280 PHYSIOLOGICAL CHEMISTRY 



flame and diluted to 10 c.c. as was the unknown, or against the picramic acid 

 standard mentioned below. 1 



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



reading of standard 

 Milligrams glucose in unknown = readin O f unknown milll g rams of glucose 



in standard. 



(b) Pearce's Modification of Lewis-Benedict Method. 2 This modification 

 entails the use of an autoclave instead of the free flame and has the advantages 

 of decreasing danger of loss and making it possible to carry out a large number 

 of estimations at one time. Proceed exactly as in the Lewis-Benedict, but use 

 6 c.c. of the the picric acid filtrate instead of 8 c.c. and instead of heating over the 

 free flame introduce into an autoclave for 15-30 minutes at about 20 pounds 

 pressure to the square inch. Compare with standard in a colorimeter. The 

 standard recommended by Lewis and Benedict may be diluted one-fourth or 

 allowed for by calculation, since 6 c.c. of filtrate are used in place of 8 c.c. 



(c) 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 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 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 finger. Weigh again immediately and determine by 

 subtraction the weight of blood taken. 



1 Permanent Standard. A solution of picramic acid makes a very satisfactory permanent 

 standard. The color is identical in quality with that formed in the method above and its 

 solution keeps perfectly. The formula of the permanent standard is: 



Picramic acid o . 064 gram 



Sodium carbonate (anhydrous) o. 100 gram 



Water to make 1000.0 c.c. 



Dissolve the picramic acid with the aid of heat in 25 to 50 c.c. of distilled water which 

 has been made alkaUne with sodium carbonate. Cool and dilute to i liter. This solu- 

 tion has the same intensity of color as that obtained^ by the proposed method with 0.64 

 mg. of sugar when the final volume of the reaction fluid is made 10 c.c. The solution should 

 be standardized against pure glucose. 



A satisfactory preparation of picramic acid may be obtained from the J. T. Baker 

 Chemical Co., Phillipsburg, N. J. 



2 Pearce: Jour. Biol. Chem., 22, 525, 1915. 



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

 tained from Warmbrunn and Quilitz, 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. The weighing must be made in a few seconds and with an accuracy of about 

 i mg. 



