🫧 Dissolved Oxygen — Winkler Method (Iodometric)
Classic iodometric (Winkler) method for dissolved oxygen determination in water. Based on oxidation of Mn(II) to Mn(IV) by oxygen, iodine liberation, and titration with sodium thiosulfate. Reference method since 1888.
Overview
Dissolved oxygen (DO) is one of the most important water quality parameters, determining the ability of aerobic organisms (fish, invertebrates, aerobic bacteria) to live. DO saturation concentration in water is ~8.5 mg/L O₂ at 20°C and atmospheric pressure. DO decrease below 4 mg/L causes oxygen stress, below 2 mg/L — anaerobic conditions (fish kill, putrefaction processes).
Winkler method, developed by Hungarian chemist Lajos Winkler in 1888, is the oldest and most accurate chemical method for DO determination. It involves binding oxygen by Mn²⁺ ions in alkaline medium, then liberating iodine (I₂) in acidic medium in amount stoichiometrically equivalent to oxygen content. Liberated iodine is titrated with sodium thiosulfate (Na₂S₂O₃) using starch as indicator (transition from blue to colorless).
Azide modification (with NaN₃) eliminates nitrite (NO₂⁻) interferences, which are common in biologically treated wastewater and some surface waters. This is the most commonly used version of Winkler method.
Despite widespread use of oxygen probes (electrochemical and optical), Winkler method remains reference method for probe calibration and analytical dispute resolution. It is required in DO₀ and DO₅ determination in BOD₅ test.
Method principle
In strongly alkaline medium, dissolved oxygen in water oxidizes manganous hydroxide Mn(OH)₂ (white precipitate) to manganic hydroxide MnO(OH)₂ (brown precipitate):
2 Mn(OH)₂ + O₂ → 2 MnO(OH)₂
After acidification with sulfuric acid, Mn⁴⁺ ions oxidize potassium iodide (KI) to free iodine:
MnO(OH)₂ + 2 I⁻ + 4 H⁺ → Mn²⁺ + I₂ + 3 H₂O
Liberated iodine (I₂) is titrated with sodium thiosulfate:
I₂ + 2 Na₂S₂O₃ → 2 NaI + Na₂S₄O₆
End point: disappearance of blue color of starch-iodine complex. In azide modification, sodium azide (NaN₃) is added, which decomposes nitrites (NO₂⁻ + N₃⁻ → N₂ + NO + N₂O), eliminating their interference in Mn²⁺ oxidation.
Applications
- DO determination in BOD₅ test (day 0 and day 5)
- Calibration and verification of oxygen probes (electrochemical and optical)
- Surface water quality monitoring — oxygen status of rivers and lakes
- Drinking water testing — aeration control
- Wastewater treatment process control — activated sludge aeration
- Limnological research — lake oxygen profile
- River self-purification capacity assessment
- Aquaculture — oxygen control in fish farming
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 0.2–20 mg O₂/L |
| Limit of quantification (LOQ) | 0.2 mg O₂/L |
| Accuracy | ±0.05 mg O₂/L (with careful work) |
| DO saturation (20°C, 1 atm) | ~8.84 mg O₂/L (pure water) |
| Winkler bottle volume | 250–300 mL |
| Interferences | NO₂⁻ (eliminated by NaN₃), Fe²⁺/Fe³⁺, free Cl₂, S²⁻ |
Standard
- Standard number
- PN-EN ISO 5813:2006
- Title (PL)
- Jakość wody — Oznaczanie tlenu rozpuszczonego — Metoda jodometryczna
- Title (EN)
- Water quality — Determination of dissolved oxygen — Iodometric method
Step-by-step procedure
1. Sample collection
Fill Winkler bottle (300 mL) with water sample — immerse bottle or use silicone tube. Do not aerate! Rinse with 3-fold volume. Close without air bubbles.
2. Fixation — MnSO₄
Add 2 mL MnSO₄ solution with pipette submerged below water surface. Remove pipette.
3. Fixation — alkaline-iodide reagent
Add 2 mL alkaline-iodide reagent with NaN₃ with submerged pipette. Close bottle with stopper (without bubbles). Mix by repeated inversion. White (no O₂) or brown (O₂ present) precipitate forms.
