Determination of SRN in Liquid (water soluble)

Urea-Formaldehyde Fertilizers

Method Forum
February 24, 2016
Albuquerque, New Mexico
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 SRN Fertilizers

Slow Release Fertilizers (SRN):

Slow Release Fertilizers ‐are solid or liquid fertilizer products that release (convert to 
a plant available form) their nutrients at a slower rate relative to a “reference 
soluble” product. This may be accomplished by biodegradation and/or by limited 
solubility and/or by hydrolysis or other recognized chemical or biochemical means. 
Some examples include solid fertilizers such as: methylene urea (MU), Magnesium 
Potassium Phosphate and bio‐solids, and liquid fertilizer such as triazone. The 
product may provide longevity claims when compared to an appropriate reference 
material.

Solid: IBDU, CDU, Nutralene

Liquid: N-Sure, NBN 30, Clear N, Last N

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 Manufacturers of Liquid Water soluble SRN Fertilizers
Helena
Koch
Yara
Tessenderlo Kerley
Arclin
Advachem
Sadepan
Agrium

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 Production

• From Urea, Formaldehyde, Formalin (or UFC)

• Or from Urea, Formaldehyde, Formalin (or UFC) and
ammonia.
• under controlled conditions to form a liquid urea-
formaldehyde condensate product.

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 Urea Formaldehyde Condensate Reaction

• Base Catalyzed

O O
Base

H2N NH2 + H H
Urea

O O

H2N NH CH2OH + CH2OH HN NH CH2OH

DMU
MMU

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 Urea Formaldehyde Condensate Reaction

• Acid Catalyzed

O O Acid
+
H2N NH2 H H

O O O O O

+
H2N NH HN NH2 H2N NH HN NH HN NH2
CH2 CH2 CH2

MDU
DMTU

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Urea + Formaldehyde + Ammonia (gas or aqua)

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HPLC Chromatogram

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 (28-0-0) UF Liquid product Composition?
• Monomethylol urea (MMU) ≈ 2%
• Dimethylol urea (DMU) ≈ 0.3%
• Methylene diurea (MDU) ≈ 1.00%
• Biuret ≈ 0.02-0.3%
• HMT ≈ 1-2%
• Free Urea 14-15%

Overall, 22% of N is from urea & 78% of N is from SRN

The minor components are about 5-8%

70% of the composition is a major component

What is this major component?

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1H‐NMR Spectra in DMSO
D2O Added H‐NMR Spectra
13C‐NMR Spectra in DMSO
Major component of crystal.  Green is unaccounted for electron density (error in structure)
Proposed Mechanism for the Formation of the Major Component

O
Urea
+ Formaldehyde + Ammonia
H 2N NH 2

O
H N N H
O + Ureaforms
H N N H +
N N-Sure
H 2N NH 2
N
N C NH 2
CH 2OH
H O

5-Hydroxymethyltriazine Urea

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 Slow Release Pattern

15
 SRN Determination

• SRN is coming from cyclic compound or chained

compounds (MMU, DMU, etc).
• Free Urea does not provide SRN
• To accurately report the SRN one has to determine
the Free Urea accurately.
• SRN is determined by subtracting the total
Nitrogen present from the amount of free Urea

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 Aqueous Urea-Formaldehyde Fertilizer Products

Each of existing products in the market claim certain

Percent of SRN from 50-80%.

Existing Method of Analyses

• AOAC Methods 959.03 (Urease, 1960)

• AOAC Method 983.01 (Liquid Chromatography, 1984)1
• AOAC Method 2003.14 (Liquid Chromatography, 2008)

