Extraction and determination of chlorophyll
2.1 Weight accurately 2 g of sample and put into mortar.
2.2 Add 0.1 g Sodium carbonate and 0.2 g quartz sand.
2.3 Macerate tissue with pestle and grind short time.
2.4 Add 90% acetone, little at time, and continue grinding until tissue is finely ground.
2.5 Filter with filter paper (Whatman No.4) and wash residue with 90% acetone.
2.6 Return residue to mortar with more 90% acetone and grind again.
2.7 Filter and wash as before.
2.8 Repeat procedure until tissue is devoid of any green and washing are colorless.
2.9 Combine the clarified extract solution and dilute to 100 ml with 90% acetone.
2.10 Calculate the chlorophyll contents by the following equations as shown below:
Total chlorophyll content (mg/L) = (7.12×A660) + (16.8×A642.5)
Chlorophyll a (mg/L) = (9.93×A660) - (0.77×A642.5)
Chlorophyll b (mg/L) = (17.6×A642.5) - (2.81×A660)