Determination of Nitrate (NO3-)

Background
The nitrate ion absorbs ultraviolet radiation at 220 nm but not at 275 nm. The nitrate absorption at
220 nm follows Beer’s law with linear behavior to 11 mg nitrogen/L solution. Because dissolved
organic matter may also absorb at 220nm, one must be able to distinguish between the absorbance
of nitrate and the other organic matter in water. This is accomplished by making a measurement of
the water sample’s absorbance at 275 nm, a wavelength that the organic matter can absorb
electromagnetic radiation but in which the nitrate cannot. Once known, an empirical correction
factor can be applied to the 220 nm measurement to distinguish the nitrate from the organic matter.
Because this is an empirical correction, this method is not recommended for waters requiring a
significant correction for organic matter. It is main use is as a screening tool for water samples in
which the organic matter is relatively constant. If it is desired to create a more accurate method,
another approach to the calibration process can be performed. This approach, known as the
method of standard additions, is beyond the scope of the intent of this lab, though your instructor
can steer you to reading information to more fully describe this approach.

Sample preparation consists of two steps. First, the sample will be filtered, to prevent scattering of
UV light by suspended particles in the water sample, which will change the measured absorbance.
The samples will next be acidified using 1 N HCl to prevent interferences due to the absorption of
either OH- or CO32-, both of which can absorb at 220 nm. The acidification should prevent
interferences from these ions up to 1000 mg CaCO3/L. Hydrochloric acid is used because Cl- does
not absorb light in 250 – 290 nm region of the spectrum. Some other ions, such as Cr6+ (as either
chromate or dichromate ion), nitrite, chlorate and chlorite, may also interfere, but these are
expected to be low or nearly non-existent in the water samples used in our analysis.

Procedure
All solution must be prepared using the nitrate-free water available in the laboratory. Do not use
tap water or regular distilled water. Exercise care when using the nitrate-free water and do not
waste it.

In a volumetric flask prepare 1.00 L of a 100 μg N/mL stock solution from solid KNO3 which has
been dried in an oven at 105 ºC for 24 hours. Note that the dried KNO3 will be provided to you.
You will need to determine the mass of KNO3 that you actually used to the nearest milligram and
calculate your exact concentration from this.

From the nitrate stock solution prepare 500.0 mL of a nitrate solution that contains 10 μg N/mL.
Note that your actual value may be slightly different depending on the actual amount of KNO3 you
used to prepare the first solution.

Prepare NO3- calibration standards in the range of 0 to 7 g N/mL (this corresponds to the addition
of between 0 and 350 μg N per 50 mL of solution). Use 50.0 mL volumetric flasks and pipets to
create these solutions. Volumes of diluted standard nitrate solution ranging between 0 and 35 mL
will be sufficient to cover this range of concentrations. To be useful, the calibration curve should
be created at least five different standard solutions (plus a “blank”) that “bracket” the expected
concentration of nitrate. Be sure to dilute carefully and mix thoroughly before using.
The water sample is prepared by first gravity filtering through acid-washed, ashless hard finish,
filter paper to filter fine precipitates to remove large particles and debris. The filtered solution will
then be filtered through a 0.45 m pore diameter membrane filter using a syringe to force the water
through the filter. Filter at least 75 mL of sample through the filter paper and then filter only as
much sample as needed through the membrane filter. To each 50 mL of clear sample, add 1 mL of
the 1 M HCl solution and mix thoroughly.

Use the Ocean Optics spectrometer and cells provided to measure the absorbance of each solution
(the blank, the calibration standards and the water sample) at 220 nm and at 275 nm. You will be
using a special sample cell for this experiment (either quartz or a special plastic), because ordinary
glass and plastic do not transmit light in the ultraviolet region of the spectrum. Handle these cells
very carefully, as they are extremely expensive and fragile. Also, be sure to keep fingerprints off
of the surfaces that light passes through, as the acids in your fingerprints can be permanently
etched into the surface of the expensive cuvette when exposed to UV light. If this happens, the
cuvettes are ruined. Please see [Link] to review proper spectrophotometer
operation. Note if your absorbance readings are much larger than 1, or if your water sample’s
absorbance is much higher than your highest standard solution, you will need to dilute your
samples accordingly.