1 Official Methods of Analysis (2000) 17th Ed., AOAC INTER NATIONAL, Gaithersburg, MD, Methods 959.03 and 983.01

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 Test products
1. LF3060 (30‐0‐0), Koch Agronomic Services, LLC, 4111 E. 37th St. N, Wichita, KS 67220, USA
2. CoRoN (28‐0‐0), Helena Chemical Company, 225 Schilling Blvd., Collierville, TN 38017
3. Arclin 28‐0‐0, Arclin Resins, Research & Technology, 4754 28th St., Springfield, OR 97477
4. Greenfeed 27‐0‐0 (light Green liquid), Plant Food Company, 38 Hightstown‐Cranbury Station Road,
Cranbury, NJ 08512
5. Greenfeed 27‐0‐0 (Dark Green liquid), Plant Food Company, 38 Hightstown‐Cranbury Station Road,
Cranbury, NJ 08512
6. NDemand 30‐0‐0, Wilbur‐Ellis Agribusiness, 3300 S. Parker Road, Suite 500, Aurora, CO 80014,
USA
7. NDemand 30L, Wilbur‐Ellis Agribusiness, 3300 S. Parker Road, Suite 500, Aurora, CO 80014, USA
8. Gradual N (30‐0‐0), Winfield Solutions, LLC, 1080 County Road West, Shoreview, MN 55126
9. N‐28 clear Fertilizer (28‐0‐0), Advachem, Route de Wallonie, Darse d’Hautrage, BE 7334 Hautrage
10. N‐Sure (28‐0‐0), Tessenderlo Kerley, Inc., 2255 N. 44th St., Suite 300, Phoenix, AZ
11. Trisert NB (26‐0‐0), Tessenderlo Kerley, Inc., 2255 N. 44th St., Suite 300, Phoenix, AZ
12. Formolene Plus (30‐0‐0), Tessenderlo Kerley, Inc., 2255 N. 44th St., Suite 300, Phoenix, AZ

18
 Analyses of Twelve Fertilizer Samples for Urea by AOAC Method 2003.14
 
Fertilizer Lab ID Total –N* % Urea‐N
Sample Mean Std. dev.
Lab 1 Lab 2
1  26‐068‐03 29.89 12.04 12.45 12.25  0.21
2  26‐051‐01 28.54 8.63 8.51 8.57  0.06
3  26‐068‐02 28.44 8.68 8.57 8.63  0.05
4  26‐076‐07 28.33 8.45 8.45 8.45  0.00
5  26‐076‐06  28.47 8.58 8.60 8.59  0.01 
6  26‐076‐08  24.95 16.08 15.70 15.89  0.19 
7  26‐079‐01 30.78 12.55 12.70 12.63  0.07
8  26‐086‐04  30.06 12.56 12.07 12.32  0.25 
9  26‐103‐02  28.01 12.09 11.36 11.73  0.37 
10  26‐103‐03 28.13 6.85 6.83 6.84  0.01
11  26‐100‐03 26.36 15.55 15.28 15.42  0.14
12  26‐010‐02  29.87 9.37 9.51 9.44  0.07 
Average  X  10.95  10.84  ‐  ‐ 
Standard Deviation  σx 2.97  2.87  ‐  ‐ 
Number of samples  n  12 12 ‐  ‐ 
Variance  σ2 8.80  8.21  ‐  ‐ 
Pearson Correlation 0.99  ‐ ‐ ‐  ‐ 
T Stat  1.30 ‐ ‐ ‐  ‐ 
P(T<t) one‐tail  0.11 ‐ ‐ ‐  ‐ 
t Critical one‐tail 1.80  ‐ ‐ ‐  ‐ 
P(T<=t) two‐tail 0.22  ‐ ‐ ‐  ‐ 
T Critical two‐tail 2.20 ‐ ‐ ‐  ‐ 
         *Note: Total‐N includes other forms of nitrogen in addition to unreacted (free) urea 

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 Schematically comparisons of HPLC Results by two Labs
18
Comparison of Urea analysis by Labs 1 & 2
16

14 Lab 1
Lab 2
12
% Urea‐N

10

4
0 2 4 6 8 10 12
Sample #

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 Urease Analyses of Twelve Fertilizer Samples & Comparisons with the HPLC Results for Urea
      % Urea‐N  % Urea‐N     % Urea‐N 
HPLC Method  Difference 
Fertilizer  Urease Method 
Total‐N   AOAC 2003.14  Urease‐HPLC 
Sample  AOAC 959.03 
 (Means from two Labs)  (Mean) 
29.89  1  13.26  12.25  +1.02 
28.54  2  9.87  8.57  +1.30 
28.44  3  10.06  8.63  +1.44 
28.33  4  9.33  8.45  +0.88 
28.47  5  9.91  8.59  +1.32 
24.95  6  15.44  15.89  ‐0.45 
30.78  7  14.81 12.63  +2.19 
30.06  8  14.08  12.32  +1.77 
28.01  9  8.89  11.37  ‐2.48 
28.13  10  8.94  6.84  +2.10 
26.36  11  16.18  15.42  +0.77 
29.87  12  11.86  9.44  +2.42 
 

