328 PHYSIOLOGICAL CHEMISTRY 



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

 tained in 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. 



6. 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 483. 



Calculation. Multiply the total nitrogen content by the factor 6.37^0 obtain 

 the protein content of the milk examined. 



7. Hart's Casein Method. 2 Introduce 10.5 c.c. of milk into a 200 c.c. Erlen- 

 meyer flask and add 75 c.c. of distilled water and 1-1.5 c - c ' f 10 per cent acetic 

 acid. 3 Mix the contents by giving the flask a vigorous rotary motion. The 

 precipitated casein is now filtered off upon a 9-11 cm. filter paper. 4 Wash out 

 the adsorbed and loosely combined acetic acid by means of cold water. Con- 

 tinue the washing of both the casein on the filter and that adhering to the flask, 

 until the wash water has reached a volume of at least 250 c.c. 



Now return the precipitate and paper to the original Erlenmeyer flask, add 

 75-80 c.c. of neutral (carbon dioxide -free) water, 10 c.c. of N/io potassium hy- 

 droxide and a few drops of phenolphthalein. Stopper the flask and shake it 

 vigorously, by hand or machine, until the casein has been brought into solution. 5 

 Rinse the stopper with neutral (carbon dioxide -free) water and titrate the alka- 

 line casein solution at once with N/io hydrochloric acid until there is a dis- 

 appearance of all red color. 6 



Calculation. Subtract the corrected 6 acid reading from the 10 c.c. of alkali 

 used. The difference is the percentage of casein in the milk. For example, if 

 it takes 6.7 c.c. of N/io hydrochloric acid to titrate the alkaline solution to the 

 end point and the check test was equivalent to 20 c.c. N/io acid the casein value 

 would be obtained as follows : 



10 (6.7+0.2) = 3.1 per cent casein 



1 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. 



2 Hart: Jour. Biol. Chem., 6, 445, 1909. 



8 In general 1.5 c.c. of acetic acid gives a clear solution which niters nicely but occasion- 

 ally, when the milk has a low casein value it is advisable to use less acetic acid. 



4 The process of filtration may be retarded through the packing of the casein mass 

 upon the filter paper. In this case conduct a fine stream of cold water against the upper 

 point of contact of filter paper and casein. By this means the casein precipitate is loosened 

 and gathers in the apex of the filter. This procedure is very essential. It is not necessary 

 to remove the casein which adheres to the interior of the flask. 



" 6 Solution is indicated by the disappearance of the white casein particles which would 

 otherwise settle to the bottom of the flask. 



6 A check test should be run parallel with the entire determination. Even with special 

 precautions as to neutrality, it is generally found that an acid check of 0.2-0.3 will be 

 obtained. This check titration should be added to the volume of acid used in titration. 



