86 METHODS FOR THE ANALYSIS OF BLOOD 



acid fumes begin to fill the test-tube. This is usually accomplished 

 in from three to seven minutes. When the fumes are unmistakable, 

 cut down the size of the flame so that the contents of the tube are 

 just visibly boiling, and close the mouth of the test-tube with a 

 watch glass or a very small Erlenmeyer flask. Continue the heat- 

 ing very gently for two minutes from the time the fumes began 

 to be unmistakable, even if the solution has become clear and 

 colorless at the end of twenty to forty seconds. If the oxidations 

 are not visibly finished at the end of two minutes, the heating must 

 be continued until the solution is nearly colorless. Such cases 

 are very rare; the oxidation is almost invariably finished within 

 the first minute. Allow the contents to cool from seventy to 

 ninety seconds and then add 15 to 25 c.c. of water. Cool further, 

 approximately to room temperature, and add water to the 35 c.c. 

 mark. Add, preferably with a pipette, 15 c.c. of the special Nessler 

 solution. 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 nitrogen (in the form of ammonium sulphate) in a 100 c.c. 

 flask. Add to it 2 c.c. of the sulphuric-phosphoric acid mixture, 

 about 50 c.c. of water, and 30 c.c. of Nessler solution. Fill to the 

 mark and mix. The unknown and the standard should be Nessler- 

 ized at approximately the same time. If the standard is set at 20 

 mm. for the color comparison, 20 divided by the reading and 

 multiplied by 30 gives the non-protein nitrogen in mg. for 100 c.c. 

 of blood. 



Preparation of Reagents for Non-protein Nitrogen Method 



Sulphuric-phosphoric Acid Solution. Mix 300 c.c. of phosphoric 

 acid, syrup (about 85 per cent HsPC^) with 100 c.c. of concentrated 

 sulphuric acid. Transfer to a tall cylinder, cover well to exclude 

 the absorption of ammonia, and set aside for sedimentation of 

 calcium sulphate. This sedimentation is very slow, but in the 

 course of a week or so the top part is clear and 50 to 100 c.c. can be 

 removed by means of a pipette. (It is not absolutely necessary 

 that the calcium should be thus removed, but it is probably a 

 little safer to have it done.) To 100 c.c. of the clear acid add 10 

 c.c. of 6 per cent copper sulphate solution and 100 c.c. of water. 



