Size sorting of citrate reduced gold nanoparticles by sedimentation field-flow fractionation

Gold nanoparticles (GNPs) have been synthesized through the citrate reduction method; the citrate/gold(III) ratio was changed from 1:1 up to 10:1 and the size of the resulting nanoparticles was measured by sedimentation field-flow fractionation (SdFFF). Experimental data showed that the GNPs size decreases in the ratio range 1:1–3:1 and then increases from 5:1 to 10:1 passing through a plateau region in between, and is almost independent of the precursor solution concentrations. In the zone of minimum diameters the synthetic process does not produce monodispersed GNPs but often multiple distributions, very close in size, are observed as evidenced by the particle size distributions (PSDs) derived from the SdFFF fractograms. UV–vis spectrophotometry, being the most common technique employed in the optical characterization of nanoparticles suspensions, was used throughout this work. A confirmation of the nucleation–aggregation–fragmentation mechanism was inferred from the cross-correlation between UV–vis and SdFFF results.     

Separation of carbon nanotubes by frit inlet asymmetrical flow field-flow fractionation

Flow field-flow fractionation (flow FFF), a separation technique for particles and macromolecules, has been used to separate carbon nanotubes (CNT). The carbon nanotube ropes that were purified from a raw carbon nanotube mixture by acidic reflux followed by cross-flow filtration using a hollow fiber module were cut into shorter lengths by sonication under a concentrated acid mixture. The cut carbon nanotubes were separated by using a modified flow FFF channel system, frit inlet asymmetrical flow FFF (FI AFIFFF) channel, which was useful in the continuous flow operation during injection and separation. Carbon nanotubes, before and after the cutting process, were clearly distinguished by their retention profiles. The narrow volume fractions of CNT collected during flow FFF runs were confirmed by field emission scanning electron microscopy and Raman spectroscopy. Experimentally, it was found that retention of carbon nanotubes in flow FFF was dependent on the use of surfactant for CNT dispersion and for the carrier solution in flow FFF. In this work, the use of flow FFF for the size differentiation of carbon nanotubes in the process of preparation or purification was demonstrated.     

Estimation of the Hamaker constants by sedimentation field-flow fractionation

van der Waals forces are one of several forces that control the adhesion between two materials. These forces are important to quantify in adhesion studies because they are always present and are always attractive. The major problem in calculating the van der Waals interaction between colloidal particles is that of evaluating the Hamaker constant. Hence, an accurately determined Hamaker constant for a given material is needed when interfacial phenomena such as adhesion are discussed in terms of the total potential energy between a particle and a substrate. In this paper, a new simple and accurate methodology for the estimation of the Hamaker constant is introduced. The results are in good agreement with those values found in literature.     

Separation of mitochondria by flow field-flow fractionation for proteomic analysis

Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.     

Solid phase immune reactions as monitored by sedimentation field-flow fractionation

Summary Sedimentation field-flow fractionation was shown to permit the precise evaluation of surface concentrations of human IgG, adsorbed to polystyrene latex spheres of different sizes. Unlike conventional techniques for measuring protein uptake by colloidal substrates, this method allowed a direct evaluation of mass adsorbed per unit area, without the need for potentially destructive labelling reactions. Thus, a four hour adsorption of IgG from a 3–10 fold excess of protein in solution yielded surface concentrations which were 1.4±0.1 mg/m² on a 272 nm latex and 1.9±0.1 mg/m² on a latex with a diameter of 142 nm. The lower value coincided with the estimated monolayer surface coverage. The IgG-PS 272 nm adsorption complex was shown to take up negligible amounts of HSA from a 10 mg/mL solution, while its specific uptake of a polyclonal rabbit anti-human IgG was 2.6 molecules per molecule of adsorbed antigen. The same ratio was found for the smaller particles. The surface concentration of adsorbed second antibody, often crucial in immunodiagnostic quantifications, was therefore found to be significantly enhanced by the increased substrate curvature presented by the smaller particles.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Tandem hollow-fiber flow field-flow fractionation

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem HF5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a "trap" constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses.     

