Marine Biology (2+1)

Estimation of chlorophyll

Principle

The water sample free of zooplankton is filtered through GF/C glass filter paper and the pigments, chlorophyll a, b and c are extracted from the phytoplankton by using 90% acetone. The resulting coloured acetone extract is measured in a spectrophotometer.

Reagents

90% acetone: Mix 90 ml of acetone with 10 ml of distilled water to get 90% acetone.

Magnesium carbonate suspension: Add 1 g of finely powdered magnesium carbonate to 100 ml of distilled water.

Procedure

Invert the polythene bottle containing the sample (free from zooplankton), into the funnel of millipore filtering equipment fitted with a 4.5 cm dia GF/C filter paper with a thin bed of magnesium carbonate suspension. The magnesium carbonate bed can be prepared by pipetting out 2ml of magnesium carbonate solution onto the glass-fiber filter paper fitted in the filtering equipment, and then applying low suction and sucking out the magnesium carbonate to a dry bed.

After the formation of a thin film of magnesium carbonate, pour 250 ml of the sample into the funnel and apply suction.

After e filtration remove the filter paper with magnesium carbonate bed and place it in a clean test-tube free of acid.

Add 5 ml of 90% acetone and grind well by using a tissue grinder.

Take 2 ml of 90% acetone and wash the pestle of the grinder inside the test tube.

Then close the test-tube with a stopper and keep it in a black bag in a refrigerator for 20-24 hours.

After that open the tube again and re-grind the contents.

Take 1 ml of acetone to wash the pestle inside the test tube.

Pour the contents of the test-tube into a centrifuge tube and carry out the centrifugation at 3000 rpm for 5 minutes.

Make up the clear supernatant extract to 10 ml with 90% acetone.

Measure the extinction of the extract at the wave length of 530 nm, 645 nm and 665 nm using a 10 cm path length cell (cuvette).

Calculation

                     Chlorophyll - a = 11.6E₆₆₅ -   1.31 E₆₄₅ - 0.14E ₆₃₀

                     Chlorophyll - b  =20.7E₆₄₅ - 4.34E₆₆₅ - 4.42E₆₃₀

                     Chlorophyll - c  = 5.15E₆₃₀ - 4.65E₆₆₅ - 16.3E₆₄₅

Here E stands for the extinction (absorbance or optical density) at different wavelengths obtained.

Example for calculation of chlorophylla

Volume of water filtered = 5 litres

The extinction ‘E’ measured using 10 cm cell:

E = 0.34     665

E = 0.09     645

E = 0.08     630

Therefore chlorophyll ‘a’ present in 5 litre sample (Ca) =

(11.6 x 0.34) – (1.31 x 0.09) – (0.14 x 0.08)

= 3.944 – 0.1179 – 0.0112

= 3.8149

                                           Ca

Chlorophyll concentration ----- mg/m3

                                            V

Where Ca is the amount of chlorophyll ‘a’ present in the filtered water

V is the volume of water in litres.

                                3.8149

Then chlorophyll a = --------- = 0.763 mg/m3

                                     5

In this way, chlorophyll ‘b’ and ‘c’ can be calculated using the equations given above.

Determination of the phaeo-pigments

Chlorophyll degradation products may at times constitute a significant fraction of the total green pigments in seawater. These degraded forms of inactive chlorophyll interfere with the spectrophotometric determination of chlorophylls because they absorb light in the same region of the spectrum as chlorophyll. Pigment samples from the aphotic zone, sediments, and samples from areas of high zooplankton grazing are likely to contain inactive chlorophyll products. Chemically these may consist predominantly of phaeophytin and phaeophorbide (phaeo-pigments) but sometimes large quantities of chlorophyllide may also be present. In the following method it is possible to obtain a measure of the total quantity of chlorophyll a and phaeophytin a plus phaeophorbide a, but not of chlorophuyllide a or the phaeophytins of all chlorophylls and their degradation products. There is probably no alternative to chromatographic methods which are generally too tedious for the routine analysis of a large number of samples. For a routine observation, however, it is often sufficient to obtain a measure of the amount of non-active chlorophyll a in terms of the quantity of phaeo-pigments  present.

Outline of the method

The extinction of an acetone extract of plant pigment is measured before and after treatment with dilute acid. The change following acidification is used as a measure of the quantity of phaeo-pigments in the original sample.

Special reagents

Reagents 1 and 2

Hydrochloric acid: Dilute 50 ml of concentrated hydrochloric acid to 100 ml with distilled water.

Procedure

Carry out procedure

Measure the extinction of the extract at 665 and 750 nm.

Add two drops of dilute hydrochloric acid to the cuvette, mix, and remeasure the extinction at 665 and 750 nm.

Subtract each 750 nm reading from the corresponding 665 nm, extinction and use the following equations to calculate the concentration of chlorophyll a and phaeo-pigments  in the sample.

                                        26.7 (665 – 665) x va o a

Chlorophyll a (mg/m3) = ----------------------------------

                                                        V x L

                                            26.7 (1.7 (665) - 665) x vao

Phaeco-pigments (mg/m3) = ----------------------------------

                                                        V x L

where 665 is the extinction at 665 nm before acidification, 0665 is the extinction at 665 nm after acidification

va is the volume (in ml) of acetone extract

V is the volume (in litres) of acetone extract

and L is the path length (in cm) of the cuvette.