Organo-mineral fertilizers - Identification of chelating agents - 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 document specifies a method for the determination by ion pair chromatography 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 organo-mineral fertilizers, having an organic matrix based on vegetal residues (cocoa shells, grape residue, soybean residue, …), algae extract, and animal meal (feather, bones, blood, ...) and 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 micronutrients beside Fe are present in organo-mineral fertilizers containing [o,o] EDDHA, [o,o] EDDHMA or HBED.
This method is applicable to a mass fraction of the metal chelated of at least 0,625 %.
NOTE 1   The substances EDDHA and EDDHMA 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]. 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.

Organisch-mineralische Düngemittel - Identifizierung von Chelatbildnern - Teil 2: Bestimmung von Fe chelatisiert mit [o,o] EDDHA, [o,o] EDDHMA und HBED oder der Menge der Chelatbildner mittels Ionenpaarchromatographie

Dieses Dokument legt ein Verfahren zur ionenpaarchromatographischen Bestimmung des von den einzelnen ortho(Hydroxy)-ortho(Hydroxy)-Isomeren der Chelatbildner [o,o] EDDHA und [o,o] EDDHMA sowie HBED chelatisierten Eisens in organisch-mineralischen Düngemitteln fest, die eine organische Matrix auf Basis von pflanzlichen Reststoffen (Kakaoschalen, Traubenreststoffe, Sojabohnenreststoffe usw.), Algenextrakt und Tiermehl (Federn, Knochen, Blut usw.) aufweisen und einen oder mehrere dieser Stoffe enthalten, außer [o,o] EDDHMA- sowie HBED-Mischungen.
Das Verfahren erlaubt die Identifizierung und die Bestimmung der Gesamtkonzentration an wasserlöslichen Eisenchelaten der genannten Chelatbildner. Nach der Derivatisierung mit Fe kann ferner auch der lösliche Gehalt an Chelatbildnern bestimmt werden, wenn in organisch-mineralischen, [o,o] EDDHA, [o,o] EDDHMA oder HBED enthaltenden Düngemitteln andere Spurennährstoffe außer Fe präsent sind.
Dieses Verfahren ist bei einem Massenanteil des chelatisierten Metalls von mindestens 0,625 % anwendbar.
ANMERKUNG 1   Die Stoffe EDDHA und EDDHMA existieren in unterschiedlichen isomeren Formen. Es sind Stellungsisomere der Hydroxyl- oder Methylgruppen (in den Stellungen ortho, meta und para) sowie Stereoisomere (meso- und dl-Racematform) bekannt. Sowohl meso- als auch dl-Racematform von [ortho,ortho] EDDHA und [ortho,ortho]. Da die para-, meta- und ortho-Methylstellungsisomere von EDDHMA eine recht ähnliche Stabilität aufweisen, könnten sie gruppiert werden: Im hier beschriebenen Verfahren werden die para-, meta- und ortho-Methylstellungsisomere [o,o] EDDHMA gemeinsam betrachtet. HBED (N,N’-di(2-hydroxybenzyl)ethylendiamin-N,N’-diessigsäure) hat keine isomeren Formen.
ANMERKUNG 2   Gegenwärtig gibt es analysereine Standards nur für [ortho,ortho] EDDHA, [ortho,ortho] EDDHMA und HBED. Da andere Stoffe nicht als Standard erhältlich sind, wurden die Auswirkungen ihres Vorhandenseins in den Proben auf die Sensitivität und die Selektivität des Verfahrens nicht untersucht.
ANMERKUNG 3   Die meso- und dl-Racematformen von [o,o] EDDHA und [o,o] EDDHMA können mit diesem Verfahren getrennt voneinander bestimmt werden.

Engrais organo-minéraux - Identification des agents chélatants - Partie 2 : Détermination du Fe chélaté par [o,o] EDDHA, [o,o] EDDHMA et HBED, ou de la quantité d'agents chélatants par chromatographie d'appariement d'ions

