Hojjatie - AAPFCO

The Specialty Liquid Fertilizer People TM
Michael Hojjatie, Ph.D.

Biuret Determination: A Collaborative (Or SLV) Studies
Proposal
AAPFCO/Method Forum
San Antonio, TX
February 22-23, 2012
• Proposal:
Liquid Chromatographic Determination of Biuret in Water-Soluble
Urea-Formaldehyde Fertilizer Products, Urea Based Fertilizer
Products and in Aqueous Urea Solutions

Biuret
Biuret is a white solid chemical compound
with the chemical formula:
It is the result of condensation of two molecules of
urea. This white solid is soluble in hot water.
Solubility in cold water 2g/100 ml @ 25 o C.

Biuret
‣ It is formed during urea production upon
heating urea at 80 Degree C and higher,
especially above its melting point
according due to the following reaction
release of ammonia:
2 CO(NH 2 ) 2 → H 2 N-CO-NH-CO-NH 2 + NH 3 ↑

Urea Reaction Possible Products

Biuret In Urea Fertilizers
• Urea has become the leading form of N fertilizers Worldwide.
• Historically there has been concern about level of biuret in urea.
• The potential adverse effect of biuret on plant growth is a concern.
• Biuret is phytotoxic.
• Biuret is mineralized by soil microorganisms at much lower rate
then urea.
• Biuret can damage seedlings.
• Foliar application of high biuret urea causes phytotoxicity.
• Crop tolerance to biuret varies according to the plant species, soil
conditions, fertilizer placement, and method of application.
• Biuret applied to soil or to plant foliage interferes with N
metabolism and protein synthesis.

Adverse Effect of Biuret
• A foliar application of urea prepared from prilled urea applied
to young pineapple and citrus plants caused leaf injury due to
biuret present in urea (G. Webster, et.al., Plant Physiol. 32 (1),
60-61(1957).
• Biuret: A form of nitrogen toxic to citrus and avocado
leaves(A. Haas, et.al., Plant Physiol. 34, 11-12 (1954).
• Biuret toxicity of urea foliage spray on citrus (W. Jones,
Science, 120, 499-500 (1954).
• Toxicity to pineapple plants of biuret found in urea fertilizers
from different sources (W. G. Sanford, et.al., Science, 120,
349-350 (1954).

Urea Nitrogen Fertilizers
Urea
UAN
Slow Release fertilizers containing Urea:
• Solid Products
Sulfur Coated urea , Polymer Coated urea , Absorbed urea
• Liquid Products (Urea based fertilizers)
Triazone (N-Sure), CoRoN, Nitamine

C of A of Urea (Source 1)

Urea Spec Sheet (source 2)

Recommended Biuret Level
Plant Physiologist recommend maximum of 0.3%
biuret in urea based fertilizers for plants and crops
safe applications.

Current Methods Available for Biuret Analyses
AOAC 960.04 (1980) for biuret in urea
Tartrate salt (NaKC 4 H 4 O 6 .4H 2 O), copper sulfate, biuret standard,
spectrophotometer.
AOAC 976.01 (1980) for biuret in fertilizers
Based on copper complex and determination by AAs in
combination with AOAC 960.04.
Biuret determination by ion exchange chromatography and
ninhydrin (J. Anim.Sci, 1967)
Amino acid analyzer, sulfonated styrene resin, ninhydrin.
UV-vis determination of biuret in hydroquinone-ureacontaining
fertilizers (Nut. Cycl. In Agrosys., 1963)
Based on copper colorimetric method.
HPLC Method (Anal.Chem, 1982)
Reversed phase HPLC in combination with copper compelexation.

‣ It is based on HPLC
Proposed Method
‣ Based on analyses of average of 5 samples/day for 20
years internally and other Labs.
‣ UV detection.
‣ Acetonitrile/water mobile phase (no buffer)
‣ Except for mobile phase no other solvents required.
‣ It can be done in 10 minutes.
‣ Low chain urea-forms could be quantified
simultaneously.

AOAC Official Method 2003.14
Liquid Chromatography
First Action 2003
HPLC Method
UV absorption detector 195 nm
Mobile Phase: 15% Acetonitrile, 85% water
Flow rate: 1.30 ml/min
Mobile phase temp: Ambient
Column temperature: 30-35 o C
Injection volume: 10 µl
HPLC Column – A 250 x 4.6 mm amine (NH2) column
with 5µ packing is used for this separation We
obtained the best results using Phenomenex amine
column.
External or standard curves be used (Urea, Biuret,
MMU, DMU, and HMTA Standards).

Biuret standard
• Like any other analytical method, a good standard is
necessary.
• Laboratory Chemical vendors (i.e., Aldrich) sells 97-98%
biuret.
• Commercial samples of biuret are contaminated with
organic impurities.
• Special care must be taken to purify biuret by repeated
crystallization and drying.

