Natural sample fractionation by FlFFF-MALLS-TEM: sample stabilization, preparation, pre-concentration and fractionation

Two flow field flow fractionation (FlFFF) systems: symmetrical (SFlFFF) and asymmetrical (ASFlFFF) were evaluated to fractionate river colloids. Samples stability during storage and colloids concentration are the main challenges limiting their fractionation and characterization by FlFFF. A pre-fractionation (<0.45 microm) and addition of a bactericide such as NaN3 into river colloidal samples allowed obtaining stable samples without inducing any modification to their size. Stirred cell ultra-filtration allowed colloidal concentration enrichment of 25-folds. Scanning electron microscope (SEM) micrographs confirmed the gentle pre-concentration of river samples using the ultra-filtration stirred cell. Additionally, larger sample injection volume in the case of SFlFFF and on channel concentration in the case of ASFlFFF were applied to minimize the required pre-concentration. Multi angle laser light scattering (MALLS), and transmission electron microscope (TEM) techniques are used to evaluate FlFFF fractionation behavior and the possible artifacts during fractionation process. This study demonstrates that, FlFFF-MALLS-TEM coupling is a valuable method to fractionate and characterize colloids. Results prove an ideal fractionation behavior in case of Brugeilles sample and steric effect influencing the elution mode in case of Cézerat and Chatillon. Furthermore, comparison of SFlFFF and ASFlFFF fractograms for the same sample shows small differences in particle size distributions.     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.     

Hyperlayer separation in hollow fiber flow field-flow fractionation: effect of membrane materials on resolution and selectivity

Hollow fiber flow FFF (HF FlFFF) has recently shown its capability to separate and characterize the size of submicrometer particles and has demonstrated the potential to be developed into a disposable flow FFF channel. In this work, HF FlFFF was used for the hyperlayer separation of micron-sized particles and the separation capability was examined by using various hollow fiber membrane materials (Polysulfones, cPVC, and PAN). From the experiments, PAN (polyacrylonitriles) showed an outstanding performance in particle separation compared to the other membranes. By orienting the fiber module in an upright direction, the upstream flow migration reduced band broadening of eluted peaks. When the efficiency of the PAN hollow fiber system was tested by varying the ratio of outflow-rate to radial flow-rate, it was found that optimum separation in hyperlayer HF FlFFF can be obtained at the ratio of about 6–7. From the examination of retention at or around steric inversion diameter, it was observed that experiments showed a good agreement with predictions by semi-empirical calculation. In hyperlayer HF FlFFF the diameter based selectivity values were shown to be 1.2–1.7 depending on the type of membranes and the field strength (the radial flow-rate) conditions.     

Hollow-fiber flow/hyperlayer field-flow fractionation for the size characterization of airborne particle fractions obtained by SPLITT fractionation

Hollow-fiber flow field-flow fractionation (HF FlFFF) was applied for the separation and size characterization of airborne particles which were collected in a municipal area and prefractionated into four different-diameter intervals >5.0, 2.5-5.0, 1.5-2.5, <1.5 microm) by continuous split-flow thin (SPLIIT) fractionation. Experiments demonstrated the possibility of utilizing a hollow-fiber module for the high-performance separation of supramicron-sized airborne particles at steric/hyperlayer operating mode of HF FlFFF. Eluting particles during HF FlFFF separation were collected at short time intervals (approximately 10 s) for the microscopic examination. It showed that particle size and size distributions of all SPLITT fractions of airborne particles can be readily obtained using a calibration and that HF FlFFF can be utilized for the size confirmation of the sorted particle fraction during SPLITT fractionation.     

Sedimentation field flow fractionation of immunoglobulin A coated polystyrene beads. Influence of carrier composition on complex characterization

The amount of immunoglobulin A (IgA) adsorbed on the surface of two different samples of polystyrene (PS) microbeads was evaluated using differential sedimentation field flow fractionation (SdFFF) analyses. For the first time, the SdFFF separations obtained by using, as mobile phase, solutions common to many biochemical procedures and applications have been compared and discussed. Good separation results were achieved in the different carriers, and the SdFFF gave equivalent mass per particle values in all carriers provided that the pH and ionic strength conditions of the eluents were well controlled. The IgA adsorption process onto PS occurred by maintaining unaltered the capacity of the PS-IgA substrate to selectively recognize anti-IgA (aIgA), as proven by elution of the ternary complex PS-IgA-aIgA and from the monitored lack of reaction when the PS-IgA was placed in contact with aIgE.     