Le présent document spécifie une méthode pour la détermination par chromatographie d’appariement d’ions du fer chélaté par chacun des isomères individuels ortho(hydroxy)-ortho(hydroxy) des agents chélatants [o,o] EDDHA, [o,o] EDDHMA et HBED dans les engrais organo-minéraux ayant une matrice organique basée sur des résidus végétaux (coques de cacao, résidus de raisins, résidus de soja …), des extraits d’algue et des farines animales (plumes, os, sang ...) et contenant une ou plusieurs de ces substances, excepté pour les mélanges d’[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 solubles peut être déterminée quand d’autres oligo-éléments que le fer sont présents dans les engrais organo-minéraux contenant de l’[o,o] EDDHA, de l’[o,o] EDDHMA ou de l’HBED.
Cette méthode est applicable à une fraction massique de métal chélaté d’au moins 0,625 %.
NOTE 1   Les substances EDDHA et EDDHMA 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 d’[ortho,ortho] EDDHA et d’[ortho,ortho]. 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 produits de référence purs pour analyse que pour [ortho,ortho] EDDHA, [ortho,ortho] EDDHMA et 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 d’[o,o] EDDHA et d’[o,o] EDDHMA peuvent être déterminées séparément par la présente méthode.

Organsko-mineralna gnojila - Identifikacija sredstev za kelatiranje - 2. del: Določevanje železa, kelatiranega z [o,o] EDDHA, [o,o] EDDHMA in HBED ali količino sredstev za kelatiranje s kromatografijo ionskih parov

General Information

Status
Published
Public Enquiry End Date
02-Jan-2022
Publication Date
09-Jan-2023
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Jan-2023
Due Date
13-Mar-2023
Completion Date
10-Jan-2023

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17789-2:2023
01-februar-2023
Organsko-mineralna gnojila - Identifikacija sredstev za kelatiranje - 2. del:
Določevanje železa, kelatiranega z [o,o] EDDHA, [o,o] EDDHMA in HBED ali
količino sredstev za kelatiranje s kromatografijo ionskih parov

Organo-mineral fertilizers - Identification of chelating agents - 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

Organisch-mineralische Düngemittel - Identifizierung von Chelatbildnern - Teil 2:

Bestimmung von Fe chelatisiert mit [o,o] EDDHA, [o,o] EDDHMA und HBED oder der
Menge der Chelatbildner mittels Ionenpaarchromatographie

Engrais organo-minéraux - Identification des agents chélatants - Partie 2 : Détermination

du Fe chélaté par [o,o] EDDHA, [o,o] EDDHMA et HBED, ou de la quantité d'agents
chélatants par chromatographie d'appariement d'ions
Ta slovenski standard je istoveten z: CEN/TS 17789-2:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17789-2:2023 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17789-2:2023
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
April 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Organo-mineral fertilizers - Identification of chelating
agents - 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

Engrais organo-minéraux - Identification des agents Organisch-mineralische Düngemittel - Identifizierung

chélatants - Partie 2 : Détermination du Fe chélaté par von Chelatbildnern - Teil 2: Bestimmung von Fe

[o,o] EDDHA, [o,o] EDDHMA et HBED, ou de la quantité chelatisiert mit [o,o] EDDHA, [o,o] EDDHMA und HBED

d'agents chélatants par chromatographie oder der Menge der Chelatbildner mittels
d'appariement d'ions Ionenpaarchromatographie

This Technical Specification (CEN/TS) was approved by CEN on 21 February 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17789-2:2022 E

worldwide for CEN national Members.
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)
Contents Page

European foreword ............................................................................................................................................ 3

Introduction .......................................................................................................................................................... 4

1 Scope .......................................................................................................................................................... 5

2 Normative references .......................................................................................................................... 5

3 Terms and definitions ......................................................................................................................... 5

4 Principle ................................................................................................................................................... 6

5 Interferences .......................................................................................................................................... 6

6 Reagents ................................................................................................................................................... 6

7 Apparatus ................................................................................................................................................ 8

8 Sampling and sample preparation ................................................................................................. 8

9 Procedure ................................................................................................................................................ 8

9.1 Preparation of the sample solution for iron chelates .............................................................. 8

9.2 Preparation of the sample solution for other micronutrient chelates .............................. 9

9.3 Preparation of the calibration solutions ...................................................................................... 9

9.4 Chromatographic analysis ................................................................................................................. 9

10 Expression of results ......................................................................................................................... 13

10.1 Fe in Fe-chelates .................................................................................................................................. 13

10.2 Chelating agent in organo-mineral fertilizers .......................................................................... 14

11 Test report ............................................................................................................................................. 15

Annex A (informative) Complete names of chelating agents ............................................................. 16

Annex B (informative) General procedure for the determination of the titrimetric purity of

the chelating agents using a photometric automatic titrator ............................................. 17

Bibliography ....................................................................................................................................................... 18

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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)
European foreword

This document (CEN/TS 17789-2:2022) has been prepared by Technical Committee CEN/TC 260

“Fertilizers and liming materials”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document has been prepared under a Standardization Request given to CEN by the European

Commission and the European Free Trade Association.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United

Kingdom.
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)
Introduction

Micronutrients are considered to be, in plant nutrition, a number of elements known to be needed in

small amounts for proper plant growth and development. The most common are Iron (Fe), Manganese

(Mn), Molybdenum (Mo), Copper (Cu), Zinc (Zn) and Boron (B).

