540 
AMERICAN JOURNAL OF BOTANY 
[Vol. 9, 
The manner of determining chlorophyll consisted in the comparison in 
a Duboscq colorimeter of 85-percent acetone extracts made from 0.25-gram 
samples of these leaves after thorough powdering in a mortar, with a standard 
extract made in the same manner from a bean-leaf powder which had been 
standardized by quantitatively determining its chlorophyll content. 
The quantitative determination of chlorophyll by the use of these raw 
acetone solutions, which contained all four pigments, chlorophyll a, chloro- 
phyll h, carotin, and xanthophyll, was possible because the shade of color 
of all the solutions closely matched that of the solution used as a standard. 
Die Rohchlorophyllosung enthalt die vier Pigmente von verschiedener Farbe und 
Farbintensitat in Mengenverhaltnissen, die von Losungsmittel beeinflusst werden und die 
von der Pflanzenart und sogar von der Ernte einer und derselben Pflanze abhangig sein 
konnen. Anndhernd gleich ist das Mengenverhdltnis der Komponenten bei den Extrakten 
einer Pflanze; man kann daher durch den Vergleich derselben die relative Bestimmung ihres 
Chlorophyllgehaltes ausfiihren. Fiir die Untersuchung von Extrakten ungleicher Farb- 
nuance, also z.B. aus verschiedenenen Pflanzen, ist es erforderlich, die Farbstoffe durch 
Verseifung mit Alkali in die indifferenten gelben und in die Alkalisalze der griinen Pigmente 
zu trennen. Die erhaltenen Chlorophyllinlosungen ermoglichen die relative Bestimmung 
des Farbwertes und ferner erlaubt ihr Vergleich mit einer alkoholischen Losung von bekann- 
tem Chlorophyllgehalt die absolute Bestimmung des Farbstoffgehalts, (Willstatter and 
Stoll, 1913, pp. 78, 79.) 
The primordial leaves used in making the standard powder were ob- 
tained from beans grown in the greenhouse from the same lot of seeds used 
in growing the plants for the experiments. 
In carrying out the quantitative determination of chlorophyll in the 
powder to be used as a standard, an indirect process is necessary, because 
mechanical difficulties interfere with quantitatively separating chlorophyll 
as a solid. The process is, briefly, as follows: Chlorophyll (the mixture of 
chlorophyll a and chlorophyll h) is obtained from a rather large but not 
exactly weighed quantity of the powder and dried. A quantitatively 
weighed amount of this chlorophyll is dissolved in absolute alcohol and 
made up to a definite volume. A quantitatively weighed amount of the 
leaf powder from which the chlorophyll has been obtained is extracted, the 
carotin and xanthophyll are removed from the extract, and the resulting 
solution, made up to a definite volume (in water and alcohol), is compared 
colorimetrically with the solution made directly from the weighed chloro- 
phyll. To facilitate removing carotin and xanthophyll from the extract 
from the powder, the chlorophyll a and chlorophyll h are saponified, giving 
the corresponding chlorophyllins (chlorophyllin a and chlorophyllin h) in 
unchanged proportion. The saponification and resulting comparison of 
chlorophylls and chlorophyllins does not introduce an error, according to 
Willstatter and Stoll (1913, p. 82). 
To isolate the chlorophyll the following steps are necessary : (i) complete 
extraction of the leaf powder with eighty-five percent acetone; (2) trans- 
ference of the pigments to petrol ether; (3) removal of the greater part of 
