Fertilizers - Determination of urea condensates using high-performance liquid chromatography (HPLC) - Isobutylidenediurea and crotonylidenediurea (method A) and methylen-urea oligomers (method B)

This document specifies methods for the determination of isobutylidenediurea (IBDU), crotonylidenediurea (CDU) (method A) and methylene-urea oligomers (MU) (method B) in fertilizers using high-performance liquid chromatography (HPLC).
The method is applicable for all fertilizers which do not contain interfering organic compounds.

Düngemittel - Bestimmung von Harnstoffkondensaten mit Hochleistungs-Flüssigchromatographie (HPLC) - Isobutylidendiharnstoff und Crotonylidendiharnstoff (Verfahren A) und Methylenharnstoff-Oligomere (Verfahren B)

Das vorliegende Dokument legt Verfahren für die Bestimmung von Isobutylidendiharnstoff (IBDU), Crotonylidendiharnstoff
(CDU) (Verfahren A) und Methylenharnstoff-Oligomere (MU) (Verfahren B) in Düngemitteln
mit Hochleistungs-Flüssigchromatographie (HPLC) fest.
Das Verfahren gilt für alle Düngemittel, die keine störenden organischen Verbindungen enthalten.

Engrais - Détermination de condensates d'urea par chromatographie liquide à haute performance (CLHP) - Isobutylidenediurea et crotonylidenediurea (méthode A) et methyleneurea-oligomeres (méthode B)

Le présent document spécifie les méthodes de dosage de l’isobutylidène diurée (IBDU), de la crotonylidène diurée (CDU) (méthode A) et des oligomères de méthylène-urée (MU) (méthode B) dans les engrais par chromatographie liquide haute performance (HPLC).
La méthode est applicable à tous les engrais qui ne contiennent pas de composés organiques interférents.

Gnojila - Določevanje kondenzatov sečnine s tekočinsko kromatografijo visoke ločljivosti (HPLC) - Izobutilidendiurea in krotonilidendiurea (metoda A) in oligomeri metilenuree (metoda B)

General Information

Status
Withdrawn
Public Enquiry End Date
09-Nov-2008
Publication Date
13-May-2009
Withdrawal Date
13-Sep-2010
Technical Committee
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
10-Sep-2010
Due Date
03-Oct-2010
Completion Date
14-Sep-2010

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SLOVENSKI STANDARD
SIST-TS CEN/TS 15705:2009
01-junij-2009
*QRMLOD'RORþHYDQMHNRQGHQ]DWRYVHþQLQHVWHNRþLQVNRNURPDWRJUDILMRYLVRNH

ORþOMLYRVWL +3/& ,]REXWLOLGHQGLXUHDLQNURWRQLOLGHQGLXUHD PHWRGD$ LQROLJRPHUL

PHWLOHQXUHH PHWRGD%
Fertilizers - Determination of urea condensates using high-performance liquid

chromatography (HPLC) - Isobutylidenediurea and crotonylidenediurea (method A) and

methylen-urea oligomers (method B)
Düngemittel - Bestimmung von Harnstoffkondensaten mit Hochleistungs-

Flüssigchromatographie (HPLC) - Isobutylidendiharnstoff und Crotonylidendiharnstoff

(Verfahren A) und Methylenharnstoff-Oligomere (Verfahren B)

Engrais - Détermination de condensates d'urea par chromatographie liquide à haute

performance (CLHP) - Isobutylidenediurea et crotonylidenediurea (méthode A) et
methyleneurea-oligomeres (méthode B)
Ta slovenski standard je istoveten z: CEN/TS 15705:2009
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 15705:2009 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST-TS CEN/TS 15705:2009
---------------------- Page: 2 ----------------------
SIST-TS CEN/TS 15705:2009
TECHNICAL SPECIFICATION
CEN/TS 15705
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
January 2009
ICS 65.080
English Version
Fertilizers - Determination of urea condensates using high-
performance liquid chromatography (HPLC) -
Isobutylidenediurea and crotonylidenediurea (method A) and
methylen-urea oligomers (method B)

Engrais - Dosage des condensats d'urée par Düngemittel - Bestimmung von Harnstoffkondensaten mit

chromatographie liquide haute performance (HPLC) - Hochleistungs-Flüssigchromatographie (HPLC) -

