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The development of reliable protocols suitable for the characterisation of the physical properties of nanoparticles in suspension is becoming crucial to assess the potential biological as well as toxicological impact of nanoparticles. Amongst sizing techniques, asymmetric flow field flow fractionation (AF4) coupled to online size detectors represents one of the most robust and flexible options to quantify the particle size distribution in suspension. However, size measurement uncertainties have been reported for on-line dynamic light scattering (DLS) detectors when coupled to AF4 systems. In this work we investigated the influence of the initial concentration of nanoparticles in suspension on the sizing capability of the asymmetric flow field-flow fractionation technique coupled with an on-line dynamic light scattering detector and a UV–Visible spectrophotometer (UV) detector. Experiments were performed with suspensions of gold nanoparticles with a nominal diameter of 40 nm and 60 nm at a range of particle concentrations. The results obtained demonstrate that at low concentration of nanoparticles, the AF4-DLS combined technique fails to evaluate the real size of nanoparticles in suspension, detecting an apparent and progressive size increase as a function of the elution time and of the concentration of nanoparticles in suspension.

Limitations of Nanoparticles Size Characterization by Asymmetric Flow Field‑Fractionation Coupled with Online Dynamic Light Scattering