Carbon Assimilation. 
245 
Chlorophyll a 
>> h 
Carotin ... 
Xanthophyll 
2 parts per 1000 
1 
~B »> 
1 
3 »> 
„ 1000 
„ 1000 
„ 1000 
In the chloroplasts these pigments are also mixed with various 
colourless substances ; fats, waxes and salts of fatty acids. Thus in 
an alcoholic extract of dried leaves chlorophyll is accompanied by 
about six times its weight of other substances. 
Willstatter has worked out methods for freeing chlorophyll 
extracts from these accompanying colourless substances, and also 
methods for isolating each of the four pigments. In all that follows, 
when we speak of chlorophyll we refer to the green pigments freed 
from the yellow ones. 
In thus being able to obtain pure pigments a very great advance 
is made. All the earlier experiments on the reactions taking place 
in the green leaf were made with extracts, such as alcoholic 
extracts of leaves, which contained many substances besides the 
pigments. 
Yet a further complication becomes obvious from Willstatter’s 
investigations, and this is also a fact which has not yet received its 
due attention in physiological researches. Moreover it is a fact not 
only important in this branch of plant physiology but in all cases 
where plant substances are extracted and purified. This is that 
by the methods of extraction the state of matter in which the 
substance generally exists may be altered. For this reason it may 
become difficult to draw conclusions from the behaviour of the 
extracted substance as to the function of the substance in its natural 
condition in the plant. 
From Willstatter’s researches it is clear that solvents which 
dissolve the pure extracted substance do not extract the substance 
from the dried leaf. For instance, the pure pigment is readily 
soluble in acetone, ether and benzol. If the dried powder of nettle 
leaves is placed in pure acetone it can remain there for half an hour 
without the acetone becoming at all coloured. But if a little water 
is added the colour immediately becomes intensely green. Neither 
ether nor benzol becomes coloured quickly when powdered nettle 
leaf is added. Yet both are immediately coloured strongly green 
when a few drops of water are added. This behaviour of chlorophyll 
suggests that chlorophyll in the leaf is in a different state of matter 
from extracted chlorophyll. 
The extracted pigment is soluble in petrol-ether as long as it 
