78 Frederic Czapek . — The Anti-ferment Reaction in 
to crystallize) Baumann recommends the preparation of the easily 
crystallizable ethylester of the acid, which gives most of the tests in the 
same manner as does the free acid. For this purpose gaseous hydrochloric 
acid was passed in large excess in an alcoholic solution of the root-tip 
substance well cooled with snow. After twenty- four hours water was 
added, the fluid made alkaline with Na 2 C 0 3 , and shaken with ether. The 
ether having been evaporated, there remained a syrupy mass containing 
embedded crystals. These crystals could finally be obtained quite pure 
by recrystallizing from hot water and washing with alcohol. The solubility 
and shape of crystals as well as the melting-point (120 0 ) agreed exactly 
with the properties of the ethylester of homogentisinic acid. The reducing 
substance of the root-tips is therefore undoubtedly to be regarded as 
homogentisinic acid. Homogentisinic acid gives a reddish colour with 
hydrogen-peroxide, and I suppose that the red colour produced in the 
tissue of Faba roots by hydrogen peroxide, as described by Pfeffer, is 
connected with the presence of homogentisinic acid. Wolkow and 
Baumann elaborated an excellent method for the quantitative estimation 
of homogentisinic acid, particularly in urine. This method can be made 
use of for our purposes without any important modifications. Baumann 
titrated with decinormal solution of silver nitrate, and determined with 
weighed quantities of absolutely pure homogentisinic acid how much 
ammoniacal silver solution is needed to oxidize I g. homogentisinic acid. 
According to this estimation i g. of homogentisinic acid corresponds fairly 
closely to 3-60-2-65 g. silver; 1 cc. decinormal solution of AgN 0 3 there- 
fore corresponds to 4-1 to 4-2 mg. of pure anhydrous homogentisinic acid. 
In determining homogentisinic acid in root-tips I proceeded as follows. 
From 100 radicles the apical 2 mm. are quickly cut off and immediately 
ground as finely as possible in a small mortar with 5 cc. distilled water 
and 2 g. of the purest glass-powder. The contents of the mortar are 
washed with a small quantity of water into a measuring-flask containing 
25 cc. The flask is then filled up to the mark and the contents shaken 
and filtered. The entire homogentisinic acid of the root-tips is now in the 
filtrate. Ten cc. of the filtrate are transferred by means of a pipette 
to a flask. Then 10 cc. of dilute ammonia 1 are added, and immediately 
afterwards a small quantity of ~ silver solution from the Curette. Generally 
1 cc. ~ silver nitrate can be added at once without risk of excess. The 
10 
mixture is now boiled. To facilitate the filtration of the silver precipitate 
the flask is allowed to cool for five minutes, then five drops of moderately 
concentrated calcium chloride solution are added, and ten drops of am- 
monia carbonate. The contents of the flask are then shaken and filtered. 
1 Commercial concentrated ammonia of sp. gr. 0-900 diluted in the proportion 1.10. 
