226 SPECTROPHOTOMETRY. 



perhaps most frequently employed. In this instrument a measured volume of 

 blood is diluted till it has the same tint as that of a standard mass of gelatin 

 coloured with carmine and picrocarmine l . There are however many other forms of 

 colorimeter designed for clinical use. 



(ii) Spectrophotometric method. All coloured substances in solution possess 

 the power of absorbing light. With a given thickness of a given substance the 

 amount of light transmitted by the solution bears to the incident light a ratio, 

 which, while it varies for different parts of the spectrum is constant for any one 

 portion, and is therefore characteristic of each substance. Hence if the absolute 

 absorbing power of a given thickness of the substance is determined once for all 

 for a given region of the spectrum under given conditions, it becomes possible to 

 determine the amount of that substance in any solution of unknown concentration 

 by examining the solution under the same conditions in the same part of the spectrum 

 and ascertaining how much light it has absorbed. Let I be the intensity of the inci- 

 dent light and I' its reduced intensity after passing through m layers of a coloured 



solution each of which reduces the initial intensity by - , then it follows that 



n 



This is true whatever be the intensity of the incident ray ; hence this intensity may 

 be taken = 1 and we have J'= . 



Again, let E denote the reciprocal of the number which represents in centimeters 

 that thickness of layer of the absorbing solution which reduces the intensity of the 

 incident ray to T V of its initial intensity during its passage through this layer 2 . 

 Then if the solution be examined in a layer which is always 1 cm. thick, this 



layer may be regarded as made up of E layers each of thickness cm. Hence if in 



hi 



the formula previously given we put n=10 and m=E, we find that the residual 

 intensity I' of light after passing through a layer 1 cm. thick is 



whence E = - log I'. 



It can also be proved that E, the coefficient of extinction, is directly proportional 

 to the amount of colouring matter present in the solution, or in other words to its 

 ' concentration ' 3 , whence if the concentration be represented by C, 



Q 



= some constant 4 A, or C = AE. 



Mi 



This constant A having been determined once for all for a given substance in a 

 solution of known concentration and for a given region of the spectrum, the 

 concentration of any solution of the same substance of unknown strength is 

 obtained by simply multiplying A by the coefficient of extinction E. 5 



1 For details see Gamgee, Physiol. Chem. Vol. i. p. 184. 



2 is called 'coefficient of extinction,' a term introduced by Bunsen and Roscoe, 

 Pogg. Annal Bd. ci. (1857), S. 235. 



? The 'concentration' is the number of grams of colouring substance dissolved 

 in 1 c.c. of fluid (Vierordt). 



4 Called the ' absorption ratio ' by Vierordt. 



5 The introduction of the Spectrophotometric method in a reliable form is due 

 to Vierordt, based upon the photochemical researches of Bunsen and Eoscoe. See 

 Vierordt, (i) " Anwend. d. Spectralapparats zur Photometric d. Absorptions- spectren 

 u. z. quant, chem. Anal.," Tubingen, 1873, and (ii) "Die quant. Spectralanal. in 

 ihrer Anwend. auf. Physiol. u. s. w.," Tiibingen, 1876. 



