218 



HAEMOGLOBIN. 



coloured liquid 1 cm. broader than that which is contained in the lower half 

 of the trough, and this is for spectrophotometric purposes exactly equivalent 

 to interposing a stratum 1 cm. broad in the path of the light impinging on one 

 (the upper) half of the slit, and no coloured liquid in the path of the light 

 reaching the other half. 



Spectrophotometric measurements are invariably made by the aid of artificial 

 light. Hitherto, oil or petroleum lamps have been used for this purpose, but 

 lately Hufner has adopted a gas lamp fitted with an Auer incandescent burner. 



We are now in a position to complete our explanation of Yierordt's 

 method. We shall assume that a spectrophotometer, such as has been 

 described, is at the disposal of the observer. The lamp is lighted and the 

 height of the flame adjusted, so as to equally illuminate the two halves of the 

 double slit ; this is seen to be the case when with equal widths of the slits 

 two superposed spectra of exactly equffl brightness are seen. The two 

 halves of the slit are then opened to the extent which is thought advisable ; 

 we shall, for convenience of description, suppose that they have been opened 

 to the extent represented by the index on the two divided circles of the 

 micrometer screws, pointing to the division 100. The observer then arranges 

 the slit in the eyepiece, so as to isolate and measure precisely the region of 

 the spectrum for which he desires to determine the coefficient of extinction. 

 In the case of haemoglobin, of oxyhsemoglobin, and of CO-hsemoglobin, he will 

 select for his observations one of the two regions which have been shown by 

 Hiifner to be specially favourable to the determination, and in which he has 

 determined the constants which he distinguishes as A and A' respectively. 



FIG. 30. A spectrophotometer with absorption trough and lamp as 

 arranged for spectrophotometric determinations by Vierordt's 

 method. 



This operation having been effected, he will again observe whether the two 

 limited spectral areas appear to be of precisely equal brightness. If this is 

 the case, the trough containing the coloured liquid is brought in front of the 

 double slit, and the height of the former is carefully adjusted, so that the 

 upper border of the glass cube appears as a line exactly coinciding with the 

 separation between the upper and the lower spectral strips. 



indispensable, book by Dr. Gerhard Kriiss and Dr. Hugo Kriiss, entitled " Kolorimetric 

 und quantitative Spektralanalyse, etc.," Hamburg u. Leipzig, 1891. Though specially 

 written for those who intend to work with Hiifner's instrument, an accurate though very 

 succinct account of spectrophotometry is contained in a pamphlet entitled " Anleitung 

 zum Gebrauche des Hiifner'schen Spectrophotometers, etc.," von Eugen Albreclit, Univer- 

 sitats-Mechaniker in Tiibingen : Tubingen, 1892. Subsequently, all Hiifner's papers on 

 spectrophotometry should be studied. 