TiO2 colloidal suspension polydispersity analysed with sedimentation field flow fractionation and electron microscopy

Sedimentation field flow fractionation (SdFFF) operated at multi gravitational field is used to analyse a highly polydisperse TiO2 colloidal suspension. From the initial sample, time dependent eluted fractions are collected and submitted to electron microscopy (EM) shape and size analysis. To assess the accuracy of FFF in determining the average size of the different fractions, these are re-introduced into the channel by means of two different procedures, the on-channel concentration of the fractions and the direct re-injection of pre-concentrated fractions (DRI). Both methods appear accurate to determine the average size of every fraction, associated to a lower recovery in the case of DRI. The fractogram band spreading characteristics of the re-introduced fractions are correlated to the particle size distribution measured by EM. After density determination of fractionated particles, the fractogram is calibrated in terms of size and size distribution using data obtained from EM for each fraction. Quantitative analyses, based on particle counting showed high recovery (80-90%) of the eluted species. However, this loss limited the possibility to extend signal information to a quantitative one.     

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.     

Size selectivity in field-flow fractionation: lift mode of retention with near-wall lift force

A simple theoretical model for the size selectivity, S(d), in the lift mode of retention in field-flow fractionation (FFF) is developed on the basis of the near-wall lift force expression. S(d) is made up of two contributions: the flow contribution, S(d,f), arising from the variation of the flow velocity at center of particle due to a change in particle position with particle size, and a slip contribution, S(d,s), arising from the concomitant change in the extent of retardation due to the presence of a nearby channel wall. The slip contribution is minor, but not negligible, and amounts to 10-20% of the overall size selectivity. It contributes to reduce S(d) in sedimentation FFF but to enhance it in flow FFF. S(d) would steadily increase with particle size if the flow profile was linear (Couette flow). Because of the curvature of the flow profile encountered in the classical Poiseuille flow, S(d) exhibits a maximum at some specific particle size. The model predicts a significant difference in S(d) between sedimentation FFF and flow FFF, arising from the different functional dependences of the field force with particle size between these two methods. The predictions are in good agreement with the various S(d) values reported in the literature in both sedimentation and flow FFF. On the basis of the model, guidelines are given for adjusting the operating parameters (carrier flow rate and field strength) to optimize the size selectivity. Finally, it is found that S(d) generally decreases with decreasing channel thickness.     

Flow field-flow fractionation for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems: a critical review

The use of flow field flow fractionation (FlFFF) for the separation and characterization of natural colloids and nanoparticles has increased in the last few decades. More recently, it has become a popular method for the characterization of manufactured nanoparticles. Unlike conventional filtration methods, FlFFF provides a continuous and high-resolution separation of nanoparticles as a function of their diffusion coefficient, hence the interest for use in determining particle size distribution. Moreover, when coupled to other detectors such as inductively coupled plasma-mass spectroscopy, light scattering, UV-absorbance, fluorescence, transmission electron microscopy, and atomic force microscopy, FlFFF provides a wealth of information on particle properties including, size, shape, structural parameters, chemical composition and particle-contaminant association. This paper will critically review the application of FlFFF for the characterization of natural colloids and natural and manufactured nanoparticles. Emphasis will be given to the detection systems that can be used to characterize the nanoparticles eluted from the FlFFF system, the obtained information and advantages and limitation of FlFFF compared to other fractionation and particle sizing techniques. This review will help users understand (i) the theoretical principles and experimental consideration of the FlFFF, (ii) the range of analytical tools that can be used to further characterize the nanoparticles after fractionation by FlFFF, (iii) how FlFFF results are compared to other analytical techniques and (iv) the range of applications of FlFFF for natural and manufactured NPs.     

Poly(l‐lactic acid)‐modified silica stationary phase for reversed‐phase and hydrophilic interaction liquid chromatography

Poly(l ‐lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(l ‐lactic acid)‐modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(l ‐lactic acid) chain. The poly(l ‐lactic acid)‐silica column was characterized in reversed‐phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(l ‐lactic acid)‐silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited “U‐shaped” curves, which was an indicator of reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography mixed‐mode retention behavior. In addition, carbonyl groups included into the poly(l ‐lactic acid) backbone work as an electron‐accepting group toward a polycyclic aromatic hydrocarbon and provide π–π interactions.     

