BLOOD ANALYSIS 299 



prepared alkali hematin. 1 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 the 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 

 alkali hematin. Examine this solution spectroscopically and observe the narrow, 

 dark absorption band lying midway between D and E. If the dilution is not 

 too great a faint band may be observed in the green extending across E and b. 



8. Acid Hematin. To some defibrinated blood add half its volume of glacial 

 acid and an equal volume of ether. Mix thoroughly. The acidified etheral 

 solution of hematin rises to the top and may be poured off and used for the spectro- 

 scopic examination. If desired it may be diluted with acidified ether in the ratio 

 of one part of glacial acetic acid to two parts of ether. A distinct absorption band 

 will be noted in the red between C and D and lying somewhat nearer C than the 

 band in the methemoglobin spectrum. Between D and F may be seen a rather 

 indistinct broad band. Dilute the solution until this band resolves itself into two 

 bands. Of these the more prominent is a broad, dark absorption band lying in the 

 green between b and F. The second, a narrow band of faint outline, lies in the 

 light green to the red side of E. A fourth very faint band may be observed lying 

 on the violet side of D. 



9. Acid Hematoporphyrin. To 5 c.c. of concentrated sulphuric acid in a test- 

 tube add 2 drops of blood, mixing thoroughly by agitation after the addition of 

 each drop. A wine-red solution is produced. Examine this solution spectroscopic- 

 ally. Acid hematoporphyrin gives a spectrum with an absorption band on either 

 side of D, the one nearer the red end of the spectrum being the narrower. 



10. Alkaline Hematoporphyrin. Introduce the acid hematoporphyrin solution 

 just examined into an excess of distilled water. Cool the solution and add potas- 

 sium hydroxide slowly until the reaction is but slightly acid. A colored precipitate 

 forms which includes the principal portion of the hematoporphyrin. The presence 

 of sodium acetate facilitates the formation of this precipitate. Filter off the 

 precipitate and dissolve it in a small amount of dilute potassium hydroxide. Alka- 

 line hematoporphyrin prepared in this way forms a bright red solution and possesses 

 four absorption-bands. The first is a very faint, narrow band in the red, midway ^ 

 between C and D ; the second is a broader, darker band lying across D, principally to 

 the violet side. The third absorption band lies principally between D and E, ex- 

 tending for a short distance across E to the violet side, and the fourth band is 

 broad and dark and lies between b and F. The first band mentioned is the faintest 

 of the four and is the first to disappear when the solution is diluted. 



i. FleischPs Hemometer (Fig. 8,9, p. 300). This is an instrument 

 used quite extensively clinically, for the quantitative determination of 

 hemoglobin. The instrument consists of a small cylinder which is pro- 

 vided with a fixed glass bottom and a movable glass cover, and which is 



1 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. 



