Dear colleague,
Can you tell me if my calculations of chlorophyll and carotenoid content are correct, I used Arnon's equations for 80% acetone.
Chlorophyll a (mg/g) = [12.7 (A663) - 2.69 (A645)]*V/1000*W
Chlorophyll b (mg/g) = [22.9 A645) - 4.68 (A663)] *V/1000*W
Total chlorophyll (mg/g) = [20.2 (A645) + 8.02 (A663)] *V/1000*W
Carotenoid (mg/g) [A480+ (0.114 A663)-(0.638 A645)] *V/1000*W
With : V: final volume of chlorophyll extracted in 80% acetone; W: fresh weight of the algal powder used.
Dear my colleague
the calculation of photosynthetic pigment concentration in plant tissue depends on the method and solvent used for extraction. so please tell me about your solvent of extraction so i 'll be happy to help you with the equation.
Prof. Dr. Saad Farouk
Egypt
Dear my colleague,
Thanks you veru much.
I used for extraction of pigments 80% acetone. I checked the equations but my problem especially in terms of caratenoid content. Here is what I found as equation:
Carotenoids (mg/g fresh wt.) = A480 + (0.114 × A663) – (0.638 × A645)
Can I used this equation by multiplying *V/1000*W
Several procedures are available for plant leaf pigment estimation. It can be expressed in mg g-1 FW or µg ml-1 and so on. Thus, accordingly equations are varied. All seems fine to me except that you have to express it in mg g-1 FW (Not mg/g). Because, you are extracting pigments from fresh leaves. In addition, you have used acetone (80%) but to me I preferred often dimethyl sulfoxide (DMSO), because it is simple and easy. In this procedure leaf pigments of fresh leaf are extracted in DMSO, using the method of Hiscox and Israelstam (1979). I shall suggest you to please check some attached files for further information.
As you have mentioned in your second message ‘my problem especially in terms of carotenoid content’ for this, please try to read paper as mentioned below for clear explanation.
Wellburn, A. R. (1994). The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313.
I have this reference, but I found different values with my data in (mg/g). Please, here are data from the DO [(0.062 (645nm); 0.169 (663nm); 0.262 (480nm)]
Can you give me this little calculation? by expressing the contents in mg/g.
Experimentation: A fine dried powder (0.5 g) was extracted using a mortar and pestle in 20 ml of 80% acetone. After centrifugation at 5000 rpm for 10 min, the supernatant is collected. This procedure was repeated several times until the supernatant becomes clear and then the solution is made up to 50 ml with 80% acetone. The absorbance of the solution was measured at 448 nm, 645 and 663 nm.
Not specifically answering your question, but I would like to point out that it is risky to rely on 3 wavelengths unless you are really sure that (1) the only pigments are Cpl a and Cpl b and the expected carotenoid, and (2) the solvent composition is exactly what the equations were derived for. Even then, in 80% acetone, I understand that Beer's law behavior is limited by oligomerization at higher concentrations. This may be OK in your case since you are extracting dry powder with 80% acetone, but is it possible your "algal" powder contains some bacteriocholorphyll or exotic bacterial carotenoids? At least take a full spectrum to verify the peaks are where expected.
Much safer is to take the full spectrum and fit it to a linear combination of the spectra of the individual components. Instructions for doing this with the free "scanedit" program are available at the sourceforge site: https://sourceforge.net/projects/scanedit/files/openspectralworks-1.0/doc/demo2.pdf. If the fit is garbage due unexpected turbidity, too much water in the acetone, or other pigments, you will know immediately. With 3 wavelengths you are working blind: you get 3 numbers even if you put chicken soup in the cuvet, and (unless one or more concentration comes out negative) you have no idea how reliable they are.
If you do choose the single-wavelengths method, or you need accurate extinction coefficients to standardize your basis spectra for full-spectrum fitting, I believe Lichtenthaler has more recent determinations (than Arnon's): Hartmut K. Lichtenthaler and Claus Buschmann. Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. IN Current Protocols in Food Analytical Chemistry (2001) F4.3.1-F4.3.8 Copyright © 2001 by John Wiley & Sons, Inc.
Dear Colleague,
Calculation of chlorophylls and carotenoid content in plants, definitely related to solvent you used for the assay.
Hartmut Lichtenthaler, distinguished professor at Karlsruhe Institute of Technology, has the best papers about photosynthetic pigments assay. I am sure you can find your answer about the calculation of chlorophylls and carotenoid in them. I attached them to you for your reference.
Best regards, Homayoon.
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