Combination of gravitational SPLITT fractionation and field-flow fractionation for size-sorting and characterization of sea sediment

A combination of gravitational split-flow thin (SPLITT) fractionation and sedimentation/steric field-flow fractionation (Sd/StFFF) has been used for continuous size-sorting of a sediment sample and for size analysis of the collected fractions. An IAEA (International Atomic Energy Agency) sediment material was separated into four size fractions (with theoretical size ranges <1.0, 1.0–3.0, 3.0–5.0, and >5.0 m in diameter) by means of a three-step gravitational SPLITT fractionation (GSF) for which the same GSF channel was used throughout. The GSF fractions were collected and examined by optical microscopy (OM) and by Sd/St FFF. The mean diameters of the GSF fractions measured by OM were within the size interval predicted by GSF theory, despite the theory assuming that all particles are spherical, which is not true for the sediment particles. The Sd/St FFF results showed that retention shifted toward shorter elution time (or larger size) than expected, probably because of the shape effect. The results from GSF, OM, and Sd/StFFF are discussed in detail.     

Determination of thickness, aspect ratio and size distributions for platey particles using sedimentation field-flow fractionation and electron microscopy

Sedimentation field-flow fractionation (SdFFF) can be used to prepare fractions of very narrow mass range for electron microscopic (EM) analysis. Assuming the particle density is the same for all particles within that fraction the equivalent spherical diameter for the particles can be calculated from SdFFF theory. Integration of the micrograph image of each particle yields an area measurement which, when used in conjunction with the equivalent spherical particle diameter (from SdFFF), provides information about the particle thickness and aspect ratio. Thus SdFFF-SEM can be used to provide detailed information about clay morphology across the particle size distribution of the sample. Three clay minerals have been studied using the methodologies outlined in this paper. The aspect ratio for the Purvis School Mine kaolinite ranged from 2.8–5.9, for RM30 illite from 11.3–24.3, and for Muloorina illite from 3.1–4.3.     

Formic acid solubilization of marine biological tissues for multi-element determination by ETAAS and ICP–AES

A simple, fast method is described for the determination of Ag, As, Cd, Cu, Cr, Fe, Ni, and Se in marine biological tissues by electrothermal atomic-absorption spectrometry (ETAAS) and Na, Ca, K, Mg, Fe, Cu, and Mn by inductively coupled plasma–atomic emission spectrometry (ICP–AES). Solubilization of the biological tissue was achieved by using formic acid with vortex mixing followed by heating to 50°C in an ultrasonic bath. Once solubilized, the tissues were diluted to an appropriate volume with water for analysis. Aliquots were sampled into a graphite furnace and ICP–AES using a conventional autosampler. The method was validated by use of biological certified reference materials from NRC, DORM-2, DOLT-2, DOLT-3, LUTS-1, TORT-2, and NIST SRMs 1566b and 2976. Simplicity and reduced sample-preparation time prove to be the major advantages to the technique.     

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.     

Coupling gravitational and flow field-flow fractionation, and size-distribution analysis of whole yeast cells

This work continues the project on field-flow fractionation characterisation of whole wine-making yeast cells reported in previous papers. When yeast cells are fractionated by gravitational field-flow fractionation and cell sizing of the collected fractions is achieved by the electrosensing zone technique (Coulter counter), it is shown that yeast cell retention depends on differences between physical indexes of yeast cells other than size. Scanning electron microscopy on collected fractions actually shows co-elution of yeast cells of different size and shape. Otherwise, the observed agreement between the particle size distribution analysis obtained by means of the Coulter counter and by flow field-flow fractionation, which employs a second mobile phase flow as applied field instead of Earths gravity, indicates that yeast cell density can play a major role in the gravitational field-flow fractionation retention mechanism of yeast cells, in which flow field-flow fractionation retention is independent of particle density. Flow field-flow fractionation is then coupled off-line to gravitational field-flow fractionation for more accurate characterisation of the doubly-fractionated cells. Coupling gravitational and flow field-flow fractionation eventually furnishes more information on the multipolydispersity indexes of yeast cells, in particular on their shape and density polydispersity.     

