Separation of Porphyrin Isomers

The separation of isomers, particularly the type I and type III isomers, is important for the differential diagnosis of certain porphyrias. For example, the copropor-phyrin excreted in the urine and feces of patients with congenital erythropoietic porphyria (CEP) is type I, while in hereditary coproporphyria it is type III.

3.2.1. Uroporphyrin I, II, III, and IV Isomers

Uroporphyrin I and III isomers can be rapidly and effectively separated by isocrat-ic reversed-phase (RP)-HPLC on octade-cylsilyl (ODS) C^ columns with 13% (vol/vol) acetonitrile in 1 M ammonium acetate buffer, pH 5.16 (adjusted with acetic acid), as eluent (Figure 2). Optimization studies have shown that the molar concentration and pH of ammonium acetate buffer significantly affected the retention and resolution of uroporphyrin isomers (18). The optimum buffer concentration was 1 M, and the best pH range for chromatography on a conventional ODS column was between 5.10 and 5.20.

For separation on a base-deactivated (BDS) C18 column, the optimum pH was 5.55 (Figure 2), although 5.16 was also suitable. BDS C18 columns are columns with

Figure 2. Separation of uroporphyrin I and III isomers. (a) On Hypersil-BDS C18 with acetonitrile:1 M ammonium acetate, pH 5.16 (9:91, vol/vol), as eluent; (b) on Hypersil-ODS with acetontrile:1 M ammonium acetate, pH 5.16 (13:87, vol/vol), as eluent; and (c) on Hypersil-BDS C^ with acetonitrile:1 M ammonium acetate, pH 5.55 (9:91, vol/vol), as eluent. Flow-rate, 1 mL/minute.

Figure 2. Separation of uroporphyrin I and III isomers. (a) On Hypersil-BDS C18 with acetonitrile:1 M ammonium acetate, pH 5.16 (9:91, vol/vol), as eluent; (b) on Hypersil-ODS with acetontrile:1 M ammonium acetate, pH 5.16 (13:87, vol/vol), as eluent; and (c) on Hypersil-BDS C^ with acetonitrile:1 M ammonium acetate, pH 5.55 (9:91, vol/vol), as eluent. Flow-rate, 1 mL/minute.

fewer residual silanol groups through exhaustive end-capping or made by different bonding procedures to those normally used for conventional ODS columns. Residual silanol groups on silica-based C18 columns interact adversely with basic compounds, causing peak tailing or broadening. In general, BDS C18 columns give better resolution and faster separation for porphyrins than conventional C18 columns.

Methanol should not be used as the organic modifier for the separation of uro-porphyrins, especially when isocratic elution is used. It causes severe peak tailing and excessive retention with loss of resolution. Methanol is a hydrogen-bonding organic modifier. A layer of methanol adsorbed onto the hydrophobic hydrocarbonaceous C18 stationary phase surface can form extensive hydrogen bonds with the 8 carboxylic acid groups of uroporphyrin. The result is long retention and peak tailing. This phenomenon is not observed for porphyrins with one or more methyl groups, since the interaction is dominated by hydrophobic interaction between the hydrophobic methyl group(s) and the stationary phase surface.

A small proportion (e.g., 10%) of ace-tonitrile can be added to methanol, and the mixture (10% acetonitrile and 90% methanol) can be used as the organic modifier. The more hydrophobic acetonitrile, which is also a nonhydrogen bonding organic modifier, will be preferentially adsorbed onto the stationary phase surface, thus preventing hydrogen bond formation.

The complete separation of uroporphyrin I, II, III, and IV isomers has not been achieved. They were resolved into three peaks in the elution order of I, III + IV, and II (7,18). The resolution was not improved by employing a BDS C18 column.

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