light. The separated tetrapyrroles are detected by their red fluorescence. In this system, Proto migrates with a retention factor (Rf) of about 0.8, while Copro and Uro migrate with Rfs of about 0.6 and 0.3, respectively (37). Although paper chro-matography is useful for separating Proto form other tetrapyrroles, it is not convenient for preparative purposes because of the difficulty of eluting the separated tetrapyrroles.

3.3.2. Chromatographic Separation and Determination by HPLC

If HPLC instrumentation is available, it can be efficiently used for the quantitative separation of Proto from other tetrapyrroles. Using large columns, it can also be conveniently used for preparative purposes.

We have successfully used the solvent system of Ho (23) for separating Proto from Uro, Copro, and other multi-caboxylic tetrapyrroles in HEAR fractions prepared from cancer cell cultures (50). The same system can be used with HEAR preparation of plant sources. With this solvent system, there is no need to convert tetrapyrroles to their methyl esters prior to chromatography. If feasible, it is always advantageous to avoid derivatization prior

Figure 5. Chlorin group. Tetrapyrroles reduced to the oxidation state of chlorophylls, i.

e., a single bond between carbons 7 and 8.



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