DISTRIBUTION OF NITROGEN 89 



B. DISTRIBUTION OF THE NITROGEN IN SEVEN 



GROUPS. 



I. Estimation of Amino Nitrogen. 



The action of nitrous acid upon amino acids and amides as a method for estimating 

 these compounds was introduced by Sachsse and Kormann [1875]. These investigators 

 employed an apparatus consisting of a small cylinder, furnished with a rubber stopper, 

 through which two tap funnels and an exit tube for the evolved gas passed. Potassium 

 nitrite was placed in the cylinder, dilute sulphuric acid in one of the tap funnels, and the 

 solution of the substance (o'6-i'o gram) in the other. The exit tube was placed under an 

 eudiometer filled with ferrous sulphate solution to absorb the nitric oxide. Air was expelled 

 from the apparatus by the decomposition of some of the nitrite with the acid. As soon as 

 the expulsion was complete, the amide solution and more acid solution were allowed to 

 enter into the cylinder and the gas collected. The ferrous sulphate solution freed the 

 mixture from nitric oxide and more was added, if necessary. Carbon dioxide was then re- 

 moved with potash and the remaining gas was measured. 



This method was exhaustively tested in the Guinness Research laboratory by Horace 

 Brown and J. H. Millar [1903]. They found that there were several serious sources of error 

 in the original method before they could apply it to their own subject of investigation. 

 These were due (i) to residual air contained in the apparatus, or in the liquids ; (2) to the 

 difficulty of absorbing the excess of nitric oxide with ferrous sulphate. Carbon dioxide 

 was used to remove the air and the evolved nitrogen, and difficulty was experienced in ob- 

 taining a steady and constant stream of this gas. Pure carbon dioxide was prepared by 

 the action of 30 per cent, sulphuric acid on normal sodium carbonate solution. A constant 

 evolution of gas was not obtained, when the two solutions were allowed to drop separately 

 into a flask, owing to the supersaturation of 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 aperture 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 accumulated 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 solutions 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 nitro- 

 meter, 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. 



The estimation of the amino nitrogen was originally performed by 

 Van Slyke [1910, I, 2 ; 191 1, I, 2, 4 ] in an apparatus very similar to 

 that used by Sachsse and Kormann and by Horace Brown and Millar. 



