- 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
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.