From: Hawk, Philip B. Practical Physiological Chemistry. P. Blakiston's Sons & Co., Philadelphia (1913) pp. 215-218.

Copyright © 1998 Richard A. Paselk



VI. Spectroscopic Examination of Blood

(For Absorption Spectra see Plates I. and II.)

Either the angular-vision spectroscope (Figs. 63 and 64, page 217) or the direct-vision spectroscope (Fig. 62, page 216) may be used in making the spectroscopic examination of the blood. For a complete description of these instruments the student is referred to any standard text-book of physics.
I. Oxyhaemoglobin.--Examine dilute (1:50) defibrinated blood spectroscopically. Note the broad absorption-band between D and E. Continue the dilution until this single broad band gives place to two narrow bands, the one nearer the D line being the narrower. These are the typical absorption-bands of oxyhaemoglobin obtained from dilute solutions of blood. Now dilute the blood very freely and note that the bands gradually become more narrow and, if the dilution is sufficiently great, they finally entirely disappear.

2. Haemoglobin (so-called Reduced Haemoglobin)--To blood which has been diluted sufficiently to show well-defined oxyhaemoglobin absorp- tion-bands add a small amount of Stokes' reagent. The blood immediately changes in color from a bright red to violet-red. The oxyhaemoglobin has been reduced through the action of Stokes' reagent 1 and haemoglobin (so-called reduced haemoglobin) has been formed. This has been brought about by the removal of some of the loosely combined oxygen from the oxyhaemoglobin. Examine this haemoglobin spectroscopically. Note that in place of the two absorption bands of oxyhaemoglobin we nowhave a single broad band lying almost entirely between D and E. This is the typical spectrum of haemoglobin. If the solution showing this spectrum be shaken in the air for a few moments it will again assume thebright red color of oxyhaemoglobin and show the characteristic spectrum of that pigment.
3. Carbon Monoxide Haemoglobin-The peparation of this pigment may be easily accomplished by passing ordinary illuminating gas2 through defibrinated ox-blood. Blood thus treated assumes a brighter tint (carmine) than that imparted by oxyhaemoglobin. In very dilute solution oxyhaemoglobin appears yellowish-red whereas carbon monoxide haemoglobin under the same conditions appears bluish-red. Examine the
1Stokes' reagent is a solution containing 2 per cent ferrous sulphate and 3 per cent tartaric acid. When needed for use a small amount should be placed in a test-tube and arnmonium hydroxide added until the precipitate wshich forms on the first addition of the hydroxide has entirely dissolved. This produces ammonium ferrotartrate which is a reducing agent.
2 The so-called water gas with which ordinary illuminating gas is diluted contains usually as much as 20 per cent of carbon monoxide (CO).

carbon monoxide haemoglobin solution spectroscopically. Observe that the spectrum of this body resembles the spectrum of oxyhaemoglobin in showing two absorption-bands between D and E. The bands of carbon

monoxide haemoglobin, however, are somewhat nearer the violet end of the spectrum. Add some Stokes' reagent to the solution and again examine spectroscopically. Note that the position and intensity of the absorption-bands remain unaltered.

The following is a delicate chemical test for the detection of carbon monoxide hremoglobin:
Tannin Test.-Divide the blood to be tested into two portions and dilute each with four volumes of distilled water. Place the diluted blood mixtures in two small flasks or large test-tubes and add 20 drops of a I0 per cent solution of potassium ferricyanide.1 Allow both solutions to stand for a few minutes, then stopper the vessels and shake one vigorously for I0-I5 minutes, occasionally removing the stopper to permit air to enter the vessel.2 Add 5-I0 drops of ammonium sulphide (yellow) and I0 c.c. of a I0 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 carbon monoxide haemoglobin, will exhibit a bright red precipitate, characteristic of carbon monoxide hsemoglobin. 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 haemoglobin.
4. Neutral Methaemoglobin.--Dilute a little defibrinated blood (I:I0) and add a few drops of a freshly prepared I0 per cent solution of potassium ferricyanide. 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 absorp- tion bands of oxyhaemoglobin. Add a few drops of Stokes' reagent to the methaemoglobin solution while it is in position before the spectroscope and note the immediate appearance of the oxyhaemoglobin spectrum which is quickly followed by that of hamoglobin.
5. Alkaline Methaemoglobin.--Render a neutral solution of methaemoglobin, 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 methaemoglobin 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 mentioned, lies between D and E somewhat nearer E.
6. Alkali Haematin-Observe the spectrum of the alkali haematin prepared in Experiment I6 on page 2I2. Also make a spectroscopic examination of a freshly prepared alkali haematin.3 The typical spectrum of alkali haematin shows a single absorption-band lying across D and mainly toward the red end of the spectrum.
1 This transforms the oxyhaemoglobin into methaemoglobin.
2 This is done to free the blood from carbon monoxide haemoglobin.
3 Alkali haematin may be prepared by mixing one volume of a concentrated potassium hydroxide or sodium hydroxide solution and two volumes of dilute (1:5) defibrinated blood.This mixture should be heated gradually almost to boiling, then cooled and shaken for a few moments in the air before examination.