🔬 Nitrites in Water
Spectrophotometric determination of nitrites (NO₂⁻) in water by diazotization with sulfanilamide and NED. An indicator of incomplete nitrification and fresh contamination.
Overview
Nitrites (NO₂⁻) are an intermediate form in the nitrogen cycle — formed during nitrification (NH₄⁺ → NO₂⁻) and denitrification (NO₃⁻ → NO₂⁻). Their presence in water indicates: fresh sewage contamination, incomplete nitrification, inadequate disinfection, or problems in water distribution system.
Nitrites are toxic — cause methemoglobinemia (cyanosis), particularly dangerous for infants. Drinking water standard: ≤0.5 mg NO₂⁻/L (Polish Ministry of Health Regulation). In natural waters concentration is usually low (<0.1 mg/L), because nitrites quickly undergo further oxidation to nitrates.
ISO 6777 method is based on Griess reaction — diazotization of sulfanilamide by nitrite ion and coupling with N-(1-naphthyl)ethylenediamine (NED), forming intensely pink-red azo dye measured at 540 nm. Method is very sensitive — detection limit 0.005–0.01 mg N/L.
Method principle
In acidic environment (pH 1.5–2.0) nitrites react with sulfanilamide (4-aminobenzenesulfonic acid) forming diazonium salt. Diazonium salt undergoes coupling reaction with N-(1-naphthyl)ethylenediamine dihydrochloride (NED), forming intensely colored pink-red azo dye.
NO₂⁻ + sulfanilamide → (H⁺) → diazonium salt Diazonium salt + NED → azo dye (pink-red)
Dye absorbance is measured at 540 nm. Reaction proceeds at room temperature, color develops within 10–20 minutes and is stable for 2 hours. Molar absorption coefficient: ~53 000 M⁻¹cm⁻¹.
Applications
- Drinking water quality control (limit ≤0.5 mg NO₂⁻/L)
- Water distribution network monitoring (secondary contamination indicator)
- Nitrification process control in wastewater treatment plants
- Surface and groundwater testing
- Pool water quality control
- Source and water intake monitoring
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 0.005–1.0 mg N/L |
| Detection limit | 0.005–0.01 mg N/L |
| Wavelength | 540 nm |
| Color development time | 10–20 minutes (stable 2 h) |
| Molar absorption coefficient | ~53 000 M⁻¹cm⁻¹ |
| Precision (CV) | 2–5% |
Standard
- Standard number
- PN-EN 26777:1999
- Title (PL)
- Jakość wody — Oznaczanie azotynów — Metoda absorpcyjnej spektrometrii cząsteczkowej
- Title (EN)
- Water quality — Determination of nitrite — Molecular absorption spectrometric method
Step-by-step procedure
1. Solution preparation
Prepare sulfanilamide solution in HCl and NED solution in water. Store in dark bottles refrigerated. Solutions valid max 1 month.
2. Calibration curve
Prepare standard series: 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1.0 mg N/L from NaNO₂ standard solution. WARNING: diluted nitrite solutions prepare fresh (unstable!).
3. Sample measurement
Measure 40 mL sample (or less at higher concentrations + fill with water to 40 mL) into 50 mL volumetric flask. Analyze sample immediately after sampling — nitrites unstable!
4. Sulfanilamide addition
Add 1 mL sulfanilamide solution. Mix. Wait 2–8 minutes (diazotization).
5. NED addition
Add 1 mL NED solution. Mix. Fill to 50 mL with deionized water. Mix thoroughly. Pink-red color develops within 10 minutes.
6. Color development
Wait 10–20 minutes for full color development. Color stable for next 2 hours — measure within this time window.
7. Absorbance measurement
Measure absorbance at 540 nm against blank (40 mL water + reagents). Read concentration from calibration curve.
8. Quality control
Control standard — deviation max ±10%. Blank — absorbance <0.005. Duplicates — RPD <10%. Curve R² >0.999.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| VIS or UV-VIS spectrophotometer | Hach DR3900, Shimadzu UV-1900, WTW photoLab 7600 | 15 000–45 000 PLN |
| Optical cuvettes 10–50 mm | Glass cuvettes 10, 20, 40, 50 mm | 50–300 PLN/pc |
| Automatic pipettes | Eppendorf Research Plus 0.1–1 mL and 1–10 mL | 800–2 000 PLN |
| Volumetric flasks 50 mL | Class A, borosilicate glass | 30–80 PLN/pc |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Sulfanilamide (4-aminobenzenesulfonamide) | 63-74-1 | Diazotizing solution: 10 g sulfanilamide in 100 mL HCl (1+1) + 800 mL water, fill to 1 L. Shelf life: 1 month refrigerated. |
| N-(1-naphthyl)ethylenediamine (NED) dihydrochloride | 1465-25-4 | Coupling reagent: 1 g NED·2HCl in 1 L water. Store in dark bottle. Shelf life: 1 month refrigerated. Discard when turns brown. |
| Hydrochloric acid HCl | 7647-01-0 | Diluted HCl (1+1) for acidifying reaction environment to pH 1.5–2.0. |
| NO₂⁻ standard solution 1000 mg N/L | 7632-00-0 | NaNO₂ in water, certified CRM. For calibration curve. WARNING: nitrite solutions unstable — prepare fresh! |
| Nitrite-free deionized water | — | Checked for NO₂⁻ absence — absorbance at 540 nm <0.005 (as blank). |
Health and safety (OHS)
- Hydrochloric acid HCl — corrosive, irritating fumes. Work under fume hood.
- NED — irritating, sensitizing. Use nitrile gloves.
- Sulfanilamide — irritating, avoid skin contact.
- Sodium nitrite NaNO₂ — toxic if ingested, oxidizer. Do not mix with acids (toxic nitrogen oxide release)!
- Safety goggles, gloves, and coat mandatory.