302 THE BLOOD. 



sides containing a layer of liquid 1 cm. thick (HOPPE-SEYLER'S hsematinom- 

 eter). The use of HOPPE-SEYLER'S colorimetric double pipette is more 

 advantageous. Other good forms of apparatus have been constructed 

 by GIACOSA and ZAN GERMEISTER. 1 Instead of an oxyhaemoglobin solu- 

 tion we now generally use a carbon-monoxide haemoglobin solution as 

 a standard liquid because it may be kept for a long time. The blood 

 solution in this case is saturated with carbon monoxide. 2 



The quantitative estimation of the blood-coloring matters by means 

 of the spectroscope may be done in different ways, but at the present 

 time the spectrophotometric method is chiefly used, and this seems to be 

 the most reliable. This method is based on the fact that the extinction 

 coefficient of a colored liquid for a certain region of the spectrum is directly 

 proportional to the concentration, so that C : E C\ : E\, when (7 and 

 Ci represent the different concentrations and E and E\ the corresponding 



C C 



coefficients of extinction. From the equation =- Fr -: it follows that for 



hi JUii 



one and the same pigment this relation, which is called the absorption 

 ratio, must be constant. If the absorption ratio is represented by A, 

 the determined extinction coefficient by E, and the concentration (the 

 amount of coloring-matter in grams in 1 cc.) by C, then C = AE. 



Different forms of apparatus have been constructed (VIERORDT and 

 HUFNER 3 ) for the determination of the extinction coefficient, which is 

 equal to the negative logarithm of those rays of light which remain 

 after the passage of the light through a layer 1 cm. thick of an absorbing 

 liquid. In regard to this apparatus the reader is referred to other text- 

 books. 



For purposes of control the extinction coefficients are determined in two dif- 

 ferent regions of the spectrum. HUFNER has selected (a) the region between the 

 two absorption-bands of oxyhaemobglobin, especially between the wave-lengths 

 554 MM and 565 MM and (6) the region of the second band, especially the inter- 

 val between the wave-lengths 531.5 MM and 542.5 MM- The constants or the 

 absorption ratio for these two regions of the spectrum are designated by HUFNER 

 by A and A'. Before the determination the blood must be diluted with water, 

 and if the proportion of dilution of the blood be represented by V, then the con- 

 centration or the amount of coloring-matter in 100 parts of the undiluted blood 



C = 100. V. A. tfand 

 C = 100. V.A'.E'. 



The absorption ratio or the constants in the two above-mentioned regions 

 of the spectrum have been determined for oxy haemoglobin, haemoglobin, carbon- 

 monoxide haemoglobin, and methsemoglobin, as follows: 



Oxyhsemoglobin A =0.002070 and A' =0.001312 



Haemoglobin AT =0.001354 and A' r =0.001778 



Carbon-monoxide haemoglobin . .'A =0.001383 and A 'c =0.001263 



Methsemoglobin Am =0.002077 and A'm = 0.001754 



J F. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 16; G. Hoppe-Seyler, ibid., 21; 

 Winternitz, ibid.; Giacosa, Maly's Jahresber., 26; Zangermeister, Zeitschr. f. Biol- 

 ogie, 33. 



2 See Haldane, Journ. of Physiol., 26. 



3 See Vierordt. Die Anwendung des Spektralapparates zu Photometric, etc. (Tubin- 

 gen, 1873), and Hiifner. Arch. f. (Anat. u.) Physiol., 1894, and Zeitschr. f. physiol 

 Chem., 3, v. Noorden, ibid., 4; Otto, Pfliiger's Arch, 31 and 36. 