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 Comparisons of the Results by HPLC (2003‐14) and by Urease
Method (Powdered Sample) 
SRN Determination
Product % %SRN
Total N Claimed Method
HPLC (2003-14) Urease
28-0-0 72 72  88 
30-0-0 60 60  77 
30-0-0 50 50  68 
28-0-0 75 50  70 
30-0-0 85 46  80 
 

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 Difficulties with Existing Methods
Urease Method Problems

The Urease method is the method most commonly used in both industry
and regulatory laboratories for urea in all types of fertilizers.
However, for UF-based fertilizers, one or more of the UF-derived
components inhibit the action of Urease enzyme. Method 959.03 is
dependent upon this quantitative action.

Method Condition:
• 20 ml of 1% Urease Solution
• Urease activity should at least hydrolyze 0.1 g of Urea

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 Analyses of Urea using Different Sources of Urease

Urease % Hydrolysis of urea from the amount used

Urease Amount
Source
0.1 g 0.3 g 0.6 g 1.0 g

1 20 ml (1% Sol.) 100.1 100.1 99.7 99.9

2 10 ml Glycerol Ext. 99.9 99.7 99.7 69.9
2 20 ml Glycerol Ext. 99.9 99.7 99.7 99.4
3 20 ml (1% Sol.) 99.6 99.7 97.6 62.7
4 20 ml (1% Sol.) 99.3 99.7 83.2 52.6
5 2 tablets 100 98.6 69.3 45.5
6 20 ml Glycerol Ext. 99.9 99.9 99.7 99.5
7 20 ml (1% Sol.) 100.3 100.1 99.9 99.6
 

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AOAC 959.03 (Urease Method) vs. AOAC 2003.14 (HPLC) Results for Analyses of Unreacted Urea in 
Urea‐Triazone Liquid Fertilizers 
amount used Sample Urease vs
Urease HPLC Urease HPLC
HPLC
1 A 0.20 0.34 20.2 33.7 60
20ml 1% Solution B 0.11 0.16 10.9 16.1 68
C 0.24 0.32 23.5 32.1 73
D 0.27 0.35 27.3 35.1 78
2 A 0.17 0.34 16.9 33.7 50
10ml Glycerol Ext. B 0.08 0.16 7.5 16.1 47
C 0.14 0.32 14.2 32.1 44
D 0.25 0.35 25.4 35.1 72
2 A 0.34 0.34 34.3 33.7 102
20ml Glycerol Ext. B 0.17 0.16 17.0 16.1 106
C 0.32 0.32 32.4 32.1 101
D 0.33 0.35 33.4 35.1 95
3 A 0.15 0.34 15.0 33.7 45
20ml 1% Solution B 0.10 0.16 9.5 16.1 59
C 0.18 0.32 17.8 32.1 55
D 0.32 0.35 32.4 35.1 92
4 A 0.18 0.34 18.1 33.7 54
20ml 1% Solution B 0.11 0.16 11.0 16.1 68
C 0.18 0.32 18.4 32.1 57
D 0.24 0.35 23.5 35.1 67
5 A 0.17 0.16 6.9 16.1 43
Two Tablets B 0.13 0.32 13.0 32.1 40
6 B 0.17 0.16 16.9 16.1 105
10 ml Glycerol Ext. C 0.33 0.32 33.1 32.1 103
6 B 0.17 0.16 17.1 16.1 106
20 ml Glycerol Ext. C 0.33 0.32 33.2 32.1 103
7 A 0.34 0.34 34.0 33.7 101
20ml 1% Solution B 0.16 0.16 16.2 16.1 101
C 0.32 0.32 32.3 32.1 101
D 0.33 0.35 33.0 35.1 94
 

