288 PHYSIOLOGICAL CHEMISTRY 



to 12.5 c.c. or to 25 c.c. depending on the depth of color. 1 At any time within 

 a hatf an hour the colored solution is compared in a colorimeter with a suitable 

 standard solution, the standard being set at a height of 15 mm. 



The standard solution may be simultaneously prepared from pure glucose 

 by treating 0.64 mg. of glucose in 4 c.c. of water with 4 c.c. of the pier ate -picric 

 acid solution and i c.c. of the carbonate, and heating for 10 minutes in boiling 

 water and then diluting to 12.5 c.c. A permanent standard solution may be 

 prepared from picramic acid or from potassium dichromate as mentioned below. 2 

 The potassium dichromate standard does not match the unknown with absolute 

 exactness, but can be employed with satisfactory results when pure picramic 

 acid is not obtainable. 



Calculation. If directions are followed exactly the calculation is as follows : 



Reading of standard 



Reading of unknown + IO = per cent f Sugar m ** Ongmal blood ' 

 Where the final dilution of the unknown is made to 25 c.c. instead of 12.5 c.c. 

 the final figure is, of course, multiplied by two. 



(b) Micro -method of Bang. Principle. Two or 3 drops of 

 blood are transferred to a small weighed piece of blotting paper and the 

 paper again weighed to determine the amount of blood. The paper is 

 then treated with a boiling acidified KC1 solution which coagulates the 

 protein and allows the sugar to diffuse out. The sugar solution thus 

 obtained is boiled with alkaline cupric chloride solution. The amount 

 of cuprous chloride solution formed by the reducing action of the 

 sugar is determined by titration with standard iodine solution. 



Procedure. Small pieces of good absorbent paper, about 16X28 mm. in 

 size, 3 weighing about 100 mg. and held by a small spring clip, are used. To one 

 of these previously weighed 4 transfer 2-3 drops (about 120 mg.) of blood obtained 

 by piercing the cleansed ringer. Weigh again immediately and determine by 

 subtraction the weight of blood taken. 



1 Occasionally the final filtrates in this or other picric acid methods develop a little 

 turbidity during heating. Unless such turbidity is fairly marked it is of no account. 

 When desired, the final colored solution may be filtered through a small folded filter into 

 the colorimeter cup. 



2 Permanent Standard. The picramic acid standard is best prepared from a stock 

 solution containing 100 mg. of picramic acid and 200 mg. of sodium carbonate per liter. 

 One hundred twenty-six c.c. of this solution are treated with i c.c. of the 20 per cent 

 sodium carbonate solution and 15 c.c. of the picrate-picric acid solution, and diluted to 

 300 c.c. with distilled water. This solution matches exactly the color obtained by treat- 

 ing 0.64 mg. of glucose, as in the above method and diluting to 12.5 c.c. A satisfactory 

 preparation of picramic acid may be obtained from the J. T. Baker Chemical Co., 

 Phillipsburg, N. J. 



The standard prepared from potassium dichromate contains 800 mg. of pure potassium 

 dichromate in a liter of water. 



3 Suitable pieces of paper, weighed, ready for use, and with clip attached, may be ob- 

 tained from Warmbrunn and QuiUtz, Berlin. A suitable paper may also be obtained from 

 Griffin and Sons, London, or Grave of Stockholm. Unless specially prepared, the paper 

 should be repeatedly washed with large volumes of hot water acidified with acetic acid to 

 remove impurities. 



4 The weighing is preferably made on a special torsion micro-balance which, as well as 

 the other apparatus used in this method, may be obtained from either of the firms mentioned 

 in Note 3, above. The weighing must be made in a few seconds a*nd with an 

 accuracy of about i mg. 



