Winkler light-dark dissolved O2 bottle

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

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 (60 mL bottle)

Prepare Chemicals

1. Winkler 1 (MnCl₂) Solution
   Dissolve 40 g MnCl₂·4H₂O in MQ water and make up to 100 ml in a volumetric flask. The reagent is stable for an extended period but should be stored protected from light.
2. KI Solution
   Dissolve 15 g KI in the minimum amount of MQ water possible (warm gently if necessary).
3. KOH Solution
   Separately, dissolve 30 g KOH in the minimum amount of MQ water possible.
4. Winkler 2 (KI+KOH) Solution
   Combine KI and KOH solutions and fill up to 100 mL.
5. Sulfuric Acid 50%
   Carefully add the same amount of concentrated sulfuric acid to MQ water (add only very small amounts, as the solution heats up strongly; cool in an ice bath if necessary). Do not forget the stir bar.
6. Sodium Thiosulfate Solution (0.01 N)
   Transfer the ampoule into a 500 ml volumetric flask and fill to 500 ml with MQ water. This yields a 0.02 mol/L solution. With proper use, the titer is 1 and remains stable for about four weeks.
7. 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.

Sample Collection

1. 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.
2. Fix Oxygen
   Add 600 µl Winkler 1 and 600 µl 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.

Analysis via titration

1. 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.
2. 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.

1. Titration - Step 1
   Add 0.02 M thiosulfate solution in small incroments until the brown-yellow color nearly disappears (light yellow remains).
2. Titration - Step 2
   Add 3-5 drops of starch solution (iodine indicator). A deep blue-black iodine-starch complex forms.
3. Titration - Step 3
   Continue titration slowly until the solution becomes colorless. A while paper placed behind the beaker/bottle can help to determine the color change. Record the volume used and concentration of the titrant.
4. 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.