302 VI. HEMOGLOBIN 



iron analyses. There is little to recommend this analysis for a routine 

 estimation of hemoglobin. 



The density of blood can be readily measured' by the copper sulfate 

 method; while this method is useful for screening blood donors and the 

 discovery of anemia (326,1380), it cannot be used for the accurate estimation 

 of hemoglobin (cf. 2169). 



A determination of hemoglobin based on its peroxidative action on 

 benzidine has been worked out {176,1274,1815). This method is probably 

 more useful for measuring the small amounts of hemoglobin present in 

 plasma, urine, or feces than for estimation of blood hemoglobin. For the 

 former purpose, a photoelectric method measuring hemoglobin derivatives as 

 pyridine hemochrome with a filter with maximum transmission at 550 mix 

 has been developed by Flink and Watson {900). Their use of blanks in 

 which the hemochrome is destroyed by hydrogen peroxide can hardly be 

 recommended, nor does the modification of this method by Greenberg and 

 Erickson {10^9) appear to be adequate. The turbidity of the solutions or 

 extracts causes a difficulty which Lowry and Hastings {1782) have tried to 

 overcome by subtraction, assuming that absorption of light due to turbidity 

 increases linearly with decreasing wavelength; Colin {^63) measured changes 

 in the light absorption produced by the conversion of oxyhemoglobin 

 into hemoglobin or of hem/'globin into hem/globin cyanide. The latter 

 method presupposes that the degree of cloudiness is not altered by the 

 reagents. It is therefore preferable to use methods in which the solutions 

 are cleared before measurement, either by half saturation with ammonium 

 sulfate {1544) or by sodium hydroxide {cf. the carbon monoxide hemochrome 

 method of Lemberg and co-workers described above). 



9.6. Special Estimations 



Carboxyhemoglobin in mixture with oxyhemoglobin can be deter- 

 mined by measuring the positions of the a band in the Hartridge 

 reversion spectroscope (1143), or spectrophotometrically by measuring 

 the ratio ioiemu./ ^560miJ. {1213, p. 91), this ratio being 1.72 for oxyhemo- 

 globin and 0.875 for carboxyhemoglobin. Whether carboxyhemo- 

 globin is the only accompanying pigment can be checked by the 

 estimation of other absorption ratios such as €555 „,^/ 6568 mM ^^ the 

 reduced solution. 



For the estimation of hemiglobin in the presence of oxyhemoglobin, 

 Heilmeyer (1213, p. 103) uses the ratio €576 mu./ «59o m/x i^i 0.4% ammonia. 

 Since at 580 m^ the curve of oxyhemoglobin falls steeply, the ratio 

 *576 mt>./ ^600 m^ would probably be better. As an alternative the ratio 

 €575 m/oi/ ^560 niM after conversion of hemiglobin to hemtglobin cyanide 

 may be determined. 



Sulfhemoglobin and hemiglobin are measured spectrophotomet- 

 rically (726) by determining the total pigments as hemiglobin cyanide 