Relative velocity profile and flow-rate in sedimentation field-flow fractionation

Although the classical retention theory is used for interpreting data or optimizing separations in sedimentation field-flow fractionation (SedFFF), as in most other field-flow fractionation techniques, the assumption of a parabolic flow profile on which this theory is based is not rigorously correct in SedFFF because of the curvature of the channel walls. In order to examine quantitatively the influence of this effect, the relative velocity profile in SedFFF is obtained by solving the Navier-Stokes equation in cylindrical coordinates. Discrepancies found in the literature about the definition of the mean velocity in such channels are discussed. Relationships between mean velocity, flow-rate and pressure gradient are given. Approximating the velocity profile by a third-degree polynomial of the radial coordinate which provides the same slope as the exact profile at a reference wall, for small values of δ, the curvature ratio (ratio of the channel thickness to the mean curvature radius), shows that the adjustable parameter of the approximate profile, ν, is equal to ± δ/3, the sign depending on whether the reference wall is the inner or outer wall. The curvature ratio appears to be a good indicator of the error made on retention when using the straight channel approximation in retention theory. The error is quite small for typical SedFFF channels. It may have to be taken into account for precise determinations if thicker channels and/or miniaturized systems are used.     

Characterization of humic materials by flow field-flow fractionation

Several humic materials are characterized by flow field-flow fractionation, including humic acids, a fulvic acid, and aqueous leachates from compost. Hydrophilic and hydrophobic fractions of a compost leachate were also examined. After characterizing molecular weight distributions, the effect of pH and salt concentration on hydrodynamic size is studied. In general, the hydrodynamic size decreases as the pH is lowered. However, humic acids form large aggregates below pH 5. Small amounts of sodium chloride have little effect on the size distributions. In contrast, a little calcium chloride reduces the hydrodynamic size of individual molecules while inducing the formation of oligomers, although severe aggregation is absent. With further additions of calcium chloride, the decrease in hydrodynamic size continues but oligomer formation subsides. Precise characterization of the unaggregated material is hindered by sample penetration through the channel membrane.     

Particle size analysis of perfluorocarbon emulsions in a complex whole blood matrix by sedimentation field-flow fractionation

The goal of this study was to show that sedimentation field-flow fractionation (SdFFF) could be used to study changes in the particle size distribution of perfluorocarbon (PFC) emulsion droplets in ex vivo whole blood samples. A PFC emulsion containing 40% w/v perfluorooctyl bromide (PFOB), 20% w/v perfluorodecyl bromide (PFDB), and 6% w/v egg yolk phospholipid (EYP) was manufactured by high pressure homogenization. The emulsion was infused intravenously to rats at a dose of 2.7 g PFC/kg body weight. Blood samples were collected at 0, 3, 6, and 24 h and analyzed (without additional sample manipulation) by SdFFF. Excellent chromatographic separation between the blood components and PFC emulsion droplets was achieved. After infusion, the particle size distribution broadened slightly. With time, the larger sized droplets were selectively removed by circulating monocytes and tissue resident macrophages of the reticuloendothelial system, causing the particle size distribution to shift to lower median diameters. SdFFF is an excellent technique for studying the in vivo stability of colloidal drug particles following intravenous administration.     

Analysis of kaolin by sedimentation field-flow fractionation and electrothermal atomic absorption spectrometry detection

Summary Electrothermal (graphite furnace) atomic absorption spectrometry (ETAAS), as off-line detector for sedimentation field-flow fractionation (SedFFF) is exploited in clay analysis. Quantitation limits of coupled SedFFF-ETAAS for the determination of a submicron kaolin sample, considered a representative model of natural water suspended particulate, are theoretically established and experimentally validated with reference to Al and Si determination by ETAAS. Complete sample recovery for a 4 μg injected kaolin sample was obtained by keeping adsorption in the SedFFF apparatus to a minimum under control. The best experimental conditions, ensuring sample integrity, were low ionic strength (Na₂CO₃, 10⁻⁵ M), pH 8 and a Teflon covered accumulation wall. Several different runs, revealing the various experimental parameters affecting quantitative recovery, are reported and the different physico-chemical processes affecting such recovery are discussed. The advantages and drawbacks of SedFFF-ETAAS coupling compared with inductively coupled plasma-mass spectrometry (ICP-MS) technique are also discussed.     

Hyperlayer hollow-fiber flow field-flow fractionation of cells

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.     

