2l8 
HAEMOGLOBIN. 
coloured liquid 1 cm. broader than that which is contained in the lower half 
of the trough, and this is for spectrophotometry 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. 
Sjiectrophotometric measurements are invariably made by the aid of artificial 
light. Hitherto, oil or petroleum lamps have been used for this purpose, but 
lately llufner lias adopted a gas lamp fitted with an Auer incandescent burner. 
We are now in a position to complete our explanation of Vierordt'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 equal 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 oxyhaemoglobin, and of C< (-haemoglobin, lie will 
select for his observations one of the two regions which have been shown by 
Httfner to be specially favourable to the determination, and in which he has 
determined the constants which lie distinguishes as A,, and A'„ respectively. 
Fig. 30. — A spectrophotometer with absorption trough and lamp as 
arranged for spectropbotometric 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. 
mid quantitative Spektralanalyse, etc.," Hamburg u. Leipzig, 1891. Though specially 
written fur those who intend to work with Hufner's instrument, an accurate though very 
succinct account of spectrophotometry is contained in a pamphlet entitled "Anleitung 
brauche des Hiifner'schen Spectrophotometers, etc.," von Eugen Albrccht, Univer- 
sitats-Mechaniker in Tubingen : Tubingen, 1 S92. Subsequently, all Hufner's papers on 
spectrophotometry should be studied. 
