5 2 4 



PHYSIOLOGICAL CHEMISTRY 



Procedure. The determination of the amino-acids is carried out as follows : 

 The solution to be analyzed, if carbonates, phosphates and ammonia are absent, 

 is made neutral to litmus (paper) and the solution titrated with formalde- 

 hyde as below. 1 In case carbonates, phosphates or ammonia are present a 

 preliminary treatment is necessary which will vary according to the quantity 

 of ammonia present. 



(a) For Small Amounts of Ammonia. Applicable to most urines. Fifty 

 c.c. of the material under examination is pipetted into a 100 c.c. measuring 

 flask and i c.c. phenolphthalein solution 2 and 2 grams of solid barium chloride 

 are added ; the whole is shaken, to saturate the solution with barium chloride ; 

 saturated barium hydroxide solution is added until the red color of the phenol- 

 phthalein develops and then an excess of 5 c.c. is added. The flask is filled 

 to the graduation mark with water, shaken and permitted to stand for 15 minutes, 

 after which it is filtered through a dry filter. Eighty c.c. of the clear red filtrate 

 (which corresponds to 40 c.c. of the liquid under examination) are placed in 

 a 100 c.c. measuring flask, neutralized to litmus and diluted to 100 c.c. with 

 freshly boiled water. Equal portions of this solution, 40 c.c. (equivalent to 

 16 c.c. of the original solution), may be taken for analysis, one for the formol 

 titration and the other for the determination of ammonia nitrogen. 3 



(b) For Large Amounts of Ammonia. After the treatment with phenol- 

 phthalein, barium chloride, and barium hydroxide, and the solution has been 

 diluted to 100 c.c. as in (a) above, the ammonia is distilled off, in vacuo. 4 



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

 sary to decolorize 5 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. 6 This control solution is colored 7 so that its 

 tint matches that of the solution to be titrated. 



To this control is added about half the volume of N/s alkali which will be 

 used in the titration of the solution under investigation and it is then titrated 

 with N/s acid to a faint red (first stage). 8 



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 



1 As a standard of comparison the litmus paper used for neutralization is contrasted with 

 a similar piece dipped in a phosphate solution having a neutral reaction (M/is KH 2 PO4 and 

 M/is NasHPOO. 



2 A solution of 0.5 gram of phenolphthalein in 50 c.c. of alcohol and 50 c.c. of water. 



3 The determination of ammonia may be dispensed with in case a separate determina- 

 tion is made. 



4 For particulars with regard to the distillation, etc., see Henriques and Sorensen: Zeit. 

 physiol. Chem., 64, 137, IQOQ. 



5 For methods see jessen-Hansen, Abderhalden's Arbeits Methoden, vol. 6, p. 262, 1912. 



6 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. 



7 Solution of Bismark brown is very satisfactory for urines. Tropaeolin O, Tropaeolin 

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



8 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. 



