Separation of overlapping spectra from evolving systems using factor analysis. 4. Fluorescence spectra of hematoporphyrin IX.

Fluorescence spectra of hematoporphyrin IX (Hp) in water and in aqueous SDS solutions are obtained in the pH range 0.1 to 13 to determine the ionisation state of the molecule as a function of pH. In water, the spectra are complicated by aggregation which is quite severe near pH 4. In aqueous SDS, the aggregation is much less violent. Factor analysis (FA) is used to identify five species in the fluorescence spectra in each series of solutions. The distribution curve of these species as a function of pH is also obtained. By comparing the spectra and the distribution curve of Hp with those of HPPEEA, an ethanolamide derivative of Hp that does not contain the carboxylic groups (Part 3), the species are identified. For Hp in water we have obtained the following species: the dication in two allotropic forms in the pH range 0 to 5; the monocation (with the charge on an imino nitrogen) in the pH range 2 to 7; and the free base in the pH range 3.5 to 13. The monocation observed by the second derivative technique revealed three subspecies. For Hp in aqueous SDS we have obtained the following species; one dication in the pH range 0 to near 4; one monocation (with the charge on an imino nitrogen) in the pH range 0.5 to 9; three free bases (with no charge on the imino nitrogen) in the pH range 4 to 13. Of the latter, one species is the neutral molecule, another is a dianion (with the charges on the carboxylic side chains), and the third one appearing at pH higher than 10 is an allotropic form of the dianion.