CEN/TS 15451:2006
(Main)Fertilizers - Determination of chelating agents - Determination of iron chelated by EDDHSA by ion pair chromatography
Fertilizers - Determination of chelating agents - Determination of iron chelated by EDDHSA by ion pair chromatography
This Technical Specification specifies a method for the chromatographic determination of the total amount of iron chelated by EDDHSA in commercial products.
Düngemittel - Bestimmung von Chelatbildnern - Bestimmung von Eisen-chelatisiertem EDDHSA mit Ionen-Paarchromatographie
Engrais - Dosage des agents chélatants - Dosage du fer chélaté par EDDHSA par chromatographie d'appariement d'ions
La présente Spécification Technique spécifie une méthode par chromatographie qui permet de déterminer la quantité totale de fer chélaté par EDDHSA dans les produits du commerce.
Gnojila - Določevanje sredstev za kelatiziranje - Določevanje železovih kelatov z EDDHSA z ionsko kromatografijo
General Information
Relations
Standards Content (Sample)
SLOVENSKI oSIST-TS prCEN/TS
15451:2006
PREDSTANDARD
marec 2006
Gnojila – Določevanje sredstev za kelatiziranje - Določevanje železovih
kelatov z EDDHSA z ionsko kromatografijo
Fertilizers - Determination of chelating agents - Determination of iron chelated by
EDDHSA by ion pair chromatography
ICS 65.080 Referenčna številka
oSIST-TS prCEN/TS 15451:2006(en)
© Standard je založil in izdal Slovenski inštitut za standardizacijo. Razmnoževanje ali kopiranje celote ali delov tega dokumenta ni dovoljeno
TECHNICAL SPECIFICATION
FINAL DRAFT
prCEN/TS 15451
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
January 2006
ICS 65.080
English Version
Fertilizers - Determination of chelating agents - Determination of
iron chelated by EDDHSA by ion pair chromatography
Engrais - Dosage des agents chélatants - Dosage du fer Düngemittel - Bestimmung von Chelatbildnern -
chélaté par EDDHSA par chromatographie d'appariement Bestimmung von Eisen-chelatisiertem EDDHSA mit Ionen-
d'ions Paarchromatographie
This draft Technical Specification is submitted to CEN members for formal vote. It has been drawn up by the Technical Committee CEN/TC
260.
CEN members are the national standards bodies of Austria, Belgium, 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.
: This document is not a Technical Specification. It is distributed for review and comments. It is subject to change without notice
Warning
and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. prCEN/TS 15451:2006: E
worldwide for CEN national Members.
prCEN/TS 15451:2006 (E)
Contents Page
Foreword.3
1 Scope .4
2 Normative references .4
3 Principle.4
4 Interferences .4
5 Reagents.4
6 Apparatus .5
7 Sampling and sample preparation.6
8 Procedure .7
9 Expression of the result.7
3+
10 Precision for Fe /EDDHSA.8
3+
Annex A (informative) Chromatogram for Fe /EDDHSA .9
3+
Annex B (informative) Spectra UV-visible for Fe /EDDHSA .10
Annex C (informative) Results of the inter-laboratory tests .11
C.1 Test samples .11
C.2 Inter-laboratory test procedure .11
C.3 Results and statistical interpretation.11
Bibliography .13
prCEN/TS 15451:2006 (E)
Foreword
This document (prCEN/TS 15451:2006) 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 Formal Vote.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
prCEN/TS 15451:2006 (E)
1 Scope
This Technical Specification 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 edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
prEN 1482-1, Fertilizers and liming materials — Sampling and sample preparation — Part 1: Sampling
prEN 1482-2, Fertilizers and liming materials — Sampling and sample preparation — Part 2: Sample
preparation
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 solid 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. 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
3+
480 nm. For Fe /EDDHSA a gradient method is required.
4 Interferences
No interferences have been detected. Iron chelates 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
3+ .
Fe /EDDHSA.
