SIST-TP CEN/TR 15106:2005

Fertilizers - Determination of chelating agents EDDHA and EDDHMA by ion pair chromatography - Comparison of non-standardized Lucena method with EN 13368-2:2001

This Technical Report covers the results from two ring tests (A and B, see Introduction) to compare the repeatability and reproducibility of the standardized method for the determination of EDDHA and EDDHMA by ion chromatography (EN 13368 2:2001) with a non-standardized alternative method using ion-pair chromatography ("Lucena-method", given in Annex A). This Technical Report also provides the results of an univariate multifactorial ANOVA test and F-test for ring test A and F-test for ring test B to verify the significance of the reproducibility and the repeatability data.

Düngemittel - Bestimmung der Chelatbildner EDDHA und EDDHMA mit Ionenpaarchromatographie - Vergleich der nicht standardisierten Lucena-Methode mit EN 13368-2:2001

Engrais - Détermination des agents chélatants EDDHA et EDDHMA par chromatographie ionique - Comparaison entre la méthode Lucena non normalisée et la norme EN 13368-2:2001

Gnojila – Določevanje sredstev za kelatiziranje EDDHA in EDDHMA z ionsko kromatografijo – Primerjava nestandardizirane metode po Luceni z EN 13368-2:2001

General Information

• Status: Published
• Publication Date: 30-Nov-2005
• ICS: 65.080 - Fertilizers
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Dec-2005
• Due Date: 01-Dec-2005
• Completion Date: 01-Dec-2005
• Ref Project: CEN/TR 15106:2005 - Fertilizers - Determination of chelating agents EDDHA and EDDHMA by ion pair chromatography - Comparison of non-standardized Lucena method with EN 13368-2:2001

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TP CEN/TR 15106:2005
01-december-2005
*QRMLOD±'RORþHYDQMHVUHGVWHY]DNHODWL]LUDQMH(''+$LQ(''+0$]LRQVNR
NURPDWRJUDILMR±3ULPHUMDYDQHVWDQGDUGL]LUDQHPHWRGHSR/XFHQL](1

Fertilizers - Determination of chelating agents EDDHA and EDDHMA by ion pair
chromatography - Comparison of non-standardized Lucena method with EN 13368-
2:2001
Düngemittel - Bestimmung der Chelatbildner EDDHA und EDDHMA mit
Ionenpaarchromatographie - Vergleich der nicht standardisierten Lucena-Methode mit
EN 13368-2:2001
Engrais - Détermination des agents chélatants EDDHA et EDDHMA par
chromatographie ionique - Comparaison entre la méthode Lucena non normalisée et la
norme EN 13368-2:2001
Ta slovenski standard je istoveten z: CEN/TR 15106:2005
ICS:
65.080 Gnojila Fertilizers
SIST-TP CEN/TR 15106:2005 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TP CEN/TR 15106:2005

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SIST-TP CEN/TR 15106:2005
TECHNICAL REPORT
CEN/TR 15106
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
May 2005
ICS 65.080
English version
Fertilizers - Determination of chelating agents EDDHA and
EDDHMA by ion pair chromatography - Comparison of non-
standardized Lucena method with EN 13368-2:2001
Engrais - Détermination des agents chélatants EDDHA et Düngemittel - Bestimmung der Chelatbildner EDDHA und
EDDHMA par chromatographie ionique - Comparaison EDDHMA mit Ionenpaarchromatographie - Vergleich der
entre la méthode Lucena non normalisée et la norme EN nicht standardisierten Lucena-Methode mit EN 13368-
13368-2:2001 2:2001
This Technical Report was approved by CEN on 7 April 2005. 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, 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
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 15106:2005: E
worldwide for CEN national Members.

