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Flow field-flow fractionation (FFF) and photon correlation spectroscopy (PCS) were used to determine the size distribution of magnetic cell sorting (MACS) microbeads. The nominal 50 nm microbeads, when coupled to monoclonal Anti-Fluorescein isothiocyanate (FITC), have an average diameter of 125 nm. FFF results showed that two different batches of microbeads had similar average diameters.

In order to better understand the factors that modulate the size and reactivity of high molecular weight organic matter dissolved in seawater, fluorescently labeled dextrans were used as the model compounds whose molecular weight distributions were monitored by flow field-flow fractionation (flow FFF or F1FFF) during incubations in seawater matrices. Two fluorescein isothiocyanate (FITC) labeled dextrans (145k and 2M Da) were incubated in whole seawater (natural microbial population and natural dissolved organic matter (DOM) present), 0.02 μm filtered seawater (all microorganisms removed, but natural DOM largely unaltered), and UV-oxidized seawater (no microorganisms or natural DOM present). Flow FFF fractograms of the two FITC-dextrans incubated in UV-oxidized seawater showed no changes, signifying that the dextrans did not undergo any alteration or aggregation. The dextrans incubated in filtered seawater with natural DOM present resulted in fractograms that are shifted to higher retention times...

Two field-flow fractionation (FFF) techniques, flow FFF and thermal FFF, have been used as complementary techniques in a study of core-shell latex particles. Different types of physicochemical information about the particles can be obtained since the fields used in the two techniques interact with different particle properties. The hydrodynamic diameters (and diameter distributions) of the latex particles were measured as a function of pH using flow FFF. Retention in thermal FFF was found to depend on shell composition. These FFF techniques, used together, are shown to be capable of characterizing both the physical size and shell composition of this important class of particles.

A flow field-flow fractionation (FIFFF) system capable of accurate and reproducible flowrate programming has been assembled and tested. This modified system consists of three computer-controllable pumps, two that regulate the incoming and outgoing cross flowrates and one that controls the channel flowrate. The system also has a computer-linked balance that allows for instantaneous measurement of the cross flowrate. Programming of the field strength (or cross flowrate) was used to decrease the analysis time and improve detectability of late eluting peaks in FIFFF analysis. A six component latex mixture, with microspheres ranging from 20 to 426 nm in size, was used as a test material. With programming, this mixture can be separated in 8 min. © 1995 John Wiley & Sons, Inc.

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.

Field-flow fractionation is coming of age as a family of analytical methods for separating and characterizing macromolecules, nanoparticles, and particulates. The capabilities and versatility of these techniques are discussed in light of the challenges that are being addressed in analyzing nanometer-sized sample components and the insights gained through their use in applications ranging from materials science to biology. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

Field and flow programming and their combination, dual programming, are shown to extend the particle size range to which a single flow/hyperlayer field-flow fractionation (FFF) run is applicable to approximately 1-50 microns. The rationale for programming flow/hyperlayer FFF (or other forms of lift hyperlayer FFF) is to expand the diameter range of micron size particles that can be resolved in a single run. By contrast, the reason for programming normal-mode FFF, the only kind of programming previously realized in FFF, is to reduce the analysis time of submicron particle samples of considerable size variability. These differences are explained in detail in relationship to the basic mechanisms governing retention in normal, steric, and lift hyperlayer FFF. Experiments are described in which field, flow, and dual programming are used to expand the accessible diameter range of flow/hyperlayer FFF. An example is shown in which 11 sizes of latex microspheres in the 2-48-microns diameter range are separated by dual programming in 11 min.

Field-flow fractionation (FFF) was conceived by J. Calvin Giddings in 1966 as a separation and characterization method for macromolecules, colloids, and particulates. Similar to chromatography, sample migration is caused by differential interaction with a field acting along an axis orthogonal to that of the transport liquid. Unlike chromatography, where separation is achieved by solutes partitioning between mobile and stationary phases, separation in FFF arises from the distribution of sample components in fluid laminae flowing at different velocities in a single phase. The different flow velocities, described by a parabolic profile, arise from the high aspect ratio of the FFF channel. Different types of fields can be used in FFF as long as they interact with some physicochemical property of the sample. The FFF channel design makes it highly suited for analyses of fragile aggregates, high-molecular-weight polymers, and gels. In comparison with packed columns, shear rates in the channel and the probability of plugging the channel are low. The ability of thermal FFF to differentiate polymers and latexes of different bulk and surface composition is unique among currently used separation techniques. The FFF family of techniques can provide a great deal of information about the sample but an initial time investment is often required for method development. In this article, the fundamental mechanism of FFF is shown at play in the separation and characterization of polymers and rubbers by the two techniques par excellence in this field: flow FFF and thermal FFF. Keywords: field-flow fractionation; flow FFF; thermal FFF; polymers; water-soluble polymers; copolymers; high molecular weight; ultrahigh molecular weight; chemical composition; thermal diffusion; diffusion coefficient; size-exclusion chromatography

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.

