88 Ingvar Jorgensen and Walter Stiles. 
(b) Quantitative Estimation of Pigments in Fresh Leaves. 
1. The Extraction of the Pigments. The chief feature of the 
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 c.c. 
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 
phaeophytin 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 
