Decomposition of Cruorine and Extraction of Hematin

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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)
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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)

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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

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Addition of an Equal Volume of Diethylether
Add 5 mL of diethylether to the watery extract of
red blood cells
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Addition of Glacial Acetic Acid
Add 1 mL of glacial acetic acid to the sample
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Mix the Acid-Ether/Blood Extract
Mix the acid-ether/blood extract gently to
avoid forming an emulsion
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First Wash of the Acid-Ether/Blood Extract
Remove aqueous layer and add 5 mL of water. Mix
gently
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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
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Addition of Ammonium Hydroxide to the
Precipitated Hematin
Add a drop of conc. ammonium hydroxide
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Decomposition of Cruorine and Extraction of
Hematin
Explain in chemical terms what happened to the
coloring matter at each step
11
Cruorine to Hematin
?
Structures Stokes didnt know