Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids |
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Authors: | Herbert C. Friedmann Eric T. Baldwin |
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Affiliation: | Department of Biochemistry, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637 USA |
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Abstract: | Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C18 spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis. |
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Keywords: | porphyrias porphyrins purification reverse-phase chromatography thin-layer chromatography urines |
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