An alcoholic solution of the resin was just neutralized with potassium
hydroxide. During the titration, the solution rapidly became dark brown.
After neutralization it was shaken with ether; the water solution
remained brown while the ether layer was nearly colorless and contained
practically no dissolved substance. A portion of the water solution of
the potassium salt on being acidified with sulphuric acid and standing
over night, deposited a slight precipitate. The solution of the
potassium salt gave a heavy precipitate with lead acetate somewhat
similar to the original lead precipitate A, and also slight precipitates
with salts of zinc, mercury, copper, and silver (with reduction). It
gave a bluish-black color with impure ferrous sulphate and a dark color
with ferric chloride. It reduced ammoniacal silver nitrate and Fehling
solution. These experiments indicated the presence of a tannin compound.
An alcoholic solution of the resin gave the same color reactions with
iron salts as did the potassium salt. To determine which one of the
tannin compounds was present was a matter of some difficulty since the
di- and tri-hydroxybenzoic acids have in general the same color
reactions. The presence of other plant substances in the solution also
interferes with the color tests, and finally, a substance which gives a
blue color with iron salts and one giving a green color may be found
together in the same plant.[19] Further tests with a solution of the
resin in dilute alcohol, and with a water solution of the acid
precipitated by adding sulphuric acid to a solution of the resin in
potassium hydroxide, led to the conclusion that the acid is gallic acid.
These tests were the following:
(1) Boiling with an excess of potassium hydroxide gave a black substance
(tauromelanic acid).
(2) The acid was not precipitated by gelatin.
(3) On addition of potassium cyanide a transitory red color appeared
which reappeared on shaking with air.
Gallic acid is distinguished from tannic acid by tests (2) and (3). At
later stages in the work the potassium, barium, and sodium salts of
gallic acid were obtained, and finally the pure acid was made by
decomposing the sodium salt with sulphuric acid and crystallizing from
water. A portion of the acid so obtained was further purified by
dissolving in absolute alcohol and pouring into absolute ether.[20] The
melting point behavior of the acid corresponds with that of gallic acid;
it melted with decomposition at about 230 deg. For further identification,
some of the acid was converted into an ester by the following process:
it was dissolved in 80 per cent. alcohol, hydrochloric acid gas was
passed in, and the solution was heated an hour on the water bath. It was
then evaporated to a small bulk, neutralized with barium carbonate and
extracted with ether. The ether, on evaporation, left the ester which
was crystallized from water and dried in a desiccator over sulphuric
acid. The anhydrous ester agreed in melting point (156 deg. to 159 deg.) and
other properties with the ester of gallic acid described by Grimaux.[21]
For the sake of comparison, an ester was made from gallic acid obtained
from another source and the two agreed in properties. A mixture of the
two esters melted within the limits given for the ester of gallic acid.
While the tests leading to the identification of gallic acid were being
made, another series of experiments was in progress. Eleven and one-half
grams of the resin obtained from lead precipitate A by decomposition
with hydrogen sulphide were treated with 0.1 n. potassium hydroxide
added from a burette until the acid was exactly neutralized. All went
into solution. On shaking with ether, some of the potassium salt
separated out and was saved for examination. The solution became brown
on exposure to air and got darker as the work proceeded. The acid in
solution as a potassium salt was precipitated out in four fractions by
adding for each fraction one-fourth the amount of 0.1 n. sulphuric acid
required to neutralize the potassium hydroxide used. The precipitates
were filtered off and examined. The first was small in amount, gummy and
hard to filter. The solution was shaken with ether after each
precipitate had been filtered off. The succeeding precipitates were in
better condition, but were not pure. All appeared to be impure gallic
acid which had become brown by absorption of oxygen. They were saved,
however, to be tested for poison. After the last fraction had separated,
the filtrate was shaken several times with ether and saved for further
examination, which will be described under "Rhamnose." This filtrate is
designated as B.
At this stage of the work a portion of the resin obtained from lead
precipitate A was tested and found to be not poisonous as already
mentioned. By this test, all the substances contained in the lead
precipitate A after its extraction with ether in the Soxhlet apparatus,
were eliminated from the possible poisonous substances. The poison must
therefore have been extracted by the ether.
A fresh portion of the original poisonous material was treated with 50
per cent. alcohol and filtered from insoluble tar. The filtrate was
precipitated in six fractions by lead acetate. The last fractions were
lighter in color and apparently much purer than the first. The sixth
lead precipitate was decomposed by hydrogen sulphide, the light-yellow
water solution was tested and found to be not poisonous. It gave the
characteristic reactions for gallic acid. The poison, if precipitated at
all by lead acetate, must have gone down in one of the preceding
fractions. Later experiments showed that it is brought down partly
mechanically and partly as a lead compound in the first precipitates.