Experiment 5

Determination of Nitrate Ion in Water

General Discussion

Nitrate nitrogen may be present in small amounts in fresh domestic

wastewater. However, it is seldom found in influents to treatment
plants because the nitrate serves as an oxygen source in the
biologically unstable wastewater. On the other hand, nitrate is often
found in the effluents of biological treatment plants, because it
represents the final form of nitrogen from the oxidation of organic
nitrogen compounds. Trickling-filter and activated sludge treatment
plant effluents may contain from 0 to 50 mg/L nitrate, depending on
the total nitrogen content of the influent, the degree of loading, and the
temperature of the sewage.

Nitrate may also be found in river water, lake water, and most
importantly in ground water. The nitrate may originate from sewage, or
in rural areas, it may be produced by fertilizer or barnyard runoff. The
U.S. Public Health Service designated safe limit for nitrate in water is
45 mg/L nitrate or 10 mg/L nitrate nitrogen. Nitrate in drinking water is
particularly dangerous to small children, infants, and fetuses.

In this experiment, nitrate will be reduced to nitrite with zinc. The

nitrite reacts with sulfanilic acid and N-1-naphthylethylenediamine to
produce a red compound. The intensity of the red color is analyzed
spectrophotometrically. The amount of zinc and the contact period are
important.

Special Apparatus

a. Spectronic 20 - spectrophotometer set at 520 nm.

b. Filter paper and vacuum filtration apparatus.

Reagents

(All of the following are prepared by the instructor or lab technician

and are available for student use in the experiment.)

a. Stock potassium nitrate solution: 0.816 g of anhydrous

KNO3 is dissolved in purified water and diluted to 1 liter to
produce a 500 mg/L nitrate solution. 100 mL of this solution is
diluted to 1 liter to produce the stock 50 mg/L nitrate solution

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 which will be used in this experiment.

b. Hydrochloric acid diluted 1:4 1 part con. HCl and 4 parts

water.

c. Sulfanilic acid: Dissolve 0.60 g of sulfanilic acid in 70 mL hot

purified water, cool, dilute to 100 mL with purified water, and mix
thoroughly.

d. Zinc: Add 1.000 g finely powdered zinc to 200 g sodium

chloride, NaCl, in a bottle and mix thoroughly by shaking for
several minutes.

g. N-1-naphthylethylenediamine dihydrochloride reagent:

Dissolve 0.60 g N-1-naphthylethylenediamine dihydrochloride in
purified water to which 1.0 mL con. HCl has been added. Dilute
to 100 mL and mix. Store in refrigerator. (Stable for about a
week.)

h. Sodium acetate solution: Prepare 100 mL of 2 M

NaC2 H3 O 2 .

Procedure

a. Preparation of standards:

Blank: Measure 50 mL of purified water and transfer to a 250-

mL erlenmeyer flask.

2.5 mg/L standard: Add 2.5 mL of stock 50 mg/L nitrate

solution to a 100-mL graduated cylinder. Add purified water
and dilute to a volume of 50 mL. Transfer to a 250-mL
erlenmeyer flask.

5.0 mg/L standard: Repeat the directions for the 2.5 mg/L
standard using 5.0 mL of 50!mg/L nitrate solution.

10.0 mg/L standard: Repeat the directions for the 2.5 mg/L
standard using 10.0 mL of 50!mg/L nitrate solution.

15.0 mg/L standard: Repeat the directions for the 2.5 mg/L
standard using 15.0 mL of 50!mg/L nitrate solution.

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b. Experimental: Use the following procedure for treating
standards as well as river, lake, well, or sewage water samples.

Note: Treated sewage effluent may require a 5-fold or a 10-

fold dilution. A 10-fold dilution can be performed by pipetting 5.0
mL of the treated wastewater (sewage) sample into a 100-mL
graduated cylinder and adding enough water to bring the volume
up to 50 mL. This 50 mL sample can then be taken through the
experimental and color development procedure. A 10-fold
dilution results in a dilution factor of 10. Remember to multiply the
concentration obtained for the diluted sample by a factor of 5 or 10.

To a 50.0-mL water sample in a 250-mL erlenmeyer flask, add

1.0 mL of dilute HCl (1:4 dilution) and 1.0 mL sulfanilic acid reagent
and mix thoroughly. In a dry 10-mL graduated cylinder, measure
one mL of the Zn/NaCl granular mixture and add it to the
erlenmeyer flask.

Swirl the flask for seven minutes. Filter with a vacuum flask after
the seven minute swirling period. Rinse the erlenmeyer flask well
with purified water and pour the filtered water sample back into the
flask.

Color Development: Add 1.0 mL N-1-

naphthylethylenediamine dihydrochloride reagent to the filtered
sample and mix. Add 1.0 mL of 2 M sodium acetate solution and
mix. Allow 5 minutes (or more) for color development.

Spectrophotometric Measurement: Measure the color intensity

with a spectrophotometer at a wavelength of 550 nm. Purified
water may be used for a blank unless the water sample is cloudy.
In that case, use a sample of cloudy water as a blank. Record the
absorbance of the colored sample.

Waste Disposal

All solutions may be rinsed down the drain with water.

Calculations

From the concentration and the absorbance of the four standards,

make a plot of absorbance as a function of concentration. Use the plot
and the absorbance of each unknown solution to determine the
concentration in mg/L nitrate ion (mg NO3 -/L ) in that sample. Also,
express the concentration in terms of mg/L of nitrate nitrogen (mg N/L)

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in the sample.

Discussion

Identify and discuss possible sources of error in this experiment.

How does each kind of error affect the final answer in terms of making it
too high, too low or either?

Also, use the concentrations of nitrate nitrogen measured in each

water sample to estimate the drinking water quality (from the
standpoint of nitrate content) of each of the samples tested.

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Report the following data:

Sources of Water Samples

1.
2.
3.
4.

Absorbance of Standards
2.5 mg/L NO3 - !!!!!!!!!!!!!
5.0 mg/L NO3 - !!!!!!!!!!!!!
10.0 mg/L NO3 - !!!!!!!!!!!!!
15.0 mg/L NO3 - !!!!!!!!!!!!!

Note: Prepare a standard curve and turn it in with the report sheet.

Absorbance of Water Samples Dilution Factor

(if any)

1. !!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!
2. !!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!
3. !!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!
4. !!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!

Concentration of Nitrate in Water Samples

as NO3 - as N
1.
2.
3.
4.

Discussion

Identify and discuss sources of error in this experiment. How

would you rate the quality of the water samples tested in this
experiment?

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