Separation Of Porphyrinogens

In some applications, it is more convenient to separate porphyrins as their methyl ester derivatives. The majority of methods reported for the separation of porphyrin methyl esters are by normal phase (adsorption) chromatography on silica gel columns (7). RP-HPLC, however, provides better resolution and, although unable to separate the type I and III isomers of uro- and hep-tacarboxylic acid porphyrin methyl esters, is able to simultaneously separate the type I and III isomers of hexacarboxylic acid, pen-tacarboxylic acid, and coproporphyrin methyl esters in a single gradient elution run. Furthermore, the polar hydroxylated porphyrins, e.g., hydroxyuroporphyrin, which are difficult to elute from a silica gel column, are easily eluted from a RP column.

The separation of a mixture of por-

The porphyrinogens are the true intermediates in the biosynthesis of heme. Por-phyrinogens are unstable to oxidation by air, especially under acidic conditions and in the presence of light, to the corresponding porphyrins, and are therefore rarely analyzed. However, studies have shown that for isomer separation, the porphyrino-gens are usually better resolved than the porphyrins (3—6). Isomerically pure por-phyrinogens are needed as substrates for enzymic experiments, and their separation may also have application in situations where the complete resolution of isomers is essential. For example, investigation of the preferred order of uroporphyrinogen decar-boxylation requires the complete separation of all four type III heptacarboxylic porphyrinogen isomers (14).

Figure 8. RP-HPLC of porphyrin methyl esters. Column, Hypersil-ODS (250 x 4.6 mm, 5 pm particle size); eluent, linear gradient elution from 70% acetonitrile in water to 100% acetonitrile in 30 minutes; flow-rate, 1 mL/minute. Peaks; 1, 2, and 3 = hydroxylated porphyrins; 4, 5, 6, 7, and 8 refer, respectively, to tetra(copro)-, penta-, hexa-, hepta-, and octa-(uro)carboxyl porphyrin; I and III denote type I and type III isomers; mp = mesoporphyrin; pp = proto-porphyrin.

Figure 8. RP-HPLC of porphyrin methyl esters. Column, Hypersil-ODS (250 x 4.6 mm, 5 pm particle size); eluent, linear gradient elution from 70% acetonitrile in water to 100% acetonitrile in 30 minutes; flow-rate, 1 mL/minute. Peaks; 1, 2, and 3 = hydroxylated porphyrins; 4, 5, 6, 7, and 8 refer, respectively, to tetra(copro)-, penta-, hexa-, hepta-, and octa-(uro)carboxyl porphyrin; I and III denote type I and type III isomers; mp = mesoporphyrin; pp = proto-porphyrin.

Porphyrinogens are easily prepared by reduction of porphyrins, usually with 3% (wt/ wt) sodium amalgam, as described below.

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