Decomposition of Cruorine and Extraction of HematinExcerpt from Stokes Section 11Preparation of a Watery Extract of Red Blood CellsAddition of an Equal Volume of DiethyletherAddition of Glacial Acetic AcidMix the Acid-Ether/Blood ExtractFirst Wash of the Acid-Ether/Blood ExtractSecond and Third Washes of the Acid-Ether/Blood ExtractAddition of Ammonium Hydroxide to the Precipitated HematinSlide 10Cruorine to HematinDecomposition of Cruorine and Extraction of HematinAn 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 11This [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 tubeAddition of an Equal Volume of DiethyletherAdd 5 mL of diethyletherto the watery extract of red blood cellsAddition of Glacial Acetic AcidAdd 1 mL of glacial acetic acid to the sampleMix the Acid-Ether/Blood ExtractMix the acid-ether/bloodextract gently to avoidforming an emulsionFirst Wash of the Acid-Ether/Blood ExtractRemove aqueous layer and add 5 mL of water. Mix gentlySecond and Third Washes of the Acid-Ether/Blood ExtractRemove aqueous layer. Add 5 mL of water andmix gentlyRemove aqueous layer. Add 5 mL of water andmix gentlyAddition of Ammonium Hydroxide to the Precipitated HematinAdd a drop of conc. ammonium hydroxideDecomposition of Cruorine and Extraction of HematinExplain in chemical terms what happened to the coloring matter at each stepCruorine to Hematin?Structures Stokes didn’t
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