212 METHODS FOR THE ANALYSIS OF BLOOD 



of hemoglobin plus methemoglobin is determined colorimetrically 

 by means of a standard solution of cyanhemoglobin. The hemo- 

 globin in the blood containing the two pigments is next determined 

 gasometrically by Van Slyke's method (this Manual, page 124). 

 This value is subtracted from that obtained for hemoglobin plus 

 methemoglobin determined together as cyanhemoglobin; the 

 difference is the methemoglobin. 



Procedure. Oxalated whole blood is used. Two c.c. of the 

 blood are placed in a 100 c.c. flask and 50 c.c. of water are added 

 which effect hemolysis in a few seconds. 0.5 c.c. of a 0.1 M (3 

 per cent) solution of potassium ferricyanide is added, and the 

 flask allowed to stand for fifteen to twenty minutes. (It was 

 found that these conditions are optimum for the complete con- 

 version of the hemoglobin to methemoglobin, only the faintest 

 visible hemoglobin band being present at the end of twenty 

 minutes with this amount of potassium ferricyanide.) Five c.c. 

 of a 0.1 per cent potassium cyanide solution are now added. The 

 change to cyanhemoglobin is immediate. Water is added to the 

 mark and the solution compared with a standard of known strength 

 in a colorimeter. The result is the hemoglobin plus methemoglobin, 

 which is expressed as gm. of " total hemoglobin " per 100 c.c. of 

 blood. 



A small portion (4 to 5 c.c.) of the blood or hemoglobin solution 

 is aerated in a funnel and its total oxygen capacity determined by 

 the Van Slyke method. Barcroft l has shown that under these 

 conditions (180 mm. of oxygen tension, mm. of carbon dioxide 

 tension, and room temperature) the hemoglobin is practically 100 

 per cent saturated. Therefore the oxygen capacity corresponds 

 to the amount of hemoglobin present, and by dividing by 1.34 

 (the volume of oxygen combined with 1 gm. of hemoglobin) one 

 obtains the gms. of hemoglobin per 100 c.c. of blood. For the 

 convenience of calculation the factors for the conversion of c.c. 

 of gas combined with 2 c.c. of blood into gms. of hemoglobin per 

 100 c.c. of blood are given in the table (modified from Van Slyke). 



Preparation of Standard. The standard is prepared from 

 fresh whole oxalated or defibrinated blood which is known to con- 

 tain no methemoglobin. The hemoglobin content (gms. per 100 

 c.c.) is determined gasometrically. Five hundred c.c. of standard 

 are made by placing 10 c.c. of the blood in a 500 c.c. flask, hemo- 



' Barcroft, J., The Respiratory Function of the Blood, Cambridge, 1914. 



