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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Laboratory Methods  /  Water Analysis  /  Chlorophyll a Concentration

Chlorophyll a Concentration

Determination of algal biomas using phytosynthetic pigments

Samples of sediment and attached algae are always contaminated by detritus and other organisms but even phytoplankton are usually intimately mixed with detritus and bacteria. It is often necessary to determine the size of the crop of algae but the contamination makes measures of organic and carbon content or dry weight meaningless. However, although each phylum differs in the precise suite of pigments its members contain, all algae have the green chlorophyll a and a collection of carotenoids, which are orange-yellow in colour. The ratio of chlorophyll a to organic content of the cells varies with the physiological state of the cells and the ration of carotenoid to chlorophyll a can give a useful index of, for example, nitrogen deficiency.

Determination of pigment content of phytoplankton

Filter a known volume (250ml–1litre) of well shaken water through a glass-fibre filter under vacuum. Suck the filter dry and transfer it to a mortar. Add a pinch of sand, 1ml of MgCO3 suspension (5%) and about 1ml of acetone from the wasbottle. Grind to a smooth paste then wash the paste carefully into a measuring cylinder or graduated centrifuge tube and make up to 10ml with acetone. Place tubes in a chilled cold box for 30mins to allow pigments to be extracted into the acetone and the solids to settle out , then transfer the supernatant to a 1cm spectrophotometer cell with a pasteur pipette. Against an acetone blank determine the absorbance at 750nm, 663nm, 480nm, 430nm, and 410nm in the spectrophotometer. The value at 750 corrects for any fine colloidal matter and should be subtracted from each of the other values.

Calculate the chlorophyll a concentration as follows:

Equation 1


where...

  • A = absorbance at 663
  • v = volume (ml) of 90% acetone used for extraction (in this case 10ml)
  • V = volume of water filtered (in litres)
  • d = path length (cm) of the spectrophotometer cell (in this case 1cm)

The value 11.0 is derived from the specific absorbance (absorbance per mole) of chlorophyll a. The units calculated are mg chlorophyll a per litre of water. Typical values for freshwater lakes range 1 - 300mg/L.

Calculate the carotenoid concentration as follows:

Equation 2

 

 

The unit here is the m Specific Pigment Unit (mSPU), which is roughly equivalent to 1mg per litre. A mixture of substances is being measured, hence the precise measure of weight connot be used.

Now calculate the ratios A480 : A663 and A430 : A410. The following table will help you to interpret these.

 

chlorotab

 

 

 

 

 

 

 

 

 

 

Phytoplankton Primary Productivity

There are two main ways of determining the rate of photosynthesis (primary productivity) of a sample of algae. One involves the uptake of radioactive CO2, the other onvolves measurement of the rate of evolution of oxygen and is documented here. The basic titration for measurement of disssolved oxygen has a variety of uses in projects (e.g. microbial or animal respiration experiments) as well as measurements of photosynthesis and is given in detail elsewhere.

Fill two clear glass-stoppered bottles (25ml) and one opaque bottle with the algal suspension. The bottles should be filled to the brim and air bubbles should not be trapped when the stoppers are inserted. Place one clear bottle and the opaque bottle on a light rack or back in the lake and record the time. To the remaining clear bottle add Winkler reagents and determine the initial oxygen concentration. After several hours remove the other bottles, record the time and determine their oxygen concentration.