28 CARBON ASSIMILATION [CH. 



solution in the solvents in which it is soluble, i.e. ethyl alcohol, acetone, 

 chloroform, ether, carbon bisulphide, pyridine and benzene. Chlorophyll 

 b, when isolated, is a green-black solid giving a pure green solution : it 

 has much the same solubilities as chlorophyll a. The two chlorophylls, 

 however, can be separated by their different solubilities in methyl 

 alcohol. Both can be obtained in microscopic crystals. 



Carotin crystallizes in orange-red crystals, and xanthophyll in yellow 

 crystals. 



In the chloroplastids these pigments occur mixed with various colour- 

 less substances, fats, waxes, and salts of fatty acids. 



When chlorophyll is spoken of, it will be understood to refer to the 

 green pigments and not to the yellow. 



The pure pigments, when isolated, are readily soluble in acetone, ether 

 and benzene. When very thoroughly dried nettle leaves are treated 

 with pure acetone, no green colour is extracted, but if a few drops of 

 water are added, the extract becomes green. Also if acetone is poured 

 on to fresh leaves, the pigment is extracted. The explanation offered 

 for these phenomena is that chlorophyll is present in a colloidal condition 

 in the cell. This point will be considered again later (see p. 85). 



The Common Nettle (Urtica) is the plant which has been used for 

 material for the extraction of chlorophyll on a large scale, and it also 

 forms very useful material for extraction on a small scale. The pigment 

 has been found to be unaltered by drying, and, since dried leaves involve 

 far less bulk and dilution of solvents, material should be dried before 

 using. Some leaves (Elder and Conifers) are spoilt by drying. From 

 dried leaves pure solvents, such as petrol ether, benzene and acetone, 

 extract very little pigment for reasons which will be mentioned later, 

 but if the solvents contain a moderate amount of water, the pigment is 

 readily soluble. About 80/o acetone is the best solvent. The nettle 

 leaves are removed from the stalks and laid on sheets of paper to dry. 

 When well air-dried they are finely powdered, and the powder further 

 dried at 30-40 C. in an incubator. The leaf-powder can be kept for a 

 considerable time in a well-stoppered bottle. 



Expt. 18. Extraction of pigment. Two grains of leaf- powder are sucked to a filter- 

 paper on a small porcelain funnel and 2-3 c.c. of 85 % acetone are added. This is 

 allowed to soak into the powder for a few minutes. The fluid is then sucked through 

 with the pump. The operation is repeated until 20 c.c. of the solvent have been 

 added, when the powder is sucked dry. A deep blue-green solution with a red 

 fluorescence is obtained which contains all the four pigments from the leaf. The 

 acetone extract thus obtained is then poured into double the quantity of petrol ether 

 contained in a separating funnel. An equal quantity of distilled water is added, this 



