CHEMICAL COMPOSITION OF PROTEIN MOLECULE 71 



which gathers in D and forces 10-15 c.c. of solution back into A. 

 The cock c is now again opened and the nitric oxide, together with 

 the air swept out of the solution, is forced out of D by liquid from A. 

 This process is repeated to ensure removal of all the air. 



By closing c and shaking D a gas space of about 20 c.c. is made, so 

 as to make room for the amino solution from B. 



G and H are filled with permanganate ; F is filled with the sul- 

 phuric acid solution up to the top of the rubber piece connecting it to C. 



Tap a is closed and tap c is opened. These manipulations require 

 about two minutes. 



The solution from B is run into D and mixed with the nitrous acid. 

 The evolution of nitrogen commences immediately ; after five minutes 

 D is thoroughly shaken to complete the evolution of the gas. 



If substances other than a-amino acids are being estimated a drop 

 of amyl alcohol from E is occasionally added and the solution shaken 

 several times per minute. Some compounds require longer than five 

 minutes for complete decomposition ; in this case the reaction is con- 

 tinued for a period of fifteen minutes to two hours. 



When the reaction is completed the gas from D and C is driven 

 into F by opening a and letting liquid run from A into D. 



The gas is then driven from F into H by raising the levelling 

 bulb. No gas must be left in the capillaries of G and the pipette. 

 By shaking the gases with the permanganate the nitric oxide is ab- 

 sorbed. The residual nitrogen is run back into F, the permanganate 

 filling G as far as the tap of F. The volume of gas in F is then 

 measured by bringing the surface of the liquid in the bulb even with 

 the meniscus. Generally, one shaking with the fresh permanganate 

 suffices to remove all the nitric oxide, but it is advisable to test if the 

 absorption is complete by returning the gas to the Hempel pipette 

 and again measuring. The weight of nitrogen corresponding to the 

 volume of gas is calculated in the usual way. The results are divided $ 

 by 2 (see equation, p. 67). 



Each milligram of arnino substance gives off from 17 to I '9 c.c. of 

 nitrogen. 



A correction should be made for the air 0*2 c.c. dissolved in 10 

 c.c. amino solution, i.e., allowing for the oxygen which combines with 

 the nitric oxide forming peroxide and is absorbed by the permangan- 

 ate, cri6 c.c. must be deducted from the volume of gas. This correc- 

 tion is equivalent to 0*09 milligrams of amino nitrogen. No correction 

 is necessary, if air-free water be used in preparing the amino solution. 