Isobutylidène diurée et crotonylidène diurée (méthode A) et Isobutylidendiharnstoff und Crotonylidendiharnstoff

oligomères de méthylène-urée (méthode B) (Verfahren A) und Methylenharnstoff-Oligomere (Verfahren

This Technical Specification (CEN/TS) was approved by CEN on 15 November 2008 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, 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: Avenue Marnix 17, B-1000 Brussels

© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15705:2009: E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
Contents Page

Foreword ..............................................................................................................................................................3

1 Scope ......................................................................................................................................................4

2 Normative references ............................................................................................................................4

3 Terms and definitions ...........................................................................................................................4

4 Sampling and sample preparation .......................................................................................................4

5 Method A: Determination of CDU and IBDU .......................................................................................4

6 Method B: Determination of methylen-urea oligomers (MU) ............................................................7

7 Precision method A and method B ................................................................................................... 12

8 Test report ........................................................................................................................................... 13

Annex A (informative) Results of the inter-laboratory tests ........................................................................ 14

Annex B (informative) Chromatogram and calibration curves method A .................................................. 16

Annex C (informative) Chromatogram and calibration curves method B .................................................. 18

Bibliography ..................................................................................................................................................... 21

---------------------- Page: 4 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
Foreword

This document (CEN/TS 15705:2009) 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 [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

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, Bulgaria, 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.
---------------------- Page: 5 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
1 Scope

This document specifies methods for the determination of isobutylidenediurea (IBDU), crotonylidenediurea

(CDU) (method A) and methylene-urea oligomers (MU) (method B) in fertilizers using high-performance liquid

chromatography (HPLC).

The method is applicable for all fertilizers which do not contain interfering organic compounds.

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 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 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987)

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.
4 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.
5 Method A: Determination of CDU and IBDU
5.1 Principle

The sample is extracted with water and, after appropriate dilution, analyzed using a suitable HPLC system.

5.2 Reagents
5.2.1 General

Use only reagents of recognized analytical grade and distilled or demineralized water (grade 3 according to

EN ISO 3696:1995).
---------------------- Page: 6 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
5.2.2 Acetonitrile, p.a., HPLC-grade;
5.2.3 Isobutylidenediurea and crotonylidenediurea, in their pure form.
5.3 Apparatus

5.3.1 Laboratory equipment and glassware, for preparation of solutions and dilutions;

5.3.2 Analytical balance, capable for weighing to an accuracy of ± 0,1 mg;
5.3.3 HPLC-system, with UV-detector;
5.3.4 Ultrasonic bath;
5.3.5 Magnetic stirrer;
5.3.6 Disposible filter, 0,45 µm.
5.4 Procedure
5.4.1 System parameters of HPLC
Analytical/separating column: silica column with C18 reverse phase
Detection wavelength: 200 nm
Eluent: acetonitrile/water: 10/90 (volume fraction)
Flow rate: 1 ml/min
Temperature: ambient temperature
Injection volume: 20 µl
5.4.2 Calibration
5.4.2.1 Stock solution IBDU ρ(IBDU) = 100 mg/l

Weigh 100/R mg of IBDU (5.2.3), where R is the purity of IBDU, into a 1 000 ml flask and add about 900 ml of

water (5.2.1). Dissolve in an ultrasonic bath (5.3.4) for about 10 min, followed by stirring on a magnetic stirrer

(5.3.5) for about 1 h. Make up to volume. Filtration is not necessary.
5.4.2.2 Stock solution CDU ρ(CDU) = 100 mg/l

Weigh 100/R mg of CDU (5.2.3) , where R is the purity of CDU, into a 1 000 ml flask and add about 900 ml of

water (5.2.1). Dissolve in an ultrasonic bath (5.3.4) for about 10 min, followed by stirring on a magnetic stirrer

(5.3.5) for about 1 h. Make up to volume. Filtration is not necessary.
E.g. LiChrosorb RP-18 7µm 250/4 mm or equivalent.
---------------------- Page: 7 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
5.4.2.3 Calibration solution

For calibration, prepare three solutions according to Table 1 using one-mark (bulb) pipettes and dilute to the

mark with water (5.2.1).

For the determination of the retention time, dilute 10 ml of the stock solution 5.4.2.1 or respectively 5.4.2.2 into

two 100 ml flasks and make up to volume with water (5.2.1).

