590 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



The drops of blood are soaked up in weighed pieces of filter paper 1 

 12 x 25 mm. in size and i mm. thick weighing about 100 mgm. and weighed. 



The weighing is most conveniently effected with a torsion balance. 2 The 

 paper containing the blood as placed for 3-5 minutes in an oven at 90- 

 100 to coagulate the proteins. The glucose contained in the blood is 

 exiracted by immersing the paper in 7 c.c. of a solution containing 150 c.c. 

 of saturated potassium chloride solution, 70 c.c. of water and 2-3 drops of 40 per 

 cent, acetic acid for 45 minutes to i hour, during which time it is raised twice 

 to the boiling-point. The solution is poured into a 50 c.c. Jena glass flask 

 with a straight neck and no rim. The paper is again boiled out with 4 c.c. 

 of the above solution in the same way and this is put into the flask. 3 c.c. 

 of the alkaline copper solution (see p. 613) are added. The flask is closed with 

 a piece of rubber tubing 3 mm. thick and 4-5 cm. long leaving 2 cm. over, 

 which can be clamped with a spring clip or a special clip. 3 Dissolved gases are 

 removed by shaking and exhausting the flask with a suction pump. The con- 

 tents of the flask are heated to boiling for 3 minutes 4 ; just before the expiration 

 of this time the tubing is closed with the cap. The flask is rapidly cooLd 

 under running water. The rubber cap is removed, and to prevent oxidation 

 a current of carbon dioxide is immediately passed through the flask by a 

 small tube w,hich is bent for convenience and attached to the flask by a band. 

 The reduced cuprous salt is titrated with -oiN iodine solution or 'oc^N 

 iodine solution which is prepared from - iN iodine solution contained in a 

 2 c.c. burette with a fine point and graduated in fiftieths of a c.c. using 2-3 

 drops of starch solution as indicator. In all cases '01 per cent, should be 

 subtracted from the final result since blood contains a substance which reduces 

 iodine. 



Gardner and Maclean state thac at least four parallel determinations 

 should be carried out and the mean of 3 or 4 satisfactory estimations taken 

 as the correct figure. 



E. LACTIC ACID. 



In order to estimate lactic acid in blood and tissues it must first be separated 

 by extraction. The analysis of the lactic acid is then made either by con- 

 version into its zinc salt, which is weighed, or by oxidation to aldehyde, which 

 is titrated. Wolf 5 has tested the various methods and finds that the deter- 

 mination as zinc salt is the most accurate ; the determination as aldehyde 

 gives about 95 per cent, of the total. In extracting the lactic acid Wolf finds 

 that the best method is to absorb the concentrated solution containing the 

 lactic acid into filter paper, rather than with a special liquid extractor. His 

 procedure closely follows that of Embden and his co-workers : 



The blood or tissue is placed in 10 volumes of cold 2 per cent, hydrochloric 

 acid and, after standing, an equal volume of 5 per cent, mercuric chloride is 

 added. (Embden used 200 c.c. defibrinated blood, or blood collected in 

 sodium fluoride solution, 200 c.c. water, 400 c.c. HC1 and 400 c.c. HgCl 2 .) 

 The proteins are precipitated and after standing at least 12 hours, are removed 

 by filtration on a Buchner funnel, the precipitate being washed several times 

 with a solution of equal parts of the acid and mercuric chloride solution. The 

 colourless filtrate is treated with hydrogen sulphide, the sulphide precipitate is 

 washed and the sulphuretted hydrogen removed from the filtrate by a current 

 of air. The acid solution is evaporated in vacua to about 10 c.c. and the 



1 No. 2640. Made by Finbruken, Stockholm. It must be boiled out several times with 

 water containing a few drops of acetic acid until a test experiment shows that it contains 

 no reducing substances. 



' Bv Hartmann and Braun or Warmbrunn and Quilitz. 



'' Made by Mekaniker Hill, Lund, Denmark. 



4 1 minute 10 seconds to i minute 25 seconds generally elapse to raise to boiling-point, 

 after which the boiling is continued for 2 minutes, 



6 J Physiol., 1914.48, 34l- 



