Total chlorophyll can be measured by leaves extraction using acetone (80%) three times then the extract absorption taken by spectrophotometer at the suitable wave length of chlorophyll.
Dear Yasaman,
In addition to the answer of Professor Dr. Abbas M. Jasim, please check theses resources.
Best wishes
SM Najim
https://www.ijsr.net/archive/v4i10/SUB159224.pdf
http://experimentjournal.com/expadmin/pdf_files/Exp_9.1._522-525.pdf
http://onlinelibrary.wiley.com/doi/10.1002/jsfa.2740050910/full
www.aslo.net/lo/toc/vol_12/issue_2/0335.pdf
More details about chlorophyll measurement were presented at Phytochemical Methods by Harborne
Dear Yasaman Etemadian
The following equations have been developed by Ritchie (2008) for determining chlorophyll (Chl) a, b, c, d, and total Chl for 90 % acetone, 100 % methanol and ethanol solvents suitable for estimating Chl in extracts, using a spectrophotometer:
Chl a = – 0.3319 A630 – 1.7485 A647 + 11.9442 A664 – 1.4306 A691
Chl b = –1.2825 A630 – 19.8839 A647 – 4.8860 A664 – 2.3416 A691
Chl c = 23.5902 A630 – 7.8516 A647 – 1.5214 A664 – 1.7443 A691
Chl d = – 0.5881 A630 + 0.0902 A647 – 0.1564 A664 – 11.0473 A691
Total Chl = 21.3877 A630 + 10.3739 A647 + 5.3805 A664 + 5.5309 A691
You can also use a portable chlorophyll meter (e.g. CCM200) to estimate chlorophyll content index (CCI) without any plant destruction.
Best Regards
Kazem Ghassemi-Golezani
In case that you use a portable chlorophyll meter (for example CCM200) to estimate chlorophyll content index (CCI), you will need to make calibrations of the chlrophyll meter by using a spectrophotometer.
Many brown algae do not have Chlorophyll b.
In the past I've used this:
Estimation of Chlorophyll: Five hundred mg of fresh leaf material was taken and ground with help of pestle and mortar with 10 ml of 80% acetone. The homogenate was centrifuged at 3000 rpm for 15 minutes The supernatant was stored. The residue were re-extracted with 5 ml of 80% acetone. The extract was utilized for chlorophyll estimation. Absorbance was read at 645 and 663 nm in the UV-spectrophotometer (See Arnon, 1949).
(12.7 × ΔA663 – 2.69 × ΔA645)
Chlorophyll a (mg/g.fr.wt.) = ------------------------------------------------- × V
a × 1000 × W
(22.9 × ΔA645 – 4.68 × ΔA663)
Chlorophyll c (mg/g.fr.wt.) = ------------------------------------------------- × V
a × 1000 × W
and
(20.2 × ΔA645 – 8.02 × ΔA663)
Total Chlorophyll (mg/g.fr.wt.) = ------------------------------------- × V
a × 1000 × W
where
ΔA = Absorbance at respective wavelength
V = Volume of extract (ml)
W = Fresh weight of the sample (g)
Estimation of Carotenoid: The carotenoid content of seaweeds were determined by the method of Kirk and Allen, 1965.The extract that was used for the chlorophyll estimation was used for carotenoid estimation also. The same chlorophyll extract was measured at 480nm in UV-spectrophotometer to estimate the carotenoid content.
Carotenoid (µg/g.fr.wt) = ΔA480 + (0.114 × ΔA663) – (0.638 × ΔA645)
where ΔA = Absorbance at respective wavelength
References
Arnon, D.I., (1949). Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris. Plant Physiol., 2: 1-15
Kirk, J.T.O., and R.L. Allen, 1965. Dependence of chloroplast pigments synthesis on protein synthetic effects on actilione. Biochem. Biophysics Res. J. Canada, 27: 523-530.
You can also use the method developed by Hendrik Kupper (Gauss Peak Spectra Analysis) that will give you a more detailed information about the carotenoid profile. For that you should use pure acetone to avoid allomerizations and artifacts introduced by water in the medium and you should freeze dry your samples. See my papers where I have applied the GPS method several times in plants but I've also done it in algae and works very well. Also see the papers from Prof. Hendrik to get the algorithm and instructions on how to apply.
I need a simple method about measuring total chlorophyll content and carotenoids using methanol as extrator. How can help me?
Dear Marcelo I advise you to use acetone method that depend on spectrophotometer. Warms
Ali Almehemdi, I already extract chlorophyll from the leaves, what I lack now are the equations for calculating the concentrations.
Marcelo Pompelli : you can get information on how to estimate chlorophyll from this old (but classical) publication. Good Luck.
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