L'l'4. SPECTROPHOTO^IETRY. 



amount of iron, existing as oxide of iron, in this residue. 1 b. Since 

 tlu' volume of oxygen which unites with a given quantit}- of ha-ino- 

 globin is known with considerable accuracy (but see above, p. 2l'l), 

 the amount of this substance may be determined by saturating its 

 solution with oxygen and then estimating the volume of the gas 

 which is united to the hiemoglobin. The determination is made 

 either by extracting the oxygen with a mercurial pump or displacing 

 it by carbon-monoxide, or estimating it in the solution by a volu- 

 metric process with sodium sulphite and indigo. 2 These methods are 

 inferior to the following. 



2. Physical. These may be again divided into two : colorimetric 

 and spectrophotometric. 



(i) Colorimetric method. The principle of this method may be 

 briefly stated as follows. A standard solution of haemoglobin is pre- 

 pared from pure crystals of the substance. The tint of the solution 

 in which the haemoglobin is to be determined is then compared with 

 that of the standard solution: if it is not the same when examined 

 under the same conditions, it must be equalised by either of the 

 methods to be next described; and from the operations necessary to 

 produce equality of tint the relative concentrations of the two solu- 

 tions may be inferred, and hence the absolute concentration of the 

 unknown solution. The methods more usually employed consist 

 either in diluting one or the other of the solutions until their tint 

 is the same when examined in layers of equal thickness (Hoppe- 

 Seyler), 3 or else in determining the different thicknesses of the fluid 

 layer of each which exhibits the same tint (Duboscq). Since in the 

 latter case the concentrations of the two solutions are inversely pro- 

 portional to the thicknesses of their layers when their tint is the 

 same, the amount of haemoglobin in the solution of unknown strength 

 can be at once inferred. 4 For clinical purposes Gower's haemo- 

 globinometer is perhaps most frequently employed. In this instru- 

 ment a measured volume of blood is diluted till it has the same tint 

 as that of a standard mass of gelatin coloured with carmine and 

 picrocarmine. 5 There are, however, many other forms of colorimeter 

 designed for clinical use. 



(ii) Spectrophotometric method. All coloured substances in solu- 

 tion possess the power of absorbing light. With a given thickness 

 of a given substance the amount of light transmitted by the solution 

 bears to the incident light a ratio which, while it varies for different 

 parts of the spectrum, is constant for any one portion, and is there- 

 fore characteristic of each substance. Hence if the absolute absorb- 



1 Pelouze, Compt. Rend. T. i. (1865), p. 880. 



* Grehant, Compt. Rend. T. LXXV. (1872), p. 495. Quiuquaud, Ibid. T. LXXVI. 

 p. 1489. Schiitzenberger et Risler, Ibid. p. 440. 



3 Hdbch. d. physiol. patkol.-chem. Anal. Aufl. 5 (1883), S. 435 



4 For a description of Duboscq's and other apparatus see G. u. H. Kniss, 

 Kolorimetrie u. quant. Spektralanal. 1891, S. 7 et seq. This work gives a most 

 excellent account of the best physical methods employed for the determination of 

 colouring substances in solution" A useful review of methods up to date (1882) is 

 given by Lambling, " Des proce'des de dosage de I'lie'raoglobine," Nancy, 1882. Of. 

 later, E. von Fleischl, Med.Jahrb. 1885, S. 425. Malassez, Arch. d. Physiol. 1886, 

 p. 257. 



5 For details see Gatngee, Physiol. Chem. Vol. i. p. 184. 



