APPENDIX 235 



absorption of oxygen) in the air when drawn back into the tube. The 

 remaining gas is nitrogen. 



KJELDAHL'S METHOD OF ESTIMATING NITROGEN 



This simple method can be used in connection with most substances of 

 physiological importance. Briefly, it consists in converting all the nitrogen 

 present into ammonia by means of sulphuric acid ; then rendering alkaline 

 with soda, and distilling over the ammonia into standard acid, the diminu- 

 tion in acidity of which measures the amount of ammonia present. 



The following modification of the original method is used in this 

 laboratory. 



About 1 gramme of the substance under investigation (or in the case of 

 urine when one wishes to make an estimation of total nitrogen, 5 or 10 c.c. 

 of that fluid) is placed in a round bottomed Jena flask of about 250 c.c. 

 capacity, and 20 c.c. of pure sulphuric acid added. Six grammes of 

 potassium sulphate and about half a gramme of copper sulphate are also 

 added. The flask should be provided with a loose balloon stopper, and 

 arranged in a sloping direction over a small flame. The mixture is heated 

 slowly until it boils. In about twenty minutes the fluid becomes nearly 

 colourless ; boiling is continued for another forty-five minutes. By this 

 time all the nitrogen will be in combination as ammonia. 



After cooling, the fluid is washed into a litre flask of Jena glass (fig. 82, A) 

 and water added until the total volume of the fluid is about 400 c.c. Add 

 then an excess of 40 per cent, caustic soda solution, 

 a few pieces of granulated zinc to avoid bumping in the 

 subsequent distillation, and immediately fit the glass 

 tube B into the neck of the flask by means of a well- 

 fitting rubber stopper. The other end of B leads into 

 the flask C which contains a measured amount (50 or 

 100 c.c.) of standard sulphuric acid ; ^ normal acid is a 

 convenient strength to use. The bulb D shown in the 

 figure guards against regurgitation, and the end of the 

 tube should dip just below the surface of the acid in C. FIG. 82. Kjeidahi' 

 The mixture in the flask is now boiled for about half an 

 hour when all the ammonia will have distilled over ; 

 the use of a condenser around the tube B is unnecessary. The acidity of the 

 standard acid is then determined by titrating with standard alkali, a few 

 drops of methyl orange being added to act as the indicator of the end of the 

 reaction (this gives a pink colour with acid, yellow with alkali). 



Example. Suppose 1 gramme of a nitrogenous substance is taken, and 

 the ammonia distilled over into 100 c.c. of i| normal sulphuric acid ( = 20 

 c.c. normal acid). This is then titrated with a corresponding solution of 

 soda, and it is found that the neutral point is reached when 60 c.c. of the 

 soda solution have been added. The other 40 c.c. must therefore have been 

 neutralised by the ammonia derived from the substance under investigation. 

 This 40 c.c. of acid = 8 c.c. of normal acid = 8 c.c. of normal ammonia = 

 8 x 0'017 = 0-136 gramme of ammonia. One gramme of the substance ana- 

 lysed, therefore, yields 0-136 gramme of ammonia, and this contains 0'112 