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CEN/TR 15106:2005 (E)
Contents page
Foreword .3
Introduction.4
1 Scope .5
2 Normative references .5
3 Test procedure for ring test A.5
4 Test procedure for ring test B.10
5 Final conclusion.17
Annex A (informative) Non-standardized method: "Fertilizers - Determination of Fe chelated by
the chelating agents EDDHA and EDDHMA in fertilizers by ion pair chromatography".18
Annex B (informative) Comments received from the participating laboratories and suggestions
of CEN/TC 260/WG 5 for improvement of the non-standardized method .27
Annex C (informative) Suggestions received from the participating laboratories of
CEN/TC 260/WG 5 after ring test B .29
Bibliography.30

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Foreword
This document (CEN/TR 15106:2005) 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.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Report: Austria, Belgium, Cyprus, Czech Republic, Denmark,
Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg,
Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and the
United Kingdom.
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Introduction
European Regulation (EC) No 2003/2003 of the European parliament and of the Council of 13th October 2003
relating to fertilizers states that the synthetic commercial iron chelates shall be water soluble products
obtained by the chemical combination of iron with a chelating agent. The iron (Fe) chelated by each chelating
agent shall be declared on the label.
EN 13368-2:2001 specifies a method for the determination of EDDHA and EDDHMA by ion chromatography.
This method determines the amount of chelated iron in Fe-EDDHA and Fe-EDDHMA commercial products. A
ring test performed by CEN/TC 260, showed that reproducibility data were poor, possibly due to the low
number of participating laboratories.
Some laboratories used a non-standardized method [1], [2], based on ion-pair chromatography. This can be
performed using common chromatography equipment and some experiences [3] suggested that it can give
adequate reproducibility and repeatability data.
CEN/TC 260/WG 5 agreed to launch a new work item in order to compare the reproducibility, repeatability and
accuracy of the two methods (standardized and non-standardized). The aim being to develope a European
Standard for the non-standardized method if better results were obtained. The results of the first ring test (ring
test A) were not significant enough to confirm that the non-standardized method is better than the method
standardized in EN 13368-2:2001.
The comments of the participating laboratories (see Annex B) suggested that the non-standardized method
could be improved with some modifications which could then result in significant differences with respect to
EN 13368-2:2001.
For this reason, WG 5 decided to carry out a second ring test (ring test B) using the method with the
suggested modifications.
This Technical Report includes the results and conclusions from the two ring tests.
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1 Scope
This Technical Report covers the results from two ring tests (A and B, see Introduction) to compare the
repeatability and reproducibility of the standardized method for the determination of EDDHA and EDDHMA by
ion chromatography (EN 13368-2:2001) with a non-standardized alternative method using ion-pair
chromatography ("Lucena-method", given in Annex A). This Technical Report also provides the results of an
univariate multifactorial ANOVA test and F-test for ring test A and F-test for ring test B to verify the
significance of the reproducibility and the repeatability data.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
EN 13368-2:2001, Fertilizers - Determination of chelating agents in fertilizers by ion chromatography - Part 2:
EDDHA and EDDHMA.
3 Test procedure for ring test A
3.1 Methods used in ring test A for the determination of chelating agents EDDHA and
EDDHMA by chromatography.
Both methods tested for the determination of EDDHA and EDDHMA are based on high performance liquid
chromatography.
The standardized method, based on ion chromatographyis is given in EN 13368-2:2001.
The non-standardized method, based on ion-pair chromatography is given in Annex A (without the
modifications presented in italics, which were only used for ring test B).
3.2 Test Samples
Four different samples were provided to all the participants. Two samples were commercial iron chelates in
solid form, one containing Fe-EDDHA and the other containing Fe-EDDHMA. The other two samples were
solutions prepared from commercially available standards.
3.3 Ring test A procedure
The test samples were sent to 15 private or official laboratories from seven countries of which only 14
provided results.
The participating laboratories were requested to carry out 2 replicate analyses of each sample according to
each method.
Table 1 shows the samples analysed by each participating laboratory.
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Table 1 — Samples analyzed by each participant laboratory

LABOR STANDARDIZED METHOD NON-STANDARDIZED METHOD
ATORY
Commercial samples Solutions from Commercial samples Solutions from
(solid form) standards (solid form) standards
EDDHA EDDHMA EDDHAEDDHMAEDDHA EDDHMAEDDHA EDDHMA
1 x x x x x x x x
2 x x x x x x x x
3 x x x x x x x x
4 - - - - x x x x
5 - - - - x - x -
6 - - - - x x x x
7 x x x x x x x x
8 x x x x x x x x
9 x x x x x x x x
10 - - - - x x x x
11 x x x x - - - -
a
12 x x x x x x x x
13 - - - - x x x x
14 x x x x x x x x
15 - - - - - - - -
TOTAL 9 9 9 9 13 12 13 12
a
only one replicate was reported