The evaluation of calibration is carried out manually or by means of a suitable PC-aided (computerized)

calculation method.
Table 1 — Preparation of calibration solutions
Amount of stock
solution
Content of IBDU Content of CDU
Parameter
IBDU/CDU
mg/l mg/l
(to be added to the
100 ml flask)
Standard1
10 10,0 10,0
Standard 2
25 25,0 25,0
Standard 3
50 50,0 50,0
5.4.3 Preparation of the test portion

Weigh 1 g of the sample grounded to < 0,2 mm to the nearest 0,1 mg and flush into a 1 000 ml volumetric

flask with water (5.2.1). Fill the flask to an amount of approximately 900 ml and treat it for 10 min in the

ultrasonic bath (5.3.4). Then make up to the mark and stir for 1 h at room temperature on a magnetic stirrer

(5.3.5). Dilute 10 ml of the solution in a 100 ml volumetric flask and filter into the HPLC injection vial through a

disposable filter (5.3.6).
5.4.4 Measurement

Measurement is performed manually or by means of an automatic sample loading system (autosampler).

5.4.5 Important annotations

IBDU is able to form urea in aqueous solution. Therefore, the measurement of the calibration and sample

solutions shall be completed within one working day.

The concentrations of CDU and IBDU in the sample solutions shall be kept within the calibration limits (5.4.2)

to ensure sufficient reproducibility.
5.5 Calculation

The calculation can be performed manually or by means of a PC using the calibration parameters in respect to

the amount used.

In the case of PC-aided (computerized) calculation and application of Table 1 regarding the amounts of stock

solution, the content of IBDU/CDU in milligrams per litre will be calculated by the system. The calculated

values are equal to the percentage mass concentration of IBDU/CDU in the analysed sample of fertilizer.

---------------------- Page: 8 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)

Following general rules for declaration in regulations to declare the content of the compounds as percentage

mass fraction of nitrogen, calculate the contents, w /w in percent (g/100 g), according to the

N(IBDU) N(CDU)
following equations:
w = w × 0,322 (1)
N IBDU
(IBDU)
w = w × 0,326 (2)
N(CDU) CDU
where

0,322 is the conversation factor for the content of IBDU in the fertilizer into nitrogen content;

0,326 is the conversation factor for the content of CDU in the fertilizer into nitrogen content.

6 Method B: Determination of methylen-urea oligomers (MU)

NOTE By the condensation of urea and formaldehyde developed no only one compound, as it is by the reaction of

urea and crotonaldehyde or isobutylaldehyde, but oligomers like methylen–diurea (MDU), dimethylen-triurea (DMTU),

trimethylen-tetraurea (TMTU) and higher oligomers. These three molecules are the most soluble in water, the higher

compounds are insoluble in hot water, but their nitrogen is available for plants by microbiological decomposition. Also urea

is always a companion of MU – oligomers.
6.1 Principle

The sample is extracted with boiling water and analyzed using a suitable HPLC system.

The methylen-urea soluble oligomers are measured and detected by the HPLC-method.

In the HPLC-diagram methylen-urea oligomers are represented by different peaks: urea, methylen-diurea,

dimethylen-triurea; trimethylen-tetraurea are, in the mean time, the most soluble and important.

6.2 Reagents
6.2.1 General

Use only reagents of recognized analytical grade and distilled or demineralized water (grade 3 according to

EN ISO 3696:1995).
---------------------- Page: 9 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
6.2.2 Acetonitrile, p.a., HPLC-grade;
6.2.3 Urea, p.a., 46,6 % of total nitrogen;

