ADVANCED CHEMICAL PHYSIOLOGY 315 



Now pour the mixture on to a filter large enough to hold it all. Folin 

 advises that a few c.c. be first poured on to the double portion of the 

 filter paper and that the remainder should be withheld until the whole 

 filter has been wet. As soon as complete wetting takes place pour in 

 the rest of the mixture and cover with a watch glass. If conditions 

 are right even the first few drops of the filtrate should be water clear ; 

 no re-filtering should be necessary. If filtrate has to be kept for any 

 time (more than two days) add a few drops of toluol as a preservative. 

 The filtrate which results is suitable for the determination of non- 

 protein nitrogen, urea, uric acid, creatinine, creatine and sugar. 



Determination of Non-protein Nitrogen. Pipette 5 c.c. of the above 

 filtrate into a hard glass test tube (200 mm. x 25 mm.), marked at 35 

 and 50 c.c., which should either be perfectly dry or just previously 

 rinsed with alcohol and drained, and add 1 c.c. diluted acid mixture 1 

 (1 part acid mixture, 1 part water). Boil vigorously over a micro 

 burner until tube is filled with dense fumes (in from three to seven 

 minutes, depending on the size of the flame), then reduce the flame 

 sharply so that speed of boiling is reduced to lowest limit. Cover mouth 

 of test tube with a watch glass and continue gentle heating for two 

 minutes from the time the tube is filled with fumes. If solution is not- 

 then perfectly colourless heating must be continued until this end is 

 attained. When colourless allow digestion mixture to cool for seventy 

 to ninety seconds, then add 1525 c.c. water, cool to room temperature 

 and finally add more water to make total volume 35 c.c. Add 15 c.c. of 

 Nessler's solution, 2 close with a clean rubber stopper and mix. If 

 solution is turbid it must be centrifuged before doing the colour value 

 with the standard. 



Folin's standard is 0-3 mg. of N. Add 3 c.c. of standard ammonium 

 sulphate solution (0-4716 gm. absolutely pure dry ammonium sulphate 

 dissolved in 1 litre water, 10 c.c. of this solution contains 1 mg. 

 of nitrogen) to a 100 c.c. volumetric flask, then 2 c.c. of the acid mixture 

 to balance acid in other test, dilute to about 60 c.c. and add 30 c.c. of 

 Nessler's solution. The unknown and the standard should be nesslerised 

 si multaneously . 



Calculation. If standard is set at 20 mm. in the colorimeter, 20 



1 Acid Mixture. To 50 c.c. of a 5 per cent, copper sulphate solution add 

 300 c.c. of 85 per cent, phosphoric acid, mix, then add 100 c.c. concentrated 

 N-free sulphuric acid, again mix. Keep free from all ammonia fumes. 



2 Nessler's Reagent. Place 150 gms. KI and 110 gms. I in a 500 c.c. flask, 

 add 100 c.c. water and an excess of metallic mercury (140-150 gms.). Shake 

 vigorously and continuously for seven to fifteen minutes or until dissolved 

 iodine has nearly all disappeared. Solution becomes hot. When red iodine 

 solution begins to become definitely pale, though still red, cool in running 

 water and continue shaking until solution assumes a greenish colour (about 

 fifteen minutes in all). Decant off the solution from the surplus mercury, 

 wash well with water, dilute solution and washings to 2,000 c.c. 



From a completely saturated solution of NaOH (55 gms. NaOH per 100 c.c.) 

 prepare an approximately accurate 10 per cent, solution. To 3,500 c.c. of this 

 10 per cent. NaOH add 750 c.c. of the double iodide solution and 750 c.c. of 

 distilled water, giving thus in all 5 litres of Nessler's solution. 



To prevent turbid mixtures on the addition of Nessler solution to the fluid to 

 be tested as a general rule the volumetric flask or test tube should be at least 

 two -thirds full before the addition of Nessler is made. The volume of Nessler 

 to be added should form about 10 per cent, of the ultimate total volume of 

 fluid and reagent. This amount is of course modified if much acid or alkali 

 is present. 



