Separation by Paper Chromatography

Although more powerful techniques have since been developed, it is still a viable and cheap alternative for the separation of Proto from more polar tetrapyrroles such as Uro and Copro by ascending chromatogra-phy (37). Essentially, the tetrapyrrole mixture, as in an aliquot of HEAR or acetone extract, is spotted at the origin of a 5 x 15 cm piece of No. 1 or No. 3 chromatogra-phy paper (Whatman, Clifton, NJ, USA), allowed to dry, and then the paper is inserted into a cut 1000-mL glass cylinder containing about 10 mL of 2,4- or 2,6-lutidine: 0.05 N NH4OH (5:3.5 vol/vol). The cylinder is capped with a piece of aluminum foil. Development is carried out under subdued light at room temperature in a ventilated hood. When the solvent front has migrated about 10 cm, the chro-matogram is viewed under a 360 nm UV

Figure 4. Protochlorophyllide group. Tetrapyrroles which incorporate the fifth isocyclic ring, ring E. LCFA =

long-chain fatty acid.



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