THE CHEMISTRY“OF THE PROTEINS 101 
esters pass over into the distillate, but they can be recovered by 
acidifying with hydrochloric acid and evaporating, when they 
are obtained as amino acid hydrochlorides. The dark brown 
residue which remains in the distilling flask is fractionally 
distilled in vacuo, leaving a mass consisting of tyrosine and 
other amino acids, which in the first method are contained in 
the mass of potassium carbonate. 
The distillation in vacuo is carried out first at the pressure 
of 10-12 mm. produced by the water pump, and then at a 
pressure of o'5 mm. produced by a Geryck pump. At 10-12 mm., 
three fractions (up to 60°, 80°, 100°) are collected, the tempera- 
ture taken being that of the water bath; the distillation is 
then continued at o'5 mm., the first fraction being that at the 
temperature of the boiling water bath; several fractions are then 
collected up to 160°, the temperature being procured by the use 
of an oil bath. The residue, which does not distil at this 
temperature, consists of a dark brown oil; it sets to a hard 
mass on cooling, and contains, amongst other substances, 
leucinimide. 
The various fractions, so obtained, consist of a mixture of 
the esters of two or more amino acids, and they are again 
fractionated in vacuo ; this is not essential for the fractions dis- 
tilling above 100° C. at o°5 mm. pressure, which contain the esters 
of phenylalanine, aspartic acid, glutamic acid, and serine. These 
esters are separated by taking advantage of their different 
solubilities in water, ether, petroleum ether; e.g. phenylalanine 
ester is soluble with difficulty in water, and is extracted from 
the aqueous solution of the esters with ether, the esters of 
aspartic and glutamic acids remaining in the water. Serine 
ester is insoluble in petroleum ether; on mixing the fraction 
containing it with petroleum ether it separates as an oil, the 
esters of the three other acids remaining in solution. 
Finally the esters must be converted into the amino acids; 
in the case of the lower-boiling fractions, they are hydrolysed 
by boiling with water under a reflux condenser for five to six 
hours until the alkaline reaction disappears; leucine may 
crystallise out during this process. The esters in the higher- 
boiling fractions which contain glutamic and aspartic acids are 
hydrolysed by boiling with baryta; if hydrolysed by water, 
they remain as acid esters. On cooling, aspartic acid may 
separate as the barium salt of the racemised acid. The 
