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

  • Inmaculada de la CalleEmail author
  • Diego Soto-Gómez
  • Paula Pérez-Rodríguez
  • J. Eugenio López-Periago


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.

Graphical Abstract


Sepia ink DLS AF4-MALLS SEM Eumelanin Particle size 



The authors thank the Ultra Trace Analysis Aquitaine UT2A/ADERA (Pau, France) for the analysis performed on DLS and AF4-MALLS and the CACTI services from Universidade de Vigo for the SEM photographs (Vigo, Spain).

Funding Information

I. De la Calle thanks Xunta de Galicia for financial support as a postdoctoral researcher of the I2C program (POS-B/2017/012-PR) and co-financed by the European Social Funding P.P. 0000 421S 140.08. D. Soto-Gómez is funded by a predoctoral Fellowship Program (FPU) from the Spanish Ministry of Education. P. Pérez-Rodríguez is funded by a postdoctoral contract from Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED481B 2017/31).

Compliance with Ethical Standards

Conflict of Interest

Inmaculada de la Calle declares that she has no conflict of interest. Diego Soto-Gómez declares that he has no conflict of interest. Paula Pérez-Rodríguez declares that she has no conflict of interest. J. Eugenio López-Periago declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Informed consent is not applicable for this study.

Supplementary material

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ESM 1 (DOCX 1540 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Departamento de Química Analítica y Alimentaria, Área de Química Analítica, Facultad de QuímicaUniversidad de VigoVigoSpain
  2. 2.Ultra Trace Analyses Aquitaine UT2A/ADERAHélioparc Pau-PyrénéesPauFrance
  3. 3.Soil Science and Agricultural Chemistry Group, Department of Plant Biology and Soil Science, Faculty of SciencesUniversity of VigoOurenseSpain
  4. 4.Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS)Université de Strasbourg / EOST, CNRSStrasbourgFrance

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