Retention Mechanism Of Porphyrins And Metalloporphyrins In Rphplc

Understanding the retention behavior and mechanism is useful in the prediction and elucidation of the possible nature of substituent groups present in unknown porphyrins.

The most dominant mechanism of retention in RP-HPLC is hydrophobic interaction. In porphyrins, this is between the side-chain substituents and the hydrophobic hydrocarbonaceous (ODS) stationary phase surface. The hydrophobic-ity of the porphyrin side-chain substituents increases in the order:

The number of the most hydrophobic substituents available for interaction, therefore, determines the relative retention of the porphyrin. This is dominated by the number of alkyl (especially methyl) groups. Thus, the elution of porphyrins in the order of uroporphyrin (8COOH), hep-tacarboxylic acid porphyrin (7COOH, ICH3), hexacarboxylic acid porphyrin

(6COOH, 2CH3), pentacarboxylic acid porphyrin (5COOH, 3CH3), copropor-phyrin (4COOH, 4CH3), mesoporphyrin (2COOH, 4CH3, 2CH2CH3), and protoporphyrin (2COOH, 4CH3, 2CH=CH2) was observed.

The hydrophobic interaction mechanism also explains the elution order of porphyrin isomers. The elution in the order of I, III, IV, and II, for coproporphyrin isomers is an example. Coproporphyrin II has two pairs of adjacent CH3 groups at positions 1 and 8 and 4 and 5, respectively, using Fischer's numbering system. These provide the largest hydrophobic surface areas available for interaction. It is therefore the longest retaining isomer. The symmetrical coproporphyrin I has no adjacent CH3 groups and, thus, has the smallest hydro-phobic surface areas available for interaction. It is, therefore, the fastest eluting isomer. Coproporphyrin III and IV isomers each have a pair of adjacent CH3 groups, at positions 1 and 8 and 2 and 3, respectively. In this situation, the relative distance between the adjacent CH3 pair and the remaining two nonadjacent CH3 groups becomes an important factor in determin-

CH2COOH<CH2CH2COOH<CH3<CH2CH3<CH=CH2

Figure 6. Separation of a standard mixture of porphyrins. Column, Hyper-sil-ODS (25 cm x 4.6 mm i.d., 5 pm particle size); solvents, 0.1% trifluoroacetic acid (solvent A) and ace-tonitrile (solvent B); elu-tion, linear gradient from 25% solvent B (75% solvent A) to 50% solvent B in 30 minutes; flow rate, 1 mL/minute; detection, ab-sorbance 404 nm. Peaks: 4, 5, 6, 7, and 8 refer, respectively, to tetra(copro)-, penta-, hexa-, hepta-, and octa(uro)carboxylic acid porphyrin; I and III denote type I and type III isomers.

ing the relative hydrophobicity. In copro-porphyrin III, these are 5 (from position 1 to 3) and 6 (from position 8 to 5) bond lengths apart, respectively. In copropor-phyrin IV, each of the adjacent CH3 groups is 5 bond lengths away (from positions 2 to 8 and 3 to 5) from their nearest nonadja-cent CH3 group. The slightly shorter distance (one bond length) between one of the adjacent pair CH3 groups and its nearest nonadjacent CH3 group (5 instead of 6 bonds apart) is sufficient to make the type IV isomer slightly more hydrophobic and, therefore, be retained longer than the type III isomers. Similar arguments apply to the separation of the penta- and hexacarboxylic acid porphyrin isomers.

The type III heptacarboxylic acid porphyrin isomers each have only a single CH3 group that dominated the hydropho-bic interaction. These isomers are, therefore, very similar in hydrophobicity and difficult to separate.

In metalloporphyrins, hydrophobic interaction between the side-chain sub-stituents and stationary phase surface is also an important retention mechanism. However, the insertion of a metal ion, which completely occupies the center of the porphyrin hole, significantly altered the electronic environment around the central N atoms. The retention of metallopor-phyrins is also dependent on the ability, and therefore the species, of the inserted

Figure 7. Separation of porphyrins in (a) the fecal extract and (b) the urine of a patient with PCT. Column, Hypersil-ODS (250 x 4.6 mm, 5 pm particle size); solvent A, 10% acetonitrile in 1 M ammonium acetate buffer (pH 5.16); solvent B, 10% acetonitrile in methanol; elution, linear gradient at 1 mL/minute from 10% to 90% solvent B in 30 minutes, followed by isocratic elution at 90% solvent B for a further 10 minutes; detection, 404 nm. Peaks; 4, 5, 6, 7, and 8 refer, respectively, to tetra(copro), penta-, hexa-, hepta-, and octa (uro)carboxylic acid porphyrin; 4-Iso = isocoproporphyrin; pp = pro-toporphyrin.

metal ion to accept axial ligands from the mobile phase. Addition of polar axial ligands leads to a decrease in the overall hydrophobicity of a molecule and, consequently, its retention (10).

phyrin methyl esters on an ODS column by gradient elution from 70% (vol/vol) acetonitrile in water to 100% acetonitrile in 30 minutes is shown in Figure 8. The system is also fully compatible with MS.

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