Flow field-flow fractionation is a powerful method for the analysis of nanoparticle size distributions, but its widespread use has been hampered by large analyte losses, especially of metal nanoparticles. Here, we report on the colloidal mechanisms underlying the losses. We systematically studied gold nanoparticles (AuNPs) during asymmetrical flow field-flow fractionation (AF4) by systematic variation of the particle properties and the eluent composition. Recoveries of AuNPs (core diameter 12 nm) stabilized by citrate or polyethylene glycol (PEG) at different ionic strengths were determined. We used online UV-vis detection and off-line elementary analysis to follow particle losses during full analysis runs, runs without cross-flow, and runs with parts of the instrument bypassed. The combination allowed us to calculate relative and absolute analyte losses at different stages of the analytic protocol. We found different loss mechanisms depending on the ligand. Citrate-stabilized particles degraded during analysis and suffered large losses (up to 74%). PEG-stabilized particles had smaller relative losses at moderate ionic strengths (1-20%) that depended on PEG length. Long PEGs at higher ionic strengths (≥5 mM) caused particle loss due to bridging adsorption at the membrane. Bulk agglomeration was not a relevant loss mechanism at low ionic strengths ≤5 mM for any of the studied particles. An unexpectedly large fraction of particles was lost at tubing and other internal surfaces. We propose that the colloidal mechanisms observed here are relevant loss mechanisms in many particle analysis protocols and discuss strategies to avoid them.

A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field.

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate.