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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 cc. by the addition of distilled water and then filtered. Into lOOcc. 

 of the filtrate a standard solution of sodium hydroxide is run until neutral to 

 phenolphthalein. These solutions are so standardized that 1 cc. 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 

 qinte clearly the casein molecule has a constant molecular weight. Fiiot, 

 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 Avith 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 ^ sodium hydroxide, or 

 1 cc. of ~ sodium hydroxide neutralizes .11315 grams of casein. From this 

 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 cc. of y 6 acid used shall correspond to percents or 

 fraction of a percent. Since 1 cc. 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. 



