Arnon (1949) gave the equations for extraction with 80% acetone and absorbances read at 645nm and 663 nm as
Chlorophyll a (μg/ml) = 12.7 (A663) - 2.69 (A645)
Chlorophyll b (μg/ml) = 22.9 (A645) - 4.68 (A663)
Total chlorophyll (μg/ml) = 20.2 (A645) + 8.02 (A663)
Lichtenthaler & Welburn (1983) gave the equations for extraction with 80% acetone read at 646 nm and 663 nm as
Chlorophyll a (μg/ml) = 12.21 (A663) - 2.81 (A646)
Chlorophyll b (μg/ml) = 20.13 (A646) - 5.03 (A663)
Lichtenthaler (1987) as cited by Royse (2012) gave the equations for 100% acetone read at 661.6 nm and 664.5 nm as
Chlorophyll a (μg/ml) = 11.24(A661.6) - 2.04 (A664.8)
Chlorophyll b (μg/ml) = 20.13 (A664.8) - 5.03 (A661.6)
Dear Stephen Oyedeji
The best reference I known in the one of
Lichtenthaler, H.K. (1987) Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes. Methods in Enzimology, 148: 350-382.
There you will find several formulae, for several extractiing methods (metanol, acetone, etc., at several concentrations, 80%, 100%).
You can find a more recente review of Lichtenthaler and Buschmann in the following link:
http://www.thyssen-web.de/assets/files/fd_documents/sp_buche/uv_vis_pigmente.pdf
Best wishes
José C. Ramalho
it is better to use80% acetone for extraction of chlorophyll s,than100% acetone. i have been using this concentration of acetone for years. The best equestions for calculating chlorophyll's are those of Holden (1965) Analytical methods: chlorophyll s. In: Chemistry and Biochemistry of plant pigments(T.W.Goodwin,ED) ACADEMIC PRESS, LONDON,461-88
Dear Ram Singh:
Are you the one extracting Chl with 100% acetone? Moreover, if extraction is from leaves, there is no way it can be 100%..If it is someone else, let this person write to me and I will get him/her to be in contact with Dr.
Porra (and others) in Australia.
Please read the article by Porra in PSRES (2002) that he wrote for me ; then I have given you info from a web site; and info from a 1966 book on "The Chlorophylls'
Govindjee
Please contact Professor Govindjee, an authority in the photosynthesis: His email address is: Govindjee
*Govindjee, Professor Emeritus, Biochemistry, Biophysics and Plant Biology, UIUC, 265 Morrill Hall, 505 South Goodwin Av, Urbana,IL 61801-3707, USA; Phone: 217-337-0627 (home); 217-333-1794 (office); fax:
217-244-7246
#Wikipedia:
#URL :
#1994-2015 Publications:
# A beginning of a family page is at: http://www.govindjee.org/
------------
###See a recent you-tube for fun and frolic--enjoy the song:
###Also see a nice clip of James Stewart in a 1938 movie…
Dear Stephen,
Checkout the attached review, maybe this can be of some help.
http://www.life.illinois.edu/govindjee/Part1/Part1_Porra.pdf
Article The chequered history of the development and use of simultan...
Because chlorophyl in vivo is not free but atached to protein complexes, it is necessary to use polar solvent together with some proportion of water (20%). One must also take care of having a limpid solution, otherwise the presence of particles in suspension may have some scattering effect. An appropriate organic solvent is acetone that prevents to some extent the transformation of chlorophyll into pheophytin (thus, it is recommended to extract pigments in cold and to avoid a too long delay before the measurement). Be aware also that a crude leaf extract contains other substances than chlorophylls (not only carotenoids, but their contribution in the red region is very low). For all these reasons, a crude extract cannot be identified to a well defined mixture of pure chlorophylls a and b. This is why the chl a+b concentration given by Lichtenthaler’s equations (therefore, the a/b ratio) is slightly different from that computed at the isobestic point used by Arnon (645 nm).
Another useful reference for estimating chlorophylls is:
Ritchie (2008) Photosynthetica , Volume 46, Issue 1, pp 115-126
Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents
I thank you all for all your responses. I have contacted Profs. Govindjee and Lichtenthaler who have recommended possible solutions to the research problem. We have started working on this in my laboratory and we hope to get a breakthrough with this new idea from the two renowned Professors.
I have contacted Profs. Govindjee and Lichtenthaler who have recommended possible ways to determining Chlorophylls a and b with either 80% acetone or 100% acetone at varying wavelengths. We have started working on this in my laboratory and we hope to get a breakthrough with this new idea from the two renowned Professors.
I have contacted Profs. Govindjee and Lichtenthaler who have recommended possible ways to determining Chlorophylls a and b with either 80% acetone or 100% acetone at varying wavelengths. We have started working on this in my laboratory and we hope to get a breakthrough with this new idea from the two renowned Professors.