NOTE Most of the laboratories which did not analyse samples by the standardized method did not have the
necessary equipment for the determination given in EN 13368-2:2001.
Test results, observations and remarks have been reported on the appropriate sheets.
3.4 Results and statistical interpretation of ring test A
3.4.1 General
Statistical calculations have been carried out using all the results, according to ISO 5725-2 [4].
Parameters of repeatability and reproducibility have been evaluated for each sample and method (mean value,
standard deviation of repeatability (s), standard deviation of reproducibility (s ), repeatability limit (r),
r R
reproducibility limit (R), relative standard deviation of repeatability (RSD ) and relative standard deviation of
r
reproducibility (RSD ).
R
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3.4.2 Statistical results of ring test A
3.4.2.1 Repeatability and reproducibility
Table 2 shows the statistical results of the ring test for repeatability and reproducibility.
Table 2 — Statistical results for ring test A

Parameter STANDARDIZED METHOD NON-STANDARDIZED METHOD
Commercial samples Solutions from Commercial samples Solutions from
standards standards
(solid form) (solid form)
SAMPLE EDDHA EDDHMA EDDHA EDDHMA EDDHA EDDHMA EDDHA EDDHMA
Number of 9 9 9 9 13 12 13 12
laboratories
Number of 0 0 2 1 1 0 1 1
outliers
Number of 9 9 7 8 12 12 12 11
laboratories
after
elimination of
outliers
Mean value 3,04 4,58 24,47 25,31 3,09 g 4,28 24,04 26,49
mg/l Fe/100 g mg/l
gFe/100 g gFe/100 g mg/l g Fe/100 g mg/l
Repeatability 0,21 0,36 0,47 1,38 0,04 0,12 0,20 0,32
limit, r mg/l
gFe/100 g gFe/100 g mg/l mg/l g Fe/100 g g Fe/100 g mg/l
RSD , % 2,5 2,8 0,7 2,0 0,5 1,0 0,3 0,4
r
Repeatability 0,07 0,13 0,17 0,49 0,02 g 0,04 0,07 0,11
standard Fe/100 g mg/l
gFe/100 g gFe/100 g mg/l mg/l g Fe/100 g mg/l
deviation
s
r
Reproducibility 0,41 1,31 2,97 7,21 0,33 1,11 2,58 4,85
limit, R mg/l
gFe/100 g gFe/100 g mg/l mg/l gFe/100 g g Fe/100 g mg/l
RSD , % 4,8 10,2 4,3 10,2 3,8 9,2 3,8 6,5
R
Reproducibility 0,15 0,47 1,27 2,57 0,12 0,37 0,92 1,73
standard mg/l
gFe/100 g gFe/100 g mg/l mg/l gFe/100 g g Fe/100 g mg/l
deviation
s
R
Horwitz value 3,4 3,2 9,9 9,8 3,4 3,2 9,9 9,8
R
Horrat R index 1,43 3,22 0,44 1,03 1,12 2,88 0,38 0,67

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3.4.2.2 Reproducibility and repeatability significance
The comparison of the reproducibility and repeatability between the two methods for each sample is given in
Table 3.
Table 3 — ANOVA test results for mean values by types of samples

PARAMETER Commercial samples Solutions from standards
(solid form)
SAMPLE EDDHA EDDHMA EDDHA EDDHMA
a a a a
Calculated F
21,83 9,04 5,57 19,15
(r)
Calculated F 1,58 1,40 1,32 2,21
(R)
Tabulated F 2,95 2,95 3,09 3,14
a
significant at α =0,05

3.4.2.3 Accuracy comparison
A univariant multifactorial ANOVA test has been developed for the comparison of the means to examine the
significance of the differences between the two test methods.
Table 4 — R- an r- values and their significance by types of samples

PARAMETER Commercial samples Solutions from standards
(solid form)
SAMPLE EDDHA EDDHMA EDDHA EDDHMA
Calculated F 139,24 8,82 95,44 274,42
(Fischer)
0,054 0,207 0,068 0,038
Significance α

