174 
L. L. Van Slyke and A. W. Bosworth in 1909 published their volumetric 
method (Technical Bulletin, N. Y. Ag. Exp. St.). The method worked out 
in their publication mentioned is briefly as follows: ‘‘A given amount of 
milk, diluted with water, is made neutral to phenolphthalein by the addition 
of a solution of sodium hydroxide. The casein is then completely pre- 
cipitated by the addition of standard acetic acid, the volume is then made 
up to 200 ce. by the addition of distilled water and then filtered. Into 100ce. 
of the filtrate a standard solution of sodium hydroxide is run until neutral to 
phenolphthalein. These solutions are so standardized that 1 ec. is equivalent 
to 1 per cent. casein, when a definite amount of milk is used. Therefore, the 
number of cubic centimeters of standard acid used, divided by 2 less the 
amount of standard alkali used in the last titration gives the percentage of 
casein in the milk.” 
This method is based on the well known facts in chemistry and shows 
quite clearly the cascin molecule has a constant molecular weight. First, 
uncombined casein is insoluble in milk serum, water or very dilute acids. 
Second, it has properties of an acid and combines with alkalies to form 
definite chemical compounds, neutral to phenolphthalein. 
Now, if we know the molecular weight of casein or its equivalent in 
terms of a standard alkali, we can at once devise a definite method for estim- 
ating the casein by titration. Casein exists in milk in a colloidal condition 
combined with bases, upon addition of an acid sufficient to combine with 
salts in combination with casein, free casein is formed, insoluble in the 
serum (it must be remembered that casein and other albuminoids are soluble 
in excess of acids, the solubility depends on the kind of acid and tempera- 
ture). There exists a definite relation between the amount of acid required 
to form free casein and the amount of casein present. It has been found that 
one gram of free casein neutralizes 8.8378 cc. of N sodium hydroxide, or 
sodium hydroxide neutralizes .11315 grams of casein. From this 
1 ce. of 7 
data the molecular weight of casein can be calculated. 
From the above facts it is easy to determine the quantity of milk re- 
quired, so that each ce. of \ 
acid used shall correspond to percents or 
fraction of a percent. Since 1 ec. of NaOH neutralizes .11315 grams of casein, 
it must require an equivalent amount of acid to set free the casein from its 
original combination in milk. If we wish to know the quantity of milk to be 
taken so that 1 cc. of acid used to separate the casein from its combinaion 
shall equal 1 per cent. of casein, we make use of the above equivalent, i.e. 
ee ee ——— 
