Quantitative HPLC Analysis of N-(n-butyl) Thiophosphoric Triamide

(NBPT) Using UV DetectionPresented By

Brandon SkinnerBarton College

School of SciencesNovember 7th, 2015

Background • Urea is a commonly used nitrogen fertilizer.• Disadvantages of using urea-based fertilizers:• Urea is easily metabolized by soil microorganisms, producing ammonia gas.• Ammonia is volatile, leading to nitrogen loss.• Multiple applications of fertilizer are required.

• Wasteful and time consuming

Background• NBPT• N-(n-Butyl) Thiophosphoric Triamide• Urease inhibitor• Prevents soil microorganisms from converting urea to ammonia• Reduces the amount of nitrogen loss• Eliminates the need for multiple applications of fertilizer

Background• An effective method to determine the concentration of NBPT in a

solution was needed.• Assure customers of the precise concentration of NBPT in their fertilizer

product

• Purpose of this project:• Develop a method to quantify the concentration of NBPT in any given solution

using a high performance liquid chromatograph (HPLC) and UV detection.

Methods• A single blind study was conducted.• Samples with concentrations of NBPT unknown to the analysts were prepared

by Dr. Garnett Whitehurst’s lab.

• Two analytical methods were developed and tested using samples with unknown concentrations of NBPT.• Calibration method• Standard addition method

Preparation of Standards• Approximately 1 gram of NBPT was diluted in 50 mL of DI water to obtain a 20 mg/mL

stock solution.• The solution was sonicated for five to ten minutes.• Calibration method

• Varying amounts of the stock solution were diluted in 25 mL of DI water to create calibration standards with the following concentrations:• 0.080 mg/mL• 0.25 mg/mL• 0.40 mg/mL• 0.60 mg/mL• 1.00 mg/mL

• Standard addition method• 2.5 mL of the stock solution was diluted in 25 mL of DI water to obtain a 2.0 mg/mL standard NBPT

solution.

Preparation of Samples• 6 samples with unknown concentrations of NBPT were prepared.• One blank sample was prepared using urea.• Approximately 1 gram of each sample was diluted in 50 mL of DI

water and sonicated for five minutes.• 1 mL of each diluted sample was diluted further in 25 mL of DI water.

Preparation of Samples• Calibration method• 4 mL of each sample and each calibration standard were pipetted into 4 mL

vials.

• Standard addition method• 3.80 mL of each sample was spiked with 0.2 mL of the 2.00 mg/mL standard

solution.• 4 mL of each sample was pipetted into 4 mL vials.

• Samples were analyzed using HPLC.

Analysis of Samples• HPLC Instrument conditions• Solvents: 10% acetonitrile, 90% HPLC grade water• UV Detector Wavelengths: 203 nm and 210 nm• Injection volume: 10 μl• Run time: 8.50 min/injection

Chromatograms• NBPT peaks were determined using the retention time of NBPT.• Average retention time of NBPT: 5.958 ± 0.073 min• The areas under the NBPT peaks were proportional to the amount of

NBPT in the sample.• Areas under each NBPT peak was calculated and recorded.

Chromatograms

5.921

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

Chromatograms

5.529

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

Data Analysis• Calibration method• A calibration curve was created using the five NBPT calibration standards.• Concentration vs. Area• The equation of the curve was determined by linear regression analysis using

a spreadsheet. • The concentrations of NBPT in the six unknown samples were calculated using

the equation for the calibration curve.

Calibration Curve (203 nm)

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.000

2000000

4000000

6000000

8000000

10000000

12000000

14000000f(x) = 12538201.6932635 x + 605402.175782676R² = 0.993002404767845

Calibration Curve for Concentration of NBPT (203 nm)

[NBPT] / (mg/mL)

Chro

mat

ogra

phic

Are

a / A

.U.

Calibration Curve (210 nm)

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.000

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

f(x) = 13396021.9783165 x + 559667.965087099R² = 0.990448321517835

Calibration Curve for Concentration of NBPT (210 nm)

[NBPT] / (mg/mL)

Chro

mat

ogra

phic

Are

a / A

.U.

Data Analysis• Calibration method• Several values fell outside of the calibration curve• Possibly due to over dilution of the less concentrated samples• Currently working on a method to improve the calibration method

• Decrease the dilution factor of the less concentrated samples

Data Analysis• Standard addition method• The area under the peaks of the spiked samples and non-spiked samples were

proportional.• Concentrations of NBPT were determined using the following formula:

• [A]u / [A]u+s = Iu / Iu+s

• [A]u = Concentration of NBPT• Iu = Area Under Peak of Sample• [A]u+s = Concentration of NBPT + Spike• Iu+s = Area Under Peak of Spiked Sample

Results (203 nm)Sample Trial 1 Trial 2 Trial 3 Trial 4 Average

Standard Deviation

Relative Standard Deviation

Actual Concentration

FJ9012NW 4.2% 3.5% 3.9% 4.1% 3.9% 0.3% 7.9% 4.0%

PK6295TM 2.0% 2.0% 1.9% 1.9% 2.0% 0.1% 3.0% 2.0%

RE7734LP 7.7% 6.6% 7.6% 7.7% 7.4% 0.5% 7.2% 8.0%

VC8375AH 16.5% 16.1% 16.8% 14.8% 16.1% 0.9% 5.5% 16.0%

ZU1472KR 31.7% 41.5% 35.3% 34.7% 35.8% 4.1% 11.5% 32.0%

081915-20 20.1% 17.6% 20.5% 21.8% 20.0% 1.8% 8.8% 20.0%

Results (210 nm)Sample Trial 1 Trial 2 Trial 3 Trial 4 Average

Standard Deviation

Relative Standard Deviation

Actual Concentration

FJ9012NW 4.1% 3.6% 3.9% 4.1% 3.9% 0.2% 6.0% 4.0%

PK6295TM 1.9% 2.0% 2.0% 1.9% 2.0% 0.1% 3.0% 2.0%

RE7734LP 7.6% 6.7% 7.6% 7.6% 7.4% 0.5% 6.1% 8.0%

VC8375AH 16.6% 16.9% 16.7% 14.5% 16.2% 1.1% 6.9% 16.0%

ZU1472KR 32.2% 38.7% 34.0% 33.1% 34.5% 2.9% 8.4% 32.0%

081915-20 19.8% 17.4% 20.3% 21.2% 19.75% 1.6% 8.3% 20.0%

Conclusions• The standard addition

• Yielded the best results • Consistent• Calculated concentrations very close to actual concentrations• Since 210 nm worked better, it will be used for all future analyses.

• Calibration method• Several values fell outside of calibration curve• Could be due to over dilution of less concentrated samples

• This analytical method has been confirmed by an independent analytical lab located in Wilson, NC.

• Future work• Repeat the method to confirm reproducibility.• Alter the calibration method to produce reliable results at lower concentrations.• Produce a standard operating procedure for routine analytical work.

Acknowledgements

• Dr. John Dogbe• Dr. Garnett Whitehurst• Barton College Science Department• SERMACS/SWRMACS