Size characterization of drug-loaded polymeric core/shell nanoparticles using asymmetrical flow field-flow fractionation

Asymmetrical flow field-flow fractionation (AsFlFFF) was used to determine the size distribution of drug-loaded core/shell nanoparticles which have a lipid core of lecithin and a polymeric shell of a Pluronic. AsFlFFF provided separation of the drug-loaded core/shell nanoparticles from smaller coreless polymeric micelles, thus allowing accurate size analysis of the drug-loaded nanoparticles without interference by the coreless micelles. It was found from AsFlFFF that the drug-loaded nanoparticles have broad size distributions ranging from 100 to 600 nm in diameter. It was also found that, after the nanoparticles had been stored for 70 days, they disappeared as a result of self-degradation. Being a separation technique, AsFlFFF seems to be more useful than transmission electron microscopy or dynamic light scattering for size analysis of core/shell nanoparticles, which have broad and bimodal size distributions. Figure Separation by AsFlFFF     

Characterisation of River Po particles by sedimentation field-flow fractionation coupled to GFAAS and ICP-MS

A procedure for elemental composition determination of water-borne river particles (Po River) on both size-fractionated and unfractionated submicron particles (0.1–1 μm) by graphite furnace atomic absorption spectroscopy (GFAAS) and inductively coupled plasma-mass spectrometry (ICP-MS) is reported. Sample fractionation was performed using sedimentation field-flow fractionation (SdFFF). The distribution of relative mass vs. particle size was determined using UV detection. Fractions were collected over a narrow size range for scanning electron microscopy. With this combination of techniques the mass, elemental composition, and shape distributions can be obtained across the size spectrum of the sample.

The size distributions of the major elements (Al, Fe) were determined by coupling both GFAAS and ICP-MS techniques to the SdFFF. The procedure was validated using a reference clay sample. Satisfactory agreement was found between both the GFAAS and ICP-MS aluminium signal and the UV detector signal. Some discrepancies were observed in the Fe/Al ratios when comparing GFAAS and ICP-MS. Thus further investigation is in order to fully assess the role of SdFFF-ICP-MS and SdFFF-GFAAS techniques for elemental characterisation of aquatic colloids. Both GFAAS and ICP-MS signals unambiguously indicate a significantly higher Fe content in the lower size range, which is consistent with previous investigations.

Trace element levels in unfractionated Po River particles, determined by both GFAAS and ICP-MS, show good agreement. The high levels of Cu, Pb, Cr and Cd found associated with the colloidal particles underlines the significance of the environmental role played by the suspended matter in rivers in both highly industrialised and intensively cultivated areas.     

Particle size analysis by sedimentation field flow fractionation. Performance and application

Recently commercial equipment using sedimentation field flow fractionation (SFFF) has become available for analysis of particulate materials in the sub-micron range. This paper describes the DuPont instrument and discusses its performance. A particular study is described on the comparison of the SFFF technique with that of quasi-elastic light scattering (QELS). The paper concludes that the instrument is capable of measuring particle size distributions with high resolution and precision, provided that no particles above the upper size limit — about 1 μm — are present.     

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation

The separation of wide molecular mass (Mr) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high Mr) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.     

Size characterization and quantification of silver nanoparticles by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry

A method for determining the size of silver nanoparticles and their quantification by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (ICP-MS) is proposed and was tested in consumer products. Experimental conditions were studied in detail to avoid aggregation processes or alteration of the original size distributions. Additionally, losses from sorption processes onto the channel membrane were minimized for correct quantification of the nanoparticles. Mobile phase composition, injection/focusing, and fractionation conditions were evaluated in terms of their influence on both separation resolution and recovery. The ionic strength, pH, and the presence of ionic and nonionic surfactants had a strong influence on both separation and recovery of the nanoparticles. In general, better results were obtained under those conditions that favored charge repulsions with the membrane. Recovery values of 83 ± 8% and 93 ± 4% with respect to the content of silver nanoparticles were achieved for the consumer products studied. Silver nanoparticle standards were used for size calibration of the channel. The results were compared with those obtained by photon correlation spectroscopy and images taken by transmission electron microscopy. The quantification of silver nanoparticles was performed by direct injection of ionic silver standard solutions into the ICP-MS system, integration of the corresponding peaks, and interpolation of the fractogram area. A limit of detection of 5.6 μg L^-1 silver, which corresponds to a number concentration of 1×10¹² L^-1 for nanoparticles of 10 nm, was achieved for an injection volume of 20 μL.