AMINO-ACID NITROGEN 57 



of an equal volume of boiled distilled water and 20 c.c. of the 

 formaldehyde mixture. This control solution is colored so that 

 its tint matches that of the solution to be titrated. 



To this control is added about half the volume of N/5 alkali 

 which will be used in the titration of the solution under investi- 

 gation and it is then titrated with N/5 acid to a faint red (first 

 stage) . 



An additional drop of N/5 alkali is added, which imparts a 

 distinct red to the solution (second stage). 



The, solution to be analyzed is now titrated to the color pro- 

 duced in the second stage of the control. The formaldehyde 

 mixture is now added; 10 c.c. for each 20 c.c. of the solution, and 

 the mixture again titrated to the second stage with N/5 alkali. 



Two drops of the N/5 alkali are now added to the control 

 solution which assumes a deep red color (third stage). Fifth 

 normal alkali is now added to the solution under examination 

 until it assumes a color corresponding to the third stage of the 

 control. This completes the titration. 



Calculation. The calculations are similar to those which 

 pertain to any acidimetry procedure. Each cubic centimeter 

 of an N/5 alkali or acid solution is equivalent to 0.0028 gm. 

 of nitrogen. An example will illustrate the procedure: 40 c.c. 

 of solution (16 c.c. of urine) required 5.10 c.c. N/5 NaOH; con- 

 trol, 0.10 c.c. N/5 NaOH; total required for amino-acids 5.00 c.c. 

 equivalent to 0.014 gm. of nitrogen. Ammonia nitrogen in 16 c.c. 

 of urine 0.007 gm. N. Then 0.014-0.007 = 0.007 gm. ammo- 

 acid nitrogen in 16 c.c. of urine. 



AMINO-ACID NITROGEN 



Van Slyke's Method for Total Ammo-Acid Nitrogen 1 



Take 25 c.c. of urine and mix with 1 c.c. of concentrated 

 sulphuric acid and heat in an autoclave at 180 (oil bath tempera- 

 ture) for one and one-half hours. Transfer to a 50 c.c. flask and 

 add 2 gms. powdered calcium hydroxide. Shake thoroughly, 

 make up to 50 c.c. and filter through a dry folded filter. Trans- 

 fer 20 c.c. of the filtrate to a Jena glass evaporating dish and con- 

 centrate to dryness on the water-bath. This requires about half 



1 Van Slyke: Jour. Biol. Chem., 1913, 16, 125. 



