Decomposition of Cruorine
and Extraction of Hematin
An experiment described in Section 11 of G. G. Stokes
“On the Oxidation and Reduction of the Colouring Matter of the Blood.”
Proceedings of the Royal Society of London, 13, 355-364 (1864)
Excerpt from Stokes Section 11
This [the decomposition of cruorine and separation of hematin]
may be easily effected on a small scale by adding to the watery
extract from blood-clots about an equal volume of ether, and
then some glacial acetic acid, and gently mixing, but not
violently shaking for fear of forming an emulsion. When enough
acetic acid has been added, the acid ether rises charged with
nearly the whole of the colouring matter, while the substance
which caused the precipitate remains in the acid watery layer
below. The acid ether solution shows in perfection the
characteristic spectrum fig. 3. When most of the acid is washed
out the substance falls, remaining in the ether near the common
surface. If after removing the wash-water a solution, even a
weak one, of ammonia or carbonate of soda be added, the
colouring matter readily dissolves in the alkali.
G. G. Stokes (1864)
Preparation of a Watery Extract
of Red Blood Cells
• Obtain a fresh sample of animal
blood in presence of an
anticoagulant, e.g. citrate or
ETDA
• Centrifuge the blood and decant
the blood plasma.
•Suspend the pellet of red blood
cells in 80 volumes of water.
•Add a 5 mL sample to a test tube
Addition of an Equal Volume of
Diethylether
Add 5 mL of diethylether
to the watery extract of
red blood cells
Addition of Glacial Acetic Acid
Add 1 mL of glacial
acetic acid to the sample
Mix the Acid-Ether/Blood Extract
Mix the acid-ether/blood
extract gently to avoid
forming an emulsion
First Wash of the
Acid-Ether/Blood Extract
Remove aqueous layer
and add 5 mL of water.
Mix gently
Second and Third Washes of the
Acid-Ether/Blood Extract
Remove
aqueous
layer. Add
5 mL of
water and
mix gently
Remove
aqueous
layer. Add
5 mL of
water and
mix gently
Addition of Ammonium Hydroxide
to the Precipitated Hematin
Add a drop of conc.
ammonium hydroxide
Decomposition of Cruorine
and Extraction of Hematin
Explain in chemical terms what happened
to the coloring matter at each step
Cruorine to Hematin
?
Structures Stokes didn’t know