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The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes.

Thermal field-flow fractionation coupled with online multiangle light scattering, differential refractive index and quasielastic light scattering (ThFFF-MALS/dRI/QELS) was used to simultaneously determine the molecular weight (MW) and composition of polystyrene-poly(n-butyl acrylate) (PS-PBA) and polystyrene-poly(methyl acrylate) (PS-PMA) copolymers. The online measurement of the normal diffusion coefficient (D) by QELS allowed calculation of the copolymer thermal diffusion coefficient (D(T)) of sample components as they eluted from the ThFFF channel. DT was found to be independent of MW for copolymers with similar compositions and dependent on composition for copolymers with similar MW in a non-selective solvent. By using a solvent that is non-selective to both blocks of the copolymer, it was possible to establish a universal calibration plot of DT versus mole fraction of one of the monomer chemistries comprising the copolymer. PS-PBA and PS-PMA linear diblock polymers were determined to vary in composition from 100/0 to 20/80 wt% PS/acrylate and ranged in MWs between 30 and 360 kDa. The analysis of a PS-PBA miktoarm star copolymer revealed a polydisperse material with a weight percent PBA of 50-75% and MW ranging from 100 to 900 kDa. The presented ThFFF-MALS/dRI/QELS method allowed rapid characterization of polymers with MW and chemical distributions in a single analysis.

The challenging task of characterizing polydisperse polymer mixtures possessing ultrahigh molecular weight (MW) polymers and microgels in organic solvents is addressed with thermal field-flow fractionation (ThFFF) and multiangle light scattering-differential refractive index (MALS-dRI) detection. In initial experiments, a 350,000 g/mol poly(methyl methacrylate) (PMMA) standard is used to evaluate the effects of temperature gradient and temperature gradient programming on the measurements. dRI baseline fluctuations caused by temperature programming were minimized by using a mobile phase heater to thermostat connecting tubing. ThFFF-MALS-dRI is then used to separate and characterize a complex polyvinyl acetate (PVAc) sample containing ultrahigh MW polymers and microgels. The open channel design employed by ThFFF allowed the PVAc sample to be analyzed with minimal sample preparation. Unfiltered PVAc sample showed components with MWs close to 10(9) g/mol and root mean square radius r(rms) values approaching 400 nm. The same sample, filtered through a 0.5 microm pore-size membrane, yielded a MW that was at least one order of magnitude lower. These results demonstrated that the common practice of prefiltering polymer samples prior to analysis can lead to erroneously low average MWs and polydispersities. The accuracy of MW and r(rms) calculated using standard light scattering equations developed for small scattering molecules and relatively high wavelengths is also examined.

Until quite recently, theories on thermophoresis of particles predicted very low thermophoretic velocities of metal particles in liquids. This prediction was based on the understanding that the very high thermal conductivities of metals relative to most liquid media resulted in quite low temperature gradients across the metal particle thereby leading to low net force on the particle. In this paper, we report the retention behavior of submicrometer size metal particles of silver (Ag), gold (Au), palladium (Pd) and platinum (Pt) suspended in both aqueous and organic (specifically, acetonitrile and tetrahydrofuran) carrier liquids in thermal field-flow fractionation (ThFFF). The dependence of the metal particle retention on various factors such as particle composition, amount of added electrolyte, carrier liquid composition, field strength, channel thickness, and carrier flow-rate is evaluated and discussed. A comparison in particle retention behavior among equal-sized metal, latex and silica particles is also provided.

The performance of thin (down to 71 microns thick) flow field-flow fractionation (flow FFF) channels was examined at both high channel flow-rates (up to approximately 47 ml/min) and cross flow-rates (up to approximately 11 ml/min). High levels of retention were observed, suggestive of good performance characteristics. Supporting this expectation, four sizes (0.04-0.30 microns) of polystyrene latex microbeads were baseline separated in under 3 min. Nonetheless, a plate height analysis showed that performance was still less than theoretically expected. Fast steric-hyperlayer separations of larger latex beads were observed in the same systems. Furthermore, it was shown that the steric inversion diameter was shifted down to approximately 0.23 micron thus expanding the size range to which this FFF mode is applicable. The steric inversion phenomenon observed using narrow latex standards was shown to be consistent with that found for a polydisperse polyvinylchloride latex.