Results
Subtract 2 times the absorbance reading of the water sample at 275 nm from the value of
absorbance for that sample at 220 nm. This operation will give a value for the experimental
absorbance due to NO3-.

From the corrected absorbances of the standard solutions create a calibration curve of absorbance
as a function of the concentration of NO3- (in mg N/L) that includes best fit line though the data.
Include the correlation coefficient for this fit.

Using the measured absorbances of your water samples, determine the concentration of nitrogen in
each your water samples in μg N/mL solution. Convert this value to the concentration of NO3- in
μg/mL solution. Don’t forget that in the determination of any real samples, you should also report
the standard deviation and confidence limits for your results.

What are the predominant sources of nitrate in natural waters? How do your results compare with
what is expected of natural water sources in the Midwest? If elevated, what ways might the nitrate
be remediated?
 Determination of Dissolved Oxygen in Water

Background
Oxygen is poorly soluble in water. Where creatures that live in the atmosphere have about 19%
oxygen available for consumption, organisms living in water have a maximum of about 0.15 %
oxygen. Moreover, as temperature or as salt content in the water increases, the dissolved oxygen
(DO) concentration decreases. DO in pure water (no salt content) ranges from 7.6 mg/L at 30◦C to
14.6 mg/L at 0◦C. Many organisms, such as the mountain trout, have specific oxygen needs, so
that they can only live in a small range of these variable O2 concentrations. The mountain trout is
only found in icy cold water with the highest concentration of DO.

DO concentrations can be determined by the Winkler, or iodometric, method. This method

involves a series of oxidation-reduction reactions starting with the oxidation of Mn2+ (added to the
sample in the form of MnSO4) by O2 in basic solution (by addition of an alkaline potassium iodide
solution, KI in NaOH) to give solid MnO2, as shown in reaction (1).

2Mn2+ (aq) + 4 OH- (aq) + O2 (aq) → 2MnO2(s) + 2 H2O (l) (1)

The formation of MnO2 stabilizes essentially locks up the dissolved O2 in a form that allows for
short term storage of the sample before it is analyzed.

To start the actual analysis of oxygen, H2SO4 and more KI are added to the solution containing the
MnO2 precipitate. At low pH, the MnO2 oxidizes I- completely into I2, as shown in reaction (2).

MnO2 (s) + 2 I- (aq) + 4 H+ (aq) → Mn2+ (aq) + I2 (aq) + 2 H2O (l) (2)

Because both reactions (1) and (2) go to completion, the I2 formed is directly related to the amount
of DO in the sample.

The I2 produced in reaction (2) is determined by titration with standardized sodium thiosulfate
(Na2S2O3), via another oxidation-reduction reaction in which thiosulfate reduces the I2 back into I-.
This reaction is illustrated in reaction (3) below.

The endpoint of the reaction is detected using a starch indicator. When I2 is present, the indicator
has a deep blue color (due to a complexation of iodine by the starch), but when no I2 is present in
solution, the solution is colorless. So, once the equivalence point is reached, there will be no I2
remaining in solution and the dark blue color that was initially present in the solution will
disappear.

2 S2O32- (aq) + I2 (aq) → S4O62- (aq) + 2 I- (aq) (3)

From the known S2O32- concentration and the volume of the S2O32- solution used to reach the
equivalence point, we can ultimately determine the number of moles of O2 that were originally
present in your sample of water. Since the volume of the original sample is known, we can
calculate the concentration of O2 in mg/L, which is what will be reported along with the
temperature of the water when it was sampled.
To prevent nitrite interference (a real possibility in fresh water samples) sodium azide (NaN3) is
added with the alkali-KI reagent to the water sample as it is taken from the source. The addition of
sulfuric acid then results in the removal of NO2- through the following series of reactions, (4) and
(5) below.

