Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines

We have employed size exclusion chromatography (SEC) to the study of the auto- and Cu²⁺-mediated oxidation of the catecholamines, dopamine, epinephrine and norepinephrine, into melanins. We observed that, due to non-size exclusion-mediated effects, the catecholamines and some of the low molecular mass intermediates generated during the oxidation reactions, could be resolved from each other and from the high molecular mass pigment generated. Thus, SEC allowed us to monitor the disappearance of the catecholamine starting compounds, the appearance and subsequent disappearance of the low molecular mass chromophores generated in the initial phase of the reactions and the appearance of the high molecular mass melanins. In the process of this research, we observed that many, mostly anionic polysaccharides (PS), enhanced both the auto- and Cu²⁺-mediated oxidation of all three catecholamines. SEC analyses of reaction mixtures involving PS suggested that very high molecular mass aggregates between PS and melanins can be generated. In addition, SEC analysis allowed us to verify the efficiency of the dialysis purification process employed to obtain pure and dried melanin materials for cell-biological studies.