BLOOD ANALYSIS 277 



It will be noted that the precipitation is not made in volumetric 

 flasks. By the process described 6 or 7 or n or 12 c.c. of blood can be 

 used, whereas with volumetric flasks one is compelled to use 5, 10 or 

 20 c.c., because flasks suitable for other volumes are not available. 

 Special graduated " blood pipets," made by the Emil Greiner Co., 

 New York, are very useful for the measurement of the blood, the tung- 

 state and the acid. 



The protein free blood filtrates are not acid enough to prevent 

 bacterial decomposition. If the filtrates are to be kept for any length 

 of time, more than two days, some preservative, a few drops of toluene 

 or xylene should be added. 



2. Determination of Non-protein Nitrogen. Principle. Nitrogen 

 is determined in a portion of the blood filtrate by a micro- Kjeldahl, 

 using a sulphuric and phosphoric acid mixture for the digestion, the 

 ammonia formed being determined colorimetrically after direct Nessleri- 

 zation of the digestion mixture. 



Procedure. Transfer 5 c.c. of the blood filtrate to a large test tube (Pyrex) 

 200 mm. X 25 mm., preferably graduated at 35 c.c. and 50 c.c. 1 The test tube 

 should either be dry or rinsed with alcohol to reduce the danger of bumping. 

 Add i c.c. fcf diluted acid mixture 2 and a quartz pebble. Boil vigorously over a 

 micro burner until the characteristic (^ise fumes begin to fill the tube. This 

 will happen in from 3 to 7 minutes, d^Bnding on the size of the flame. When 

 the test tube is nearly full of fumes rRTuce the flame sharply so that the speed 

 of the boiling is reduced almost to the vanishing point. Cover the mouth of the 

 test tube with a watch glass. Continue the gentle heating for 2 minutes, count- 

 ing from the time the test tube became filled with fumes. If the oxidations are 

 not visibly finished at the end of two minutes the heating must be continued until 

 the solution is nearly colorless. Usually the solution becomes colorless at the 

 end of 20 to 40 seconds. At the end of 2 minutes remove the flame and allow 

 the digestion mixture to cool for 70 to 90 seconds. Then add 15 to 25 c.c. of 

 water. Cool further approximately to room temperature and then fill to the 

 35 c.c. mark with water. Add 15 c.c. of Nessler's solution (see final section 

 on Reagents and Solutions). Insert a clean rubber stopper and mix. If the 

 solution is turbid, centrifuge a portion before making the color comparison with 

 the standard. 



The standard most commonly required is 0.3 mg. of N. Add 3 c.c. of stand- 

 ard ammonium sulphate solution (containing i mg. of N per 10 c.c., made by 

 dissolving 0.4716 gm. of specially purified ammonium sulphate (see note p. 511) 

 in one liter of ammonia-free distilled water) to a 100 c.c. volumetric flask. 

 Add to it 2 c.c. of the phosphoric sulphuric acid mixture, to balance the acid in the 



1 These may be obtained from Emil Greiner, New York. 



2 Made by diluting regular acid mixture with an equal volume of water. The regular 

 acid mixture is made as follows: To 50 c.c. of a 5 per cent copper sulphate solution add 

 300 c.c. of 85 per cent phosphoric acid and mix. Add 100 c.c. of concentrated sulphuric 

 acid free from the least trace of ammonia and mix. Keep well protected to prevent ab- 

 sorption of ammonia from the air. 