5 Reagents
5.1 General
a) All reagents shall be of recognised analytical grade.
b) All water used for the preparation of eluent, standards, and sample solutions should be water type 1
according to EN ISO 3696.
c) When 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 the required concentration in the solution.
5.2 Sodium hydroxide solution
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
prCEN/TS 15451:2006 (E)
Dissolve 4 g of NaOH in pellet form in a 1 litre volumetric flask with water free of carbon dioxide. Dilute to the
mark and homogenize.
5.4 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 500 ml with water.
3+
5.6 Fe /EDDHSA solution
ρ(Fe) = 100 mg/l
Dissolve 0,7594 g of iron(III)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 is (1050 ± 30) mg Fe/l
As the Fe(NO ) ·9H O is deliquescent it shall be added in solution of a known concentration.
3 3 2
Dissolve 0,233 0 g×100/R, where R is the complexometric purity of the standard in percentage of the acid
form H EDDHSA obtained by photometric titration with a Fe(III) standard in a 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). After complete dissolution, add 25 ml of the Fe solution 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 cellulose
filter and make up to volume (250 ml) with water.
5.7 Eluent for the determination
Two solutions are used to make a linear gradient eluent:
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 (5.4 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 µm membrane filter (6.3 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 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 µm-membrane filter (6.3 b).
NOTE 1 TBACl or TBABr may be used, providing that pH is adjusted to 6,0 w
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Fertilizers - Determination of chelating agents - Determination of iron chelated by
EDDHSA by ion pair chromatography
Düngemittel - Bestimmung von Chelatbildnern - Bestimmung von Eisen-chelatisiertem
EDDHSA mit Ionen-Paarchromatographie
Engrais - Dosage des agents chélatants - Dosage du fer chélaté par EDDHSA par
chromatographie d'appariement d'ions
Ta slovenski standard je istoveten z: CEN/TS 15451:2006
ICS:
65.080
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL SPECIFICATION
CEN/TS 15451
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
November 2006
ICS 65.080
English Version
Fertilizers - Determination of chelating agents - Determination of
iron chelated by EDDHSA by ion pair chromatography
Engrais - Dosage des agents chélatants - Dosage du fer Düngemittel - Bestimmung von Chelatbildnern -
chélaté par EDDHSA par chromatographie d'appariement Bestimmung von Eisen-chelatisiertem EDDHSA mit Ionen-
d'ions Paarchromatographie
This Technical Specification (CEN/TS) was approved by CEN on 27 May 2006 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, 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 STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15451:2006: E
worldwide for CEN national Members.
Contents Page
Foreword.3
1 Scope .4
2 Normative references .4
3 Principle.4
4 Interferences .4
5 Reagents.4
6 Apparatus .6
7 Sampling and sample preparation.6
8 Procedure .7
9 Expression of the result.8
3+
10 Precision for Fe /EDDHSA.8
11 Test report .9
3+
Annex A (informative) Chromatogram for Fe /EDDHSA .10
3+
Annex B (informative) Spectra UV-visible for Fe /EDDHSA .11
Annex C (informative) Results of the inter-laboratory tests .12
Annex D (informative) Complete names of chelating agents .14
Bibliography .15
Foreword
This document (CEN/TS 15451:2006) has been prepared by Technical Committee CEN/TC 260 “Fertilizers
and liming materials”, the secretariat of which is held by DIN.
This document has been prepared 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 announce this Technical Specification: Austria, Belgium, 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 the United Kingdom.
1 Scope
This Technical Specification 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 edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
prEN 1482-2, Fertilizers and liming materials — Sampling and sample preparation — Part 2: Sample
preparation
EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696:1995)
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 solid 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. 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
3+
480 nm. For Fe /EDDHSA a gradient method is required.
4 Interferences
No interferences have been detected. Iron chelates 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
3+ .
Fe /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, and sample solutions should be water type 1
according to EN ISO 3696.
c) When 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 the required concentration in the solution.
