.478 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



THE REACTIONS OF HEMOGLOBIN IN DEFIBRIN- 



ATED BLOOD. 



SPECTROSCOPIC EXAMINATION OF HEMOGLOBIN 

 AND ITS DERIVATIVES. 



Haemoglobin and its derivatives show typical absorption bands 

 when their solutions are examined with a spectroscope. The forma- 

 tion of the derivatives is thus readily observed and they are thus easily 

 identified. 



Construction of a Spectroscope. 



A spectroscope consists essentially of 3 parts, a collimator or tube furnished 

 with a slit at one end and a convex lens at the other so as to produce a beam 

 of parallel rays, a prism and a telescope to focus the light for observation. 

 There is usually also a scale of wave lengths attached to the telescope so as 

 to be able to note the position of any absorption bands. The beam of white 

 light of parallel rays which reaches the prism from the collimator is resolved by 

 the unequal refraction of its constituents at each bent surface into a band of 

 light of several colours. This band of light is termed a spectrum, the colours 

 being red, orange, yellow, green, blue, violet and indigo. 



A spectrum obtained from a beam of sunlight shows a series of vertical 

 dark lines the Fraunhofer's lines. Thus in the red region three lines are 

 seen ; they are known as the A, B, C lines ; in the yellow region one line, 

 the D line ; in the green three, the E, , F lines ; in the violet two, the G and 

 H lines. These dark lines are due to the absorption of light in these regions 

 of the spectrum by the passage of the light through certain volatile substances 

 in the sun's atmosphere. The D line is due to sodium vapour as can be 

 shown by passing ordinary light through sodium vapour; a line or rather 

 two lines very close together, are seen in the D position. The other lines are 

 due to the vapours of other elements. The colour of light after passing through 

 a coloured liquid is the colour which is not absorbed by the liquid. 



When light is passed through a solution of haemoglobin it is absorbed in 

 certain regions. These regions become visible when the light is passed through 

 a prism. They are the absorption bands. 



In an ordinary spectroscope the field of vision is at an angle to the 

 original source of light, but for convenience a direct vision spectroscope is used. 

 Such a spectroscope consists of a series of prisms of crown and flint glass. 

 The light which enters is refracted at each prism and finally emerges in the 

 same direction as the original beam of light. 



An absorption band has a certain width and a point of maximum absorp- 

 tion which does not alter with different concentrations of the solution ; this 

 point of maximum absorption is determined in very accurate work and noted 

 on the line of wave lengths. The width of the band alters with different con- 

 centrations and is therefore not registered. For ordinary work the absorption 

 bands visible to the eye only are noted, but for detailed work the absorption 

 bands in the ultra-violet region, which are not visible to the eye, are also 

 determined by photographing on special plates. 



The Absorption Spectra of Haemoglobin and its Derivatives. 



A solution of I volume of defibrinated blood, or blood, is gradually 

 diluted with 30 volumes of water and examined with a spectroscope. 

 The concentrated solution appears dark red to the naked eye, but 

 light will be found to pass through the red region of the spectrum 

 between the Fraunhofer's lines C and D. On diluting, light will 

 pass through the green region and between the red and green 

 regions a dark absorption band will be seen. On diluting further, 