A new particle sizing and counting method based on the coupling of flow field-flow fractionation (FFF) with dual-sensor, single particle optical sensing (SPOS) detection is reported. The integration of FFF and SPOS systems was accomplished by means of a dilution interface that preserved the resolution of FFF-separated particles. Analysis of a model mixture of polystyrene latex standards of different diameters established that the FFF-SPOS system can resolve particles into discrete peaks for subsequent particle counting. Application of this method for the analysis of a colloidal silica standard demonstrated its use for materials commonly employed as CMP abrasives. Further development and refinement of the technique will enable compositional and structural analyses of heterogeneous large particle populations constituting commercial CMP slurries.

This review summarizes developments and applications of flow and thermal field-flow fractionation (FFF) in the areas of macromolecules and supramolecular assemblies. In the past 10 years, the use of these FFF techniques has extended beyond determining diffusion coefficients, hydrodynamic diameters, and molecular weights of standards. Complex samples as diverse as polysaccharides, prion particles, and block copolymers have been characterized and processes such as aggregation, stability, and infectivity have been monitored. The open channel design used in FFF makes it a gentle separation technique for high- and ultrahigh-molecular weight macromolecules, aggregates, and self-assembled complexes. Coupling FFF with other techniques such as multiangle light scattering and MS provides additional invaluable information about conformation, branching, and identity.

A simple interface based on an oscillating capillary nebulizer (OCN) is described for direct deposition of eluate from a thermal field-flow fractionation (ThFFF) system onto a matrix-assisted laser desorption/ionization (MALDI) probe. In this study, the polymer-containing eluent from the ThFFF system was mixed on-line with MALDI matrix solution and deposited directly onto a moving MALDI probe. The result was a continuous sample track representative of the fractionation process. Subsequent off-line MALDI-mass spectrometry analysis was performed in automated and manual modes. Polystyrene samples of broad polydispersity were used to characterize the overall system performance. The OCN interface is easy to build and operate without the use of heaters or high voltages and is compatible with any MALDI probe format.

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.

Self-assembled cationic lipid-DNA complexes have shown an ability to facilitate the delivery of heterologous DNA across outer cell membranes and nuclear membranes (transfection) for gene therapy applications. While the size of the complex and the surface charge (which is a function of the lipid-to-DNA mass ratio) are important factors that determine transfection efficiency, lipid-DNA complex preparations are heterogeneous with respect to particle size and net charge. This heterogeneity contributes to the low transfection efficiency and instability of cationic lipid-DNA vectors. Efforts to define structure-activity relations and stable vector populations have been hampered by the lack of analytical techniques that can separate this type of particle and analyze both the physical characteristics and biological activity of the resulting fractions. In this study, we investigated the feasibility of flow field-flow fractionation (flow FFF) to separate cationic lipid-DNA complexes prepared at various lipid-DNA ratios. The compatibility of the lipid-DNA particles with several combinations of FFF carrier liquids and channel membranes was assessed. In addition, changes in elution profiles (or size distributions) were monitored as a function of time using on-line ultraviolet, multiangle light scattering, and refractive index detectors. Multiangle light scattering detected the formation of particle aggregates during storage, which were not observed with the other detectors. In comparison to population-averaged techniques, such as photon correlation spectroscopy, flow FFF allows a detailed examination of subtle changes in the physical properties of nonviral vectors and provides a basis for the definition of structure-activity relations for this novel class of pharmaceutical agents.

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.

Flow/hyperlayer field-flow fractionation (FFF) has been successfully applied to the rapid size-based fractionation and characterization of various 3-μm and 5-μm commercial high-performance liquid chromatography silica supports. Because highj-resolution information on both average particle diameter and size distribution was obtained in less than 3 min by using a relatively simple apparatus, the method is attractive for the routine characterization of chromatographic packing material. Flow/hyperlayer FFF is not only faster than most sedimentation methods, it is ideally suited for the characterization of porous particles since separation is based solely on diameter and is independent of density.