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%.