Variations in Quantity of Leaf Pigments. 41 



(b) Quantitative Estimation of Pigments in Fresh Leaves. 



1. The Extraction of the Pigments. The chief feature of tlie 

 quantitative estimation of the pigments in fresh leaves is the 

 primary treatment of them with watery acetone, and subsequent 

 extraction with acetone containing only a low percentage of water. 



The first treatment with weak acetone softens the leaves, 

 removes plant acids, inhibits enzyme action, and at the same time 

 removes no chlorophyll. 



The details of the method are as follows : — In a mortar, 25 cm. 

 in diameter, are put 40 grams of fresh leaves with 50 c.c. 40% 

 acetone ; the leaves are quickly mashed with 0'5 gram of quartz 

 sand. This serves the double purpose of facilitating disintegration 

 of the leaf and of further diluting the leaf substances. 



There are now added 100 c.c. 30% acetone, and the whole 

 filtered on a Buchner funnel through a thin layer of talc, which 

 keeps back the slimy protoplasmic matter. 



After sucking the residue dry with the pump it is washed with 

 30% acetone until the filtrate runs off colourless. The watery acetone 

 is then sucked through. 



The leaf substance is now treated with pure acetone and again 

 sucked dry. This is repeated until the acetone has removed all 

 the pigment. Complete extraction will require from 400 to 600 c.c. 

 acetone ; towards the end of the extraction 5 to 10% of water is 

 added to the solvent. 



As the extract is obtained it is poured, in quantities of 100 to 

 200 c.c, into 200 to 250 c.c. of ether, and the acetone washed out 

 with distilled water. 



Finally the whole of the ether solution is dried with anhydrous 

 sodium sulphate. The extract is then divided into two equal parts, 

 one of which is used for the determination of the green, the other 

 for the determination of the yellow pigments. 



2. Separation of the Chlorophyll Components. The ether 

 solution so obtained from 20 grams fresh leaves is converted into 

 phseophytin by treatment with 0'5c.c. 2 N alcoholic hydrochloric 

 acid in a boiling flask. 



The ether is then evaporated off, first in a vacuum in the cold 

 and then at 60" for a short time under very low pressure. The residue 

 is dissolved in 1 to 2 c.c. of pyridine and heated on a steam bath. 

 While still heated there is added to the pyridine solution 25 to 30 c.c. 

 of boiling, concentrated potash solution in methyl alcohol. This is 



