Resolving measurement artifacts of field-flow fractionation coupled to conventional dynamic light scattering detection by using phase and spatially-resolved dynamic light scattering to improve size accuracy.

The use of dynamic light scattering (DLS) as an online detector in asymmetrical flow field-flow fractionation (AF4 ‒ DLS) for size characterization is known to introduce measurement bias for particles with hydrodynamic size exceeding ∼100 nm. This study demonstrates that the accuracy of hydrodynamic size measurements in AF4 ‒ DLS is significantly improved by applying advanced phase and spatially-resolved DLS (PhaSR-DLS) detection. PhaSR-DLS overcomes two limitations of conventional DLS: (i) overestimation of particle size due to 'number fluctuations' at low particle concentrations, causing 'U-turn' shaped size profiles and (ii) underestimation of particle size due to the flow through the detector flow cell. These improvements are demonstrated by analyzing mixtures of 100 nm and 200 nm polystyrene particles at different concentrations and detector flow rates using AF4 coupled online to both PhaSR-DLS (AF4 ‒ PhaSR-DLS) and conventional DLS (AF4 ‒ DLS) simultaneously for comparison. Accuracy improvements with AF4 ‒ PhaSR-DLS are most significant at the highest flow rate (0.80 mL/min), where the large biases are removed or significantly reduced (from -19% to 0% error for the 100 nm standard and from -36% to -5% error for the 200 nm standard). Furthermore, using direct concentration measurements from AF4 ‒ PhaSR-DLS, it is shown that accurate particle size can be achieved at concentrations as low as 108 particles/mL. Since the measurement accuracy depends on the particle size, particle concentration, and detector flow rate, these findings show that use of PhaSR-DLS in AF4 can significantly improve accuracy and robustness for a range of applications using nanoparticles of different compositions and morphologies.