3. 2.

Determination of dissolved oxygen in water using the Winkler method

The Winkler method, determines the dissolved oxygen concentration through a series of
oxidationreduction reactions. First, Mn2+ (as MnSO4) is added to a 250- or 300-mL sample. Next,
the alkaliiodide reagent (KI in NaOH) is added. Under these caustic conditions, if oxygen is
present in the water sample, the Mn2+ will be oxidized to Mn4+, which precipitates as a brown
hydrated oxide. This reaction is relatively slow and the solution must be shaken several times to
complete the reaction.
This reaction can be represented by the following equations:

The titration is complete when all of the I2 has been converted to I-. The endpoint of this titration
can be determined through colorimetric indicators. The most common indicator is starch, which
turns from deep blue to clear. The DO concentration can be determined using the following
equation, which also reflects the series of redox reactions in the equations given above:

Several modifications of the Winkler method have been developed to overcome interferences. The
azide modification, the most common modification, effectively removes interference from nitrite,
which is commonly present in water samples from biologically treated wastewater effluents and
incubated biochemical oxygen demand samples. Nitrite interferes by converting I- to I2, thus

overestimating the dissolved oxygen in the sample. This is illustrated in the following equations:

Note that N2O2 is oxidized by oxygen, which enters the sample during the titration procedure and
is converted to NO2- again, establishing a cyclic reaction that can lead to erroneously high results.
This final result yields oxygen concentrations that are far in excess of the amounts that would
normally be expected.
Nitrite interference can easily be overcome through the addition of sodium azide (NaN3). Azide is
generally added with the alkaliKI reagent, and when sulfuric acid is added, the following
reactions result in the removal of NO2- :

Other methods can also be used to remove ferrous iron (the permanganate modification), ferric
iron (the potassium fluoride modification), and suspended solids (the alum flocculation
modification). We will be using only the azide modification in this laboratory practical.
You will be given one water sample for titration using the Winkler method. For this laboratory
exercise you do not have to be concerned with preservation of the sample or sample-handling
practices, but in the real world many precautions need to be taken. Most important is the
preservation of field samples that need to be analyzed in the laboratory. Two approaches are used
to preserve samples for later DO determination. First, you can fix your samples using the
procedures described below and then perform the titration when the samples are brought to the
laboratory. Samples should be stored in the dark and on ice until titration. This preservation technique will
allow you to delay the titration for up to 6 hours. However, this procedure may give low results for samples
with a high iodine demand. In this case it is advisable to use the second option, which is to add 0.7 mL of
concentrated sulfuric acid and 0.02 g of sodium azide. When this approach is used, it is necessary to add 3
mL of alkaliiodide reagent (below) rather than the usual 2 mL. In addition, avoid any sample treatment or
handling that will alter the concentration of DO, including increases in temperature and the presence of
atmospheric headspace in your sample container. You will titrate your samples using the procedures
described below. Conduct at least three careful titrations.

Safety Precautions: Avoid skin and eye contact with caustic and acidic solutions. If contact
occurs, rinse your hands and/or flush your eyes for several minutes. Seek immediate medical
advice for eye contact. Use concentrated acids in the fume hood and avoid breathing their vapors.
Sodium azide is a toxin and should be treated as such.

Chemicals and Solutions

Manganese sulphate: Dissolve 48 g of MnSO4.4H2O, 40 g of MnSO42H2O, or 36.4 g of MnSO4
H2O in about 80 mL of deionized water. Filter the solution and dilute to 100 mL.
Alkaliiodideazide reagent. Dissolve 50 g of NaOH (or 70 g of KOH) and 13.5 g of NaI (or 15
g of KI) in deionized water and dilute to 100 mL. Add 1 g of NaN3 dissolved in 4 mL of deionized
water.
Concentrated H2SO4. (1.0 mL of this solution is equivalent to approximately 3 mL of alkali
iodideazide solution.)
Starch solution. Dissolve 2 g of laboratory-grade soluble starch and 0.2 g of salicylic acid (as a
preservative) in 100 mL of hot distilled water. Allow to cool before use. Refrigerate.
Sodium thiosulphate titrant, 0.0250 M: . Dissolve 6.205 g of Na2S2O35H2O in deionized water.
Add 1.5 mL of 6 M NaOH or 0.4 g of solid NaOH and dilute to 1.0 L. Standardize with biiodate
solution.
Standard potassium biiodate solution, 0.00210 M. Dissolve 0.8124 g of KH(IO3)2 in deionized
water and dilute to 1.000 L.
Glassware
For each student group: three Erlenmeyer flasks, 25-mL buret, 20.00-mL pipet, Pasteur pipets,
three 1.00-mL pipets (at least one of these should be a wide-bore pipet for the viscous azide
reagent)
Procedure
Standardization of Sodium Thiosulphatee Titrant
Note: The thiosulphate titrant may already have been standardized by your demonstrator. If so,
skip to step 5.
1. Dissolve approximately 2 g of KI (free of iodate) in an Erlenmeyer flask containing 100 to 150
mL of deionized water.
2. Add 1 mL of 6 M H2SO4 or a few drops of concentrated H2SO4 and pipet 20.00 mL of standard
biiodate solution into the flask. Recall from the reactions given in the theory section that I2 will be
formed from the reaction when any DO is present.
3. Titrate the liberated I2 with thiosulphate titrant until a pale straw (yellow) color is reached. Add
a few drops of starch indicator, which will result in a blue color, and continue the titration to the
endpoint, which is clear.
4. If all solutions were made properly, 20.00 mL of the biiodate solution will require 20.00 mL of
thiosulfate titrant. If this result is not achieved, calculate the exact molar concentration of your
titrant.

Titration of Water Samples

5. To a 250- or 300-mL sample bottle, add 1 mL of MnSO4 solution, followed by 1 mL of alkali
iodideazide reagent. If your pipets are dipped into the sample (as they should be), rinse them
before returning them to the reagent bottles. If the solution turns white, no DO is present.
6. Stopper the sample bottles in a manner to exclude air bubbles 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.
7. Add 1.0 mL of concentrated H2SO4.
8. Restopper 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.
9. Titrate 200 mL of the sample with sodium thiosulphate solution (0.025 M), stirring the contents
of the flask until the yellow-brown colour fades to a pale straw colour. Add a few drops of starch
solution and a blue colour will develop. Continue titrating a drop at a time until the blue colour
disappears
10. Repeat the titration for two more samples and determine the mean value, standard deviations
and relative standard deviations of the dissolved oxygen level for the water sample.
Calculation
For titration of 200 mL of sample with 0.025 M sodium thiosulphate:
1 ml Na2S2O3 solution=1 mg L-1 dissolved oxygen
If sodium thiosulphate is used at a strength other than 0.025 M, and if the sample volume titrated
is other than 200 ml), the dissolved oxygen in the sample may be calculated from the following
formula:

___________________________________________________________________________References: American Public Health Association, APHA (1998). Standard Methods for the
Examination of Water and Wastewater. 20th ed. 1220pp. American Public Health Association,
Washington DC, USA.APHA (1999)
Csuros M. (1997) .Environmental Sampling and Analysis, CRC press