3.5 Conclusions for ring test A
3.5.1 General
Laboratories have provided more results by non-standardized method than by EN 13368-2.
In this ring test more results have been obtained for the standardized method than were available in the ring
test carried out by CEN/TC 260 in the past. This gives a better estimate of the repeatability and reproducibility
data.
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3.5.2 Repeatability and reproducibility
The non-standardized method shows better repeatability for all samples, even for the solid samples where the
complete procedure was applied (i.e. sample preparation and analytical determination). s is significantly lower
r
for the non-standardized method.
There are no significant differences regarding s between the two methods, but the tendency is that the non-
R
standardized method shows lower reproducibility values for all samples, even for the solid samples where the
complete procedure shall be applied (i.e. sample preparation and analytical determination).
3.5.3 Accuracy comparison
There are no significant differences between the methods at α =0,05 as shown by the ANOVA test results (see
Table 4) except for Fe-EDDHMA in solutions.
3.5.4 Final conclusions of the ring test A:
The results show good correlation between the two methods.
Data are not significant enough to confirm that the non-standardized method is better than the standardized
method. Based on the comments of the participating laboratories (see Annex B), the non-standardized
method could be improved with some modifications, and then it might result in significant differences with
respect to the standardized method.
For this reason, ring test on the non-standardized method should be repeated with the following modifications
(see modifications given in italics in Annex A):
 Preparation of the standards:
 Iron nitrate is deliquescent and therefore it shall be added as a solution of a known concentration.
 To determine the titrimetric purity of the chelating agent standards if there is any doubt on the actual
purity.
 For the dissolution of the standards it is important to use CO free fresh alkali solution.
2

 Sample preparation
 To remove the filtration with cellulose filters.

 Calibration curves:
 To use six standards and two replicates for each standard.
 Significant statistical negative intercept could be caused due to the contamination of the column.

 Integration:
 If overlapping occurs, baseline corrections in the integration should be made (see Figure A.4).

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These modifications are included in Annex A in italics in the relevant positions.
4 Test procedure for ring test B
4.1 Method used in ring test B for the determination of chelating agents EDDHA and
EDDHMA by chromatography.
The samples were tested only by the non-standardized method including the modifications suggested by
CEN/TC 260/WG 5 and included in Annex A in italics .
4.2 Test Samples
Seven different samples have been provided to all the participants. Five samples were commercial iron
chelates in solid form, three containing Fe-EDDHA and two containing Fe-EDDHMA. The other two samples
were solutions prepared from commercially available standards, one containing Fe-EDDHA and the other
containing Fe-EDDHMA.
4.3 Ring test B procedure
The test samples were sent to 16 private and official laboratories from 8 countries, of which only 14 provided
results.
The participating laboratories were requested to carry out 2 replicate analyses on each sample using the non-
standardized method including the modifications in italics. The results of the two replicates of each sample
should be reported to two decimal places.
Table 5 shows the samples that were tested by each participating laboratory.

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Table 5 — Participating laboratories providing results

NON-STANDARDIZED METHOD
Commercial samples
Solutions from
LAB.
standards
(solid form)
EDDHA-1 EDDHA-2 EDDHA-3 EDDHMA-1 EDDHMA-2 EDDHA EDDHMA
1 x x x x x x x
2 x x x x x x x
3 x x x - - x -
4 x x x x x x x
5 x x x x x x x
6 x x x x x x x
7 x x x x x x x
8 x x x x x x x
9 x x x x x x x
10 x x x x x x x
11 x x x - - x -
12 x x x x x x x
13 x x x x x x x
a
14 x x x x x x x
15 - - - - - - -
16 - - - - - - -
TOTAL 14 14 14 14 12 13 12
a
only one replicate was provided

Test results, observations and remarks have been reported on the appropriate sheets.
4.4 Results and statistical interpretation for ring test B
4.4.1 General
Statistical calculations have been made using all the results, according to ISO 5725-2 [4].
Parameters of repeatability and reproducibility have been evaluated for each sample (mean value, standard
deviation of repeatability (s ), standard deviation of reproducibility (s ), repeatability limit (r), reproducibility limit
r R
(R), relative standard deviation of repeatability (RSD) and relative standard deviation of reproducibility
r
(RSD )).
R
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4.4.2 Statistical results
4.4.2.1 Repeatability and reproducibility
Table 6 shows the statistical results of the ring test on repeatability and reproducibility.
Table 6 — Statistical results of ring test B