Characterization of heavy-metal-containing seepage water colloids by flow FFF, ultrafiltration, ELISA and AAS

Heavy-metal-containing humic colloids from seepage water samples of three different municipal waste disposal plants were characterized in terms of molecular weight, hydrodynamic radius and heavy metal content. The size distribution of the colloids was determined with ultrafiltration (UF) and flow field-flow fractionation (flow FFF). The humic colloids in the seepage water samples were characterized using an off-line coupling of flow FFF with an enzyme-linked immunosorbent assay (ELISA) for humic substances. The heavy metals in the different size fractions obtained by UF and flow FFF were determined using atomic absorption spectroscopy (AAS). The colloid size distributions obtained with UF showed a maximum of the distribution in the range 1–10 nm. Seepage water samples with high colloid concentrations had a second maximum in the range 0.1–1 m. The determination of colloid size with flow FFF gave different colloid size distributions for the three waste disposal seepage waters, whereas water from the oldest disposal plant showed the smallest colloid size with a maximum at 0.9 nm and water from the most recent plant showed the largest colloid size with a maximum at 1.3 nm. The determination of particle classes with regard to the chemical composition using a scanning electron microscope with energy dispersive X-ray fluorescence detector (SEM/EDX) showed that the particles can be divided into five classes: silicates, insoluble salts, iron(hydr)oxides, carbonates and organic colloids (humic colloids). Flow FFF/ELISA off-line coupling showed that the most frequently occurring colloids of the seepage waters were humic colloids and investigation of the UF-size-fractions with AAS showed that up to 77% of the total mass of a heavy metal element can be bound to particles, especially to humic colloids. Additionally, the distributions of the heavy metals Fe, Cu and Zn were investigated with flow FFF/AAS off-line coupling. These results also showed that a substantial amount of these heavy metals (up to 46%) was bound to humic colloids.     

Sedimentation field flow fractionation and flow field flow fractionation as tools for studying the aging effects of WO₃ colloids for photoelectrochemical uses

WO₃ colloidal suspensions obtained through a simple sol–gel procedure were subjected to a controlled temperature aging process whose time evolution in terms of particle mass and size distribution was followed by sedimentation field flow fractionation (SdFFF) and flow field flow fractionation (FlFFF). The experiments performed at a temperature of 60 °C showed that in a few hours the initially transparent sol of WO₃ particles, whose size was less than 25 nm, undergoes a progressive size increase allowing nanoparticles to reach a maximum equivalent spherical size of about 130 nm after 5 h. The observed shift in particle size distribution maxima (SdFFF), the broadening of the curves (FlFFF) and the SEM–TEM observations suggest a mixed mechanism of growth-aggregation of initial nanocrystals to form larger particles. The photoelectrochemical properties of thin WO₃ films obtained from the aged suspensions at regular intervals, were tested in a biased photoelectrocatalytic cell with 1 M H₂SO₄ under solar simulated irradiation. The current–voltage polarization curves recorded in the potential range 0–1.8 V (vs. SCE) showed a diminution of the maximum photocurrent from 3.7 mA cm⁻² to 2.8 mA cm⁻² with aging times of 1 h and 5 h, respectively. This loss of performance was mainly attributed to the reduction of the electroactive surface area of the sintered particles as suggested by the satisfactory linear correlation between the integrated photocurrent and the cyclic voltammetry cathodic wave area of the W(VI) → W(V) process measured in the dark.     

Size-based analysis of incinerator fly ash using gravitational SPLITT fractionation, sedimentation field-flow fractionation, and inductively coupled plasma-atomic emission spectroscopy

Fly ash has been regarded as hazardous because of its high adsorption of toxic organic and/or inorganic pollutants. Fly ash is also known to have broad distributions of different chemical and physical properties, such as size and density. In this study, fly ash emitted from a solid waste incinerator was pre-fractionated into six sub-populations by use of gravitational SPLITT fractionation (GSF). The GSF fractions were then analyzed by sedimentation field-flow fractionation (SdFFF) and ICP–AES. SdFFF analysis showed the fly ash has a broad size distribution ranging from a few nanometers up to about 50 µm. SdFFF results were confirmed by electron microscopy. Inductively coupled plasma–atomic emission spectroscopy (ICP–AES) analysis of the GSF fractions showed the fly-ash particles contain a variety of inorganic elements including Ca, Si, Mg, Fe, and Pb. The most abundant in fly ash was Ca, followed by Si then Mg. No correlations were found between trace element concentration and particle size.     

