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For determination of chlorophyll a and b, and total carotene content, the modified method of Dere
et was used. The weighted sample, having been added diethyl ether (50 mL for each gram) was dissolved in an ultrasonic bath for one minute. It was then homogenized for 30 seconds with homogenizer, and again in the ultrasonic bath for one minute.
The homogenate was centrifuged for 10 minutes at 3000 rpm. The supernatant was separated and the
absorbances measured at 400 – 700 nm in an UV spectrophotometer. Chlorophyll a showed the maximum absorbance at 660 nm, chlorophyll b at 642.5 nm, and total carotene at 470 nm. All analyses were repeated three times. The amount of these pigments was calculated according to the formulas given below (Eqs. 2–5):
Chlorophyll a = 9.93· A660 – 0.78· A642.5
Chlorophyll b = 17.60· A642.5 – 2.81· A660
Chlorophyll a+b = 7.12· A660 – 16.80· A642.5
Total carotene = (1000· A480 – 0.52· Chl a – 7.25· Chl b) / 226
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