Particle Size Characterization of Sepia Ink Eumelanin Biopolymers by SEM, DLS, and AF4-MALLS: a Comparative Study

Cephalopod ink is a complex mixture of bioactive substances with technical properties of interest in many fields (e.g., biophysics, ecology, environment, biomedicine, food technology, cosmetics, or fine arts). It was previously reported that organic nanoparticles may naturally appear in this mixture. Thus, the particle size determination of these biopolymers is interesting from the point of view of food nanotechnology and nanotoxicology. In this work, the particle size of purified eumelanin microspheres from commercial sepia ink was successfully measured by three techniques: Scanning electron microscopy (SEM), dynamic light scattering (DLS), and asymmetric-flow field-flow fractionation linked to multi-angle laser-light scattering (AF4-MALLS). This study shows the potential and differences of the application of these techniques in terms of sample preparation, conditioning, introduction, and principles for particle size characterization of natural organic nanoparticles in foods. Thus, this methodology can be a model for the characterization of other natural and engineered organic nanoparticles in this matrix type. DLS and AF4-MALLS provide the size corresponding to the hydrodynamic diameter, which is usually larger than the size of the dense core provided by SEM (without hydration or solvation layer). Additionally, SEM informs about the particles morphology, showing a quasi-spherical shape for particles between 100 and 140 nm. DLS and AF4-MALLS indicate particles of hydrodynamic diameter in the range of 180–260 nm. Furthermore, the absolute molar mass of particles has been measured by MALLS.