🌿 Chlorophyll a in Water
Spectrophotometric determination of chlorophyll a in surface waters after acetone extraction and filtration. Key indicator of eutrophication of water bodies and water trophic state.
Overview
Chlorophyll a is the main photosynthetic pigment present in all photoautotrophic organisms — algae, cyanobacteria, and aquatic plants. Its concentration in water is a widely used indicator of phytoplankton biomass and degree of eutrophication of water bodies. Trophic classification of waters is largely based on chlorophyll a concentration: oligotrophic waters (<2.5 µg/L), mesotrophic (2.5–8 µg/L), eutrophic (8–25 µg/L), and hypereutrophic (>25 µg/L).
The spectrophotometric method according to PN-ISO 10260 involves concentrating phytoplankton on glass fiber filter (GF/C or GF/F), extracting pigments with acetone (90%), and measuring absorbance at 665 nm (chlorophyll a absorption maximum) with turbidity correction at 750 nm. Acidification of extract with HCl allows distinguishing active chlorophyll a from its degradation products (pheophytin a).
Chlorophyll a monitoring is required by Water Framework Directive (2000/60/EC) as element of biological assessment of surface water status. In Poland, this parameter is mandatory in ecological status assessment of lakes and dam reservoirs (Journal of Laws 2019 item 2149).
Alternatively, chlorophyll a can be determined fluorometrically (more sensitive, LOD ~0.1 µg/L) or chromatographically (HPLC — separation of individual pigments). However, the spectrophotometric method remains reference method due to simplicity and low cost.
Method principle
Phytoplankton from defined water volume (0.5–2 L) is concentrated on glass fiber filter (GF/C, porosity 1.2 µm or GF/F, 0.7 µm) by vacuum filtration. Filter with residue is homogenized in 90% acetone and extracted in darkness at 4°C for 12–24 hours. Extract is clarified by centrifugation (500 g, 20 min). Absorbance is measured at 665 nm (chlorophyll a) and 750 nm (turbidity correction). Then extract is acidified with 0.1 M HCl (0.1 mL per 10 mL extract) and after 90 seconds absorbance is measured again at 665 and 750 nm. Difference in absorbance before and after acidification allows calculation of active chlorophyll a and pheophytin a concentrations. Calculations: Chl a [µg/L] = 26.7 × (A665₀ - A665a) × V_extract / (V_sample × l), where l — optical path length.
Applications
- Eutrophication monitoring of lakes and dam reservoirs (WFD 2000/60/EC)
- Trophic classification of surface waters (oligotrophic → hypereutrophic)
- Ecological status assessment of waters — biological element (Journal of Laws 2019 item 2149)
- Algal and cyanobacterial bloom monitoring (toxin risk)
- Water quality control at surface water intakes
- Limnological and oceanographic research
Key parameters
| Parameter | Value |
|---|---|
| Measurement wavelength | 665 nm (chlorophyll a), 750 nm (background correction) |
| Measurement range | 0.5–500 µg/L (with filtration volume adjustment) |
| Limit of quantification (LOQ) | 0.5–1.0 µg/L |
| Extraction solvent | 90% aqueous acetone |
| Extraction time | 12–24 hours at 4°C, darkness |
| Trophic classification | <2.5 µg/L oligo | 2.5–8 meso | 8–25 eu | >25 hyper |
Standard
- Standard number
- PN-ISO 10260:2002
- Title (PL)
- Jakość wody — Pomiar parametrów biochemicznych — Spektrofotometryczne oznaczanie stężenia chlorofilu a
- Title (EN)
- Water quality — Measurement of biochemical parameters — Spectrometric determination of the chlorophyll a concentration
Step-by-step procedure
1. Water sample collection
Collect representative water sample (1–5 L) from established depth. Store in darkness at 4°C. Filter within 4 hours of collection.
2. Filtration
Place GF/C filter (47 mm) on vacuum filtration apparatus. Add pinch of MgCO₃ to filter. Filter measured sample volume (0.5–2 L) under vacuum (<0.3 bar).
3. Acetone extraction
Transfer filter with residue to centrifuge tube. Add 10 mL 90% acetone. Homogenize filter with Ultra-Turrax (30 s). Close, wrap with aluminum foil.
4. Incubation in darkness
Place tubes in refrigerator (4°C) for 12–24 hours in darkness. Acetone penetrates cells and extracts pigments.
5. Extract clarification
Centrifuge extract (500 g, 20 min) or filter through GF/F filter. Transfer clear extract to clean vial.
6. Absorbance measurement (before acidification)
Measure absorbance at 665 nm and 750 nm against 90% acetone (blank). Record values A665₀ and A750₀.
7. HCl acidification
Add 0.1 mL 0.1 M HCl to cuvette with 10 mL extract. Gently mix by inversion. Wait exactly 90 seconds.
8. Absorbance measurement (after acidification)
Measure absorbance at 665 nm and 750 nm. Record values A665a and A750a. Acidification converts chlorophyll → pheophytin.
9. Calculations
Chl a [µg/L] = 26.7 × [(A665₀ - A750₀) - (A665a - A750a)] × V_extract [mL] / (V_sample [L] × l [cm]). Pheophytin a = 26.7 × [1.7 × (A665a - A750a) - (A665₀ - A750₀)] × V_e / (V_p × l).
10. Quality control
Chlorophyll a standard in acetone (known concentration) — recovery 90–110%. Acetone blank A665 < 0.005. Duplicate: CV < 15%.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| UV-Vis spectrophotometer | Shimadzu UV-1900i, Hach DR 6000, Agilent Cary 60, Thermo Evolution 201 | 15,000–80,000 PLN |
| Vacuum filtration apparatus | Sartorius, Millipore, 47 mm funnel + suction flask + membrane pump | 2,000–5,000 PLN |
| Glass fiber filters GF/C | Whatman GF/C 47 mm (1.2 µm) or GF/F (0.7 µm) | 1–3 PLN/pc |
| Laboratory centrifuge | Eppendorf 5804R, Hettich Universal 320R, min. 500 g | 10,000–40,000 PLN |
| Tissue homogenizer | IKA T10 basic Ultra-Turrax, glass Potter homogenizer | 3,000–8,000 PLN |
| Laboratory refrigerator (4°C) | Liebherr LKPv 6520, POL-EKO CHL 700 | 5,000–15,000 PLN |
| Glass cuvettes 1–5 cm | Hellma, Starna, optical borosilicate | 100–500 PLN/pc |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Acetone (C₃H₆O) 90% | 67-64-1 | Analytical grade, 900 mL acetone + 100 mL deionized water, extraction solvent |
| Hydrochloric acid (HCl) 0.1 M | 7647-01-0 | For extract acidification — chlorophyll a and pheophytin a differentiation |
| Magnesium carbonate (MgCO₃) | 546-93-0 | Addition to filter before filtration — prevents chlorophyll degradation |
| Chlorophyll a standard | 479-61-8 | Pure chlorophyll a from Anacystis nidulans or spinach, for calibration, Sigma-Aldrich C6144 |
| Deionized water | — | For 90% acetone preparation and rinsing |
Health and safety (OHS)
- Acetone — flammable (flash point -20°C), vapors form explosive mixtures, work under fume hood!
- Hydrochloric acid — corrosive, irritant vapors, use in ventilated area
- Water samples — potentially contain cyanobacterial toxins (microcystins), gloves
- Safety glasses, lab coat, and nitrile gloves mandatory
- Collect acetone waste in flammable organic waste container