Comparative Toxicity of Fly Ash: An In Vitro Study

Fly ash produced during coal combustion is one of the major sources of air and water pollution, but the data on the impact of micrometer-size fly ash particles on human cells is still incomplete. Fly ash samples were collected from several electric power stations in the United States (Rockdale, TX; Dolet Hill, Mansfield, LA; Rockport, IN; Muskogee, OK) and from a metallurgic plant located in the Russian Federation (Chelyabinsk Electro-Metallurgical Works OJSC). The particles were characterized using dynamic light scattering, atomic force, and hyperspectral microscopy. According to chemical composition, the fly ash studied was ferro-alumino-silicate mineral containing substantial quantities of Ca, Mg, and a negligible concentration of K, Na, Mn, and Sr. The toxicity of the fly ash microparticles was assessed in vitro using HeLa cells (human cervical cancer cells) and Jurkat cells (immortalized human T lymphocytes). Incubation of cells with different concentrations of fly ash resulted in a dose-dependent decrease in cell viability for all fly ash variants. The most prominent cytotoxic effect in HeLa cells was produced by the ash particles from Rockdale, while the least was produced by the fly ash from Chelyabinsk. In Jurkat cells, the lowest toxicity was observed for fly ash collected from Rockport, Dolet Hill and Muscogee plants. The fly ash from Rockdale and Chelyabinsk induced DNA damage in HeLa cells, as revealed by the single cell electrophoresis, and disrupted the normal nuclear morphology. The interaction of fly ash microparticles of different origins with cells was visualized using dark-field microscopy and hyperspectral imaging. The size of ash particles appeared to be an important determinant of their toxicity, and the smallest fly ash particles from Chelyabinsk turned out to be the most cytotoxic to Jukart cells and the most genotoxic to HeLa cells.     

Speciation of nickel,copper, zinc,and manganese in different edible nuts: a comparative study of molecular size distribution by SEC–UV–ICP–MS

Molecular size distribution patterns of Cu, Mn, Ni, and Zn were determined in several nut species by size-exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP–MS) for detection. The molecular weight (MW) fractionation of the different metals was performed with a Superdex Peptide column, injecting 100 L of the extracted solutions. The association of the elements with different MW fractions was observed with sequential detection by UV and ICP–MS. Various separation conditions were evaluated to obtain proper resolution and reproducible results with the size-exclusion column. Complete MW information of the elemental fractions in the nut samples was obtained within a retention time of 30 min. Fractionation of the above mentioned elements was done in nine different nut species commonly found in commercial markets. Variability of the fractionation patterns for two different extraction media, 0.05 mol L^–1 NaOH and 0.05 mol L^–1 HCl, was evaluated for every nut sample. Differences in the elemental fractionation patterns were found depending on the extraction procedure, nut species, and the type of element studied. It was also observed that the elements studied showed predominant association with high MW fractions when extracted with basic solution whereas with acidic extraction media only low MW fractions were obtained.     