If an organo-mineral fertilizer contains a substance, or one of the substances in the mixture, which is

intended to enhance the long term availability to plants of micronutrients in the EU fertilizing product,

that substance is either a chelating agent or a complexing agent.
The chelating agents are divided into two groups :
— Group 1: EDTA, DTPA, HEEDTA, IDHA and [S,S]-EDDS;

— Group 2: Chelating agents present in UVCB (unknown or variable composition, complex reaction

products or biological materials) chelates including [o,o] EDDHA, [o,p] EDDHA, [o,o] EDDHMA, HBED

and EDDHSA.

This document defines the test method to be used in order to identify and determine the content of UVCB

micronutrient chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED in organo-mineral fertilizers (product

function category (PFC) 1(B) according to Regulation (EU) 2019/1009 [4]).
Abbreviated terms are described in Annex A.
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)
1 Scope

This document specifies a method for the determination by ion pair chromatography 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 organo-mineral fertilizers, having an organic matrix based on vegetal

residues (cocoa shells, grape residue, soybean residue, …), algae extract, and animal meal (feather, bones,

blood, ...) and 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 micronutrients beside Fe are present in organo-mineral

fertilizers containing [o,o] EDDHA, [o,o] EDDHMA or HBED.

This method is applicable to a mass fraction of the metal chelated of at least 0,625 %.

NOTE 1 The substances EDDHA and EDDHMA 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]. 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 are referred to in the text in such a way that some or all of their content

constitutes requirements 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 12944-1, Fertilizers and liming materials — Vocabulary — Part 1: General terms

EN 12944-2, Fertilizers and liming materials — Vocabulary — Part 2: Terms relating to fertilizers

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 12944-1 and EN 12944-2 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)
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.
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 can occur with

some equipment depending on the column used. The use of a different column of the same type can solve

this problem.
6 Reagents
All reagents shall be of recognized analytical grade.

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.
6.1 Water.

All water used should conform to EN ISO 3696, be degassed and be free of organic contaminants.

6.2 Sodium hydroxide solution, substance concentration 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 CO from the atmosphere should be carefully avoided. Otherwise, the dissolution of

chelating agents (see 6.6, 6.7 and 6.8) can be incomplete.
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 (6.1).

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 (6.1).
6.5 Iron-nitrate solution, mass concentration ρ(Fe) = 1 050 mg/l.
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SIST-TS CEN/TS 17789-2:2023
CEN/TS 17789-2:2022 (E)

Dissolve 0,759 4 g of ferric nitrate 9-hydrate (Fe(NO ) ·9H O) in 100 ml of water. Check (for example by

3 3 2
AAS) that the Fe concentration in this solution amounts 1 050 mg/l ± 30 mg/l.

NOTE As the Fe(NO ) ·9H O is deliquescent it will be added in solution of a known concentration.

3 3 2
6.6 Fe-[o,o] EDDHA solution, ρ(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/SP g (where SP 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 m
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17789-2:2021
01-december-2021
[Not translated]

Organo-mineral fertilizers - Identification of chelating agents - 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

Organisch-mineralische Düngemittel - Identifizierung von Chelatbildnern - Teil 2:

Bestimmung von Fe chelatisiert mit [o,o] EDDHA, [o,o] EDDHMA und HBED oder der
Menge der Chelatbildner mittels Ionenpaarchromatographie
Ta slovenski standard je istoveten z: FprCEN/TS 17789-2
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17789-2:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17789-2:2021
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kSIST-TS FprCEN/TS 17789-2:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17789-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
October 2021
ICS 65.080
English Version
Organo-mineral fertilizers - Identification of chelating
agents - 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
Organisch-mineralische Düngemittel - Identifizierung
von Chelatbildnern - Teil 2: Bestimmung von Fe
chelatisiert mit [o,o] EDDHA, [o,o] EDDHMA und HBED
oder der Menge der Chelatbildner mittels
Ionenpaarchromatographie

