Application

Note: 51862
Analysis of Nitrate Nitrogen (NO3-) in Water by
the EPA Approved Brucine Method
Gordon Bain, Ph.D., Michael W. Allen, Ph.D., Nicole Kreuziger Keppy, Thermo Fisher Scientific, Madison, WI, USA

Introduction Sample Work-up Procedure

Key Words In this experiment quantitative analysis of nitrate nitrogen 1. Measure a known, consistent volume (1–5 mL is a typical
(NO3-) was performed by the Brucine method. Data was range) of sample into a calibrated tube or flask
• Brucine Method
acquired and processed using the Thermo Scientific 2. Dilute with deionized water to a total volume of 10 mL
• Nitrate Evolution Array UV-Visible spectrophotometer and 3. If samples are saline, add 2 mL sodium chloride solution
• UV-Visible Thermo Scientific VISIONcollect software. 4. Add 10 mL sulfuric acid solution and mix well by
shaking while cooling in flowing water.
• Water Analysis Background Do not stopper the tube/flask.
The Brucine method for nitrate is approved by the United CAUTION: This step is exothermic. The contents will
States EPA as Method No. 352.1.1 When a water sample heat up and pressure will result inside the container if
containing nitrate ion is treated with Brucine in sulfuric it is stoppered.
acid, a yellow solution results. The concentration of 5. Add 0.5 mL brucine in sulfanilic acid solution and mix
nitrate nitrogen may be calculated based upon the by shaking
absorbance of the solution at 410 nm. 6. Heat 25 minutes at 100 °C in a water bath
7. Cool with flowing water, dilute to 25 mL with deionized
Experimental water, stopper and invert several times to mix thoroughly
Reagents and Apparatus 8. Pour ca. 3 mL of solution into the 10 mm pathlength
cuvette and record the absorbance using the
• Nitrate ion standard solution (0.001 mg NO3- N/mL)
spectrophotometer
• Sodium chloride solution (30% w/v)
– Dissolve 30 g NaCl in 100 mL deionized water Instrument Parameters
• Sulfuric acid solution (4+1)
Data collection was carried out with VISIONcollect
– 4:1 = Sulfuric acid (H2SO4):deionized water software using the parameters displayed in Figure 1.
– CAUTION: Wear appropriate protective clothing and The Curve Zero Offset and Curve Order settings specify
observe proper protocols for diluting sulfuric acid a linear data fit that is not forced through the origin.
• Brucine-Sulfanilic acid solution
– Dissolve 1 g brucine dihydrate (C23H26N2O4 • 2H2O) and
0.1 g sulfanilic acid (H2NC6H4SO3H • H2O) in 3 mL conc.
HCl. Dilute to 100 mL with deionized water
• Evolution™ Array™ UV-Vis spectrophotometer with
VISIONcollect™ software
• Cuvette (10 mm pathlength)

Standards and Measurement Procedure

1. Prepare a series of nitrate standards from the 0.001 mg/mL
NO3- standard. Each of the standards should be an
accurately measured volume between 1 mL and 10 mL
of stock diluted to a total volume of 10.0 mL. Also
prepare a 0 ppm reagent blank. Between four and six
standards are generally considered to be sufficient to
establish a robust calibration curve.
2. Treat each standard according to the procedure
described in the sample work-up procedure below
3. In Quantification Standard mode
a. Measure the 0 ppm standard as the blank
b. Measure the absorbance of the remaining standards Figure 1: Experimental parameters for Nitrate assay
4. In Quantification Sample mode, measure the absorbance
of the unknown sample(s) and calculate the concentration
Results Conclusion In addition to these
offices, Thermo Fisher
Figure 2 shows the software screen for the standard solution In this experiment quantitative analysis of nitrate nitrogen
Scientific maintains
data collection. Full spectra of all standards are displayed (NO3-) in water was successfully performed with the
a network of represen-
in the Standard Spectrum window (upper left). Details of Evolution Array spectrophotometer and VISIONcollect
tative organizations
the standards used and their measured absorbance values software. Rapid acquisition of full-range spectra and
throughout the world.
at the analytical wavelength (410 nm) are shown at the excellent instrument sensitivity made the analysis quick,
bottom of the screen and a calibration curve is plotted accurate and reproducible.
(upper right). The equation of the line of best fit is
calculated by the least squares method and displayed References
below the Standard Spectrum. 1. Electronic: http://www.epa.gov/waterscience/methods/method/files/352_1.pdf.
A sample containing an unknown amount of nitrate Date acquired 12-12-2009.
Africa-Other
was prepared according to the procedure and measured in +27 11 570 1840
duplicate. The results were displayed as shown in Figure 3. Australia
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Figure 2: Standard solution data +81 45 453 9100
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Figure 3: Sample solution data

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