THE CHEMISERY OF : THE PROTEINS 99 
out, are filtered off after boiling with charcoal and the hydro- 
chlorie acid solution concentrated under reduced pressure. At 
this stage, if glutamic acid be present in considerable quantities, 
as in the case of caseinogen, it is separated as hydrochloride. 
The concentrated solution is therefore saturated with gaseous 
hydrochloric acid and allowed to stand in a cold place, when 
crystallisation occurs. The glutamic acid hydrochloride is 
filtered off, after mixing the solution with an equal volume of 
ice-cold alcohol; it is purified by dissolving in water, boiling 
with charcoal and again separating by saturating the solution 
with gaseous hydrochloric acid. 
The next stage consists in the esterification of the amino acids. 
The acid solution is concentrated as far as possible in vacuo, the 
residue covered with absolute alcohol, and gaseous hydrochloric 
acid passed in, till the solution is saturated ; it is finally warmed 
on the water bath. For every kilogram of protein three litres 
of absolute alcohol are required, as in the process of esterifica- 
tion a large amount of water results, which is harmful to the 
reaction. In order to complete the esterification the alcoholic 
solution is concentrated in vacuo to a thick syrup at a tempera- 
ture not above 50° C., a fresh quantity of absolute alcohol is 
added, and the liquid again saturated with gaseous hydrochloric 
acid. This process may be repeated a second time. If glycine 
be present in the protein in considerable amounts, as in gelatine, 
it is isolated now as glycine ester hydrochloride. To effect this, 
a crystal of this compound is added to the solution, and it is 
allowed to stand for twelve hours at o° C., when it crystallises out. 
It is filtered off whilst cold and washed with ice-cold alcohol, 
and is obtained pure after one recrystallisation from alcohol. A 
further separation of glycine ester hydrochloride occurs on 
concentrating the mother liquor, saturating with hydrochloric 
acid gas, and again allowing to stand. Almost all the glycine 
can be removed from the mixture in this way. The filtrate 
contains the ester hydrochlorides of the remaining amino acids, 
and it is concentrated in vacuo as much as possible at a 
temperature of 40°C. From this mass the esters are extracted 
by one of two methods. 
The first and older method, which has been the most 
frequently employed, consists in adding about an equal volume 
of water and one and a half volumes of ether to the residue, 
thoroughly cooling in a freezing mixture, and adding strong 
