic retention time 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 silica column and an aqueous solution of TBA+ (tetrabutylammonium) and acetonitrile as eluent. The detection is based on photometry at 280 nm. For metal chelates different from Fe, a derivatization method may be used to form the Fe chelates, and then the chelating agent can be determined by the isocratic ion-pair high-performance liquid chromatography here presented. NOTE For additional information, see [5], [6] and [7]. 5 Interferences No interferences have been detected. Iron chelates with HBEP, EDDHSA, EDTA, DTPA, CDTA, HEEDTA, [p,p] EDDHA, [o,p] EDDHA, IDHA as well as the chelating agents do not interfere since they are separated from Fe-[o,o] EDDHA, Fe-[o,o] EDDHMA or Fe-HBED. Fe-[o,o] EDDHA does not interfere with Fe-[o,o] EDDHMA or Fe-HBED. In the case that Fe-[o,o] EDDHMA and Fe-HBED are present in the same sample, an overlapping of the meso isomer of the Fe-[o,o] EDDHMA and Fe-HBED peaks may occur with some equipment depending on the column used. The use of a different column of the same type can solve this problem. NOTE For the complete names of the abbreviations of chelating agents used in this document, see Annex C. 6 Reagents 6.1 General. All reagents shall be of recognized analytical grade. Water used for the preparation of eluents, standards, and sample solutions shall conform to EN ISO 3696, grade 1 and shall be degassed and free of organic contaminants. If products 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 exactly the required concentration in the solution. If there is any doubt of the purity of the standard, it is necessary to determine it. NOTE For this determination, a titrimetric method can be used. See Annex B for a general method using an automatic titrator. Manual titration could also be adequate. 6.2 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of NaOH in pellet form in a 1 l volumetric flask with water (6.1). Dilute to the mark and homogenize. The incorporation of CO2 from the atmosphere should be carefully avoided. Otherwise, the dissolution of chelating agents (see 6.6, 6.7 and 6.8) can be incomplete. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 8 6.3 Hydrochloric acid solution, c(HCl) = 1,0 mol/l. Dilute 88 ml of hydrochloric acid (mass fraction 35 % HCl) to 1 000 ml with water. 6.4 Hydrochloric acid solution, c(HCl) = 0,1 mol/l. Dilute 50 ml of hydrochloric acid 1,0 mol/l (6.3) to 500 ml with water. 6.5 Iron-nitrate solution, (Fe) = 1 050 mg/l. Dissolve 0,759 4 g of ferric nitrate 9-hydrate (Fe(NO3)3·9H2O) in 100 ml of water. Check (for example by AAS) that the Fe concentration in this solution amounts 1 050 mg/l ± 30 mg/l. NOTE As the Fe(NO3)3·9H2O is deliquescent it will be added in solution of a known concentration. 6.6 Fe-[o,o] EDDHA solution, (Fe) = 100 mg/l. Depending on the availability, either 6.6.1 or 6.6.2 procedures can be used to prepare the standard stock solution. 6.6.1 Preparation from Fe-[o,o] EDDHA. Dissolve 5/P g (where P is the purity of the solid standard in percentage of Fe chelated), in 50 ml of water (6.1) in a 100 ml beaker and make up to 500 ml in a volumetric flask with water. The standard obtained in this way may be stored in darkness for one year. 6.6.2 Preparation from [o,o] EDDHA. Dissolve 0,322 1 g (see 6.1) of ethylenediamine-NáN"-di[(ortho-hydroxyphenyl)acetic acid] in 350 ml of water (6.1) and 27 ml of NaOH (6.2) in a 500 ml beaker. Add 50 ml of the Fe solution (6.5) to the chelating agent solution, stirring for about 5 min. Adjust the solution to pH 7,0 with NaOH solution (6.2). 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 500 ml in a volumetric flask with water (6.1). The standard obtained in this way may be stored in darkness for one year. 6.7 Fe-[o,o] EDDHMA solution, (Fe) = 100 mg/l. Dissolve 0,347 1 g (see 6.1) of ethylenediamine-N,N’-di[(ortho-hydroxy-para-methylphenyl)acetic acid] [the paramethyl isomer of [o,o] EDDHMA, (see Note 1 in Clause 1)] in 350 ml of water (6.1) and 27 ml of NaOH (6.2) in a 500 ml beaker. Add 50 ml of the Fe solution (6.5) to the chelating agent solution, stirring for about 5 min. Adjust the solution to pH 7,0 with NaOH solution (6.2). 