Characterization of particle size and size distribution of multi-sized polymer lattices by centrifugation plus quasielastic light scattering

 A method for characterizing the particle size and size distribution of multi-sized polymer lattices was developed by combining quasielastic light scattering (QELS) with a centrifuge. Lattices were first fractionated by centrifugation and the different populations of particles were separated in successive steps. The size of these particles was measured by QELS, and the mass fraction of the particles was determined gravimetrically. The particle size and size distribution of several blends of monodisperse lattices and two industrial multi-sized lattices have been measured by this method. The results show that the particle sizes obtained using this method are in good agreement with the expected particle diameters, and that the relative amounts of the different groups of particles in the blends can be accurately determined. The efficiency of centrifuge-QELS was also confirmed by comparison with other techniques such as transmission electron microscopy (TEM), QELS, field-flow fractionation (FFF) and capillary hydrodynamic fractionation (CHDF). However, this method is not suited for the analysis of continuous, broad distributions or mixtures with a high number of different populations. It is better suited for distributions with a small number of families of particles, and then can be used for preparative propose on a laboratory scale. Received: 9 October 1996 Accepted: 7 July 1997     

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.     

Characterization of functionalized styrene-butadiene rubber by flow field-flow fractionation/light scattering in organic solvent

Flow field-flow fractionation (FlFFF) using an organic solvent as mobile phase has been effectively utilized for the separation and characterization of functionalized styrene-butadiene rubbers (SBR) that are polymerized and followed by coupling reaction in solution. Separation of broad molecular weight SBR was accomplished by an asymmetrical FlFFF channel in THF under field programming and the molecular weight distribution (MWD) of the SBR sample was determined by on-line measurement of light scattering. In this study, FlFFF has been utilized to characterize high-MW functionalized SBR from the low-MW non-functionalized molecules which were used for coupling reaction to produce high-MW functionalized SBRs, and to determine the coupling number of the functionalized SBRs depending on the type of the coupling reagents. The resulting MWD of the SBR samples prepared by the different coupling reagents (SnCl(4) and a polydimethylsiloxane compound) were compared.     

无固定相分离-电感耦合等离子体质谱法在环境中痕量金属纳米颗粒分析中的应用

环境中金属纳米颗粒的分析检测不仅需要关注其浓度和化学组成,还需要对其形状、粒径和表面电荷等进行表征.此外,环境中金属纳米颗粒的分析需要解决其低赋存浓度以及复杂基质干扰的难题.无固定相分离技术与电感耦合等离子体质谱(ICP-MS)的在线联用,具有较强的颗粒分离能力和较低的元素检出限,能够快速准确地提供金属纳米颗粒的粒径分...     

Application of flow field-flow fractionation for the characterization of macromolecules of biological interest: a review

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.     

Characterization of winemaking yeast by cell number-size distribution analysis through flow field-flow fractionation with multi-wavelength turbidimetric detection

Yeasts are widely used in several areas of food industry, e.g. baking, beer brewing, and wine production. Interest in new analytical methods for quality control and characterization of yeast cells is thus increasing. The biophysical properties of yeast cells, among which cell size, are related to yeast cell capabilities to produce primary and secondary metabolites during the fermentation process. Biophysical properties of winemaking yeast strains can be screened by field-flow fractionation (FFF). In this work we present the use of flow FFF (FlFFF) with turbidimetric multi-wavelength detection for the number-size distribution analysis of different commercial winemaking yeast varieties. The use of a diode-array detector allows to apply to dispersed samples like yeast cells the recently developed method for number-size (or mass-size) analysis in flow-assisted separation techniques. Results for six commercial winemaking yeast strains are compared with data obtained by a standard method for cell sizing (Coulter counter). The method here proposed gives, at short analysis time, accurate information on the number of cells of a given size, and information on the total number of cells.