Negotiating system's band broadening in hydrodynamic chromatography for the determination of particle-size...

Nanoparticles (NPs) are widely applied in various fields. Determination of the particle-size distribution (PSD) is important for understanding NP properties. Hydrodynamic chromatography (HDC) can resolve submicron NPs and provide mean NP size after calibration based on size-certified polystyrene (PS) standards. However, assessment of the NP PSDs through observed HDC peak widths is hampered by contribution of the analytical system to band broadening (σ_sys²).

This work proposes a workflow for PSD determination from HDC profiles acquired with a conventional LC-UV system. HDC demonstrated high repeatability and efficiency with elution-time RSDs below 0.02 % and reduced plate heights of 1.5-2.3. However, due to the small elution-time window, the achievable NP-size resolution was confined. The σ_sys² was assessed as a function of elution time by subtracting the PSD contribution to the peak variance (σ_PSD²) of PS NPs from the observed peak variance (σ_app²). PSD widths of NP samples were determined by subtracting σ_sys² from the determined σ_app² and a lower limit for reliable PSD assessment was established. Leave-one-out cross-validation (LOOCV) using the PS NPs, confirmed the suitability of the PSD-determination method. Based on blends of PS NPs, a deconvolution approach was developed to resolve NP populations in mixtures. The applicability of the deconvolution and σ_sys²-correction approach was evaluated by the analysis of various industrial polymer NPs. Size populations were successfully resolved while providing PSD widths for each.

A simple HDC-UV method comprising deconvolution and negotiation of system's band broadening allows for the size and PSD-width determination of polymeric NPs, even when showing multimodal distributions.