Porphyrinogens In Rphplc

Although hydrophobic interaction is undoubtedly still the mechanism governing the retention of porphyrinogens in RP-HPLC, the reversal in the elution order observed for many of the porphyrinogen isomers in comparison to porphyrins requires explanation. For example, while coproporphyrin isomers were eluted in the order I, III, IV, and II based on the hydrophobic interaction hypothesis, the corresponding coproporphyrinogen isomers were eluted in the order of I, II, III, and IV, an apparent contradiction to the hydrophobic interaction hypothesis. However, reduction of the methine bridges of the rigid porphyrin macrocycle resulted in a relatively flexible porphyrinogen mole cule, which is able to adopt various conformations. In the flexible coproporphyrino-gen molecules, the small CH3 group in each isomer may be subjected to varying degrees of steric hindrance or shielding by the larger propionic acid groups, depending on the adopted conformation. This alters the expected hydrophobic surface areas available for interaction, which results in a change in elution order. Since the conformations of the various groups of por-phyrinogens under RP-HPLC conditions are unknown, it makes prediction of the elution order difficult.

8. HPLC DETECTORS FOR PORPHYRINS, METALLO-PORPHYRINS, AND PORPHYRINOGENS

Porphyrins and metalloporphyrins have an intense absorption band at the 400 nm region (Soret band). Detection at the Soret band region with an UV-visible detector set at 400 to 405 nm allows the simultaneous detection of all porphyrins and metal-loporphyrins.

Porphyrins also have intense red fluorescence, and a fluorescence detector set at excitation and emission wavelengths of 400

to 415 nm and 600 to 620 nm, respectively, provides a highly sensitive and specific method of detection. Heme is a nonfluo-rescence compound and cannot be detected fluorimetrically.

The porphyrinogens do not fluoresce and have only relatively weak UV absorption at the 220 to 240 nm region. They can be detected with a UV detector set at 220 nm, but are best detected electrochem-ically with an amperometric or coulomet-ric detector because of their ease of oxidation. Porphyrins and metalloporphyrins are also electro-active and can be detected with an electrochemical detector.

In terms of sensitivity, specificity, and ability to positively identify and characterize compounds, the best "detector" currently available is the mass spectrometer. On-line HPLC-ESIMS and tandem mass spectrometry (MS/MS) provide unsurpassed specificity for the analysis of tetrapyrroles.

The methyl esters of porphyrins are usually used in MS analysis because they ionize better in the ion source than free acid por-phyrins. Porphyrins with a higher number of carboxylic acid groups, e.g., uro- and hep-tacarboxylic acids porphyrins, are more difficult to ionize than those with a lesser number of carboxylic acid groups, such as copro-and protoporphyrins. With the recent devel

Figure 9. Separation of heptacar-boxyl porphyrinogen isomers.

Column, Hypersil-ODS (250 x 4.6 mm, 5 pm particle size); elu-ent, acetonitrile:methanol:1 M ammonium acetate, pH 5.16 (7:3:90, by vol); flow-rate, 1 mL/minute; detection, ampero-metric at +0.70 V.

Figure 9. Separation of heptacar-boxyl porphyrinogen isomers.

Column, Hypersil-ODS (250 x 4.6 mm, 5 pm particle size); elu-ent, acetonitrile:methanol:1 M ammonium acetate, pH 5.16 (7:3:90, by vol); flow-rate, 1 mL/minute; detection, ampero-metric at +0.70 V.

opment and introduction of high sensitivity high resolution hybrid electrospray ionization orthogonal quadrupole-time of flight mass spectrometer (e.g., Q-Tof II; Micromass, Altrincham, Cheshire, England, UK), however, all free acid porphyrins can now be analyzed with great sensitivity without the need for derivatization.

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