A modified procedure for the preparation of protoporphyrin IX dimethyl ester from hemoglobin.

Protoporphyrin IX dimethyl ester, a crystalline compound with a definite melting point, is generally used to identify protoporphyrin IX. Various methods of preparation (l-4) have been described in the literature. As indicated by Grinstein and Watson (5, S), application of chromatographic separation is necessary in order to obtain a pure product. By following the procedures thus far described in the literature the same conclusion has been reached. However, by modifying the procedure as heretofore described, we have been able to obtain the product in a pure state without the necessity of applying chromatographic separation. A solution of crude protoporphyrin IX was prepared from defibrinated blood by Hamsik’s procedure (7) with some modifications. Either citric acid or salicylic acid can be used in the place of oxalic acid. The solution of crude protoporphyrin was then successively extracted with ether and 10 per cent hydrochloric acid and converted into the dimethyl ester, as described below. For crystallization of the ester it has been found that a mixture of acetone and ether is more satisfactory than chloroform and methanol. This procedure has been successfully employed to prepare crystalline protoporphyrin IX dimethyl ester from a sample of red blood cells as small as 0.15 ml. In the case of small samples, the amounts of materials used are proportionally reduced and the time of reaction shortened.