I am currently working with flowering plants, as I need to quantify the content of chlorophyll (particular subtype, e.g. chlorophyll a) in a specific leaf after its extraction, I was wondering whether anyone could give me a hand on the extraction protocol for the chlorophyll (looking for a high purity extraction).
Thank you very much in advance.
If you need just to quantify Chl a in higher plant leaves, you can use a simple extraction with an organic solvent such as 100% acetone with a subsequent spectrophotometric assay (e.g. according to Lichtenthaler 1987) since only Chls a and b are expected to be there. It important to prevent the formation of the phaeophytin in this case. This could be done by addition of 200 mg CaCO3 per sample prior to homogenization. For more details, see the file link attached.
Article A Spectrophotometric Analysis of Pigments in Apples
A method for preparing chlorophyll-a comprising the step of treating an undisrupted intact chlorella with an organic solvent. By treating the chlorella with 90-100% ethanol to extract chlorophyll-a, whereby obtaining a chlorophyll-a extract. For extraction of chloralin treating the chlorophyll-a with an acid to eliminate Mg2+ from the chlorophyll-a, whereby obtaining pheophytin , treating the pheophytin a with a base to obtain chlorin e6. biomass is treated with acetone 2-3 times in order to extract chlorophyll-a, the biomass is filtrated or centrifuged, the extract is evaporated, treated with acid in order to remove magnesium ion out of the chlorophyll molecule and to hydrolyze phytyl ester group. Methyl alcohol being added for concurrent esterification, the reaction mass is treated with water, pheophorbide α derivative is extracted with chlorous methylene, the extract is neutralized, washed with water, evaporated, chromatographed on aluminium oxide. Methylpheophorbide α is crystallized out of the mixture of chlorous methylene-methanol and the resulting pheophorbide α derivative is reacted with strong inorganic base in the presence of oxygen in pyridine-diethyl ether-n-propanol, the reaction mass is treated with water. The water phase is acidified till pH 4, unstable chlorin is extracted with chlorous methylene, the extract is evaporated, unstable chlorine is redissolved in tetrahydrofurane, the solution is evaporated, this procedure is repeated till absorption at 700 nm stops to increase
If you need just to quantify Chl a in higher plant leaves, you can use a simple extraction with an organic solvent such as 100% acetone with a subsequent spectrophotometric assay (e.g. according to Lichtenthaler 1987) since only Chls a and b are expected to be there. It important to prevent the formation of the phaeophytin in this case. This could be done by addition of 200 mg CaCO3 per sample prior to homogenization. For more details, see the file link attached.
Article A Spectrophotometric Analysis of Pigments in Apples
If you want pure chla for making a standard the use of flowering plants is not the best way, because the accessory pigment composition and O2 reactivity upon extraction require comploex preparative HPLC under N2. If you want chla Content you can use intact leaf reflectance as in Spectrum Tech Spad 502 that allows to follow aging on the same spot in attached leaf as well heterogeity in shaded portions, or follow the actual procedure in BBA.. Apply Care to prevent oxidations with extra sodium ascorbate. Copy available from UCLA http://www.chem.ucla.edu/dept/Faculty/merchant/pdf/Porra_1989.pdf
Extraction protocol for chlorophyll usually petroleum ether is good solvent for chlorophyll extraction . One can use n-hexane also for extraction of chlorophyll from green plant leaves.
Hi, Ping Kwan! We have used 80% acetone extract of leaf material according to Lichtenthaler for most studies which gives fairly good results. However, use of ethanol extract is also a very good option, appears more accurate as ethanol is less volatile and easier to handle although may take a little longer (see Knudson et al. ,1977). Calculations can be done on leaf area, fresh as well as dry weight basis.
Knudson, L.L., T.W. Tibbitts and G.E. Edwards. 1977. Measurement of ozone injury by determination of leaf chlorophyll concentration. Plant Physiol., 60: 606-608.
Extraction with 80% acetone is a commonly used method. According to Porra et al (1989) extraction with 80% acetone is not complete for chlorophyll b ( this is if you would be looking at chlorophyll a and b separately). With methanol, chlorophyll b is extracted completely, resulting in a better determination of the chlorophyll a:b ratio.
Hello greetings,
I am also recommended the method prescribed by Lichtenthaler 1987. All the best.
I have used the method described by Arnon (1949), but this http://www.thyssen-web.de/assets/files/fd_documents/sp_buche/uv_vis_pigmente.pdf address, there are very detailed description. Good luck.
A method by Wintermans and De Mots, using 96% ethanol, I have followed to extract chlorophyll. Leaves were choped into small pieces, put in test tube, add 96% ethanol and put in waterbath (70 oC). Before measurement for absorbances using UV-vis, leaves were ground, filtered and centrifuged.
I think that you must precise your question. Do you need chlorophyll for standard prepartion purpose or do you need a method to prepare a chlorophyll extract. Please give also details about purity. Regarding to which kind of molecules.
Put your leaves in 100% DMSO for a few minutes and heat to about 60 deg celsius. No need for maceration. Cannot remember the refernce right now but it works great and does not smell!
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