6.2.4 Methylen-diurea (MDU), synthesized and purified by a special laboratory, 42,4 % of total nitrogen;

6.2.5 Dimethylen-triurea (DMTU), synthesized and purified by a special laboratory, 41,2 % of total nitrogen;

6.2.6 Trimethylen-tetraurea (TMTU), synthesized and purified by a special laboratory, 40,6 % of total

nitrogen.
6.3 Apparatus

6.3.1 Laboratory equipment and glassware, for preparation of solutions and dilutions;

6.3.2 Analytical balance, capable for weighing to an accuracy of ± 0,1 mg;
6.3.3 Technical balance, capable for weighing to an accuracy of ± 0,01 g;
6.3.4 HPLC-system, equipped with an UV-detector;
6.3.5 Ultrasonic bath;
6.3.6 Magnetic stirrer;
6.3.7 Disposable filter, 0,45 µm.
6.4 Procedure
6.4.1 System parameters of HPLC
Analytical/separating column NH column, 5 µm, 250 mm × 4,6 mm
A guard-column is recommended.
Detection wavelength 195 nm (Diode Array detector)
Eluent acetonitrile:water 85/15 (volume fraction)
Flow rate 1 ml/min
Temperature 60 °C
Run time 30 min
Injection volume 20 µl
2) e.g. Supelcosil LC-NH or equivalent.
---------------------- Page: 10 ----------------------
SIST-TS CEN/TS 15705:2009
CEN/TS 15705:2009 (E)
6.4.2 Calibration
6.4.2.1 Stock solution of urea, ρ ≈ 1 000 mg/kg

Weigh (6.3.2) 100/R mg of urea (6.2.3), where R is the purity of urea, to the nearest 0,1 mg and put into an

empty and dry 100 ml volumetric fl
...

SLOVENSKI STANDARD
kSIST-TS prCEN/TS 15705:2008
01-oktober-2008
*QRMLOD'RORþHYDQMHNRQGHQ]DWRYVHþQLQHVWHNRþLQVNRNURPDWRJUDILMRYLVRNH

ORþOMLYRVWL +3/& ,]REXWLOLGHQGLXUHDLQNURWRQLOLGHQGLXUHD PHWRGD$ LQROLJRPHUL

PHWLOHQXUHH PHWRGD%
Fertilizers - Determination of urea condensates using high-performance liquid

chromatography (HPLC) - Isobutylidenediurea and crotonylidenediurea (method A) and

methylen-urea oligomers (method B)
Düngemittel - Bestimmung von Harnstoffkondensaten mit Hochleistungs-

Flüssigchromatographie (HPLC) - Isobutylidendiharnstoff und Crotonylidendiharnstoff

(Verfahren A) und Methylenharnstoff-Oligomere (Verfahren B)

Engrais - Détermination de condensates d'urea par chromatographie liquide à haute

performance (CLHP) - Isobutylidenediurea et crotonylidenediurea (méthode A) et
methyleneurea-oligomeres (méthode B)
Ta slovenski standard je istoveten z: prCEN/TS 15705
ICS:
65.080 Gnojila Fertilizers
kSIST-TS prCEN/TS 15705:2008 en,fr,de

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

---------------------- Page: 1 ----------------------
kSIST-TS prCEN/TS 15705:2008
---------------------- Page: 2 ----------------------
kSIST-TS prCEN/TS 15705:2008
TECHNICAL SPECIFICATION
FINAL DRAFT
prCEN/TS 15705
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2008
ICS 65.080
English Version
Fertilizers - Determination of urea condensates using high-
performance liquid chromatography (HPLC) -
Isobutylidenediurea and crotonylidenediurea (method A) and
methylen-urea oligomers (method B)

Engrais - Dosage des condensats d'urée par Düngemittel - Bestimmung von Harnstoffkondensaten mit

chromatographie liquide haute performance (CLHP) - Hochleistungs-Flüssigchromatographie (HPLC) -

Isobutylidène diurée et crotonylidène diurée (méthode A) et Isobutylidendiharnstoff und Crotonylidendiharnstoff

oligomères de méthylène-urée (méthode B) (Verfahren A) und Methylenharnstoff-Oligomere (Verfahren

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

Committee CEN/TC 260.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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.

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
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. prCEN/TS 15705:2008: E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
Contents Page

Foreword..............................................................................................................................................................3

1 Scope ......................................................................................................................................................4

2 Normative references ............................................................................................................................4

3 Terms and definitions ...........................................................................................................................4

4 Sampling and sample preparation.......................................................................................................4

5 Method A: Determination of CDU and IBDU .......................................................................................4

6 Method B: Determination of methylen-urea oligomers (MU) ............................................................7

7 Precision method A and method B....................................................................................................12

8 Test report ............................................................................................................................................13

Annex A (informative) Results of the inter-laboratory tests .........................................................................14

Annex B (informative) Chromatogram and calibration curves method A ...................................................16

Annex C (informative) Chromatogram and calibration curves method B ...................................................18

Bibliography ......................................................................................................................................................21

---------------------- Page: 4 ----------------------
kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
Foreword

This document (prCEN/TS 15705:2008) 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 TC Approval.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.
---------------------- Page: 5 ----------------------
kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
1 Scope

This document specifies methods for the determination of isobutylidenediurea (IBDU), crotonylidenediurea

(CDU) (method A) and methylene-urea oligomers (MU) (method B) in fertilizers using high-performance liquid

chromatography (HPLC).

