346 PHYSIOLOGICAL CHEMISTRY 



8. Hart's Casein Method. 1 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 - of 10 per cent acetic 

 acid. 2 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. 3 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. 4 

 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 5 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 casern 



9. Casein. Mix about 20 grams of milk with 40 c.c. of a saturated solution 

 of magnesium sulphate and add the salt in substance until no more will dissolve. 

 The precipitate consists of casein admixed with a little fat and lacto-globulin. 

 Filter off the precipitate, wash it thoroughly with a saturated solution of magnesium 

 sulphate, 6 transfer the filter paper and precipitate to a Kjeldahl digestion flask, and 

 determine the nitrogen content according to the directions given in a previous 

 experiment (6). 



Calculation. Multiply the total nitrogen by the factor 6.37 to obtain the casein 

 content. 



10. Lactalbumin. To the filtrate and washings from the determination of 

 casein, in Experiment 8, add Almen's 7 reagent until no more precipitate forms. 

 Filter off the precipitate and determine the nitrogen content according to the direc- 

 tions given under Proteins, page 345. 



Calculation. Multiply the total nitrogen by the factor 6.37 to obtain the lactal- 

 bumin content. 



11. Lactose. To about 350 c.c. of water in a beaker add 20 grams of milk, 

 thoroughly, acidify the fluid with about 2 c.c. of 10 per cent acetic acid and 



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



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

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



3 The process of nitration 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. 



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

 otherwise settle to the bottom of the flask. 



5 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. 



6 Preserve the filtrate and washings for the determination of lactalbumin (Expt. 10). 



7 Almen's reagent may be prepared by dissolving 5 grams of tannic acid in 240 c.c. of 

 50 per cent alcohol and adding 10 c.c. of 25 per cent acetic acid. 



