Instrument and Method to Determine the Electrophoretic Mobility of Nanoparticles and Proteins by Combining Electrical and Flow Field-Flow Fractionation

Abstract

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.

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…The most recent technique in the family is the electrical asymmetric flow FFF (EAF4) where, in addition to the secondary flow, an electric field is generated across the channel. In EAF4 the separation is ruled both by size and electrophoretic mobility, which allows the simultaneous determination of size and particle surface charge [23].…”

Size-exclusion chromatography of metal nanoparticles and quantum dots

“…A novel nanoparticle characterization method was recently introduced, namely electrical asymmetrical flow FFF. The underlying principles and instrumentation, along with application examples, can be found in reference [63] and in the supporting information accompanying that paper.…”

Characterization of Gold Nanorods Conjugated with Synthetic Glycopolymers Using an Analytical Approach Based on spICP-SFMS and EAF4-MALS

“…When combined with an adequate detection approach, it provides information on particle size, size distribution, shape, and chemical composition (stoichiometry) of the particles studied [ 25 ]. In addition, electrical asymmetric-flow field-flow-fractionation hyphenated to a multi-angle light scattering detector (EAF4-MALS) combines high-resolution separation with surface charge (Zeta potential) measurement [ 26 , 27 ]. As such, it presents a promising tool for characterization of GNRs conjugated with synthetic glycopolymers.…”