The method is applicable for all fertilizers which do not contain interfering organic compounds.

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 1482-2, Fertilizers and liming materials — Sampling and sample preparation — Part 2: Sample

preparation

EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987)

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

ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method

for the determination of repeatability and reproducibility of a standard measurement method

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.
4 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.
5 Method A: Determination of CDU and IBDU
5.1 Principle

The sample is extracted with water and, after appropriate dilution, analyzed using a suitable HPLC system.

---------------------- Page: 6 ----------------------
kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
5.2 Reagents
5.2.1 General

Use only reagents of recognized analytical grade and distilled or demineralized water (grade 3 according to

EN ISO 3696:1995).
5.2.2 Acetonitrile, p.a., HPLC-grade.
5.2.3 Isobutylidenediurea and crotonylidenediurea, in their pure form.
5.3 Apparatus

5.3.1 Laboratory equipment and glassware, for preparation of solutions and dilutions.

5.3.2 Analytical balance, capable for weighing to an accuracy of ± 0,1 mg.
5.3.3 HPLC-system, with UV-detector.
5.4 Procedure
5.4.1 System parameters of HPLC
Analytical/separating column: silica column with C18 reverse phase
Detection wavelength: 200 nm
Eluent: acetonitrile/water: 10/90 (volume fraction)
Flow rate: 1 ml/min
Temperature: ambient temperature
Injection volume: 20 µl
5.4.2 Calibration
5.4.2.1 Stock solution IBDU ρ(IBDU) = 100 mg/l

Weigh 100/R mg of IBDU, where R is the purity of IBDU, into a 1 000 ml flask and add about 900 ml of water

(5.2.1). Dissolve in an ultrasonic bath for about 10 min, followed by stirring on a magnetic stirrer for about 1 h.

Make up to volume. Filtration is not necessary.
5.4.2.2 Stock solution CDU ρ(CDU) = 100 mg/l

Weigh 100/R mg of CDU, where R is the purity of CDU, into a 1 000 ml flask and add about 900 ml of water

(5.2.1). Dissolve in an ultrasonic bath for about 10 min, followed by stirring on a magnetic stirrer for about 1 h.

Make up to volume. Filtration is not necessary.
E.g. LiChrosorb RP-18 7µm 250/4 mm or equivalent.
---------------------- Page: 7 ----------------------
kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
5.4.2.3 Calibration solution

For calibration, prepare three solutions according to Table 1 using one-mark (bulb) pipettes and dilute to the

mark with water (5.2.1).

For the determination of the retention time, dilute 10 ml of the stock solution 5.4.2.1 or respectively 5.4.2.2 into

two 100 ml flasks and make up to volume with water (5.2.1).

The evaluation of calibration is carried out manually or by means of a suitable PC-aided (computerized)

calculation method.
Table 1 — Preparation of calibration solutions
Amount of stock
solution
Content of IBDU Content of CDU
Parameter
IBDU/CDU
mg/l mg/l
(to be added to the
100 ml flask)
Standard1
10 10,0 10,0
Standard 2
25 25,0 25,0
Standard 3
50 50,0 50,0
5.4.3 Preparation of the test portion

Weigh 1 g of the sample grounded to < 0,2 mm to the nearest 0,1 mg and flush into a 1 000 ml volumetric

flask with water (5.2.1). Fill the flask to an amount of approximately 900 ml and treat it for 10 min in the

ultrasonic bath. Then make up to the mark and stir for 1 h at room temperature on a magnetic stirrer. Dilute

10 ml of the solution in a 100 ml volumetric flask and filter into the HPLC injection vial through a 0,45 µm

disposable filter.
5.4.4 Measurement

Measurement is performed manually or by means of an automatic sample loading system (autosampler).

