SPECTROPHOTOMETRIC DATA. 235 



of light (as determined by the extinction-coefficient) in a definite spectral 

 region, exerted by a mixture of two or more colouring matters, is the sum of 

 the extinction-coefficients of each of its coloured constituents; and that in 

 the case of a solution containing two colouring matters, if we are acquainted 

 with the optical constants of each in two and the same spectral regions, we are 

 able by the spectrophotometer to determine the relative and absolute amount 

 of each constituent. In a similar manner we should, according to theory, 

 be able to determine the amounts of three or of x colouring matters coexisting 

 in a solution, if we were acquainted with the value of A in three and the same, 

 or in x and the same spectral regions. The immense importance of a method 

 which permits of the accurate determination of oxy- and reduced haemoglobin 

 in blood, and which furnishes us with essential data for calculating the 

 amount of oxygen present in combination with haemoglobin, makes it 

 necessary that we should explain the nature of the very simple calculations 

 which enable us, from the determination of the extinction-coefficients in two 

 spectral regions, to effect a determination which, so far as I know, cannot 

 be carried out with any pretence to scientific accuracy, or even with any claim 

 to be presumably correct, by any other process whatsoever. 



We shall assume that, by following methods which we shall not attempt 

 to describe, but for which the reader is referred to Hiifner's original papers, 

 blood has been diluted with O'l per cent, of aqueous solution of NaOH, 

 under conditions which preclude the possibility of contact with oxygen, and 

 that in the diluted blood solution the extinction-coefficients have been deter- 

 mined in the first and in the second regions selected by Hiifner. These 

 extinction- coefficients of a mixture of two colouring matters, we shall represent 

 by E and E.' 



Let A r be the absorption relation of (reduced) haemoglobin in the first 



region (A 554 - A 556). 

 A' r that of the same body in the second spectral region (A 531*5 - 



A 542-5). 

 A the absorption relation of oxyhsemoglobin in the first spectral 



region. 

 A' that of the same body in the second spectral region. 



Then the percentage of (reduced) haemoglobin, which we may designate x, will 

 be found by the equation 



x __A r A' r (E'A' -EA ) 



A A r A A r 



and the percentage of oxyhaemoglobin by the following equation 



_A A' (EA r -E'A' r ) 



y A' A 44' 



j A r <a. A r 



Having thus determined by spectrophotometry the amount of oxyhaemoglobin 

 by weight existing in a known volume, say 100 c.c. of blood, we can ascertain 

 the volume of the respiratory oxygen measured at C. and 760 mm. pressure 

 (which could, but probably with less accuracy, be likewise determined with the 

 aid of the mercurial pump and subsequent analyses of the gases boiled out of the 

 blood) by multiplying each gramme of oxyhsemoglobin found by 1-338 (or 1'34). 

 In this manner Hiifner, having determined the relative and absolute amounts of 

 haemoglobin and oxyhaemoglobin in the blood, drawn simultaneously from the 

 main artery and vein of a limb, ascertained the amount of oxygen in each. 

 There is a strong presumption that determinations of oxygen made in this manner 

 are nearer the truth than those which the more complex and laborious methods 

 by means of the mercurial pump and gas analysis are capable of giving. In the 

 process of raising the blood to a temperature of at least 40 C. in the exhausted 



