Environmental Engineering Laboratory

By: Le Ngoc Thuan

Chlorophyll-a

The concentration of photosynthetic pigments is used extensively to estimate phytoplankton

biomass, all green plants contain chlorophyll-a., which constitutes approximately 1 or 2%of
the dried weight of planktonic algae.

Apparatus:

Membrane filter funnel, Filtration apparatus with reservoir and coarse (40- to 60- µm).

Procedure:

1. Filtrate 200ml sample through filter with filtration apparatus and

2. Remove filter to a 100ml bottle (Schott Duran).
3. Add 10 ml of acetone solution (9:1), rinse completely filter into the solution.
4. Cover the bottle with a stopper and foil
5. Incubate bottle at 20oC for at least 1 day.
6. Take out and grain filter with a glass bar to extract chlorophyll-a.
7. Transfer acetone solution to centrifuge tube and operate centrifugation at 6000 rpm
for 10 minutes.
8. Absorbance reading the supernatant at 750nm, 663nm, 645nm, 630nm
Prepare one blank sample, or absorbance reading with acetone solution 9:1 as blank
sample.

Caculation:

Y x V1 (ml)
Chlorophyll-a (mg/m3) =
V2 (L)

Where: Y= 11.64 X1 – 2.16 X2 + 0.10 X3

X1 = OD663 – OD750 (optical density)
X2 = OD645 – OD750
X3 = OD630 – OD750
V1 = volume of extract, ml
V2 = volume of sample, L
Reagent:
- Acetone solution 9:1, = 9V acetone :1V dW