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 500 ml in a volumetric flask with water (6.1). The standard obtained in this way may be stored in darkness for one year. 6.8 Fe-HBED solution, (Fe) = 100 mg/l. Dissolve 0,347 1 g (see 6.1) of N,N’-bis(2-hydroxybenzyl)-ethylenediamine-N,N’-diacetic acid (HBED) or 0,379 7 g if HBED·HCI is used in 350 ml of water (6.1) and 27 ml of NaOH (6.2) in a 500 ml beaker. Add 50 ml of the Fe solution (6.5) to the chelating agent solution, stirring for about 5 min. Adjust the solution to pH 5,0 with HCI solution (6.4) or NaOH solution (6.2). 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 500 ml in a volumetric flask with water (6.1). The standard obtained in this way may be stored in darkness for one year. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 9 6.9 Eluent for the determination. Add 20 ml of TBAOH (mass fraction 40 % Tetrabutylammonium hydroxide solution in water) to 600 ml of water (6.1). Adjust to pH 6,0 with hydrochloric acid solution (6.3 and 6.4). Add 300 ml of acetonitrile (HPLC grade) and make up to volume in a 1 l volumetric flask with water. Filter through a 0,2 µm membrane filter (7.4 b) and degas. TBACl or TBABr may be used, providing that pH is adjusted to 6,0 with NaOH or HCl. 7 Apparatus Usual laboratory equipment, glassware and the following: 7.1 Magnetic stirrer. 7.2 Chromatograph, equipped with: a) an isocratic pump delivering the eluent at a flow rate of 1,5 ml/min; b) an injection valve with a 20 µl injection loop; c) a C-18 column; internal diameter:3,9 mm; column length: 150 mm; dp = 5 µm1); d) a C-18 guard column (recommended); e) a UV/VIS-detector with a 280 nm-filter; f) an integrator. 7.3 Balance, with an accuracy of ± 0,1 mg. 7.4 Membrane filters, including: a) micro membrane filters resistant to aqueous solutions, with porosity of 0,45 µm; b) micro membrane filters resistant to organic solutions (e.g. polyamide 66 micro membrane filters), with porosity of 0,2 µm. 8 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 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 grind the sample to a particle size less than 1 mm.
1) SYMMETRYTM C18, from WATERS, LiChroCART® Purospher® RP-18, from MERCK or equivalent are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 10 9 Procedure 9.1 Preparation of the sample solution for iron chelates Weigh, to the nearest 0,1 mg, 200 mg of the iron chelate into a 250 ml beaker. Add 200 ml of water (6.1). Stir using a magnetic stirrer for 1 h. Transfer quantitatively into a 250 ml volumetric flask. Dilute to the mark with water and homogenize. For samples declaring more than 5 % of chelated Fe, use a 500 ml volumetric flask. For liquid samples, weigh an amount equivalent to 200 mg of solid. 9.2 Preparation of the sample solution for other micronutrient chelates Weigh, to the nearest 0,1 mg, 1 g of the chelate into a 250 ml beaker. Add 200 ml of water (6.1). Stir using a magnetic stirrer for 1 h. Transfer quantitatively into a 250 ml volumetric flask. Dilute to the mark with water (6.1) and homogenize. Filter through a paper filter. Pipette 20 ml of the solution into a 50 ml beaker. Add 10 ml of the iron(III) solution (6.5), homogenize, and allow to stand for 15 min. Adjust the pH to 9,0 ± 0,1 with sodium hydroxide solution (6.2) and allow to stand for another 15 min. Transfer quantitatively into a 100 ml volumetric flask. Dilute to the mark with water (6.1) and homogenize. For samples declaring more than 5 % of chelated metals, use in the first step a 500 ml volumetric flask. For liquid samples, weigh an amount equivalent to 200 mg of solid. 