244 Some New Constituents of Milk. III 
hydrochloric acid. Owing to the fact that its acid compounds 
are soluble in water it can also be obtained from milk serum 
from which casein has been precipitated by acid and the coagu- 
lable proteins by heat. . 
From the purified casein which has been prepared in this lab- 
oratory we have obtained large volumes of dilute alcoholic ex- 
tracts from which several hundred grams of this unique protein 
have been isolated in the following manner. 
Casein was precipitated with dilute hydrochloric acid from 
centrifugated milk, filtered out, pressed, suspended in water, and 
dissolved, while actively stirring, by gradually adding dilute 
sodium hydroxide solution. From the resulting solution, which 
was but slightly alkaline to litmus, the casein was precipitated by 
dilute acetic acid. After twice reprecipitating in this way the 
casein was again suspended in water, dissolved by adding very 
dilute sodium hydroxide solution, precipitated by highly diluted 
hydrochloric acid, and filtered out on cheese-cloth. A sample 
of the filtrate when evaporated to dryness left a residue, chiefly 
sodium chloride, which dissolved completely in water, gave no 
biuret reaction, and no reduction with Fehling’s solution. 
The moist casein was then suspended in about an equal vplume 
of 92 per cent alcohol, stirred for a short time, filtered out on 
cheese-cloth, and the extract filtered clear through paper pulp. 
In this way we obtained 43 liters of the first alcoholic extract, 
which was acid to litmus and contained 43 per cent by weight of” 
alcohol and 350 gm. of solids, much of which was sodium chloride. 
The casein was again stirred up with more 92 per cent alcohol 
and 16 liters of a second extract were obtained containing 54 per 
cent. of alcohol and 113 gm. of solids. ’ 
Of the first alcoholic extract 40 liters were concentrated to 
one-half, under diminished pressure, below 75°. The turbid 
concentrated extract on standing deposited on the bottom of the 
jars a transparent syrupy sediment, A. From this the nearly 
clear solution was decanted and concentrated to about 15 liters 
in open dishes. On cooling over night to room temperature a 
coherent deposit, B, formed. 
A and B were dissolved in a little warm 70 per cent alcohol 
and the somewhat turbid solution was poured into several volumes 
of distilled water whereby an opaque colloidal “solution” was 