Profs. Govindjee and Lichtenthaler recommended possible ways to determining Chlorophylls a and b with either 80% acetone or 100% acetone at varying wavelengths. We have started working on this in my laboratory and we hope to get a breakthrough with this new idea from the two renowned Professors.
Based on the recommendation of Profs. Govindjee and Lichtenthaler for possible ways to determining Chlorophylls a and b with either 80% acetone or 100% acetone, we have started work my laboratory and we hope to get a breakthrough with these new ideas.
Dear colleague,
I have also a question:
Does anyone tell me if my calculations of chlorophyll and carotnoids contents are correct with the used the following equations :
(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 res. @Nabila Benhammou,
To answer your question, I will refer to the words of Dr. HARTMUT K. LICHTENTHALER, and I quote 'The extinction coefficients and the equations used and established by Arnon (1949) are not correct. They provide only a rough estimate of Chl a and b levels and yield inaccurate Chl b values, and, consequently, incorrect values for the Chl a/b ratio. They have been redetermined by Lichtenthaler (1987) using the extinction coefficients of Smith and Benitez (1955) for pure Chl a and Chl b in diethyl ether, which were found to be correct in the red absorption maxima at 661 and 642 nm, respectively, for purified Chls. The relative absorptions of Chl a and Chl b at other wavelengths in other organic solvents have been redetermined using modern high-resolution spectrophotometers' (Hartmut K. Lichtenthaler & Buschmann, 2005)
doi.org/10.1002/0471709085.ch21
Most of your questions have their answers in, e.g., Lichtenthaler's articles, already cited by others. I would, therefore, make more general comments. In vivo, photosynthetic pigments form complexes with other biological components, at the first place proteins. A good solvent should break these links while preventing chlorophyll degradation (most common products are pheophorbide and pheophytin). In addition, all solvents are not equally effective in extracting chl b vs. chl a, carotenoids vs. chl, xanthophylls vs. carotenes, etc. The traditional 80% acetone is a good compromise, but other solvents may also be considered: 100% acetone, ethanol, methanol, diethyl ether, DMSO... However, the stability of pigments varies with solvents and it is better to make quantitative determinations shortly after extraction, keeping mixtures in cold, darkness, and closed vessels. Be sure also that your spectrophotometer is correctly calibrated (for example, with specific blue rays of a UV discharging lamp), as chlorophyll peaks are steep. For each pigment, or average pigment mixture (carotenoids), the extinction coefficients at given wavelengths have been published: see again Lichtenthaler (notice also that isosbestic points are useful for a rapid calculation of total chlorophylls: Arnon, 1949, with 80% acetone, but in principle, this is true for any other solvent).
Dear res. Nabila Belyagoubi-Benhammou
Yes, as professor Lichtenthaler mentioned: 'one of the reasons why the method of Arnon (1949), using 80% (v/v) acetone, is prone to inaccuracy is because the specific absorption coefficients were originally taken from Mackinney (1941), who used pure acetone' (Lichtenthaler & Wellburn, 1983).
Reference articles:
Lichtenthaler, H. K., & Wellburn, A. R. (1983). Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Liverpool.
Lichtenthaler, Hartmut K. (1987). Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes. Methods in Enzymology, 148(C), 350–382. https://doi.org/10.1016/0076-6879(87)48036-1
Lichtenthaler, Hartmut K., & Buschmann, C. (2001). Extraction of Photosynthetic Tissues: Chlorophylls and Carotenoids. Current Protocols in Food Analytical Chemistry, 1(1), F4.2.1-F4.2.6. https://doi.org/10.1002/0471142913.faf0402s01
Lichtenthaler, Hartmut K., & Buschmann, C. (2005). Chlorophylls and Carotenoids: Measurement And Characterization by UV-VIS Spectroscopy. Handbook of Food Analytical Chemistry, 2–2, 171–178. https://doi.org/10.1002/0471709085.ch21
Akshatha Banadka: See attached- I don't seem to be able to make it legible in this editor.
Please, find this link!
https://clu-in.org/download/ert/2030-R00.pdf
Good luck
Hey Sowmya Radhakrishna,
just a curiosity.. what is the value 1000 in the formula standing for?
Carolina Cozzi-
In Radhakrishna's equation, the quantity in square brackets gives the concentration of CplA in mg/L. Multiplying this by the volume of the extract in L would thus give the mg of CplA in the total extract. Dividing that by the fresh weight in g would give mg/g fresh weight, no 1000 factor needed. But if you measure the volume in ml instead of L, you must divide by 1000 for liters. So the equation as written is for extract volume in ml, fresh weight in g.