Robust streamlined two-dimensional offline coupling of asymmetrical flow field-flow fractionation and capillary electrophoresis for the separation and quantitation of a five-component submicron particle mixture

Characterization of submicrometer particles by size and surface charge is critical to understanding their property and functionality in industrial formulations. Although the recently reported offline coupling of asymmetrical flow field-flow fractionation and capillary electrophoresis (AF4×CE) shows success in separating a five-component submicrometer particle mixture based on size and mobility, it is a long and tedious process requiring extensive method development and instrument expertise. Moreover, it suffers from low throughput and ambiguity in large particle identification, limiting its widespread acceptance and application in industry. Here we report a new AF4×CE-laser-induced fluorescence (LIF) method which involves minimal method development and eliminates both the large sample injection and subsequent on-capillary stacking in CE separation, to significantly streamline separation and improve analysis throughput. Comprehensive characterization of eight fluorescently labeled five-component submicrometer particle mixture standards, at the concentration ratio of 200 : 200: 133 : 133: 334 and the total concentration level of 1000-10,000 mg/L, and a random sample can be performed within several days. In the lowest-concentration standard, as few as 2.97 × 10² particles at the peak center can be detected on the two-dimensional plot for the 500 nm particles, indicating extremely high detection sensitivity. By eliminating the overstacking issue, large particles can be unambiguously identified on the 2D plot based on the migration time. AF4-light scattering (LS) quantitation demonstrates good accuracy for the artificial five-component sample even when the five components are not completely separated. Moreover, AF4×CE-LIF quantitation is explored for future more challenging mixtures requiring higher separation capacity and resolution.