The Specialty Liquid Fertilizer PeopleTM

2. AOAC Collaborative Study Liquid Chromatographic Determination of Urea in Water-Soluble Urea-Formaldehyde Fertilizer Products and in Aqueous Urea Solutions: Collaborative Study. 2003-14 (First Action 2003) 346 HOJJATIE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 2, 2004 AGRICULTURAL MATERIALS MICHAEL M. HOJJATIE, DEAN E. ABRAMS, and THOMAS M. PARHAM, JR Tessenderlo Kerley, Inc., R&D Laboratories, 2480 W. Twin Buttes, Sahuarita, AZ 85629 Collaborators: J. Balthrop; R. Beine; V. Dickinson; J. Hartshorn; S. Herald; G. Latimer; J. Padmore; S. Pleasants; K. Riter; R. Roser; G. Schmunck; R. Sensmeier; V. Smith; L. Taylor; G. Volgas

3. Slow Release Nitrogen Fertilizers Types of SRN containing Urea: Solid Products Sulfur Coated, Polymer Coated, Absorbed Liquid Products Triazone (N-Sure), CoRoN, Nitamine

4. 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 1 Official Methods of Analysis (2000) 17th Ed., AOAC INTER NATIONAL, Gaithersburg, MD, Methods 959.03 and 983.01

5. Difficulties with Existing Methods for Liquid Urea-Formaldehyde Fertilizer Products A Typical HPLC Chromatogram of Liquid

6. 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

7. Hydrolysis of Various Amount of Urea by Selected Sources of Urease Enzyme

8. Urease Method Results Often Not Reproducible Type of Urease is important (powder, Tablet, Liquid form) Urease activities varies with different sources

9. 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

10. Comparison of HPLC Chromatograms

11. Urea Formaldehyde Condensate Product

12. Urea Formaldehyde Condensate Product Acidic Conditions

13. Triazone Formation In presence of Ammonia:

14. Reaction Pathways Proposed Mechanism:

15. Triazone AAPFCO Definition (N-33, Official 1989) Triazone is a water soluble compound of formula C5H11N5O2 [5-(N-methyl)-urea-1,3, 5- triazin-2-one or 5-methyleneureido-2- oxohexahydro-s-triazine] which contains at least 40% total Nitrogen. Triazone is a Slow or Controlled Release Nitrogen Fertilizer AAPFCO Definition (T-29, Official 1985) Slow or controlled release fertilizer means a fertilizer containing a plant nutrient in a form which delays its availability for plant uptake and use after application, or which extends its availability to the plant significantly longer than a referenced “rapidly available nutrient fertilizer” such as ammonium nitrate or urea Ammonium phosphate or KCl.

16. Slow Release N Triazone

17. Triazone Triazone (N-Sure®) vs. Urea

18. SRN Determination N-SURE 28-0-0 (72% SRN claimed) FORMOLENE-PLUS 30-0-0 (60% SRN claimed) N-SURE-PRO 30-0-0 0-0-0 (50% SRN claimed) CoRoN 28-0-0 (70% SRN claimed) NITRO-30 30-0-0 (85% SRN claimed) * Not determined by urease

19. AOAC Official Method 2003.14Liquid ChromatographyFirst 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 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

20. HPLC Chromatograms of Urea Standard and Compound

21. 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

22. Calculation Urea Factor is determined from Standard Urea Peak area average (at least two injections) (UF = Average Area/Weight of Standard X 0.01) Amount of Urea in the sample is determined from Peak area of Urea and the Urea Factor (UF) (% Urea in the sample = area of the Urea peak / weight / UF) Amount of N from Urea is calculated (% Urea X 0.4665) = N from Urea Total Nitrogen is determined by Block Digestion-Titration- Kjeldhal SRN = Total N – Urea N

23. Calculation Cont. MMU, DMU, and HMTA also is determined from their standards peak area factor (Factor = area/wt x 0.01)

24. Example

25. Urease Method and HPLC Method 2003-14 Comparison

26. Conclusion HPLC Method 2003-14 Provides more accurate measurement of Free Urea in Urea-Formaldehyde Liquid Fertilizers and in Aqueous Urea Solutions over the previous AOAC Methods. It provides more accurate reporting of the SRN in these products.