Dear Colleague,
I would like to know the best equation for estimation of total chlorophyll in aquatic plants. I found more than one, and I do not know what is the correct equation. Also, what is the correct optical densities of the extracts using spectrophotometer. I found research use the three wave length (665, 645 and 630 nm), another one use only two wave length (649 and 665 nm). If there references I would be thankful.
Thank you in advance.
sara
Hi,
I have been using the equations published by Porra et al. (1989) in BBA vol. 975, pp. 384-394. The paper is analyzing very thoroughly different solvents and extraction conditions.
The optical density must naturallybbe in the linearity range of your spectrophotometer. Typically I do not go above 0.5 Abs. units for the strongest absorption peak.
Dear Dr. Stefan and Dr. Kathryn,
Thank you very much for your suggestions. Also, I would like to know if I used ethanol instead acetone for extraction of total chlorophyll content that change in wavelengths and OD in equation.
Yes, definitively! All this is covered in the reference I recommended
Stefan
I absolutely agree with Stefan Falk. I'm working with Chlamydomonas and I use the the equations published by Porra et al. (1989) for 80% acetone. There are two wave length for Chl a and Chl b in this publication that you have to use, but I use one more - 750 nm as a "blank" for previous two wave length. Anyway you have to mix you samples with acetone very well by Vortex and after that to centrifuge them by MiniSpin in max speed about 10-15 min. And of course you have to do at least three repetitions of each samples.
PS. If you change solvent, it changes wave length for Chl a and Chl b.
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