MILK 345 



5. Ash. Heat the dry solids from 2-5 grams of milk, obtained according to 

 the method just given, over a very low flame 1 until a white or light gray ash is 

 obtained. Cool the dish in a desiccator and weigh. (This ash may be used hi 

 testing for borates according to directions on page 341.) 



6. Proteins: Nephelometric Determination of Proteins, Casein, 

 Globulin, and Albumin in Milk. Method of Kober. 2 Principle. The 

 proteins are precipitated with sulphosalicylic acid and the precipitate 

 estimated nephelometrically (see discussion of nephelometric methods, 

 page 294). 



Procedure. Five c.c. of milk are carefully measured into a 250 c.c. flask 

 and after adding 200 c.c. of distilled water and 10 c.c. of decinormal sodium 

 hydroxide solution, water is added to the mark and the mixture shaken. Ten 

 c.c. are put with exactly 2 c.c. of ether in a centrifuge tube which is then tightly 

 stoppered with a cork and vigorously shaken. Allow to separate and withdraw 

 5 c.c. of the aqueous layer without contamination with ether. Dilute to 50 c.c. 

 Take 10 c.c. of this solution and add 10 c.c. of 3 per cent sulphosalicylic acid. A 

 suspension of casein is obtained which can be matched accurately with the 

 following standard : i volume (5 c.c.) of a o.oi per cent casein solution 3 to which 

 is added 2 volumes (10 c.c.) of 3 per cent sulphosalicylic acid. 



The protein obtained with this reagent is not all casein, and hi order to obtain 

 the exact amount of casein the casein is precipitated according to the "official 

 method' 1 or the method of Hart given below and the amount of precipitate ob- 

 tained hi an aliquot portion of the filtrate, by adding 4 volumes of the reagent, 

 is determined nephelometrically. This fraction, for want of a better name called 

 the "globulin and albumin fraction," is subtracted from the gross casein, to 

 give the amount of casein precipitated by the "official method." 



The ether used in extracting the fat increases the volume of the solution and 

 hence a factor allowing for this must be used. For 10 c.c. of diluted milk and 

 2 c.c. of ether the factor is 0.910. 



7. Proteins. Introduce a known weight of milk (5-10 grams) into a 500 c.c. 

 Kjeldahl digestion flask and add 20 c.c. of concentrated sulphuric acid and about 

 0.2 gram of copper sulphate. Expel the major portion of the water by heating 

 over a low flame and finally use a full flame and allow the mixture to boil one to 

 two hours. Complete the determination according to the directions given under 

 Kjeldahl Method, page 504. 



Calculation. Multiply the total nitrogen content by the factor 6.37 4 to obtain 

 the protein content of the milk examined. 



1 Great care should be used in this ignition, the dish at no time being heated above a 

 faint redness, as chlorides may volatilize. 



2 Kober: /. Am. Chem. Soc., 35, 1585, 1913. 



3 Standard Casein Solution. Dissolve with stirring o.i gram of casein or its equivalent 

 in i c.c. of o.i N NaOH, add 95 c.c. of distilled water, add 2 c.c. of toluene, shake thoroughly 

 and make up to 100 c.c. This is the stock solution which keeps three or four days or longer. 

 The standard solution is made up fresh every day by making 10 c.c. of the stock solution 

 up to 100 c.c. with water. The standard is controlled by total nitrogen estimations using 

 the factor 6.38 for casein. 



4 The usual factor employed for the calculation of protein from the nitrogen content is 

 6.25 and is based on the assumption that proteins contain on the average 16 per cent of 

 nitrogen. This special factor of 6.37 is used to calculate the protein content from the total 

 nitrogen, since the principal protein constituents of milk, i.e., casein and lactalbumin, 

 contain about 15.7 per cent of nitrogen. 



