PRECIPITATION OF PROTEINS tCM 



have previous! y been heated on the water-bath, so that the nitrate does 

 not respond to HELLER'S test. Now warm a larger weighed or meas- 

 ured quantity of the liquid on the water-bath, and add gradually the 

 required quantity of acetic acid, with constant stirring, and continue 

 heating for some time. Filter, wash with water, extract with alcohol 

 and then with ether, dry, weigh, incinerate, and weigh again. With 

 proper work the filtrate should not give HELLER'S test. This method 

 serves in most cases, and especially so in cases where other bodies are 

 to be quantitatively estimated in the nitrate. 



The precipitation by means of alcohol may also be used in the quan- 

 titative estimation of proteids. The liquid is first carefully neutralized, 

 treated with some NaCl if necessary, and then alcohol added until the 

 solution contains 70-80 vol. per cent anhydrous alcohol. The precipitate 

 is collected on a filter after 24 hours^ extracted with alcohol and ether, 

 dried, weighed, incinerated, and again weighed. This method is only 

 applicable to liquids which do not contain any other substances, like 

 glycogen, which are insoluble in alcohol. 



In both of these methods small quantities of proteid may remain in 

 the filtrates. These traces may be determined as follows: Concentrate 

 the filtrate sufficiently, remove any separated fat by shaking with ether, 

 and then precipitate with tannic acid. Approximately 63 per cent of 

 the tannic-acid precipitate, washed with cold water and then dried, may 

 be considered as proteid. 



* In many cases good results may be obtained by precipitating all the 

 proteid with tannic acid and determining the nitrogen in the washed pre- 

 cipitate by means of KjELDAiLLis method. On multiplying the quantity 

 of nitrogen found by 6.25 we obtain the quantity of proteid. 



The removal of proteids from a solution may in most cases be per- 

 formed by boiling with acetic acid. Small amounts of proteid which 

 remain in the filtrates may be separated by boiling with freshty pre- 

 cipitated lead carbonate or with ferric acetate, as described by HOF- 

 MEisTER. 1 If the liquid cannot be boiled, the proteid may be precipi- 

 tated by the very careful addition of lead acetate, or by the addition of 

 alcohol. If the liquid contains substances 'which are precipitated by 

 alcohol, such as glycogen, then the proteid may be removed by the alter- 

 nate addition of potassium-mercuric- iodide and hydrochloric acid (see 

 Chapter VIII, on Glycogen Estimation), or by trichloracetic acid as 

 suggested by OBERMAYER and FRANKEL. 2 Recently MICHAELIS and 

 RONA have suggested a method for the removal of proteids by using 

 kaolin, colloidal ferric hydrate or a mastic emulsion. The principle of 

 these methods has already been given on page 96 and in regard to the 

 practical execution of the method we refer to the works there cited. 



In the precipitation of proteid as well as the quantitative estimation by means 

 of heat, it must be borne in mind, as shown by SPIRO, S that several nitrogenous 

 substances, such as piperidine, pyridine, urea, etc., disturb the coagulation of the 

 proteids. 



1 Zeitschr. f . physiol. Chem., 2 and 4. 



2 Obermayer, Wien. med. Jahrb., 1888; Frankel, Pfliiger's Arch., 52 and 55. 



3 Zeitschr. f. physiol. Chem., 30. 