Leaching Studies of Inorganic and Organic Compounds from Fly Ash

Fly ash is produced in massive quantities by fossil fuel based power plants and waste incinerators, and contains high levels of potentially toxic chemicals. Various leaching tests exist to determine the available fractions, but the outcome is strongly dependent on the experimental conditions, and these have not yet been harmonised at the international level. In order to test existing protocols for heavy metals, several intercomparisons were organised within the framework of an EU-INCO project "ANALEACH", in which seven institutes from five countries participated. Two existing reference materials were made available for the project and test batches of two new fly ash reference materials were produced. Availability tests, leaching tests and pH-stat tests were studied and critical steps in the procedures were identified. Fly ashes can also contain large amounts of inorganic sulphur and nitrogen compounds, and the determination and leaching behaviour of these compounds were also studied. In one intercomparison for metals, inorganic S and N-compounds were also included. A five-step leaching test was optimised for fly ash in order to link metal fractions to different types of binding. Column leaching experiments were carried out to investigate leaching from fly ash into soil, mimicking the effects of (acid) rain on fly ash deposited on topsoil after atmospheric transport. The major fraction of the leached metal ions was retained by the soil. Also large numbers of organic compounds (including many toxic ones) were identified in fly ash extracts, especially in city waste incinerator ash. Leaching procedures based on ultrasonic extraction were developed for organic compounds and an intercomparison exercise was organised. In a field study at the river Nitra(Slovakia) numerous organic pollutants were found at elevated levels downstream from a major fly ash dump site.     

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.     

Sedimentation field flow fractionation monitoring of bimodal wheat starch amylolysis

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.     

Investigating the relationship between cell cycle stage and diosgenin-induced megakaryocytic differentiation of HEL cells using sedimentation field-flow fractionation

Differentiation therapy could be one strategy for stopping cancer cell proliferation. A plant steroid, diosgenin, is known to induce megakaryocytic differentiation in human erythroleukemia (HEL) cells. In recent studies, the use of sedimentation field-flow fractionation (SdFFF) allowed the preparation of subpopulations that may differ in regard to sensitivity to differentiation induction. The specific goal of this study was to determine the relationship between cell cycle stage and sensitivity to megakaryocytic differentiation induction of HEL cells. After first confirming the capacity of diosgenin to specifically select targets, hyperlayer SdFFF cell sorting was used to prepare fractions according to cell cycle position from crude HEL cells. The sensitivities of these fractions to diosgenin-induced differentiation were then tested. The coupling of SdFFF cell separation to imaging flow cytometry showed that G1-phase cells were more sensitive to differentiation induction than S/G2M-phase cells, confirming the relationship between cell status at the start of induction, the extent of the biological event, and the potential of SdFFF in cancer research.     

Observation of salt-induced β-lactoglobulin aggregation using sedimentation field-flow fractionation

Sedimentation field-flow fractionation (SdFFF) was applied in order to characterize particle sizes of β-lactoglobulin aggregates induced by Ca²⁺ or Zn²⁺. Aggregation induced by Zn²⁺ was faster than that induced by Ca²⁺. Effects of Zn²⁺ and β-lactoglobulin concentrations, as well as contact time, on the aggregation of β-lactoglobulin were examined. All factors exhibited a combined effect on the size of aggregates, whereby larger aggregates were obtained at increased concentrations of Zn²⁺ and β-lactoglobulin. At fixed concentrations of 2% (w/v) β-lactoglobulin and 10 mM Zn²⁺, the particle size of the aggregates increased from 0.19 μm (at 15 min) to 0.38 μm (at 2880 min). Further, a hyphenated technique of SdFFF and inductively coupled plasma–optical emission spectrometry (ICP–OES) was used to examine whether intermolecular ionic bridges take part in salt-induced β-lactoglobulin aggregation. With SdFFF–ICP–OES, protein–cation–protein cross-linkages were observed for β-lactoglobulin aggregation induced by Zn²⁺, but not for that induced by Ca²⁺.      

Chemical characterization of density separated submicrometer coal fly ash

A new geometrical particle size separation method in the range of submicrometer has been developed for coal fly ash. Coal fly ash particles prefractionated less than 5m in geometrical diameter were fractionated with a coupling of supersonical dispersion in methyl alcohol and filtration through antistatic Nucleopore filter into >1.0, 1.0–0.8, 0.8–0.6m.Submicrometer (1.0–0.6m) and micrometer coal fly ash (25–20m) were separated into six density fractions and their compositions measured by SEM-EDX were compared. For the both size fractions, for the low density fractions (<2.8 g/cm³) consisted mainly of aluminosilicates and the fractions of 2.8–3.2 g/cm³ consisted of oxides of aluminum, silicon and calcium, and in the fraction above 3.2 g/cm³ iron was rich. In submicrometer fly ash, phosphor and sulfur concentrated to the particles rich in calcium. In the heaviest fraction, the particles containing over 30 wt% titanium as oxide observed among the particles rich in iron. These fractions were revealed not as a single component, but as mixtures of several components.     

