200 PRACTICAL PHYSIOLOGY 



it with about ten times its volume of water. Place some of this behind 

 the slit of the spectroscope, preferably in a flat-sided vessel about 1 cm. 

 thick, but a test-tube will answer fairly well. It will be noticed that 

 the whole of spectrum is blocked out except a portion of the red end. 



Dilute this solution carefully. At a certain stage some of the green 

 will be evident (see Spectrum 3 in Chart), there being a wide absorption 

 band between the red and green. On diluting still further, this wide 

 absorption band will resolve itself into two bands (Spectrum 2). These 

 two bands are both on the blue side of the D-line, and their centres 

 correspond to A 579 and X 543-8. Note carefully the position of these 

 centres on the scale and the width of the bands. Observe also the 

 limits of the visible spectrum at the red and blue ends. 



On diluting still further it may be possible to cause the band on the 

 blue side to disappear, whilst the band on red side is still just 

 appreciable (Spectrum 1). 



2. The visible Spectrum of Haemoglobin (reduced Haemoglobin). 

 If some diluted defibrinated blood be left standing undisturbed for 24 

 hours, the oxyhaemoglobin will lose its oxygen. This result may be 

 arrived at more rapidly by treating some diluted defibrinated blood 

 which shows fairly wide oxyhaemoglobin bands with a reducing 

 reagent, such as ammonium sulphide or Stokes' reducing fluid. 1 If 

 ammonium sulphide be used, the mixture should be warmed. It will 

 now be noticed that the blood loses its bright scarlet appearance and 

 becomes more purple in tint. Examine this by the spectroscope, 

 and it will be found that the two bands of oxyhaemoglobin have 

 disappeared, and are replaced by one band, the centre of which is 

 between the two bands of oxyhaemoglobin. The band is a broad one, 

 shading off" more gradually on the red side, and the darkest part corre- 

 sponds in wave-length to A 550 (Spectrum 4 in Chart). 



3. The visible Spectrum of Carbon-Monoxide Haemoglobin. If a 

 stream of carbon monoxide, or even of coal-gas, be passed through some 

 diluted defibrinated blood, the scarlet tint is changed to a carmine 

 or cherry colour. The oxygen is replaced by carbon monoxide. 

 Examined spectroscopically the blood shows two bands differing from 

 those of oxyhaemoglobin in being slightly shifted towards the blue 

 end. The two bands have centres corresponding in wave-length to 

 A 575 and A 540 approximately (Spectrum 5). 



The proportion of red and blue unabsorbed at the ends of the spectrum 

 is different in oxyhaemoglobin and CO-haemoglobin, there being more 



1 2 gms. of ferrous sulphate are dissolved with 3 gms. tartaric acid in 100 cc. of 

 water. Ammonia is added till the solution is alkaline. Stokes' fluid must be 

 freshly prepared. 