NaN3 (aq) + H+ (aq) → HN3 (aq) + Na+ (aq) (4)

HN3 (aq) + NO2- (aq) + H+ (aq) → N2 (g) + N2O (g) + H2O (l) (5)

Sample Collection
The following steps are for collecting the samples. As you will not be collecting the samples
yourself, these are included here for your information only.

Prepare a manganese(II) sulfate solution by dissolving 480 g of MnSO4.4H2O (or 400 g of

MnSO4.2H2O, or 364 g of MnSO4.H2O) in about 800 mL of deionized water. Filter the solution
and dilute to 1.0 L. Prepare the alkali-iodide-azide reagent by dissolve 500 g of NaOH (or 700 g
KOH) and 135 g of NaI (or 150 g KI) in deionized water and dilute to 1.0 L. Add 10 g of NaN3
dissolved in 40 mL of deionized water.

Samples should be collected as close to analysis as possible. Collected samples should be stored
with little or no head space, on ice and in the dark and care should be taken to avoid shaking
samples.

To a 250-300 mL plastic sample bottle almost filled with the water sample, add 1 mL of MnSO4
solution, followed by 1 mL of alkali-iodide-azide reagent (this is best done with graduated two
graduated cylinders). If the solution turns white, no DO is present.

Gently squeeze the sample bottle to raise the sample’s level in the bottle to its lip. Seal the sample
bottle so that no air bubbles are present and mix by inverting the bottle rapidly a few times. When
the precipitate has settled to half the bottle volume, repeat the mixing and allow the precipitate to
resettle.

Procedure
All solutions can be disposed of down the drain.

Prepare the sodium thiosulfate titrant by dissolving 6.205 g of Na2S2O3.5H2O in deionized water.
To this solution add 0.4 g of solid NaOH. Once the NaOH has dissolved dilute to 1.0 L. This
solution is approximately 0.0250 M, but because the solid can be oxidized by oxygen in air during
storage, we will need to standardize it with biiodate.

Dissolve 0.81 g of KH(IO3)2 in deionized water (measured to the nearest milligram) and dilute to
1.000 L. This solution should be 0.00210 M in KH(IO3)2 and will be our standard biiodate
solution.
Approximately 2 g of KI (measure to the nearest 0.001 g) is dissolved in 100 – 150 mL deionized
water in an Erlenmeyer flask and 1 mL of 6 M H2SO4 is added to the resulting solution. Pipet
20.00 mL of the standard biiodate solution into the flask; I2 will be formed from the reaction.

Titrate the liberated I2 with the thiosulfate titrant until a pale straw (yellow) color is reached. Add
a few drops of starch indicator (already prepared for you by dissolving 2 g of lab-grade soluble
starch and 0.2 g of salicylic acid as a preservative in 100 mL of hot distilled water and cooled
before use). The solution should turn blue. Continue the adding titrant until the blue color is gone,
which is the endpoint. If the solutions were made correctly with pure reagents, 2 g of KI and 20.00
mL of KH(IO3)2 should require 20.00 mL of the Na2S2O3 titrant to reach the endpoint. Determine
the concentration of thiosulfate and repeat the titration until three concentrations agree within
about 3% of each other.

Once the Na2S2O3 has been standardized, open the sealed sample bottle and add 1 mL of
concentrated H2SO4. Reseal the bottle and mix by inverting the bottle rapidly and dissolve the
precipitate. You may open the bottle and pour the sample at this point since the DO and reagents
have been “fixed” and will not react further.

Titrate 200 mL of the water sample with your standard thiosulfate solution as in the
standardization (first titrate to a pale straw color, add starch indicator, and titrate to the clear
endpoint). Take care with these titrations! Only enough samples will be collected for each group
to do four titrations. Once your allotment of samples is gone, there will be no more available!

Determine the DO concentration in the water samples and repeat the titrations at least twice more
or until three concentrations agree within about 3%.

