74 



drop of Canada balsam and cover. Tetrahedral crystals of oxy- 

 hsemoglobin will form after a time. The slide may be kept. 



(2) Spectroscopic Examination of Haemoglobin and its Derivatives. 

 (a) With a small, direct-vision spectroscope look at a bright part of 

 the sky or a white cloud. Focus by pulling out or pushing in the eye- 

 piece until the numerous fine dark lines (Fraunhofer's lines), running 

 vertically across the spectrum, are seen. Narrow the slit by moving 

 the milled edge till the lines are as sharp as they can be made. Note 

 especially the line D in the orange, the lines E and 6 in the green, 

 and F in the blue. Always hold the spectroscope so that the red is 

 at the left of the field. Now dip an iron or platinum wire with a 

 loop on the end of it into water, and then into some common salt or 

 sodium carbonate, and fasten or hold it in the flame of a fishtail burner. 

 On examining the flame with the spectroscope, a bright yellow line 

 will be seen occupying the position of the dark line D in the solar 

 spectrum. This is a convenient line of reference in the spectrum, and 

 in studying the spectra of haemoglobin and its derivatives, the position 

 of the absorption bands with regard to the D line should always be 

 noted. The dark lines in the solar spectrum are due to the absorption 

 of light of a definite range of wave-lengths by metals in a state of vapour 

 in the sun's atmosphere, and of course no dark lines are seen in the 

 spectrum of a gas-flame. Put some defibrinated blood into a test-tube. 



Fig. 20. Direct Vision Spectroscope ot Simple Type. A , slot in which a pin on the 

 eyepiece C slides in focussing the spectrum; B, milled head, by the rotation 

 of which the slit is narrowed or widened. 



Fasten it vertically in a clamp in front of the flame and examine it 

 with the spectroscope, holding the latter in one hand with the slit close 

 to the test-tube, and focussing the eyepiece with the other. Or arrange 

 the spectroscope, test-tube and gas-flame on a stand as in Fig. 21. 

 Nothing can be seen till the blood is diluted. Pour a little of the blood 

 into another test-tube, and go on diluting till, on focussing, two bands of 

 oxyhesmoglobin are seen in the position indicated in Fig. 13, p. 51 . Draw 

 the spectrum; then dilute still more, and observe which of the bands 

 first disappears. Now put 5 c.c. of the blood into another test-tube, 

 and dilute it with four times its volume of water. Take 5 c.c. of this 

 dilution, and again add four times as much water, and so on till the 

 solution is only faintly coloured. Note with what degree of dilution 

 the bands disappear. Then examine each of the solutions with the 

 spectroscope and draw its spectrum. 



(b) Make a solution of blood which shows the oxyhaemoglobin bands 

 sharply. Add some ammonium sulphide solution to reduce the oxy- 

 haemoglobin. Heat gently to about body temperature. A single, 

 ill-defined band now appears, occupying a position midway between 

 the oxyhEemoglobin bands, and the latter disappear. This is the 

 band of reduced hemoglobin (Fig. 13). 



(c) Carbonic Oxide Hemoglobin. Pass coal-gas through blood for 



