504 PHYSIOLOGICAL CHEMISTRY 



In case the solution is deeply colored, as in protein digests, it may be neces- 

 sary to decolorize 1 before the titration is attempted. 



Final Titration. For the final titration a volume of from 20-40 c.c. which con- 

 tains approximately 0.025 gram of nitrogen is the most desirable. A control 

 solution is run composed of an equal volume of boiled distilled water and 20 c.c. 

 of the formaldehyde mixture. 2 This control solution is colored 3 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 investigation and it is then titrated 

 with N/5 acid to a faint red (first stage). 4 



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 produced 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. 5 



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 gram of nitrogen. An example will illustrate the pro- 

 cedure: 40 c.c. of solution (16 c.c. of urine) required 5.10 c.c. N/5 NaOH; 

 control, o.io c.c. N/5 NaOH; total required for amino-acids 5.00 c.c. equivalent 

 to 0.014 gram of nitrogen. Ammonia nitrogen in 16 c.c. of urine 0.007 gram N. 

 Then 0.0140.007 = 0.007 gram amino-acid nitrogen in 16 c.c. of urine. 



Interpretation. The excretion of total amino-acid nitrogen by a 

 normal adult averages between 0.4 to i.o gram per day or from 2 to 6 

 per cent of the total nitrogen. Free amino-acid nitrogen (see Van 

 Slyke procedure) is considerably less than this, ordinarily 0.5 to i.o 

 per cent of the total nitrogen. The amount may be largely increased 

 in disorders associated with tissue waste as typhoid, in pronounced 

 atrophy of the liver, acidosis, etc. 



2. Benedict-Murlin Modification. 6 Principle. In this method the ammonia 

 is removed by means of phosphotungstic acid, and excess acid as well as carbonates 

 and phosphates carried down with barium. 



1 For methods see Jessen-Hansen, Abderhalden's Arbeits Methoden, vol. 6, p. 202, 1912. 



2 The formaldehyde solution is freshly prepared for each set of determinations as follows: 

 to 50 c.c. of commercial formaldehyde (formol) (30-40 per cent) add i c.c. of the phenol- 

 phthalein solution. N/5 alkali is then added until the mixture acquires a faint red color. 

 The volume of the formaldehyde used will vary with the volume of the solution to be ana- 

 lyzed; approximately 10 c.c. of the formalin solution are added for each 20 c.c. of the un- 

 known solution. 



3 Solution of Bismark brown is very satisfactory for urines. Tropseolin O, Tropseolin 

 OO, -nitro-phenol, methyl orange or alizarin sulphonate, may be used. 



4 This procedure is recommended in order that the final volume of the control and the 

 unknown solutions shall be approximately the same when the process is complete. 



5 This is best accomplished by adding alkali until the color is deeper than that of the 

 control, then acid again until lighter and finally alkali to the desired color. 



6 Benedict and Murlin: four. BioL Chem., 16, 385, 1913. 



