In this article we will discuss about the tests for spectrophotometric determination of chlorophyll and carotenoids.
Extraction of Chlorophyll and Carotenoids:
Although chlorophyllous and carotenoid pigments may be extracted readily in organic solvents, for many algae, extraction is not complete unless the cells are ruptured.
Therefore, filters should be placed with forceps into the base of a glass for drinking with 3 to 4 ml of 90% alkaline acetone solution (1 lit. acetone = 100 ml dist. water + 2 drops NH4OH soln.). Millipore filters will dissolve immediately. Decant the supernatant and then centrifuge at 3,000 to 4,000 rpm for 5 mins. Store the supernatant in dark cold place, if necessary.
Test Procedure:
1. Measure the extinction coefficient of your pigment extracts in 90% alkaline acetone solution at 750 (for turbidity blank), 665, 664, 663, 647, 630 and 480 nm. Absorption at 750 nm is subtracted from each of the other values to correct for the presence of turbidity and coloured materials.
The pigments and phaeophylins absorb very little light at 750 nm. The spectrophotometer should be brought to 100% transmission at each wavelength by referencing against a matched cell containing only 90% acetone.
When the cells are exactly matched, fill both cells with 90% acetone and determine the “blank” correction at each wavelength. Correct all extinction co-efficient values by this amount. Record extinction values to the nearer 0.001 in the range of 0 to 0.04 and to the nearest 0.005 for extinctions of coefficients exceeding 0.04.
2. Add 0.1 ml of 1 N HC1 per ml of extract directly to the spectrophotometric cell, reseal and mix thoroughly. Allow to rest in the cuvette-holder for 5 mins.
3. Re-measure the extinction co-efficient values at 750, 665 and 663 nm.
4. Rinse the cells copiously with alkaline acetone and shake dry prior to use with the next sample.
Calculations:
(a) Monochromatic method (Lorenzen, 1967):
(b) Dichromatic method for mixed phytoplankton communities containing chlorophylls a and b and equal amount of chlorophylls C, and C2 with 90% acetone solvent (Jeffery and Humphrey, 1975):
From the data the amount of pigments per cell of phytoplankton can be estimated:
µg Chl in extract = (mg Chl/ml) (vol.of extract, ml)
µ ml of Chl in extract = µg Ch1 in extract/mol weight in chl
where the formula weight are Chl a = 893.5 and Chl b = 907.5 and Chl c = 610
µ ml of Chl/cell = µmol Chl in extract/ number of cells in sample
Plant carotenoids (Strickland and Parsons, 1972):
Car (µ Spu/lit or mSPU/m3) = (4.0) (E4800) (V)/(V) (Z)
where SPU = Specified plant pigment units approximating the mg
E4800= A480 – [ (A750)]
Use the factor of 10.0 when the algae consists primarily of Chrysoplyta Pyrrophyta or both.
When the algae consists predominantly of members of chlorophyta and/or cyanophyta, then use the following equation:
