68 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



the liquid with the gas. A steady stream of carbon dioxide was 

 obtained by allowing the liquids to mix in a piece of wide glass tubing, 

 which was drawn out at its lower extremity so that only a small aper- 

 ture remained and closed at its upper end by a small cork through 

 which tubes connected with reservoirs of acid and carbonate passed ; 

 two small apertures in its sides allowed for the escape of the gas. 

 This wide tube was placed in the vessel used for the generation of the 

 carbon dioxide. The mixture of acid and carbonate solutions accumu- 

 lated to a height of about 3 cm. in the tube, and then dropped into 

 the flask, which could be emptied by an attached syphon tube. The 

 carbon dioxide entered the apparatus through a special trap. Air 

 contained in the apparatus was removed by a stream of carbon dioxide 

 and steam, which was made to enter through this trap. All the solu- 

 tions used were made with air-free water, charged with carbon dioxide ; 

 the same water was used for washing purposes. 



The carbon dioxide was removed by the potash solution contained 

 in a Lunge nitrometer, with which the exit tube of the apparatus was 

 connected. The excess of nitric oxide was removed by means of 

 oxygen, prepared by the electrolytic decomposition of water, and 

 passed into the nitrometer, and the excess of oxygen by passing the 

 gas in the nitrometer into a double Hempel pipette containing 

 pyrogallol dissolved in 60 per cent, potash solution. 



Allowing one to two hours for the reaction of the nitrous acid upon 

 the amino acids, satisfactory results were obtained with glycine, alanine, 

 phenylalanine, leucine, aspartic acid, glutamic acid and asparagine. 

 The results with tyrosine were not satisfactory, but they found that it 

 reacted quantitatively after bromination. 



For the application of this method to the differentiation of the 

 various forms of nitrogen in proteins we are indebted to Van Slyke, 

 who not only has devised a much simpler apparatus for the estimation 

 of the ammo-nitrogen, but also has devised a means for carrying out 

 the estimation of the two groups of monoarnino acids, the several 

 diamino acids and cystine in one series of operations. 