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 Urease Method Results

• Often Not Reproducible

• Type of Urease is important (powder, Tablet,
Liquid form)
• Urease activities varies with different
sources
• Interference from ureaform components

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 AOAC HPLC Method 983.01
Comparisons of results for sample #10, #11, and #12 by two HPLC Methods
Sample # 10  Sample # 11  Sample #12 
(28‐0‐0)  (26‐0‐0)  (30‐0‐0) 

% Free Urea 
(AOAC 2003.14)  (AOAC 983.1) 

Lab 1   Lab 2   Lab 3  

13.6  13.9  15.8 
30.5  30.3  30.2 
21.9  21.7  23.7 
 

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 AOAC HPLC Method 983.01

Method 983.01 is not reliable for determination of urea in

the cited class of fertilizers because in most, if not all,
cases, there is co-elution of a non-urea peak with
The urea peak.
Mobile Phase: Water
Column: ODS
Detector: Refractive Index

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 Comparisons of the analytical results for Grade 26‐0‐0 by AOAC 983.1 & AOA 2003.14

Samples  %  %  %  % 
Average
Sample ID  Analyzed by  Grade  Biuret  Biuret  Biuret  Biuret  STDEV 
% Biuret 
Lab‐1  Test 1  Test 2  Test 3  Test 4 

By 2003‐14 Method 
25‐012‐05  Sulfur coated urea 39‐0‐0  0.911  0.943  0.979  0.933  0.942 0.025
25‐068‐06  Triazone Fertilizer 26‐0‐0  0.118  0.124  0.131  0.139  0.128 0.008
25‐070‐01  Fertilizer “A”  15‐15‐15  0.668  0.689  0.762  0.708  0.707 0.035
25‐070‐02  Fertilizer “B”  25‐11‐10  0.296  0.356  0.440  0.416  0.377 0.056

By 983.1 Method 
25‐012‐05  Sulfur coated urea 39‐0‐0  0.795  0.795  0.794  0.791  0.794 0.002
25‐068‐06  Triazone Fertilizer 26‐0‐0  5.793  5.854  5.856  5.814  5.829 0.027
25‐070‐01  Fertilizer “A”  15‐15‐15  0.560  0.560  0.557  0.555  0.558 0.002
25‐070‐02  Fertilizer “B”  25‐11‐10  0.299  0.302  0.303  0.300  0.301 0.002

By SRIC* Method (same as 2003‐14) 
25‐012‐05  Sulfur coated urea 39‐0‐0  0.934  0.965  0.987  0.974  0.965 0.019
25‐068‐06  Triazone Fertilizer 26‐0‐0  0.120  0.131  0.130  0.134  0.129 0.005
25‐070‐01  Fertilizer “A”  15‐15‐15  0.663  0.711  0.734  0.727  0.709 0.028
25‐070‐02  Fertilizer “B”  25‐11‐10  0.286  0.367  0.388  0.382  0.356 0.041
*
Shanghai Research Institute of Chemical Industry Testing Center 

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 AOAC Official Method 2003.14
Liquid Chromatography
First Action 2003, Final Action 2008

HPLC Method

• UV absorption detector 195 nm

• Mobile Phase: 85% Acetonitrile, 15% water
• Flow rate: 1.30 ml/min
• Mobile phase temp: Ambient
• Column temperature: 30-35 oC
• Injection volume: 10 l
• HPLC Column – A 250 x 4.6 mm amine (NH2) column with 5 packing is
used for this separation We obtained the best results using Phenomenex .
• External Urea, Biuret, MMU, DMU, and HMTA Standards

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Conclusions
The AOAC Official Methods 959.03 (Urease Method) and 983.01 (HPLC
Method) are frequently inaccurate and therefore not suitable for
measuring the amounts of free and unreacted urea in liquid urea‐
formaldehyde condensate products. AOAC Official Method 2003.14
consistently provides more accurate analytical results for measuring the
contents of free urea in these classes of fertilizers.
The results have been accepted as a manuscript for the publication in
the Journal of AOAC International and will appear in the next issue.

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