B. Extraction of pigments

Ten to twenty animals were removed from the aquarium, placed on a clean towel for about 5 minutes and gently squeezed a few times to dry out sea water from page 2coelenteron and body wall. They were then ground in a mortar with acid washed sand. Anhydrous sodium sulphate was then added until the mixture became fairly dry, and lastly the solvent of one part methanol to three parts petroleum ether (40-60°C m.p.) was added. The mixture is best left to soak overnight, but 30 minutes in the dark will give fairly good results. For the extraction of actiniochrome however, glycerine was used (with freshly ground material) as the pigment extraction medium.

The solvent extract was separated from the solid debris by suction filtration through two thicknesses of Whatman No. 1 on a Buchner funnel. The residue was re-extracted by adding more solvent. The filtered extract was collected in a separating funnel and allowed to stand for 15 minutes. Two distinct layers were obtained. The upper hydrocarbon layer was of an intense brown (epiphasic pigments) and the bottom layer was green (hypophasic pigments). The two layers were separated, placed in a dessicator in the dark and allowed to evaporate to dryness at room temperature. A few drops of petroleum ether or acetone were added to the dry residues to redissolve the pigments for thin layer chromatographic separation.