NITRITE (NO2–) and NITRATE (NO3–)
MEASUREMENTS
1. OBJECTIVE AND IMPORTANCE OF EXPERIMENT
Nitrogen exists in four forms that are of interest in water resources. These are ammonia
(NH4+), nitrite (NO2–), nitrate (NO3–), and organic nitrogen.
Chemists working with wastes and freshly polluted waters learned that most of the nitrogen is
originally present in the form of organic (protein) nitrogen and ammonia. As time progresses,
the organic nitrogen is gradually converted to ammonia nitrogen, and later on, if aerobic
conditions are present, oxidation of ammonia to nitrite and nitrate occurs. Waters in which
most of the nitrogen was in the form of nitrate were considered to have been polluted a long
time previously and therefore offered a little threat to the public health.
In 1940, it was found that drinking waters with high nitrate content often caused
methemoglobinemia in infants. For this reason, the U.S. E.P.A. has set a maximum
contaminant level requiring that the nitrate-nitrogen concentration not exceed 10 mg/L and
the nitrite-nitrogen concentration not exceed 1 mg/L in public water supplies.
1.1 Nitrite Nitrogen (NO2––N)
Nitrite is determined through formation of a reddish purple azo dye produced at pH 2.0 to 2.5
by coupling diazotized sulfanilamide with N-(1-naphthyl)-ethyl-enediamine dihydrochloride
(NED dihydrochloride).
The applicable range of the method for spectrophotometric measurements is 0,01 to 1 mg
NO2––N/L. Higher NO2– concentrations can be determined by diluting a sample.
Free chlorine, nitrogen trichloride (NCl3), Sb3+, Au3+, Bi3+, Fe3+, Pb2+, Hg2+, Ag+,
chloroplatinate (PtCl62–), and metal vanadate (VO32–) interfere. Suspended solids must be
removed by filtration.
To prevent bacterial conversation of NO2– to NO3– or NH3, store at 4oC.
1.1.1. EXPERIMENTAL PROCEDURE
[Link]. Materials and Equipment
Spectrophotometer (provides 540 nm wavelength and 1 cm light path or longer)
1 cm cuvette
50 mL volumetric flasks
Pipette
0.45 μm filter paper (if necessary)
Filtration tool
Nitrite-free water (distilled water)
1 N HCl (if necessary)
Color reagent
Stock nitrite solution (dissolve 1.232 g NaNO2 in water and dilute to 1000 mL = 250
mg N / L. Preserve with 1 mL CHCl3 or prepare it daily)
[Link]. Steps of the Experiment
Take 50 mL sample and 50 mL nitrite free water for blank to the volumetric flasks.
If sample contains suspended solids, filter through a 0.45μm filter paper.
If sample pH is not between 5 and 9, adjust to that range with 1 N HCl or NH4OH as
required.
Add 2 mL color reagent and mix.
Between 10 min and 2 h after adding color reagent to samples and standards, measure
absorbance at 543 nm.
Pour blank into a cuvette and measure the absorbance.
Repeat it for all samples and standards.
Draw a calibration curve.
ABS KONS (mg/L)
0.03 0.01
0.153 0.05
0.306 0.1
0.612 0.2
0.912 0.3
1.212 0.4
1.475 0.5
NO2-N (04.04.2011)
0.6
0.5 y = 0.3361x - 0.0028
R2 = 0.9995
0.4
KONS (mg/L)
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
ABS
[Link]. Calculations
Put the absorbance in the equation above. Calculate the concentration.
1.2. Nitrate Nitrogen (NO3––N)
Measurement of UV absorption at 220 nm enables rapid determination of NO3–. Because
dissolved organic matter also may absorb at 220 nm and NO3– does not absorb at 275 nm, a
second measurement made at 275 nm may be used to correct the NO3– value. However, this
method is not recommended if a significant correction for organic matter absorbance is
required.
Sample filtration is intended to remove possible interference from suspended particles.
Acidification with 1 N HCl is designed to prevent interference from hydroxide or carbonate
concentrations up to 1000 mg CaCO3 / L. Chloride has no effect on the determination.
Dissolved organic matter, surfactants, NO2–, and Cr6+ interfere. Various inorganic ions not
normally found in natural water, such as chlorite and chlorate, may interfere.
1.2.1. EXPERIMENTAL PROCEDURE
[Link]. Materials and Equipment
Spectrophotometer (for use at 220 nm and 275 nm wavelength and 1 cm light path or
longer)
1 cm cuvette
50 mL volumetric flasks
Pipette
0.45 μm filter paper (if necessary)
Filtration tool
Nitrite-free water (distilled water)
1 N HCl
Stock nitrate solution (Dry potassium nitrate (KNO3) in an oven at 105oC for 24 h.
Dissolve 0.7218 g in water and dilute to 1000 mL = 100 mg NO3– - N / L. Preserve
with 2 mL CHCl3 / L. This solution is stable for at least 6 months.)
[Link]. Steps of the Experiment
Take 50 mL sample and 50 mL nitrite free water for blank to the volumetric flasks.
If sample contains suspended solids, filter through a 0.45μm filter paper.
Prepare a blank and NO3– calibration standards in the range 0 to 7 mg NO3– - N / L by
diluting to 50 mL.
Add 1 mL HCl solution and mix thoroughly.
Pour blank into a cuvette and measure the absorbance.
Repeat it for all samples and standards.
Use a wavelength of 220 nm to obtain NO3– reading and a wavelength of 275 nm to
determine interference due to dissolved organic matter.
Draw a calibration curve.
[Link]. Calculations
Subtract two times the absorbance reading at 275 nm from reading at 220 nm to obtain
absorbance due to NO3–.
If correction value is more than 10% of the reading at 220 nm, do not use this method.
ABS KONS. (mg/L)
0.028 0.1
0.05 0.2
0.105 0.4
0.211 0.8
0.362 1.4
0.517 2
1.425 6
1.619 7
NO3-N (14.04.2011)
8
y = 4.3132x - 0.09
7
R2 = 0.999
5
KONS (mg/L)
0
0 0.5 1 1.5 2
ABS
An example: Abs at 275 is 0.015 and abs at 220 nm is 0.98.
The real abs is = 0.98 – (0.015 x 2) = 0.98 – 0.03 = 0.95
0.03 is not more than 10% of 0.98 so this method is suitable for that
sample.