302 PHYSIOLOGICAL CHEMISTRY 



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 dro^s 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 17 on page 269. Also make a spectroscopic examination of a freshly 

 prepared alkali hematin. 4 The typical spectrum of alkali hematin shows a single 

 absorption band lying across D and mainly toward the red end of the spectrum. 



7. Reduced Alkali Hematin or Hemochromogen. Dilute the alkali hematin 

 solution used in the last experiment (6) to such an extent that it shows no absorption 

 band. Now add a few drops of Stokes' reagent or ammonium sulphide and note 

 that tne greenish-brown color of the alkali hematin solution is displaced by a 

 bright red color. This is due to the formation of hemochromogen or reduced 



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 P er 

 cent may be detected by his test. 



s This transforms the oxyhemoglobin into methemoglobin. 



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



4 Alkali hematin may be prepared by mixing one volume of a concentrated potassium 

 hydroxide or sodium hydroxide solution and two volumes of dilute (i : 5) defibrinated blood. 

 This mixture should be heated gradually almost to boiling, then cooled and shaken for 

 a few moments in the air before examination. 



