🧪 PAH in water
Determination of 15 polycyclic aromatic hydrocarbons in water by HPLC with fluorescence detection after liquid-liquid extraction. Sensitivity > 0.005 µg/L.
Overview
Polycyclic aromatic hydrocarbons (PAH) are a group of over 100 organic compounds consisting of two or more condensed aromatic rings. Many of them are carcinogenic — benzo(a)pyrene is IARC Group 1 carcinogen, and over a dozen others are classified as probable or possible carcinogens.
PN-EN ISO 17993:2005 specifies the method for determining 15 selected PAH in drinking, ground, and surface water: naphthalene, acenaphthylene (UV only), acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(ghi)perylene, indeno(1,2,3-cd)pyrene.
Permissible concentrations in drinking water (Journal of Laws 2017, item 2294): benzo(a)pyrene ≤ 0.01 µg/L, sum of PAH (4 compounds) ≤ 0.1 µg/L. These standards are very restrictive and require techniques with extreme sensitivity, hence the need for fluorescence detection.
Method principle
The method consists of three stages: (1) liquid-liquid extraction, (2) HPLC chromatographic separation, (3) fluorescence detection.
1. Extraction: PAH from aqueous sample are extracted to cyclohexane or dichloromethane in a separatory funnel. Extract is evaporated and reconstituted in acetonitrile (HPLC solvent).
2. HPLC chromatography: Separation of 15 PAH on C18 column (reversed phase) with gradient elution water/acetonitrile. Analysis time 30–60 min. Each PAH elutes at different retention time.
3. Fluorescence detection (FLD): PAH exhibit strong native fluorescence — they absorb UV radiation and emit in the visible range. Fluorescence detector measures emission at programmed wavelengths (different Ex/Em pairs for different PAH groups), ensuring extreme sensitivity (LOD ~ 0.001 µg/L) and selectivity.
Naphthalene and acenaphthylene — weakly fluorescent — are alternatively determined by UV-DAD detector.
Applications
- Drinking water quality control (benzo(a)pyrene ≤ 0.01 µg/L, sum PAH ≤ 0.1 µg/L)
- Monitoring of surface waters — priority substances (Directive 2013/39/EU)
- Analysis of groundwater (contamination from old gasworks, coking plants)
- Monitoring of stormwater (exhaust emissions, PM2.5 dust)
- Control of industrial wastewater (refineries, coking plants)
- Environmental studies of post-industrial sites (brownfields)
Key parameters
| Parameter | Value |
|---|---|
| Number of determined PAH | 15 (EPA/ISO list) |
| Limit of quantification (FLD) | 0.005 µg/L (drinking and groundwater) |
| Limit of quantification (FLD, surface water) | 0.01 µg/L |
| Detection method | Fluorescence (FLD) + UV-DAD (naphthalene, acenaphthylene) |
| HPLC column | C18 reversed phase, 250 × 4.6 mm, 5 µm |
| Analysis time | 30–60 min/sample (gradient) |
Standard
- Standard number
- PN-EN ISO 17993:2005
- Title (PL)
- Jakość wody — Oznaczanie 15 wielopierścieniowych węglowodorów aromatycznych (WWA) w wodzie metodą HPLC z detekcją fluorescencyjną po ekstrakcji ciecz-ciecz
- Title (EN)
- Water quality — Determination of 15 polycyclic aromatic hydrocarbons (PAH) in water by HPLC with fluorescence detection after liquid-liquid extraction
Step-by-step procedure
1. Sampling
Collect in dark glass bottles (protection from PAH photodegradation). Fill without headspace. Transport in dark, 4°C. Analysis within 7 days.
2. Liquid-liquid extraction
Transfer 1 L sample to 2 L separatory funnel. Add 50 mL cyclohexane. Shake 10 min. Separate phases. Repeat 2× with 25 mL. Combine extracts.
3. Drying and concentration
Filter extract through anhydrous Na₂SO₄. Evaporate on rotary evaporator to ~1 mL (bath temp < 40°C, vacuum). Exchange solvent to ACN.
4. Reconstitution
Dissolve residue in 1 mL acetonitrile (ACN). Transfer to HPLC vial with insert. Centrifuge 5 min/10000 rpm (optionally 0.22 µm PTFE filter).
5. HPLC analysis
Inject 10–50 µL on C18 PAH column. Gradient ACN/H₂O: 50% → 100% ACN in 30–50 min. FLD detection with Ex/Em switching. UV-DAD for naphthalene/acenaphthylene.
6. Identification and calculations
Identification based on retention time (±2%). Quantification from calibration curve (5–7 points). Account for extraction recovery (surrogates added).
7. Quality control
Blank sample (HPLC water through extraction). Fortified sample (spike). CRM (PAH-certified water). Recovery 60–120%.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with FLD detector | Agilent 1260/1290 Infinity + FLD, Waters Alliance + FLD 2475, Shimadzu Nexera + RF-20A | 200 000–500 000 PLN |
| UV-DAD detector (additional) | For determining naphthalene and acenaphthylene | 50 000–120 000 PLN |
| HPLC C18 column for PAH | Waters PAH C18, Agilent ZORBAX Eclipse PAH, Supelcosil LC-PAH | 1 500–3 500 PLN |
| Liquid-liquid extraction system | 1–2 L separatory funnels, rotary evaporator (Buchi R-300) | 15 000–40 000 PLN (evaporator) |
| Autosampler | Agilent G7167B, Waters 2707 | included with HPLC |
| Degasser and gradient pump | Quaternary gradient pump system — standard in HPLC | included with HPLC |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Acetonitrile (ACN) HPLC grade | 75-05-8 | HPLC gradient grade purity (Merck LiChrosolv), mobile phase |
| Water HPLC grade | — | Ultrapure water, degassed, type I (18.2 MΩ·cm) |
| Cyclohexane | 110-82-7 | Purity for pesticide/PAH analysis. Extraction solvent |
| 15/16 PAH mixture standard | — | Certified EPA 610 PAH Mix solution (Supelco), 10–2000 µg/mL in ACN |
| Anhydrous Na₂SO₄ | 7757-82-6 | Calcined (400°C, 4h), for drying organic phase |
| Dichloromethane (DCM) | 75-09-2 | Alternative extraction solvent, pesticides grade purity |
Health and safety (OHS)
- PAH (benzo(a)pyrene) — carcinogenic! Avoid skin contact. Double nitrile gloves
- Cyclohexane — flammable, irritating. Work in fume hood, away from ignition sources
- Acetonitrile — toxic, flammable. Metabolized to HCN. Work in fume hood
- Dichloromethane — possible carcinogen. Vapors heavier than air. Fume hood mandatory
- PAH standard solutions — diluted but contain carcinogens. Gloves, avoid spillage
- Organic waste — collect separately (halogenated and non-halogenated)