558 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



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Estimation of Total Nitrogen. (Micro-Kjeldahl.) 



(a) By Titration. 



i c.c. of urine + i c.c. of water + i c.c. cone. H 2 SO 4 + i gm. K 2 SO 4 + a 

 pebble to prevent bumping are placed in a test tube 

 and boiled over a micro-burner for about 6 minutes, 

 but not less than 2 minutes after the mixture has 

 become clear. The oxidation is greatly facilitated 

 by placing over the test tube a broken 25 c.c. pipette 

 bent as shown in Fig. 78 and with its shorter end 

 pushed into the bulb. The bent end is connected 

 with a suction pump. The water is in this way pre- 

 vented from condensing on the sides of the test tube 

 from which it can run back upon the sulphuric acid. 

 To prevent air currents affecting the micro-burner 

 small beakers without bottoms serve as convenient 

 shields. 



The contents are allowed to cool for about 3. 

 minutes until they become viscous, but they are not 

 allowed to solidify. 6 c.c. of water are added, at 

 first drop by drop, and then more rapidly. 



5 or 10 c.c. of :iN acid and a drop of alizarin 

 red are placed in the receiving test tube. 



3 c.c. of saturated caustic soda, or 4 c.c. of 

 40 per cent, soda, are added to the sulphuric acid 

 by sucking it up in the tube which reaches to the 

 bottom of the test tube and through which air is 

 passed. The ammonia is aspirated into the standard 

 acid for 2 minutes with a slow current of air and 8 minutes with a rapid 

 current, or longer if the air current be not powerful. (The time must be de- 

 termined with the apparatus to be used.) 



The contents of the flask after washing out the tube with a small quantity 

 of water are titrated with "02 N alkali. 



(b) Colorimetrically. 



Normal human urine contains 5-20 mgm. of nitrogen per i c.c. This 

 amount is too great for estimation by the colorimetric method ; the urine 

 must therefore be diluted so that i c.c. contains -75 to 1*5 mgm. of nitro- 

 gen: 



If the sp. gr. is over 1018, 5 c.c. are diluted to 50 c.c. 



If the sp. gr. is under 1018, 5 c.c. are diluted to 25 c.c. 



i cc. of the diluted urine is oxidised and its ammonia collected in 2- 

 c.c. of *iN acid and 20 c.c. of water in a 100 c.c. measuring flask if the blowing 

 method is adopted ; in 2 c.c. of 'iN acid + 8 c.c. water if the suction method 

 is used and transferred to a 100 c.c. measuring flask. 



The solutions are diluted to about 60 c.c. 



A quantity of pure ammonium sulphate solution corresponding to i mgm. 

 of N is measured out and diluted to about 60 c.c. in another 100 c.c. 

 measuring flask. 5 c.c. Nessler reagent, diluted with 25 c.c. of water, are 

 added to each flask. The maximum colour develops in half an hour. The 

 flasks are filled to the mark, the contents are mixed and the colorimetric 

 comparison is made in a Duboscq colorimeter. The standard is set at 20. 

 The result is given by 



standard reading _ 20 _ ufine 



unknown reading x 



FIG. 78. 

 (After Folin.) 



