Protoporphyrin Heme and Related Compounds

Dicarboxylic acid porphyrins, heme, and related compounds are much more hydro-phobic than the other porphyrins described above. They require a much higher proportion of organic modifier for elution. Since acetonitrile is immiscible with 1 M ammonium acetate above the proportion of about 35%, it cannot be used as the sole organic modifier for the separation of this group of compounds. Either a mixture of acetoni-trile:methanol (10:90) or methanol alone can be used instead. Methanol is completely miscible with 1 M ammonium acetate.

The separation of dicarboxylic por-phyrins and metalloporphyrins by RP-HPLC has been described (10). A typical separation of protoporphyrin and heme-related compounds on a Hypersil-ODS

Figure 3. Separation of type I and type III pentacarboxylic acid porphyrin isomers. Column, Hypersil-ODS; eluent (a), 45% acetonitrile:methanol (10:90, vol/vol) in 1 M ammonium acetate, pH 5.16; eluent (b), 19% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16. The letters a, b, c, and d denote the positions of methyl groups on rings A, B, C, and D, respectively.

Figure 3. Separation of type I and type III pentacarboxylic acid porphyrin isomers. Column, Hypersil-ODS; eluent (a), 45% acetonitrile:methanol (10:90, vol/vol) in 1 M ammonium acetate, pH 5.16; eluent (b), 19% (vol/vol) acetonitrile in 1 M ammonium acetate, pH 5.16. The letters a, b, c, and d denote the positions of methyl groups on rings A, B, C, and D, respectively.

column with 86% (vol/vol) methanol in 1 M ammonium acetate buffer, pH 5.16, as eluent is shown in Figure 5.

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