Parameter Solutions from standard Commercial iron chelates (solid form)
Sample EDDHA EDDHMA EDDHA-1 EDDHA-2 EDDHA-3 EDDHMA-1 EDDHMA-2
Number of
14 12 14 14 14 12 12
laboratories
Number of
0 2 0 1 0 0 1
outliers
Number of
laboratories
14 10 14 13 14 12 11
after elimination
of outliers
24,91 25,42 3,08 4,98 3,80 4,84 4,22
Mean value
mg/l mg/l g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g
0,29 0,26 0,05 0,09 0,07 0,12 0,04
Repeatability
limit, r
mg/l mg/l g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g
RSD % 0,42 0,37 0,57 0,65 0,61 0,89 0,38
r
Repeatability
0,10 0,09 0,02 0,03 0,02 0,04 0,02
Standard
mg/l mg/l g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g
Deviation (s )
r
1,81 2,07 0,38 0,43 0,36 0,69 0,62
Reproducibility
limit, R
mg/l mg/l g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g
RSD % 2,60 2,90 4,39 3,05 3,35 5,12 5,24
R
Reproducibility
0,65 0,74 0,14 0,15 0,13 0,25 0,22
Standard
mg/l mg/l g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g g Fe/100 g
Deviation (s )
R
Horwitz Value R 9,9 9,8 3,4 3,1 3,3 3,2 3,2
Horrat R index 0,26 0,30 1,30 0,97 1,02 1,62 1,63

4.4.2.2 Comparison between ring test A results and ring test B results.
Table 7 shows the results of the F-test for standard deviation under repeatability in order to compare ring
test A with ring test B non standardized method results.
NOTE The samples used for ring test A were different from the samples used for ring test B, therefore the
comparison has been made using overall s in order to compare methods instead of samples.
r
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Table 7 — F-test for standard deviation under repeatability comparing
non standardized ring test A and B

s
r
Comparison
non standardized A-non standardized B
Fe-EDDHA liquid
F 2,15
calculated
F 2,77
α=0,05
tabulated

(13,11)
Fe-EDDHMA liquid
F 1,42
calculated
F 3,14
α=0,05
tabulated

(10,9)
Commercial sample Fe-EDDHA
F 2,44
calculated
F 2,54
tabulated α=0,05

(38,11)
Commercial sample Fe-EDDHMA
F 1,71
calculated
F 2,29
tabulated α=0,05

(11,21)
No significances were observed at α =0,05

Table 8 shows the results of the F test for standard deviation under reproducibility (s ) in order to compare
R
ring test A with ring test B non standardized method results.
NOTE The samples used for ring test A were different from the samples used for ring test B, therefore the
comparison has been made using overall s in order to compare methods instead of samples.
r
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Table 8 — F-test for standard deviation under reproducibility (s ) comparing non standardized method
R
results in ring test A and B.

s
R
Comparison
non standardized A-non standardized B
Fe-EDDHA liquid
F 2,03
calculated
F 2,64
tabulated α=0,05

(11,13)
Fe-EDDHMA liquid
a
F 5,52
calculated
F 3,14
tabulated α=0,05

(10,9)
Commercial sample Fe-EDDHA
F 0,72
calculated
F 2,54
tabulated α=0,05

(38,11)
Commercial sample Fe-EDDHMA
a
F 2,82
calculated
F 2,29
α=0,05
tabulated

(11,21)
a
Significant α=0,05


4.4.2.3 Comparison between EN 13368-2:2001 results and ring test B results.
Table 9 shows the results of the F-test for standard deviation under repeatability (s ) in order to compare ring
r
test B results with EN 13368-2:2001 method results obtained in ring test A.
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Table 9 — F-test for standard deviation under repeatability (s ) comparing non standardized ring test B
r
and EN 13368-2:2001

s
r
Comparison
EN 13368-2:2001-non standardized B
Fe-EDDHA liquid
F 2,59
calculated
F 2,92
α=0,05
tabulated

