PROTEINS 89 



one-fifth of D. For convenience, A is etched with a mark to measure this amount. 

 The acid is run into D cock c being turned so as to let the air escape from D. 

 Through A one now pours sodium nitrite solution (30 grams NaNC>2 to 100 c.c. 

 H 2 0) until D is full of solution and enough excess is present to rise a little above 

 the cock into A. It is convenient to mark A for measuring off this amount also. 

 The gas exit from D is now closed at c, and, a being open, D is shaken for a few 

 seconds. The nitric oxide, which instantly collects, is let out at c, and the shaking 

 repeated. The second crop of nitric oxide which washes out the last portions of air, 

 is also let out at c. D is now connected with the motor and shaken till all but 20 c.c. 

 of the solution have been displaced by nitric oxide and driven back into A . A mark 

 on D indicates the 20 c.c. point. One then closes a and turns c and/ so that D and 

 F are connected. The above manipulations require between one and two minutes. 



2. Decomposition of the Amino Substance. Of the amino solution to be analyzed 

 10 c.c. or less, as the case may be, are measured off in B. Any excess added above 

 the mark can be run off through the outflow tube. The desired amount is then run 

 into D y which is already connected with the motor, as shown in Fig. 34. It 

 is shaken when a-amino acids are being analyzed for a period of three to five 

 minutes. With -amino acids, proteins or partially or completely hydrolyzed 

 proteins, we find that at the most five minutes vigorous shaking completes the 

 reaction. Only in the case of some native proteins which, when deaminized form 

 unwieldy coagula that mechanically interfere with the thorough agitation of the 

 mixture, a longer time may be required. In case a viscous solution is being analyzed 

 and the liquid threatens to foam over into F, B is rinsed out and a little caprylic 

 alcohol is added through it. For amino substances such as amino purins, requiring 

 a longer time than five minutes to react, one merely mixes the reacting solutions 

 and lets them stand the required length of time, then shakes about two minutes to 

 drive the nitrogen completely out of solution. 



When it is known that the solution to be analyzed is likely to foam violently, 

 it is advisable to add caprylic alcohol through B before the amino solution. B is 

 then rinsed with alcohol and dried with ether or a roll of filter paper before it 

 receives the amino solution. 



3. Absorption of Nitric Oxide and Measurement of Nitrogen. The reaction being 

 completed, all the gas in D is displaced into F by liquid from A and the mixture of 

 nitrogen and nitric oxide is driven from F into the absorption pipette. 1 The driving 

 rod is then connected with the pipette by lifting the hook from the shoulder of d and 

 placing the other hook, on the opposite side of the driving rod, over the horizontal 

 lower tube of the pipette. The latter is then shaken by the motor for a minute, 

 which, with any but almost completely exhausted permanganate solutions, com- 

 pletes the absorption of nitric oxide. The pure nitrogen is then measured in F. 

 During the above operations a is left open, to permit displacement of liquid from D 

 as nitric oxide forms in D. 



Blank determinations, performed as above except that 10 c.c. of distilled water 

 replaces the solution of amino substance, must be performed on every fresh lot 

 of nitrite used. Nitrite giving a much larger correction than 0.3 to 0.4 c.c. should 

 be rejected. 



The room temperature and the barometric pressure must be noted. The 



x The solution in the absorption pipette is 40 grams KMNO 4 and 25 grams KOH in a 

 liter. 



