VI. Spectroscopic Examination of
(For Absorption Spectra see Plates
I. and II.)
- From: Hawk, Philip B. Practical Physiological Chemistry.
P. Blakiston's Sons & Co., Philadelphia (1913) pp. 215-218.
- Copyright © 1998 Richard A. Paselk
- 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
- 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.
- © R. Paselk
- Last modified 22 July 2000