580 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



(4) Estimation of Ammonia (p. 560). 



This estimation is the most difficult, as blood decomposes rapidly and 

 gives too high values. The analysis has to be made with hundredths of a milli- 

 gram instead of tenths. It cannot be estimated in the colorimeter in the 

 ordinary way and the colour of the Nessler reagent is yellow or yellow-green. 



The Duboscq colorimeter is modified by attaching to one side an iris 

 diaphragm, putting the unknown into a i o cm. polarimeter tube and removing the 

 glass prism. Excess of Nessler reagent must be avoided (the green tint is due 

 to excess) and dilutions to the final volume must be made with water absolutely 

 free from ammonia prepared by adding bromine water and a few drops of con- 

 centrated caustic soda solution. 



10 c.c. of the filtrate from systemic blood 

 or 5 c.c. ,, portal or mesenteric blood 



are placed in a test tube with 2-3 c.c. of oxalate-carbonate solution and 5 c.c 

 of toluene. 



The air current is continued for 20-30 minutes and the ammonia is 

 collected in 5-6 drops of ! iN acid and i c.c. of water. Splashing may be 

 hindered by introducing into the test tube a small funnel with broken stem. 



The solution is Nesslerised with i c.c. of dilute reagent diluted to 10 c.c. 

 in a measuring flask and put in the polarimeter tube. Two standard solutions 

 containing *5 and i mgm. of nitrogen diluted to 100 c.c. are used. 



The unknown remains stationary so that the standard is adjusted. 



The microscope diaphragm is used and it is fastened to the instrument 

 by two screw clamps on to the top of the colorimeter platform on which the 

 cup stands. A new zero-point must be established. 



(5) Estimation of Urea (p. 560). 



5 c.c. filtrate (cat's blood), or 10 c.c. filtrate (human blood), are placed 

 in a test tube with a drop of dilute acetic acid and 2 or 3 drops of paraffin, 

 and the alcohol is removed by evaporation in vacuo. The test tube is closed with 

 a 2 -holed rubber stopper ; a tube with a capillary several inches long and reach- 

 ing to the bottom is placed in one hole and a bent tube to attach to the pump 

 in the other. It is placed in warm water and the vacuum is started ; in 10- 

 30 minutes the alcohol is removed. The capillary is broken off and left 

 in the test tube. 



The hydrolysis is effected by heating for 8-10 minutes with 2 c.c. of 25 

 per cent, acetic acid and 7 gm. of potassium acetate. 



The ammonia is collected, Nesslerised with 3 c.c. reagent and diluted to 

 10 c.c. 



The hydrolysis may also be effected with urease (p. 561). 



Estimation of Uric Acid (p. 561). 



10-50 c.c. of blood are drawn and placed in a weighed bottle contain- 

 ing -i gm. of finely powdered potassium oxalate and shaken immediately to 

 prevent clotting. The bottle is weighed to determine the amount of blood. 



This oxalate blood is poured into 5 times its weight of boiling 'oiN acetic 

 acid (10 c.c. N acid diluted to 1000 c.c.) and the mixture raised to boiling. The 

 proteins are coagulated. The precipitate is filtered off whilst the solution is 

 hot, returned with a spatula to the flask, covered with 200 c.c. of boiling 

 water for 5 minutes and filtered off through the same filter as before. The 

 filtrate is clear if no clotting has taken place. 



If clotting has occurred, the solution cannot be heated to boiling but is 

 filtered sooner. The clot is broken up with a glass rod, put into a mortar, 

 ground up with hot water and put on to the filter. It is washed with 200 c.c. 

 of water. The filtrate is reddish, but on boiling and filtering a clear solution 

 will result. 



