SPECTROPHOTOMETRIC, ETC. METHODS OF ESTIMATION 299 



methods have been recommended by many other workers {206,376, 

 383M2;1213, p. 73; 1316,1517,2223,2]^25,2706,2728). 



How large a proportion of hem('globin and sulfhemoglobin are included in 

 this estimation depends on the wavelength chosen for tlie analysis. At 520 m/i 

 these two abnormal blood pigments have the same extinction coefficient as 

 oxyhemoglobin {20l>). Calculation from their absorption curves shows that 

 at 540 ra^i 100% of the oxyhemoglobin. 50% of hem/globin, and 65% of 

 sulfhemoglobin would be measured, and at 576 m/x 100% of oxyhemoglobin, 

 33% of hem/globin, and oo^c of sulfhemoglobin. 



The method is readily adaptable to use with photoelectric equipment. 

 Provided filters are available with suitable transmission bands, any of the 

 better quality commercial photoelectric colorimeters, or even simple ones 

 of the Evelyn type, can with care be made to give satisfactory results. 

 However, no permanent glass filter with maximum transmission in a narrow 

 band corresponding to the a band of hemoglobin has yet been produced, most 

 of those available having a rather broad transmission band in the 5'-20 to 

 540 m/i region. Calibration is therefore on an empirical basis, and not 

 necessarily linear. A decided improvement would be introduced by the 

 production of a simple photoelectric spectrophotometer, using a fixed mono- 

 chromator transmitting a 10 to 15 mju band of center 576 my.; this would give 

 greater specificity and sensitivity for oxyhemoglobin. As an alternative, a 

 Hquid filter transmitting in this region may be used with existing instruments; 

 such a filter is obtained with saturated solutions of copper chloride in alcohol 

 and cobaltous sulfate in water in separate 5-mm. vessels (393). 



The sum of oxyhemoglobin and heni/globin can be measured 

 spectrophotometrically as hemiglobin cyanide (138,726,1947). Since 

 the absorption curve of the compound does not depend on pH this is 

 preferable to measuring the absorption of hemjglobin itself. 



The first absorption band of pyridine hemochrome is so sharp that 

 it lends itself well to the measurement of spectrophotocolorimetry, 

 with pyridine hemochrome prepared from crystalline hemin as a 

 standard. The method has been applied repeatedly for hematin 

 estimation and has been recommended for hemoglobinometry by 

 Rimington (2267). 



It measures the sum of all hemoglobin derivatives, except choleglobin. It 

 is simple, but not entirely reliable. Autoxidation of the dithionite produces 

 hydrogen peroxide with consequent danger of oxidation of the hemochrome. 

 This danger is avoided in the method of Lemberg and co-workers (1710) by 

 saturation of the solution with carbon monoxide before conversion into 

 denatured globin carbon monoxide hemochrome with dithionite and sodium 

 hydroxide. This has been found more reliable than the transformation into 

 pyridine hemochrome. The method measures the sum of all hemoglobin 

 derivatives, including sulfhemoglobin, but not choleglobin. It can also be 

 applied to measure the latter, by reading at 630 m/x. The intensity of the 