(6,13)
Fe-EDDHMA liquid
a
F 27,24
calculated
F 3,29
α=0,05
tabulated

(7,9)
Commercial sample Fe-EDDHA
a
F 8,94
calculated
F
α=0,05 2,19
tabulated

(8,38)
Commercial sample Fe-EDDHMA
a
F
15,47
calculated
F α=0,05 2,42
tabulated

(8,21)
a
Significant α=0,05

NOTE Samples used for ring test A are different from samples used for ring test B, therefore the comparison has
been made using overall s in order to compare methods instead of samples.
r
Table 10 shows the results of the F-test for standard deviation under reproducibility (s ) in order to compare
R
ring test B results with results using EN 13368-2:2001.
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Table 10 — F-test for standard deviation under reproducibility (s ) comparing non standardized ring
R
test B and standardized method according to EN 13368-2:2001

s
R

Comparison EN 13368-2:2001-non standardized B
Fe-EDDHA liquid
F 2,69
calculated
F α=0,05 2,92
tabulated

(6,13)
Fe-EDDHMA liquid
a
F
12,19
calculated
F α=0,05 3,29
tabulated

(7,9)
Commercial sample Fe-EDDHA
F 1,13
calculated
F α=0,05 2,19
tabulated

(8,38)
Commercial sample Fe-EDDHMA
a
F
3,95
calculated
F α=0,05 2,42
tabulated

(8,21)
a
Significant α=0,05

NOTE Samples used for ring test A are different from samples used for ring test B, therefore the comparison has
been made using overall s in order to compare methods instead of samples.
R
4.5 Conclusions for ring test B
4.5.1 Conclusions on the comparison between ring test A results and ring test B results (only for
non-standardized method)
4.5.1.1 Repeatability and reproducibility
There are no significant differences (α = 0,05) for the Fe-EDDHA samples so the modifications introduced in
the test method do not affect the repeatability or the reproducibility.
There are no significant differences in the repeatability (α = 0,05) for the EDDHMA samples but there are
significant improvements in the reproducibility (α = 0,05) for Fe-EDDHMA liquid and commercial samples.
These improvements are related to the specifications given for the integration of chromatographic peaks.
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4.5.2 Conclusions on the comparison between ring test B results and ring test A results for
standardized method (EN 13368-2:2001)
4.5.2.1 Repeatability and reproducibility
There are significant improvements for all values in repeatability for EDDHA and EDDHMA using the non
standardized method.
There are significant improvements for reproducibility for EDDHMA using the non standardized method.
4.5.3 Final conclusions from the ring test B:
 Non standardized test with the modifications introduced in ring test B is to be preferred.
5 Final conclusion
The non standardized test method has been shown to provide the best results, therefore it is proposed to
publish it as European Standard and take it into account when reviewing Regulation 2003/2003 in order to
propose the substitution of EN 13368-2:2001.
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Annex A
(informative)

Non-standardized method: "Fertilizers - Determination of Fe chelated by
the chelating agents EDDHA and EDDHMA in fertilizers by ion pair
chromatography"
Introduction
This Annex gives the non-standardized method used for the determinations. The modifications included in the
ring test B, which were not applied during ring test A, are shown in italics.
A.1 Scope
This method specifies the procedure for the chromatographic determination of the total amount of each
individual ortho(hydroxy)-ortho(hydroxy) isomer of the chelating agents EDDHA and EDDHMA in fertilizers,
containing one or both of these substances. The method allows the identification and the determination of the
total water soluble fraction of each of these chelating agents. It does not distinguish between the free form and
the metal bound form of the chelating agents in non iron fertilizers.
NOTE The substances EDDHA (ethylene diamine di(hydroxy phenyl) acetic acid) and EDDHMA (ethylene diamine
di(hydroxy methyl phenyl)acetic acid) exist under 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 EDDHMA are considered together.
At present, analytically pure standards only exist for ortho-ortho EDDHA and ortho-ortho EDDHMA. 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. For EDDHMA, the method
was initially developed with samples of iron EDDHMA of uncertain purity and composition, any standard
product being unavailable then. Now a pure standard is available.
The meso- and the dl-racemic forms of EDDHA and EDDHMA can be determined separately by this method.
This procedure applies to EC fertilizers covered by Regulation (EC) No 2003/2003 with. It is applicable to a
content of at least 0,001 % of the chelated metal.
A.2 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 an apolar solid phase (stationary phase). The strength of the retention depends on
the molecular size and its acidity. Thus, 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
hydroxide) and acetonitrile as eluent. The detection is based on photometry at 280 nm.
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A.3 Interferences
No interferences have been detected. Iron chelates w
...