The effect of preparation procedures on the morphology of melanin from the ink sac of Sepia officinalis

The structure of melanin extracted from the ink sac of the cuttlefish Sepia officinalis was examined for different methods of isolation and purification of the pigment. Scanning electron microscopy (SEM) images of Sepia eumelanin prepared by different procedures establish that multi-μm-sized aggregates reported by previous workers are generated by their sample preparation, and that the dominant constituents of Sepia melanin are ∼150 nm spherical granules. Brunauer-EmmettTeller (BET) measurements reveal that Sepia eumelanin from Sigma (prepared by spray drying the pigment) has a surface area of 14.3 m²/g. Pigment extracted directly from the fresh ink sac and then freeze-dried has a surface area of 21.5 m²/g, while CO₂-supercritically dried has a surface area of 37.5 m²/g. This is consistent with SEM images showing that the process of freeze-drying produces aggregates, but to a lesser extent than spray drying. Supercritical drying of the sample produces suspensions of the individual ∼150 nm granule, which is more reflective of the natural pigment. BrunauerEmmett-Teller surface area analysis and Barrett-Joyner-Halenda (BJH) pore volume analysis indicate that the surface of the granules is not smooth and the interior of the granules is not porous, but rather the aggregates of granules are porous. Ultra-high resolution SEM and atomic force microscopy (AFM) images show the granules are easily deformed and are comprised of smaller constituents. De-aggregation of the granules by sonication and ultra-filtration reveal a range of structures depending on the pore size of the membrane used. The implications of these results on quantifying photochemical properties and kinetic reaction rate constants of melanin are discussed.