SPECTROPHOTOMETRIC CONSTANTS. 223 



sources of information. It is absolutely essential to work with Hiifner's 

 spectrophotometer in a perfectly darkened room. 



Before commencing photometric measurements, the observer will ascertain 

 whether the analysing Mchol is in the position in which it allows the polarised 

 beam to pass unabsorbed. He will then fill the absorption-trough, and isolate 

 and measure the spectral region for which the extinction-coefficient is to 

 be determined. 



On now looking through the eyepiece two spectral strips will be seen, 

 separated by a sharp horizontal line ; these spectral strips will be of unequal 

 brightness ; the upper, being a portion of the spectrum of the polarised 

 beam, will be much less luminous than the lower. The composite glass 

 plate in front of the slit of the collimator is now moved inwards in the 

 direction of the beam of the unpolarised light, so as to diminish its intensity, 

 until the upper and the lower spectral strips appear of precisely the same 

 brightness. 



The trough containing the coloured liquid under investigation is now 

 brought into position, the upper surface of the glass cube in the trough being 

 placed about 1 mm. below the plane passing through two horizontal angles of 

 Albrecht's glass body. On now examining the spectra, it is at once seen that 

 the lower of the two is darker than the upper. The analysing Nichol is then 

 carefully rotated until equality in the intensity of the two spectral strips is 

 attained ; the angle through which the prism has been moved is then deter- 

 mined ; several, say five, sets of readings being made in two opposite quadrants 

 of the large divided circle. The mean of these readings gives the value 



Of C/). 1 



The spectrophotometric constants of oxyhsemoglobin. It was 



previously stated that it is usual to determine the photometric constants 

 of colouring matters in two spectral regions, those regions being chosen 

 in which the variations in the absorption of light are most rapidly affected 

 by variations in the concentration of the colouring matter. 



The reasons for determining in the first instance at least two values 

 for A (which we shall distinguish as A and A'), and subsequently, each 

 time that a determination is made, ascertaining the value of e in the 

 same two regions (the two extinction-coefficients being distinguished 

 as e and e', or in the case of oxyhaemoglobin as e and e ') are the 

 following: (1) If we know the value of A and A' for any body, we are 

 able to make two independent estimations when determining the 

 concentration of a solution of the same body of unknown strength, the 

 one estimate acting as a check on the other. (2) The knowledge of the 

 value of A and A', for each of two colouring matters co -existing in 

 solution, is a necessary condition to being able to determine spectro- 

 photometrically the amount of each constituent when occurring together. 

 (3) In the case of oxyhsemoglobin, haemoglobin, and CO-haemoglobin, the 



quotient - is absolutely characteristic of each substance, and affords a 



valuable check on the purity of the colouring matter in solution and on 

 the accuracy of the analysis. 



Hiifner's most recent determinations 2 of the spectrophotometric 

 constants of oxyhaainoglobin, made with his perfected spectrophotometer, 

 have led to the results shown below. The two values of A are, as has 



1 Hiifner's spectrophotometer is constructed by, and can be obtained from, the original 

 maker, Herr Eugen Albrecht, Universitats-Mechaniker in Tubingen. 



2 Hiifner, "Photometrische Constanten des Oxyhamoglobins,"^rc7t./. PhysioL, Leipzig, 

 1894, PhysioL Abth., S. 134 et seq. 