5.2 Sodium hydroxide solution
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 carbon dioxide. Dilute to the
mark and homogenize.
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 500 ml with water.
5.6 Iron (III) nitrate solution
ρ(Fe) = 1 050 mg/l
Dissolve 0,7594 g of iron (III) nitrate nonahydrate [Fe(NO ) ·9H O] in 100 ml of water. Transfer to a 100 ml-
3 3 2
volumetric flask. Dilute to the mark with water and homogenize. Check (for example by AAS) that the Fe
concentration of this solution is (1 050 ± 30) mg/l.
3+
As the Fe(NO ) ·9H O is deliquescent, for preparation of the Fe /EDDHSA solution (5.7) it shall be added in
3 3 2
solution of a known concentration.
3+
5.7 Fe /EDDHSA solution
ρ(Fe) = 100 mg/l
Dissolve 0,233 0 g×100/P, where P is the complexometric purity of the standard in percentage of the acid
form H EDDHSA obtained by photometric titration with a Fe(III)standard in a 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). After 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 cellulose
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:
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 (5.4 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 µm membrane filter (6.3 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 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 µm-membrane filter (6.3 b).
NOTE 1 TBACl or TBABr may be used, providing that pH is adjusted to 6,0 with NaOH or HCl.
NOTE 2 Tetrabutylammonium bisulfate should be avoided because peak retention times and shape may significantly
change.
6 Apparatus
Usual laboratory equipment, glassware, and:
6.1 Magnetic stirrer
Magnetic stirrer with magnets.
6.2 Chromatograph
Chromatograph equipped with:
a) gradient pump delivering the eluent at a flow rate of 1,5 ml/min (see Table 1);
b) injection valve with a 20 µl injection loop;
1)
c) C18 column; 150 mm×3,9 mm ID; dp = 5 µm ;
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 table
Time Flow rate A B
Curve type
min ml/min % %
1,5 100 0 Linear
5 1,5 100 0 Linear
6 1,5 0 100 Linear
11 1,5 0 100 Linear
12 1,5 100 0 Linear
20 1,5 100 0 Linear
6.3 Membrane filters
a) Micromembrane filters resistant to aqueous solutions, with porosity of 0,45 µm;
b) Micromembrane filters resistant to organic solutions, (e.g.: nylon micromembrane filters) with porosity of
0,2 µm.
7 Sampling and sample preparation
Sampling is not part of the method specified in this document. A recommended sampling method is given in
prEN 1482-1.
TM
1) SYMMETRY C18, from WATERS cat.nº: WAT054205, or equivalent 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.
Sample preparation shall be carried out in accordance with prEN 1482-2.
NOTE For the size reduction of samples with a high amount of chelating agents, it is not recommended to use a high
speed laboratory mill. It is more convenient to grind the sample in a mortar to a particle size less than 1 mm.
8 Procedure
8.1 Preparation of the sample solution of iron chelates
Weigh 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 water and homogenize.
8.2 Preparation of the calibration solutions
3+
Pipette a volume (V ml) (see Table 2) of the Fe /EDDHSA standard solution (5.7) in six 100 ml-volumetric
flasks respectively. Make up to volume with water and homogenize.
3+
Table 2 — Preparation of the calibration curve of Fe /EDDHSA
Concentration of Fe in the form
of
V
Solution
3+
Fe /EDDHSA
ml
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 µm-membrane filter (6.3 a). Inject
the standard solutions (8.2.) into the chromatographic system (6.2). Measure the retention times and the
3+
areas of the Fe /EDDHSA chromatographic peaks for all solutions. Two chromatographic peaks appear for
3+ 3+
Fe /EDDHSA, corresponding with the different forms of Fe /EDDHSA (see Annex A). Draw the calibration
3+
graph with the added values of the two chromatographic peaks of the standard solutions of Fe /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 used, 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.
3+
NOTE Normally the two chromatog
...
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