4. Precipitate sedimentation
Let bottle stand for precipitate settling (min. 5 min). Mix again and let settle again. Precipitate should settle below half of bottle.
5. Acidification
Add 2 mL concentrated H₂SO₄. Close and mix by inversion until precipitate completely dissolves. Solution becomes yellow-brown (iodine).
6. Aliquot sampling
Measure 203 mL (corresponds to 200 mL original sample after deducting reagent volumes) into Erlenmeyer flask.
7. Thiosulfate titration
Titrate with Na₂S₂O₃ 0.025 M from burette to pale yellow color. Add 1 mL starch solution (blue color). Continue titration until blue color disappears.
8. Volume reading
Read consumed Na₂S₂O₃ volume with accuracy ±0.05 mL.
9. Calculations
DO [mg O₂/L] = V(Na₂S₂O₃) × M(Na₂S₂O₃) × 8000 / V_sample. For Na₂S₂O₃ 0.025 M and 200 mL sample: 1 mL thiosulfate ≈ 1 mg O₂/L.
10. Thiosulfate titer verification
Standardize thiosulfate against K₂Cr₂O₇ 0.025 M + KI in H₂SO₄ medium (darkness, 5 min). Titrate as sample. Correction factor: f = V_theor/V_actual.
11. Quality control
Saturated deionized water (aerated 1 h at 20°C) — DO ≈ 8.8 mg/L (±0.3). Duplicate: difference < 0.2 mg/L.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Winkler bottles 250–300 mL | Glass bottles with ground glass stopper and water seal, DURAN | 30–60 PLN/pc |
| Burette 25 mL class A | Glass burette with PTFE stopcock, 0.05 mL graduation | 150–400 PLN |
| Automatic pipettes | Eppendorf Research Plus 1–5 mL, Brand Transferpette | 800–2,000 PLN |
| Erlenmeyer flasks 250 mL | DURAN borosilicate, for titration | 15–30 PLN/pc |
| Magnetic stirrer | IKA C-MAG MS 4 (optional, for titration) | 1,500–4,000 PLN |
| Laboratory thermometer | Glass or digital thermometer, accuracy ±0.1°C | 50–500 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Manganous sulfate (MnSO₄·H₂O) | 10034-96-5 | Solution 480 g/L MnSO₄·4H₂O in water, alkaline reagent |
| Alkaline-iodide reagent with azide | — | NaOH 500 g + NaI 135 g + NaN₃ (CAS 26628-22-8) 10 g per 1 L water. WARNING: NaN₃ highly toxic! |
| Concentrated sulfuric acid (H₂SO₄) | 7664-93-9 | For sample acidification — precipitate dissolution and I₂ liberation |
| Sodium thiosulfate (Na₂S₂O₃) 0.025 M | 7772-98-7 | Standardized solution for titration, stabilized with NaOH (0.4 g/L) |
| Soluble starch (indicator) | 9005-25-8 | 1% solution in water (boil), end point indicator (blue → colorless) |
| Potassium dichromate (K₂Cr₂O₇) | 7778-50-9 | 0.025 M, for sodium thiosulfate titer verification |
| Potassium iodide (KI) | 7681-11-0 | For titer verification, analytical grade, free from iodates |
| Sodium azide (NaN₃) | 26628-22-8 | Nitrite interference elimination — HIGHLY TOXIC, fatal if swallowed |
Health and safety (OHS)
- Sodium azide (NaN₃) — HIGHLY TOXIC, fatal if swallowed (LD50 ~27 mg/kg), reacts with acids releasing toxic HN₃!
- Concentrated sulfuric acid — highly corrosive, severe burns, add carefully to water
- NaOH solution (alkaline reagent) — corrosive, chemical burns to skin and eyes
- Liberated iodine — irritant, stains skin, avoid vapor inhalation
- Potassium dichromate — carcinogenic (Cr⁶⁺), use in fume hood, gloves
- Safety glasses, lab coat, and nitrile gloves MANDATORY at every step