276 



PHYSIOLOGICAL CHEMISTRY 



FIG. 87. 



DILUTING 



PIPETTE. 



(Folin 



and Wu: 



Jour.. Biol. 



Chem., 



May, 



1919). 



sugar, and chlorides. About 10 c.c. of blood are needed for 

 the combined determinations. 



i. Preparation of the Protein Free Blood Filtrate. 



Principle. The total proteins of the blood are removed 

 by precipitation with tungstic acid (formed by the interac- 

 tion of sodium tungstate and sulphuric acid) and nitration. 

 The nitrate contains all of the constituents of the blood 

 determined by this system. 



Procedure. To prevent coagulation 20 mgs. of potassium 

 oxalate per 10 c.c. of blood should have been used. Use of much 

 larger amounts of oxalate or the use of citrate interferes with 

 deproteinization, and interferes more or less with the uric acid 

 determination. 



( Transfer a measured quantity (5 to 15 c.c.) of oxalated blood 

 to a flask having a capacity of fifteen to twenty times that of the 

 volume taken. Lake the blood with seven volumes of water. 

 Add one volume of 10 per cent solution of sodium tungstate 1 

 (Na 2 WO 4 2H 2 O) and mix. 



Add from a graduated pipette or burette, slowly and with shaking 

 one volume of two-thirds normal sulphuric acid. 2 Close the mouth 

 of the flask with a rubber stopper and shake. If tJM^jnditions are 

 right, hardly a single airlMtble will form as a resi ^Bthe shaking. 

 Let stand for 5 minutes ; the^rior of the coaguraH^Proually changes 

 from bright red to dark brown. If this change in color does not 

 occur, the coagulation is incomplete, usually because too much 

 oxalate is present. In such an emergency the sample may be 

 saved by adding 10 per cent sulphuric acid, one drop at a time 

 shaking vigorously after each drop, and continuing until there is 

 practically no foaming and until the dark brown color has set in. 



Pour the mixture on a filter large enough to hold it all. This 

 filtration should be begun by adding only a few c.c. of the mixture 

 down the double portion of the filter paper and withholding the 

 remainder until the whole filter has been wet. Then the whole of 

 the mixture is poured on the funnel and covered with a watch glass. 

 If the filtration is made as described the very first portion of the 

 filtrate should be clear as water and no re-filtering is necessary. 



1 A IQ 'per cent solution of sodium tungstate. Some sodium tungstates, 

 though labeled c.p., are not serviceable for this work. They usually contain 

 too much sodium carbonate. The c.p. sodium tungstate made by the 

 Primos Chemical Company, Primos, Pa., is satisfactory. 



2 A two-thirds normal sulphuric acid solution, 35 g. of concentrated c.p. 

 sulphuric acid diluted to a volume of i liter, will usually be found to be cor- 

 'rect; but it is advisable, indeed necessary, to check it up by titration. The 

 two-thirds normal acid is intended to be equivalent to the sodium content of 

 the tungstate so that when equal volumes are mixed substantially the whole 

 of the tungstic acid is set free without the presence of an excess of sulphuric 

 acid. The tungstic acid set free is nearly quantitatively taken up by the pro- 

 teins and the blood nitrates obtained are, therefore, only slightly acid to 

 congo red paper. 