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 260.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17789-2:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17789-2:2021
FprCEN/TS 17789-2:2021 (E)
Contents Page

European foreword ...................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 5

5 Interferences .................................................................................................................................................... 6

6 Reagents ............................................................................................................................................................. 6

7 Apparatus .......................................................................................................................................................... 7

8 Sampling and sample preparation ........................................................................................................... 8

9 Procedure .......................................................................................................................................................... 8

9.1 Preparation of the sample solution for iron chelates ........................................................................ 8

9.2 Preparation of the sample solution for other micronutrient chelates ........................................ 8

9.3 Preparation of the calibration solutions ................................................................................................ 9

9.4 Chromatographic analysis ........................................................................................................................... 9

10 Expression of results .................................................................................................................................. 12

10.1 Fe in Fe-chelates ........................................................................................................................................... 12

10.2 Chelating agent in organo-mineral fertilizers ................................................................................... 13

11 Test report ...................................................................................................................................................... 14

Annex A (informative) Complete names of chelating agents .................................................................... 15

Annex B (informative) General procedure for the determination of the titrimetric purity of the

chelating agents using a photometric automatic titrator ............................................................. 16

Bibliography ................................................................................................................................................................ 17

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European foreword

This document (FprCEN/TS 17789-2:2021) has been prepared by Technical Committee CEN/TC 260

“Fertilizers and liming materials”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.

This document has been prepared under a Standardization Request given to CEN by the European

Commission and the European Free Trade Association.
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Introduction

Micronutrients are considered to be, in plant nutrition, a number of elements known to be needed in

small amounts for proper plant growth and development. The most common are Iron (Fe), Manganese

(Mn), Molybdenum (Mo), Copper (Cu), Zinc (Zn) and Boron (B).

If an organo-mineral fertilizer contains a substance, or one of the substances in the mixture, which is

intended to enhance the long term availability to plants of micronutrients in the EU fertilizing product,

that substance is either a chelating agent or a complexing agent.
The chelating agents are divided into two groups :
— Group 1: EDTA, DTPA, HEEDTA, IDHA and [S,S]-EDDS;

— Group 2: Chelating agents present in UVCB (unknown or variable composition, complex reaction

products or biological materials) chelates including [o,o] EDDHA, [o,p] EDDHA, [o,o] EDDHMA, HBED

and EDDHSA.

This document defines the test method to be used in order to identify and determine the content of UVCB

micronutrient chelated by [o,o] EDDHA, [o,o] EDDHMA and HBED in organo-mineral fertilizers (product

function category (PFC) 1(B) according to Regulation (EU) 2019/1009 [4]).
1 1
Abbreviated terms are described in Annex A.
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1 Scope

This document specifies a method for the determination by ion pair chromatography 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 organo-mineral fertilizers, having an organic matrix based on vegetal

residues (cocoa shells, grape residue, soybean residue, …), algae extract, and animal meal (feather, bones,

blood, ...) and 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 micronutrients beside Fe are present in organo-mineral

fertilizers containing [o,o] EDDHA, [o,o] EDDHMA or HBED.

This method is applicable to a mass fraction of the metal chelated of at least 0,625 %.

NOTE 1 The substances EDDHA and EDDHMA 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]. 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 are referred to in the text in such a way that some or all of their content

constitutes requirements 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 12944-1, Fertilizers and liming materials and soil improvers - Vocabulary - Part 1: General terms

EN 12944-2, Fertilizers and liming materials and soil improvers - Vocabulary - Part 2: Terms relating to

fertilizers
3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 12944-1 and EN 12944-2 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
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

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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.
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 can occur with

some equipment depending on the column used. The use of a different column of the same type can solve

this problem.
6 Reagents
All reagents shall be of recognized analytical grade.

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.
6.1 Water.

All water used should conform to EN ISO 3696, be degassed and be free of organic contaminants.

6.2 Sodium hydroxide solution, substance concentration 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 CO from the atmosphere should be carefully avoided. Otherwise, the dissolution of

chelating agents (see 6.6, 6.7 and 6.8) can be incomplete.
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 (6.1).

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 (6.1).
6.5 Iron-nitrate solution, mass concentration ρ(Fe) = 1 050 mg/l.

Dissolve 0,759 4 g of ferric nitrate 9-hydrate (Fe(NO ) ·9H O) in 100 ml of water. Check (for example by

3 3 2
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.

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6.6 Fe-[o,o] EDDHA solution, ρ(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/SP g (where SP 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-N,N′-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 wi
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