Extraction of chlorophyll

Hiscox, J.D. and Israelstam, G.F. (1979). A method for the extraction of chlorophyll from tissue without maceration. Canadian Journal of Botany 57 1332-1334. 

The extraction of chlorophylls and carotenoids from water-containing plant materials requires polar solvents, such as acetone, methanol, or ethanol, that can take up water. These extracts must then be transferred to a solvent such as diethyl ether in order to be stored stably. Samples with very high water content, such as juices and macerated plant material, are usually freeze-dried first, and can then be extracted directly with diethyl ether. After extraction, solutions are clarified and diluted to an appropriate volume to measure chlorophyll content by UV-VIS spectrophotometry. Absorption coefficients and equations needed for quantitative determination are given in unitf4.3. 

This protocol has been developed for extraction of chlorophyll from leaf samples, but it can also be used for other plant food samples. The authors recommend, however, that the beginner initially perform the procedure using green leaf samples before extending its use to other types of samples. 

For freeze-dried samples, extract the dry sample ( 0.3 to 1 g dry weight starting from 2 to 3 g fresh weight) with diethyl ether and apply the equations given for diethyl ether (UNIT F4.3). Extraction of freeze-dried plant material with diethyl ether is performed by grinding in a mortar. It is also possible to use 80% acetone or 100% methanol, but diethyl ether has proved to be an excellent solvent for quantitative extraction of chlorophylls and carotenoids from freeze-dried plant material. 

The solvents that are used for extraction of chlorophylls from leaves or other biological samples are similar to those used for HPLC (unit F4.4). For example, leaves are usually ground in the presence of acetone to extract chlorophylls. After filtration, the solvent is evaporated in vacuo or under a stream of nitrogen (but not air, to avoid oxidation). One additional purification step prior to HPLC (applicable to both Basic Protocols 1 and 2) is to dissolve the chlorophyll residue in hexane and then wash the hexane with water/methanol (1 1 v/v). The hexane may be removed prior to HPLC or simply diluted with mobile phase prior to analysis. 

Suberized walls.. In cork, wounded areas of plants, endo-dermis. Alcoholic extract of chlorophyll, in the dark, imparts a green color. Alcannin and Sudan III impart a red coloration. Converted into yellowish droplets and granular masses upon heating with a strong solution of KOH. Sulpuhric acid is resisted. 

The walls of cork cells resist the action of concentrated sulphuric acid. They are colored green, when in contact with alcoholic extract of chlorophyll for several days in the dark. 

Figure 23. Extraction of chlorophyll from fresh leaves with acetone (1) and acetonitrile (2).

Chlorophyll extraction and spectroscopic measurements are applied to assess the degree of integrity of the chlorophyll in photobiontic cells in lichens. The extraction of chlorophyll is usually performed by dimethyl sulfoxide (DMSO) or acetone. The exposure of lichen thalli to chemical pollutants, either under controlled conditions or under natural conditions in the presence of air pollutants, leads in many cases to a degradation of chlorophyll. 

Oquist, G. and Samuelsson, G., Sequential extraction of chlorophyll from chlorophyll-protein complexes in lyophilized pea thylakoids with solvents of different polarity. 

Van Buren, J.P., Extraction of chlorophylls a and b from different binding sites on thylakoid chlorophyll-proteins, J. Agric. Food Chem., 33, 204, 1985. 

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