5.4.5 Important annotations

IBDU is able to form urea in aqueous solution. Therefore, the measurement of the calibration- and sample

solutions shall be completed within one working day.

The concentrations of CDU and IBDU in the sample solutions shall be kept within the calibration limits (5.4.2)

to ensure sufficient reproducibility.
5.5 Calculation

The calculation can be performed manually or by means of a PC using the calibration parameters in respect to

the amount used.

In the case of PC-aided (computerized) calculation and application of Table 1 regarding the amounts of stock

solution, the content of IBDU/CDU in milligrams per litre will be calculated by the system. The calculated

values are equal to the percentage mass concentration of IBDU/CDU in the analysed sample of fertilizer.

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kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)

Following general rules for declaration in regulations to declare the content of the compounds as percentage

mass fraction of nitrogen, calculate the contents, w /w in percent (g/100 g), according to the

N(IBDU) N(CDU)
following equations:
w = w × 0,322 (1)
N IBDU
(IBDU)
w = w × 0,326 (2)
N(CDU) CDU
where

0,322 is the conversation factor for the content of IBDU in the fertilizer into nitrogen content;

0,326 is the conversation factor for the content of CDU in the fertilizer into nitrogen content.

6 Method B: Determination of methylen-urea oligomers (MU)

NOTE By the condensation of urea and formaldehyde developed no only one compound, as it is by the reaction of

urea and crotonaldehyde or isobutylaldehyde, but oligomers like methylen–diurea (MDU), dimethylen-triurea (DMTU),

trimethylen-tetraurea (TMTU) and higher oligomers. These three molecules are the most soluble in water, the higher

compounds are insoluble in hot water, but their nitrogen is available for plants by microbiological decomposition. Also urea

is always a companion of MU – oligomers.
6.1 Principle

The sample is extracted with boiling water and analyzed using a suitable HPLC system.

The methylen-urea soluble oligomers are measured and detected by the HPLC-method.

In the HPLC-diagram methylen-urea oligomers are represented by different peaks: urea, methylen-diurea,

dimethylen-triurea; trimethylen-tetraurea are, in the mean time, the most soluble and important.

6.2 Reagents
6.2.1 General

Use only reagents of recognized analytical grade and distilled or demineralized water (grade 3 according to

EN ISO 3696:1995).
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kSIST-TS prCEN/TS 15705:2008
prCEN/TS 15705:2008 (E)
6.2.2 Acetonitrile, p.a., HPLC-grade.
6.2.3 Urea, p.a., 46,6 % of total nitrogen.

6.2.4 Methylen-diurea (MDU), synthesized and purified by a special laboratory, 42,4 % of total nitrogen.

6.2.5 Dimethylen-triurea (DMTU), synthesized and purified by a special laboratory, 41,2 % of total nitrogen.

6.2.6 Trimethylen-tetraurea (TMTU), synthesized and purified by a special laboratory, 40,6 % of total

nitrogen.
6.3 Apparatus

6.3.1 Laboratory equipment and glassware, for preparation of solutions and dilutions.

6.3.2 Analytical balance, capable for weighing to an accuracy of ± 0,1 mg.
6.3.3 Technical balance, capable for weighing to an accuracy of ± 0,01 g.
6.3.4 HPLC-system, equipped with an UV-detector.
A guard-column is recommended.
6.4 Procedure
6.4.1 System parameters of HPLC
Analytical/separating column NH column, 5 µm, 250 mm × 4,6 mm
Detection wavelength 195 nm (Diode Array detector)
Eluent acetonitrile:water 85/15 (volume fraction)
Flow rate 1 ml/min
Temperature 60 °C
Run time 30 min
Injection volume 20 µl
6.4.2 Calibration
6.4.2.1 Stock solution of urea, ρ ≈ 1 000 mg/kg

Weigh 100/R mg of urea (6.2.3), where R is the purity of urea, to the nearest 0,1 mg and put into an empty and

dry 100 ml volumetric flask, weighed before to the nearest 0,01 g. Add 50 ml of water (6.2.1) and dissolve the

urea in an ultrasonic bath for about 10 min. Make up approximately to the mark with water (6.2.1) and

homogenize. Weigh the full flask to the nearest 0,01 g and record the net weight. Store at room temperature,

well closed. This stock solution is stable for one week.
2) Supelcosil LC-NH or equivalent.
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