The Effect of Varying Concentrations of Oxyhemoglobin on Its Light Absorption

The validity of any method for determining the concentration of a colored substance by measuring its absorption of monochromatic light depends upon the ability of that substance to obey certain physical laws. In our discussion of the spectrophotometric determination of oxyhemoglobin and certain of its derivatives (Ray, Blair, and Thomas, 1932) it was assumed that these substances obeyed such fundamental laws, since the relationships required for the determination had never been questioned, although the method had been in use for over 40 years. The particular laws essential in this determination are the laws of Lambert and of Beer. Lambert’s law states that light is absorbed by a homogeneous medium in such a way that absorption is directly proportional to the intensity of the light at any point un$er consideration. Thus,