Winkler light-dark dissolved O2 bottle

• Page authors: Anabel von Jackowski
• Responsible curator: Anabel von Jackowski

Titration of 100 mL sample using the semi-automatic Titronic300 (SI Analytics GmbH, Germany)

Oxygen Concentration
Approach: Winkler titration (iodometric)
Context: discrete measurement
Spatial scale: mL
Temporal scale: in situ
Units: mmol O2 L-1
Community captured: all
Co-measurements: temperature, salinity, depth

Method Overview

Dissolved O₂ measures the gas concentrations of oxygen from the depths of interest. This measurement can be measured using the Winkler method via iodometric titration because dissolved oxygen does not directly oxidize iodide to iodine and a multi-step reaction in which manganese acts as an intermediate “transfer” agent. .

Step-by-Step Protocol

Preparing Chemicals

1. Winkler 1 (Manganese(II) chloride)
   The reagent is stable for an extended period but should be stored protected from light.
   • Option A) Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask.
   • Option B) Dissolve 100 g Mn(II)Cl2 in 250 ml of deionized water.
2. Winkler 2 (Alkaline iodide solution)
   
   • Option A) Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary). Separately, dissolve 30 g KOH in the minimum amount of MQ water possible. Combine KI and KOH solutions and fill up to 100 mL.
   • Option B) Dissolve 75 g KOH in a small amount of deionized water and then add 100 g of KI and fill up to a volume of 250 ml.
3. Sulfuric Acid 50%
   Carefully dilute 98% sulfuric acid with MQ water ATTENTION! Add sulphuric acid slowly to the water, not water to the acid; the mixture must be cooled while diluting! Wear safety glasses!!
4. Sodium Thiosulfate Solution (0.02 M)
   Dissolve 49.5 Na2S2O3 (pentahydrate) in 1L of deionized water to make 0.2 M thiosulphate solution. The 0.2 M solution is then diluted 1:10 (0.02 M). With proper use, the titer is 1 and remains stable for about four weeks.
5. Starch Solution
   Dissolve 1 g soluble starch in 100 ml MQ water with heating. Stable for at least 10 days if stored in a refrigerator.
6. Standard Solution
   Under warming dissolve 325.0 mg potassium hydrogen iodate KH(IO3) and fill up to a volume of 1000 ml with deionized water to make 0.833∙10-3 M iodate solution.

Prepare Bottles

8. Measure Bottle factor
   • Note: This can also be done after sampling but is needed to calculate oxygen concentration

Sample Collection

9. Sample Preparation
   • Place Winkler 1 and 2 solutions close to where you are taking your sample
   • Write down the number of bottle you will fill with water
   • Have the bottle you will use and a tube ready for sampling
10. Sample for oxygen
    Fill the Winkler bottle quickly with sample water by inserting a tube to the bottom of the bottle. Let water flow slowly through until the volume has been replaced ~3 times and avoid air bubbles. Remove the hose while water is still flowing.
11. Fix Oxygen
    Add Winkler 1 and Winkler 2 (= 1/100 of the sample volume each) just below the neck (~0.5–2 cm) and close the bottle bubble-free. Shake the bottle vigorously for ≥30 seconds. After 30–60 minutes, a fine brown precipitate (manganese oxide) should form. The fixed samples can be stored in the dark (or wrapped in aluminum foil) at 4°C for up to 12 hours or slightly longer.
    • Note: For a 60 mL bottle, this would be 600 µl Winkler 1 + 600 µl Winkler 2
    • Note: For a 100 mL bottle, this would be 1 ml Winkler 1 + 1 ml Winkler 2

Analysis via titration

12. Dissolving the Precipitate
    Add a few drops of 50% sulfuric acid, avoiding disturbance of the precipitate. Close the bottle. Gently swirl until the precipitate dissolves.
13. Transfer Sample options
    • Option A) Transfer the acidified sample into a sufficiently large beaker. Pipette two aliquots of 5 ml each (into separate beakers with stir bars) and titrate (duplicate determination).
    • Option B) Remove the clear supernatant (oxygen-free) down to ~1 cm above the manganese oxide precipitate using vacuum or carefully with a pipette.
    • Option C) No transfer to a beaker is required; titration can be done directly in the Winkler bottle.
14. Titration - Step 1
    Add 0.02 M thiosulfate solution in small increments until the brown-yellow color nearly disappears (light yellow remains).
15. Titration - Step 2
    Add 3-5 drops of starch solution (iodine indicator). A deep blue-black iodine-starch complex forms.
16. Titration - Step 3
    Continue titration slowly until the solution becomes colorless. A white paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
17. Disposal
    Remove the stir bar and dispose of the acidic contents of the bottle and beaker down the drain with running water. Rinse the Winkler bottles and allow them to dry.