Checked by C. R. Noller and G. A. Smith.
1. Procedure
In a
5-l. round-bottomed flask equipped with a thermometer, reflux condenser, and dropping funnel are placed
4 l. of glacial acetic acid and
1 g. of sodium chloride. The acid is heated to boiling on a sand bath until the
sodium chloride is in solution, and then 1 l. of defibrinated blood (Note
1) is added in a thin stream from the dropping funnel over a period of about 30 minutes. The blood should not touch the sides of the flask. During this time the temperature is kept at 100–105°, and heating is continued for 10 minutes after all the blood has been added. The flame is then removed and the mixture allowed to cool and stand overnight.
The precipitated
hemin is removed by centrifuging (Note
2). If the centrifuging is carried out in 100-ml. tubes, each lot of tubes is centrifuged 10 minutes, the supernatant liquid is decanted, more of the mixture added, and the centrifuging repeated. The
hemin is allowed to accumulate in the tubes until all the mixture has been centrifuged, after which it is stirred with a
glass rod and washed from the several tubes into one with
75 ml. of 50% aqueous acetic acid. After centrifuging and decanting, the
hemin is washed successively in the same manner with two 75-ml. portions of distilled water, one
50-ml. portion of 95% ethanol, and one
50-ml. portion of ether. After the
ether has been decanted the
hemin is transferred to a
watch glass by means of a
rubber policeman and about
5 ml. of ether. After evaporation to dryness
3.5–4.5 g. of crude product is obtained.
For recrystallization,
5 g. of the crude
hemin is placed in a
100-ml. Erlenmeyer flask,
25 ml. of pyridine is added, and the flask is shaken until the
hemin has dissolved.
Forty milliliters of chloroform is added, and the flask is stoppered with a cork and shaken for 15 minutes; the cork is carefully removed from time to time to release the pressure. The solution is then filtered with slight suction through a
small Büchner funnel, and the
Erlenmeyer flask and filter are washed with
15 ml. of chloroform.
During the shaking
350 ml. of glacial acetic acid is heated to boiling in a
600-ml. beaker under a hood, and
5 ml. of a saturated sodium chloride solution and
4 ml. of concentrated hydrochloric acid are added. The flame is extinguished, and the filtered
hemin solution poured in a steady stream with stirring into the hot mixture; the suction flask is rinsed with
15 ml. of chloroform. After the mixture has stood for 12 hours, the crystals are filtered with suction on a small Büchner funnel and washed with
50 ml. of 50% aqueous acetic acid, 100 ml. of distilled water,
25 ml. of ethanol, and
25 ml. of ether. Suction is continued until the crystals are dry, when they can be readily removed. The recovery is
75–85%.
2. Notes
1. Fresh blood obtained from a slaughter house is defibrinated by whipping it with a stiff vegetable-fiber brush followed by filtration with suction through a
large Büchner funnel. The blood is stirred during the filtration to prevent settling of the erythrocytes. Beef blood was used for checking this preparation.
2. The
hemin may be removed by filtration but it is usually so finely divided that centrifuging is easier and less loss results.
3. Discussion
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