246 PRACTICAL PHYSIOLOGY 



Adjustment of the Spectroscope. It is necessary to have an exact focus of 

 the image of the slit. In the small direct vision spectroscope this may be 

 obtained by directing the instrument towards a white cloud, and moving the 

 eye-piece till the various Fraunhof er lines are clearly denned, or, in absence of 

 daylight, obtaining a clear image of the upper and lower edges of the slit, 

 i.e. of the upper and lower edges of the spectrum. The slit should not be too 

 widely open. If the source of light include a sodium flame, a clear image of 

 the D-line will be obtained when the slit is in focus. 



1. The Visible Spectrum of Oxyhaemoglobin. Take some defibrin- 

 ated blood which has been thoroughly shaken with air, and dilute 

 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 the 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 



FIG. 179. Small direct vision spectroscope. 



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 A 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 the red side is still just 

 visible (Spectrum 1). 



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



If some diluted defibrinated blood be left standing undisturbed 

 for twenty-four hours, the oxyhsemoglobin will lose its oxygen. 

 This result may be arrived at more rapidly by treating some diluted 

 defibrinated blood which shows fairly wide oxyhsemoglobin 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 



1 Two grms. of ferrous sulphate are dissolved with 3 grms. tartaric acid in 

 100 c.c. of water. Ammonia is added till the solution is alkaline. Stokes' 

 fluid must be freshly prepared. 



