THE QUANTITATIVE DETERMINATION OF XANTHO- 
PHYLL BY MEANS OF THE SPECTROPHOTOMETER 
AND THE COLORIMETER 1 
F. M. SCHERTZ 
Biochemist in Soil Fertility Investigations , Bureau of Plant Industry , United States 
Department of Agriculture 
INTRODUCTION 
The quantitative determination of 
carotin has been discussed in a previous 
paper. 2 Methods 'which have been 
found applicable for the determination 
of carotin have been found to be 
equally suitable for the determination 
of xanthophyll. The greatest difference 
in the absorption of the two pigments 
is that the bands in xanthophyll are 
located farther toward the violet end 
of the spectrum than are those for the 
same concentration of carotin. Ac¬ 
curate quantitative data on the spectral 
transmissive properties of xanthophyll 
have been obtained by the Bureau of 
Standards and will be published soon. 
This paper is concerned only with the 
spectrophotometric and the colori¬ 
metric methods of determining xantho¬ 
phyll when in solution. 
THE SPECTROPHOTOMETRIC 
METHOD 3 
Since carotin and xanthophyll solu¬ 
tions are very similar as to their light¬ 
absorbing qualities, the mercury line 
435.8 my was used as in the case of 
carotin. The results given in Table I 
and in Figure 1 were obtained with 
different samples of xanthophyll pre¬ 
pared as described below. 
In general, the method of prepara¬ 
tion of the xanthophyll used in the 
work described in this paper is the 
same as that used by Willstatter and 
Stoll. 4 The complete details of the 
preparation are to be given with data 
on the physical properties of the pig¬ 
ment in a later paper. Briefly, the 
method is as follows: By means of 
acetone the xanthophyll is extracted 
from dried cowpea leaves. It is 
separated from the accompanying caro¬ 
tin and chlorophyll by means of methyl 
alcohol extractions from a solution of 
the pigments in petroleum ether. The 
xanthophyll is extracted from the 
methyl alcohol by means of ether, 
which is then evaporated to a few 
cubic centimeters and methyl alcohol 
added. The xanthophyll is then re¬ 
crystallized from methyl alcohol several 
times. Finally, it is dissolved in chloro¬ 
form and precipitated from this by the 
addition of petroleum ether. It is 
removed from the mother liquor by 
filtering on a hardened filter, dried in a 
vacuum desiccator and quickly weighed. 
A solution of the pigment (0.042 gm. per 
liter) is made at once and readings 
taken the same day. 
The xanthophyll used to obtain the 
data in experiment No. 1 and in experi¬ 
ment No. 2 was recrystallized from 
methyl alcohol and then precipitated 
from chloroform by the slow addition 
of petroleum ether. The results in 
experiment No. 3 were obtained by 
preparing a sample in the same way as 
in experiment No. 1 and experiment 
No. 2. The xanthophyll from which 
the sample in experiment No. 3 was 
taken was then dissolved in chloro¬ 
form and precipitated by the addition 
of petroleum ether, thus giving the 
sample for experiment No. 4. The 
sample in experiment No. 5 was 
obtained from the xanthophyll of the 
sample of experiment No. 4 by dis¬ 
solving it in chloroform and precipi¬ 
tating from petroleum ether. The 
xanthophyll used in the preparation of 
the sample for experiment No. 6 was 
obtained by dissolving the xantho¬ 
phyll of experiment No. 5 in chloroform 
and precipitating it therefrom by add¬ 
ing petroleum ether. It was assumed 
that dissolving the xanthophyll in 
chloroform and precipitating it by the 
deceived for publication June 7, 1924; issued April, 1925. 
2 SCHERTZ, F. M. THE QUANTITATIVE DETERMINATION OF CAROTIN BY MEANS OF THE SPECTROPHOTOM¬ 
ETER AND THE COLORIMETER. Jour. Agr. Research 26: 383-400, illus. 1924. 
3 All spectrophotometric data given in this paper were obtained on the Konig-Martens spectrophotom¬ 
eter at the Bureau of Standards, U. S. Dept, of Commerce. 
4 Willstatter, R. M., and Stoll, A. untersuchungen Aber chlorophyll; methoden und ergeb- , 
nisse. 424 p., illus. Berlin. 1913. 
Journal of Agricultural Research, 
Washington, D. C. 
( 253 ) 
Vol. XXX, No. 
Feb. 1, 1925 
Key No. G-475 
