A simple qualitative test to distinguish between protoporphyrin IX or its esters and porphyrins containing no vinyl group.

Since a satisfactory procedure of preparing pure protoporphyrin IX dimethyl ester from hemoglobin (1) has been worked out, it is interesting to investigate direct reduction of protoporphyrin IX dimethyl ester into mesoporphyrin IX dimethyl ester. Although these compounds, the melting points of which are too close to be of use for their differentiation, can be distinguished by HCl number (2) or absorption spectra, the simple and rapid qualitative test here described has proved useful in the above investigation. Protoporphyrin IX dimethyl ester used in this experiment was prepared by a simplified modification of the procedure (1) previously described. A sample of 5 ml. of red blood cells was refluxed with 100 ml. of 10 per cent oxalic acid in acetone for half an hour. The reaction mixture was filtered and washed with a little acetone. The colorless residue was discarded and the solution was then worked up by the procedure outlined in the previous paper. When in place of the 10 per cent oxalic acid solution 5 per cent and 1 per cent solutions of oxalic acid in acetone were tried, the yields of protoporphyrin IX dimethyl ester were 5 mg. and 3 mg. respectively per ml. of red blood cells. A procedure for preparing protoporphyrin IX dimethyl ester recently described by Grinstein and Camponovo (3) was also tried and modified. A sample of 5 ml. of red blood cells was stirred with 100 ml. of 1 per cent oxalic acid in methanol. The dark solut,ion separated from the residue by centrifuging was shaken with 5 gm. of stannous chloride and mixed with an equal volume of methanol previously saturated with hydrogen chloride gas. The reaction mixture was allowed to stand at room temperature for 1 to 2 hours and was worked up as usual. By this modified method a pure product was obtained without the necessity of chromatographic purification, the yield being 2.8 mg. per ml. of red blood cells. Mesoporphyrin IX dimethyl ester was also directly prepared from red blood cells. The acetone solution resulting from refluxing 5 ml. of red