122 Hemoglobin Determination 
obtained from glass blowers require recalibration before use. It 
has been found that water may be used for this calibration, as 
pipettes which have been calibrated with both mercury and water 
check sufficiently well. Theadvantage of having the pipette cali- 
brated to contain 0.10 cc. as well as 0.05 cc. is obvious. In bloods 
with a low hemoglobin content, 0.05 cc. may not be sufficient to 
give the color necessary for accurate color comparison in the | 
colorimeter. A pipette of this type, and used in the manner 
described, is capable of measuring 0.05 cc. of blood with an 
accuracy of 0.2 per cent. 
Ammonia Solution—Ammonia solutions, containing 4 cc. of 
strong ammonia in 1 liter of water, suggested by Krogh (5) are 
used, because the full color of the carbon monoxide hemoglobin 
develops at once. 
Saturation with Carbon Monoxide.—Ordinary illuminating gas 
as a source of carbon monoxide has proven entirely satisfactory. 
It was thought that there might be substances other than carbon 
monoxide in the gas which might form hemoglobin compounds and 
interfere with the determination. Accordingly, pure carbon 
monomide was prepared by heating oxalic acid with concentrated 
sulfuric acid and passing the gas produced through sodium hy- 
droxide to free it from carbon dioxide. As far as could be de- 
termined on comparison of the two solutions in the colorimeter 
the colors were identical. Oxyhemoglobin solutions are very 
unstable. Hence it is necessary, after transferring the blood to 
the ammonia solution, to saturate with carbon monoxide within 
an hour. After saturation with carbon monoxide, the solution 
may, on carefully stoppering and protecting from light, be placed 
in the ice box and the determination made at leisure. Saturation 
of the blood should be carried out under a hood. If the labora- 
tory does not possess a hood, the saturation may be accomplished 
under a funnel, attached to a small water vacuum pump, to 
remove the gas. 
Standard Hemoglobin Solution.—Haldane’s standard of a 1 per 
cent solution of a blood having an oxygen capacity of 18.5 per 
cent is used. It has been shown by Haldane and Smith (7), 
Butterfield (8), Barcroft (9), and others, that the oxygen capacity 
of the blood depends upon its hemoglobin content. A blood of 
18.5 per cent oxygen capacity contains approximately 14 gm. 