Results
Calculate the mean, standard deviation and confidence limit of the results of your titration. The
concentration of O2 should be reported in mg O2 per L solution. Compare your results with that
expected for standard fresh water at the temperature in which your sample was taken (this can be
calculated from Henry’s Law or looked up in reference books).
 Determination of Ions Using an Ion Selective Electrode

Introduction
An ion-selective electrode (ISE) incorporates a special, ion-sensitive membrane, which may be
glass, a crystalline inorganic material or an organic ion-exchanger. The membrane interacts
specifically with the ion of choice, allowing the electrical potential of the half cell to be controlled
predominantly by the ion’s concentration. ISEs are available for measuring more than 20 different
cations (e.g., Ag+, Na+, K+, Ca2+, Cu2+) and anions (e.g., F-, Cl-, S2-, CN-).

The potential of the ISE is measured against a suitable reference electrode (another half cell which
gives a complete electrochemical cell) using an electrometer or pH meter. The electrode potential
is related to the logarithm of the concentration of the measured ion by the form of the Nernst
equation given as Eqn. 1. In Eqn. 1 n is the ion charge (negative for anions), [X] is the
concentration of the species of interest, and the factor 2.303 RT/F has a theoretical value of 59 mV
at 25 °C.

RT
E  E 0  2.303 log[ X ] (1)
nF

The Nernst equation is only valid for very dilute solutions or for solutions where the ionic strength,
I, is constant. Ionic strength is defined by Eqn. 2, where Zi is the charge on an ion and Ci is its
concentration. In this analysis the ionic strength will be maintained by adding another salt to the
solution in excess. This supporting electrolyte will be NaCl for a fluoride analysis and NaNO3 for
a chloride analysis.

1
I
2
 Z i2Ci (2)

Procedure
The following procedure assumes that you are doing a fluoride analysis. If you are performing a
chloride analysis, substitute NaCl for NaF and use NaNO3, instead of NaCl, as the supporting
electrolyte.

Dry the NaF solid for 1 hour at 100 °C (already done for you) to remove any water that has
adhered to the solid’s surface.

Accurately prepare 100 mL of a 0.1 NaF solution by weighing out about 0.42 g of NaF into a
100-mL volumetric flask. Dissolve the salt in deionized water, dilute to the mark and mix well.
This is your fluoride stock solution.

Prepare 100 mL of a 0.01 M NaF solution by dilution of the fluoride stock solution using
volumetric glassware.

Weigh out 7.55 g of KCl on a top-loading balance and dissolve in 100 mL of deionized water. This
solution is 1 M in KCl.
Prepare standard solutions in four 100-mL volumetric flasks as follows:

Flask Number Volume of 0.01 M NaF (mL) Volume of 1 M KCl (mL)

I 1.00 10.00
II 2.00 10.00
III 5.00 10.00
IV 10.00 10.00

Dilute each flask to volume with deionized water and mix well.

Add 1.00 mL of your unknown, then 10.00 mL of KCl to a 100 mL volumetric flask. Dilute to the
100 mL mark with deionized water.

Pour about 30 mL of either a standard or unknown solution into a clean, dry 100 mL beaker and
immerse the electrodes in the solution to a depth of not more than 2 cm. Measure the electrode
potential (in mV) with the voltmeter. CAUTION! Do not touch the ISE membrane. Rinse it with
deionized water between measurements and then with a small volume of the new solution. Do not
wipe it dry.

When you finish, rinse the electrodes with deionized water. Leave the reference electrode in the
appropriate storage solution. The F- ISE should be stored dry and loosely capped. DO NOT force
the cap onto the electrode tip!

Results
Prepare a calibration curve in Excel relating log[NaF] in the standard solutions to the measured
potential of each solution in mV. Determine the best line through the four standard solution
experimental points. The slope of the calibration curve should be close to -59 mV.

Utilizing the calibration curve, determine the concentration of NaF in your original unknown
solution.