220 HEMOGLOBIN. 
efficient by the long-continued labours of its author, differs from Yierordt's in 
the mode by which the equalisation of the intensity of two beams of light is 
brought about, the difference in mode requiring a spectrophotometer which 
differs in important respects from the instrument already described. 
In Hiifner's spectrophotometer there is a siwjle slit, the width of which, 
after it lias been once adjusted, is never varied. 
The light which reaches one-half of this slit has been polarised by a small 
Nichol's prism (the polariser), whilst that which reaches the other half (which 
in the determination of the value of e passes through the thicker stratum of 
coloured liquid) is unpolarised. "When these two beams of light fall upon the 
refracting prism of the spectroscope, they are refracted and furnish two super- 
posed spectra, of which that corresponding to the polarised beam is naturally 
much less intense than the other. Before making any observations of e, the 
two spectra must be equalised, this being done by interposing a wedge of 
smoke-tinted glass in the path of the unpolarised beam. Equality of both 
spectra having been obtained, if a coloured medium be placed in the path of 
the unpolarised beam, its spectrum will be correspondingly reduced. Equality 
is, however, restored b} T rotating a second Xichol's prism (the analyser) which 
is in the path of the beams issuing from the refracting prism, and the rotation 
of which diminishes the intensity of the polarised beam alone. When equality 
in the illumination of both spectra has been restored, the angle (<£), through 
which the analysing Nichol has been rotated, is measured in two opposed 
quadrants of a divided circle provided with a vernier, and from the value of 
(f> that of /' is calculated. 
If the original intensity of the light =1, and the intensity of the un- 
absorbed light which has traversed the coloured medium be represented by 
I', then 
/' = cos' 2 <£ ; 
If the layer of coloured liquid investigated lie always = 1 (e.g. 1 cm.), then 
as e = - log /', 
e = - log COS 2 </» 
The following example will illustrate the mode of procedure and the steps 
of the calculation in an actual experiment for the determination of the ex- 
tinction-coefficient of blood, carried out with Hiifner's spectrophotometer: — 
1 c.c. of defibrinated blood of the ox was diluted to 160 c.c. with a 0*1 
per cent, aqueous solution of Xa(OH). The absorption-trough was filled with 
some of the perfectly clear red liquid thus obtained. The spectral region (?*), 
for which e was determined, was one of the two in which Hiifner has, in his 
most recent experiments, determined the constant A of oxyhsemoglobin (i.e. 
a portion of the region between the bands a and /3 of oxyhemoglobin). 
r = A557-5-X568-7 
(.Mean of ten measurements) <j> =61 -. s 7 
Converting the decimal 
fractions of a degree into 
seconds ^ = 61 o2' 
It has been stated that with Hiifner's spectrophotometer 
/' = cos 2 </> 
and e = - log cos-<£ 
In the above experiment 
e— - log eos 2 61°52' 
"= — 2 log cos61°52' 
"= -2 (0-67350-1) 
"= -1-34700 + 2 
" = 0-653 
