506 PHYSIOLOGICAL CHEMISTRY 



filtrate to a Jena glass evaporating dish and concentrate to dryness on the water- 

 bath. This requires about half an hour. The residue is moistened with i c.c. 

 of 50 per cent acetic acid to bring the calcium hydroxide and carbonate into 

 solution, and is then washed into a 10 c.c. flask and filled up to the mark. One 

 can use the entire solution for determination of the amino -nitrogen in the large 

 amino -apparatus, or use 2 c.c. portions for the micro-apparatus. (See Van 

 Slyke Apparatus, Figs. 34 and 35, p. 88 in Chapter IV on Proteins.) 



The length of time which the nitrous acid solution should be shaken in order 

 to drive off all the amino -nitrogen depends somewhat on the temperature. 

 When the latter is 15-20 the time should be five to four minutes; for 20-25 it 

 is three minutes, for 25-30, two and a half to two minutes. It is preferable 

 that the solution should be shaken vigorously with a motor and the time kept 

 down to these limits, for the sake not only of rapidity but of accuracy. 



Van Slyke's Method for Free Amino-Acid Nitrogen. To 25 c.c. of urine 1 

 in a 50 c.c. flask add urease solution and allow to stand for one and one-half 

 times the interval which has been found necessary to effect the maximum de- 

 composition of urea, as observed by titration of the ammonia. The last traces 

 of urea are decomposed. At the end of the digestion period 10 c.c. of a 10 per 

 cent suspension of calcium hydroxide are added, the mixture shaken and made 

 up to 50 c.c. Then filter, evaporate, and complete the determination according 

 to the method outlined under total amino-acid nitrogen, above. 



Creatinine 



Folin's Colorimetric Method. Principle. This method is based 

 upon the characteristic property possessed by creatinine, of yielding a 

 certain definite color-reaction in the presence of picric acid in alkaline 

 solution. 



Procedure. Place 10 c.c of urine in a 500 c.c. volumetric flask, add 15 cc. of a 

 saturated solution of picric acid and 5 c.c. of a 10 per cent solution of sodium hy- 

 droxide, shake thoroughly and allow the mixture to stand for five minutes. During 

 this interval pour a little N/2 potassium bichromate solution 2 into each of the two 

 cylinders of the colorimeter (Duboscq's, see Fig. 153, p. 486) and carefully adjust 

 the depth of the solution hi one of the cylinders to the 8 mm. mark. A few prelimi- 

 nary colorimetric readings may now be made with the solution in the other cylinder, 

 in order to insure greater accuracy hi the subsequent examination of the solution of 

 unknown strength. Obviously the two solutions of potassium bichromate are 

 identical hi color and in their examination no two readings should differ more than 

 0.1-0.2 mm. from the true value (8 mm.). Four or more readings should be made 

 in each case and an average taken of all of them exclusive of the first reading, which 

 is apt to be less accurate than the succeeding readings. In time as one becomes 

 proficient hi the technic it is perfectly safe to take the average of the first two 

 readings. 



At the end of the five-minute interval already mentioned, the contents of the 

 500 c.c. flask are diluted to the 500 c.c. mark, the bichromate solution is thoroughly 

 rinsed out of one of the cyclinders and replaced with the solution thus prepared and 

 a number of colorimetric readings are immediately made. 



1 See note 5, ^ page 505. 



2 This solution contains 24.55 grams of potassium bichromate to the liter A pure 

 creatinine standard is to be preferred, see p. 507. 