98% ALDRICH BIURET

COMMERCIAL UREA

Biuret Standard
• This was prepared in house from repeated crystallization of
commercially available sample (Aldrich 97%):
195-196 o C (Aldrich sample 185-190 o C).
• Elemental Anal :
• %Theoretical: (C, 23.30; H, 4.85; N, 40.77)
• % Found: (C, 23.36; H, 4.84; N, 40.87)
• MP:

BIURET STANDARD

Analyses Biuret
Standard Curve

HPLC of Biuret Standard & Urea

HPLC of Urea

Biuret Analyses by HPLC
# Name Origin Grade % Biuret % Urea
1 Source 1 28-0-0, 70%SRN 0.3 11.73
2 Source 2 30-0-0 0.1 12.23
3 Source 3 28-0-0, 72%SRN 0.1 7.30
4 Source 4 30-0-0, 60%SRN 0.2 11.25
5 Source 5 26-0-0, 33%SRN 0.2 16.92
6 Source 6 29.5-0-0,60%SRN 0.1 8.26
7 Source 7 30-0-0, 50%SRN 0.2 13.90
8 Source 8 26-0-0-0.5B, 33%SRN 0.2 16.11
9 Urea- Source 1 46-0-0 0.78 (0.5-1%)
10 Urea Source 2 46-0-0 1(0.7-1.5)%
11 Urea Source 3 46-0-0 0.4(0.4%)
12 Urea Source 4 46-0-0 0.2 (0.1-0.5%)
13 Urea Source 5 46-0-0 0.7(0.5-1%)

Urea-Form Analyses

Urea Formaldehyde Condensate Product
O
O
Base
H 2 N
Urea
NH 2
+
H
H
O
O
H 2 N
NH
CH 2 OH
+
CH 2 OH
HN
NH
CH 2 OH
DMU

UREA FORMALDEHYDE CONDENSATE
PRODUCT
Acidic Conditions
O
O
Acid
H 2 N
NH 2
+
H
H
O
O
O
O
O
H 2 N
NH
HN
NH 2
+
H 2 N
NH
HN
NH
HN
NH 2
CH 2
CH 2
CH 2
MDU
DMTU

MMU Standard Curve

MDU Standard Curve

DMU Standard Curve

HMT Standard Curve

Triazone Formation
In presence of Ammonia:
O
O
H 2 N
NH 2
+ +
H
H
NH 3
O
H
N
N
H
N
R
R = H, CH 2 NHCONH 2

Proposed Mechanism:
Reaction Pathways
N
H N H H COH
CH 2 OH N CH 2 OH
H
CH 2 OH
Ammonia
Formaline
Trimethylolamine
N N
N
HMTA
+
O
H 2 N
NH 2
O
O
H
O
H N N H
N N H +
H 2 N NH 2
N
N
CH 2 OH
N C NH 2
H O
5-Hydroxymethyltriazine Urea 5-Methylureatriazone

Urea Based Liquid Fertilizers Analyses

Examples (we have done more than 4000 sample analyses)
Preliminary Report of Analysis
Nitamine N-Sure Formolene-Plus
Total Nitrogen, Wt. %: 29.53 28.1 29.84
Urea Nitrogen, Wt. %: 12.23 6.95 12.17
% of Total Nitrogen from Urea: 41.42 24.7 40.8
SRN, Wt., %: 17.30 21.15 17.67
% of Total Nitrogen From SRN: 58.58 75.3 59.2
Triazone Nitrogen, Wt. %: 9.50 10.1 10.6
% of Total Nitrogen from Triazone: 32.18 35.95 35.52
Biuert, Wt., % 0.10

Examples
Lab Sample ID 23-079-01 23-079-02 23-079-03 Manufactured Specification
Product Name Arc-1 Arc-2 Arc-3 N-SURE
Grade 28-0-0 28-0-0 28-0-0 28-0-0
Source Springfield, OR Springfield, OR Springfield, OR - -
Batch # Formulation # 1 Formulation # 2 Formulation # 3 - -
Sample Date 01/17/2012 01/19/2012 01/19/2012 Desired Limit
Total Nitrogen, (Wt. %) 27.7% 28.0% 28.7% 28.0 – 28.2% 27.6 – 28.4%
Urea Nitrogen, (Wt. %) 8.2% 8.3% 8.2% - -
% of Total Nitrogen from Urea 29.7% 29.5% 28.7% 28 % < 31%
SRN, (Wt. %N) 19.5% 19.8% 20.5%
% of Total Nitrogen from SRN 70.3% 70.5% 71.3% 72 % > 69%
Triazone Nitrogen, (Wt. %) 10.9% 10.1% 10.9% - -
% of Total Nitrogen from Triazone 39.4% 36.0% 38.0% - -
Biuret, (wt %) 0.1 0.1 0.1 0.1 % 0.25
MMU, (wt %) 3.1 1.0 0.9 2.0 % < 4.0 %
MDU, (wt %) 1.4 3.0 2.9 2.0 %
DMU, (wt %) 0.7 0.9 0.9 0.8 % < 2.0 %
MMU + DMU, (wt %) - - - - < 6.0 %
HMT, (wt %) 0.2 0.4 0.9 1.0 % 4.0
Total Soluble K 2 O, (Wt %) 0.8 0.5 0.7 0.6 % -
Specific Gravity, ( 20/20°C) 1.286 1.274 1.287 - -
pH [Full Strength) 10.1 10.6 10.6 9.0 – 10.5 8.5 – 11.0
Appearance Clear, Colorless Clear, Colorless Clear, Colorless Clear, Blue -
Turbidity 5 NTU 3 NTU 3 NTU 5 < 20 NTU
Refractive Index (@ 25°C) 1.477 1.474 1.482 - -

Conclusion
• HPLC Method Provides accurate measurement for biuret in
different urea grades.
• It is simple and fast when standard curve is built (

That’s all Folks