576 
Journal of Agricultural Research 
Vol. XXX, No. & 
disulphide solution, while the others 
are not adsorbed but pass through if 
more carbon disulphide is added. It 
appears as if several xanthophylls 
were present. 
ISOLATION AND PURIFICATION OF 
XANTHOPHYLL 
In order that the purity of the prepa¬ 
ration of xanthophyll used in the work 
herein described may be unquestioned, 
and also to aid those who are desirous 
of obtaining the pure pigment, it is 
considered advisable to describe very 
fully the method which has been 
adapted from those described by Will- 
statter and Stoll { 12 ). 
The best source of xanthophyll is 
dried green leaves. The leaves may 
be kept for several months and the 
xanthophyll extracted when desired. 
Although the leaves of almost any green 
plant would be suitable, cowpea leaves 
were used in this investigation. The 
leaves are carefully spread out to dry, 
away from sunlight. A large oven 
with screen trays may be used, or a 
large screen on which the leaves may 
be stirred. Room temperature is 
hardly sufficient, but for drying small 
quantities it serves very well. Rapid 
drying is preferable, though great care 
must be used not to get the tempera¬ 
ture too high (above 40° C.), since too 
high a temperature destroys a large 
percentage of the pigments present in 
the leaves. The drying will take from 
12 to 24 hours, or a little longer if large 
quantities of leaves are used. 
As soon as the leaves are dried they 
are placed in a large pebble mill—the 
mill in use in the Office of Soil Fertility 
Investigations is about 38 cm. in 
diameter and 45 cm. in length—and 
ground 12 to 24 hours. When ground 
to powder, they are removed from the 
mill and placed in either glass or tin 
containers. Since the carotinoid pig¬ 
ments oxidize very readily, the mate¬ 
rial should be kept in sealed containers. 
Leaf material may be kept in this 
manner for several years, and used as 
desired for the preparation of xan¬ 
thophyll. 
The method here described will be 
suitable for obtaining 0.5 to 0.8 gm. of 
pure xanthophyll from 2 kgm. of the 
dried leaves. A quantity of leaves 
sufficient to produce this amount of 
xanthophyll is about all that can be 
easily handled with ordinary labora¬ 
tory apparatus, and even then some 
special apparatus will be desirable. 
Pure chlorophyll and carotin may 
also be prepared (these processes will 
not be described here) at the same time 
from the extract by a little more labor, 
if the worker desires the pigments. 
The method of preparing pure xan¬ 
thophyll is essentially that given by 
Willstatter and Stoll, but certain modi¬ 
fications which are very necessary for 
good results will be added. 
Two kilograms of the leaf powder 
are placed upon a large porcelain 
Buchner funnel (25 to 30 cm. in diam¬ 
eter) to which a filter paper has first 
been fitted. The leaf powder is spread 
out carefully but not compressed. The 
funnel has previously been fitted to a 
4-liter filter flask which is connected to 
the vacuum pump. At first 80 per cent 
acetone is allowed to soak into the dry 
leaf powder, which is stirred with a 
spatula or glass rod in order to hasten 
this part of the process. As soon as the 
80 per cent acetone has thoroughly 
moistened the leaf powder, suction is 
applied and more acetone is poured on. 
If the leaves are compressed or suction 
is applied before the leaf powder is 
thoroughly moistened with the aqueous 
acetone, filtration will be greatly re¬ 
tarded. The green acetone extract is 
sucked off and more acetone added in 
liter portions until approximately all of 
the leaf pigments have been extracted. 
About 6 to 8 liters of 80 per cent acetone 
will be required for the extraction, 
which should be completed in from 30 
to 60 minutes. 
About half of the acetone extract is 
now poured into a 7-liter separatory 
funnel and 2 liters of distilled petroleum 
ether are added. The whole is shaken 
thoroughly, and about 250 c. c. of 
distilled water added in order to cause 
a separation of an aqueous acetone 
layer, which is drawn off. The re¬ 
mainder of the acetone extract is added 
to the separatory funnel, shaken, and 
the acetone layer is again separated by 
adding another 250 c. c. portion of 
water. The aqueous acetone solution 
contains the yellow flavones which are 
soluble cell sap pigments. 
To the petroleum ether solution re¬ 
maining in the funnel 1,000 c. c. of 
80 per cent (by volume) acetone is 
added and shaken thoroughly. This 
solution is allowed to stand a minute 
and run off. The impurities are thus 
removed and only a very little of the 
xanthophyll is lost. The acetone is now 
partially removed by four successive 
washings, each with 250 c. c. of dis¬ 
tilled water. Four washings are suffi¬ 
cient, for if more of the acetone is 
washed out the xanthophyll will crys¬ 
tallize during subsequent washings with 
methyl alcohol and the separation of 
the xanthophyll from the chlorophyll 
will then become much more difficult. 