9.3 Preparation of the calibration solutions Pipette V ml (see Table 1) of the Fe-[o,o] EDDHA (6.6) or Fe-[o,o] EDDHMA (6.7) or Fe-HBED (6.8) standard solution into six 100 ml volumetric flasks. Make up to volume with water (6.1) and homogenize. Table 1 — Composition of the calibration solutions Solution V ml Fe concentration in the rac chelate mg/l Fe concentration in the meso chelate mg/l Total Fe concentration in chelate mg/l 1 5 2,5 2,5 5,0 2 10 5,0 5,0 10,0 3 20 10,0 10,0 20,0 4 30 15,0 15,0 30,0 5 40 20,0 20,0 40,0 6 50 25,0 25,0 50,0 NOTE 1 As the standard chelates solutions should be 50 % meso- and 50 % dl-racemic optical isomers, the concentration of the total iron chelate is the sum of the concentrations given in Table 1. NOTE 2 Since HBED does not present optical isomers the total concentrations can be used. SIST EN 13368-2:2018
EN 13368-2:2017 (E) 11 9.4 Chromatographic analysis Immediately before injection, all solutions shall be filtered through a 0,45 µm membrane filter [7.4 a)]. Inject the calibration solutions (see 9.3) into the chromatographic system (7.2). Measure the retention times and the areas of the two Fe-[o,o] EDDHA or Fe-[o,o] EDDHMA isomers (meso and dl-racemic) or Fe-HBED peaks for all solutions. For each appropriate iron chelate, draw two calibration graphs with the values of the peak areas of the calibration solutions versus the iron concentration (mg/l) in the form of the corresponding chelating agent isomer. For Fe-HBED, only one graph is needed. See Figures 1 to 4. NOTE See NOTE 1 in 9.3. A statistically significant negative intercept can be caused by the contamination of the column with Fe oxide precipitates. In that case, the column should be replaced or cleaned. Inject the sample solution (see 9.1 or 9.2). Identify the chelating agent by the retention time of the obtained peaks (see Figures 2, 3 and 4). Measure the areas of the isomer peaks for each chelating agent. Determine the concentration of the iron chelated by each optical isomer (mg Fe/l) using the corresponding calibration graph. In Figure 3 the typical chromatogram of a commercial product containing Fe-[o,o] EDDHMA is shown. Besides meso and dl-racemic isomers of para-methyl ortho-hydroxyl Fe-[o,o] EDDHMA two other isomers, assigned to other methyl positional isomers, are found (peak 3 and peak 4). Since they have similar stability to the para-methyl isomers (see NOTE 1 in Clause 1), their area should be added to the nearest (1 or 2) peak area.
Key 1 dl-racemic Fe-[o,o] EDDHA 2 meso Fe-[o,o] EDDHA X time in minutes Figure 1 — Typical chromatogram of a Fe-[o,o] EDDHA standard solution with an Fe content of 25 mg/l SIST EN 13368-2:2018
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Key 1 dl-racemic Fe-[o,o] EDDHA 4 para-hydroxy para-hydroxy isomer by-product 2 meso Fe-[o,o] EDDHA X time in minutes 3 ortho-hydroxy para-hydroxy isomer
Figure 2 — Typical chromatogram of a commercial Fe-[o,o] EDDHA product SIST EN 13368-2:2018
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Key 1 dl-racemic Fe-[o,o] EDDHMA 3 and 4 methyl positional isomers of Fe-[o,o] EDDHMA 2 meso Fe-[o,o] EDDHMA X time in minutes Figure 3 — Typical chromatogram of a commercial Fe-[o,o] EDDHMA product
Key X time in minutes Figure 4 — Typical chromatogram of a commercial Fe-HBED product If overlapping occurs, baseline correction in the integration should be performed (see Figure 5). SIST EN 13368-2:2018
EN 13368-2:2017 (E) 14
Key X time in minutes Figure 5 — Example of baseline correction 10 Expression of the results 10.1 Fe in Fe-chelates 10.1.1 Fe chelated by [o,o] EDDHA or [o,o] EDDHMA in Fe-[o,o] EDDHA or Fe-[o,o] EDDHMA chelates Calculate the mass fraction, wFe, in percent of the sample of Fe chelated by [o,o] EDDHA or [o,o] EDDHMA in the fertilizer according to Formula (1). -4racmFe+=××10ccwVm (1) where crac and cm are the Fe concentrations of the Fe chelated by the racemic or meso isomers respectively determined with the calibration graphs, in milligrams per litre; m is the mass of the test portion, in grams; V is the volume of the sample solution (9.1), in millilitres. If the sample contains both Fe-[o,o] EDDHA and Fe-[o,o] EDDHMA, the Fe chelated is the sum
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