298 PHYSIOLOGICAL CHEMISTRY 



ox-blood. Blood thus treated assumes a brighter tint (carmine) than that im- 

 parted by oxyhemoglobin. In very dilute solution oxyhemoglobin appears 

 yellowish red whereas carbon monoxide hemoglobin under the same conditions 

 appears bluish red. Examine the carbon monoxide hemoglobin solution spec- 

 troscopically. Observe that the spectrum of this body resembles the spectrum 

 of oxyhemoglobin in showing two absorption bands between D and E. The 

 bands of carbon monoxide hemoglobin, however, are somewhat nearer the violet 

 end of the spectrum. Add some Stokes' reagent to the solution and again ex- 

 amine spectroscopically. Note that the position and intensity of the absorption 

 bands remain unaltered. 



The following is a delicate chemical test 1 for the detection of carbon monoxide 

 hemoglobin : 



Tannin Test. Divide the blood to be tested into two portions and dilute each 

 with 4 volumes of distilled water. Place the diluted blood mixtures in two small 

 flasks or large test-tubes and add 20 drops of a 10 per cent solution of potassium 

 ferricyanide. 2 Allow both solutions to stand for a few minutes, then stopper the 

 vessels and shake one vigorously for 10-15 minutes, occasionally removing the 

 stopper to permit air to enter the vessel. 3 Add 5-10 drops of ammonium sulphide 

 (yellow) and 10 c.c. of a 10 per cent solution of tannin to each flask. The contents 

 of the shaken flask will soon exhibit the formation of a dirty olive-green precipitate, 

 whereas the flask which was not shaken and which, therefore, still contains car- 

 bon monoxide hemoglobin, will exhibit a bright red precipitate, characteristic of 

 carbon monoxide hemoglobin. This test is more delicate than the spectroscopic 

 test and serves to detect the presence of as low a content as 5 per cent of carbon 

 monoxide hemoglobin. 



4. Neutral Methemoglobin. Dilute a little defibrinated blood (i : 10) and 

 add a few drops of a freshly prepared 10 per cent solution of potassium ferricya- 

 nide. Shake this mixture and observe that the bright red color of the blood is 

 displaced by a brownish red. Now dilute a little of this solution and examine it 

 spectroscopically. Note the single, very dark absorption band lying to the left 

 of D, and, if the dilution is sufficiently great, also observe the two rather faint 

 bands lying between D and E in somewhat similar positions to those occupied by 

 the absorption bands of oxyhemoglobin. Add a few drops of Stokes' reagent to 

 the methemoglobin solution while it is in position before the spectroscope and note 

 the immediate appearance of the oxyhemoglobin spectrum which is quickly fol- 

 lowed by that of hemoglobin. 



5. Alkaline Methemoglobin. Render a neutral solution of methemoglobin, 

 such as that used in the last experiment (4), slightly alkaline with a few drops of 

 ammonia. The solution becomes redder in color, due to the formation of alkaline 

 methemoglobin and shows a spectrum different from that of the neutral body. In 

 this case we have a band on either side of D, the one nearer the red end of the 

 spectrum being much the fainter. A third band, darker than either of those men- 

 tioned, lies between D and E somewhat nearer E. 



6. Alkali Hematin. Observe the spectrum of the alkali hematin prepared in 

 Experiment 18 on page 266. Also make a spectroscopic examination of a freshly 



1 Sand (U geskrift for Laeger, 76, 1721, 1914; Abst. /. A. M. A., Nov. 21, 1914) proposes 

 a potassium iodide test for carbon monoxide hemoglobin in blood. He claims 0.125 per 

 cent may be detected by his test. 



2 This transforms the oxyhemoglobin into methemoglobin. 



a This is done to free the blood from carbon monoxide hemoglobin. 