Method development of combining pressurized liquid extraction and off-line HPLC fractionation with a porous graphite carbon column for the analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in environmental samples

A method was developed for analysis of PCDD/Fs in fly ash and soil samples by using the combination of pressurized liquid extraction (PLE) and off-line HPLC fractionation with a porous graphite carbon (PGC) column, followed by analysis using HRGC/HRMS. The samples were automatically extracted by PLE. A multi-layer silica column was used for the preliminary cleanup. Then off-line fractionation was applied for the separation of PCDD/Fs and the collected PCDD/Fs fraction was analyzed by HRGC/HRMS. The use of solvent back-flushing at 70°C successfully solved the carry-over problem of PGC column. The procedure of single extraction and cleanup provided advantages of high automation and significant reduction of solvent and time compared to conventional methods. The method was validated by certified materials of fly ash and industrial sandy soil, and applied for the analysis of fly ash samples collected from three two-ton medical waste incinerators in Beijing and one farm soil sample collected in the vicinity of the incinerators.     

Development of a downscale sedimentation field flow fractionation device for biological event monitoring

Classically described as a macroscale size-density based method, Sedimentation field flow fractionation (SdFFF) has been successfully used for cell sorting. The goal of this study was to develop a new SdFFF device for downscale applications, in particular for oncology research to rapidly monitor chemical biological event induction in a cell line. The development of a downscale SdFFF device required reduction of the separation channel volume. Taking advantage of a newly laboratory designed apparatus, channel volume was successfully decreased by reducing both length and breadth. To validate the apparatus and method, we used the well-known model of diosgenin dose-dependent induction of apoptosis or megakaryocytic differentiation in HEL cells. After a minute scale acquisition of a reference profile, the downscale device was able to perform fast, early, significant and reproducible monitoring of apoptosis and differentiation, two important biological mechanisms in the field of cancer research.     

燃烧福建无烟煤的循环流化床锅炉飞灰及其未燃炭分析

以三个燃烧福建无烟煤的商业CFB锅炉电厂飞灰为研究对象，分析飞灰的粒径和含碳量分布；观察并区分了飞灰中煤炭、烟怠、半焦和灰渣等颗粒的表面形貌；测定了飞灰炭的反应活性并与入炉煤作比较；并研究了飞灰炭的来源。结果表明，采用单级分离装置的CFB锅炉，粒径在0.0385mm～0.0500mm的飞灰质量最多，未燃炭份额也最高；而采用双级分离装置的CFB锅炉，飞灰质量分布和飞灰炭份额的峰值则出现在粒径为0.0500mm~0.0750mm处。飞灰主要由三种形貌的颗粒组成，颗粒状的未燃炭、絮团状的灰渣、和介于两者之间的半焦。与入炉煤相比，飞灰炭的反应活性较高，主要来源于入炉煤中的细粉和燃烧早期因破碎和磨蚀而产生的、来不及在炉膛中燃尽的细小含炭颗粒。入炉煤中易破碎、反应性较高的亮煤是构成电厂CFB锅炉飞灰炭的主要成分。     

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.     

Dimensional and elemental characterization of suspended particulate matter in natural waters: quantitative aspects in the integrated ultrafiltration, splitt-flow thin cell and inductively coupled plasma–atomic emission spectrometry approach

The present study investigates the quantitative aspects of an analytical procedure for the trace element characterization of suspended particulate matter (SPM) in natural waters. The procedure consists of the following steps: (1) ultrafiltration (UF) concentration; (2) splitt-flow thin (SPLITT) cell fractionation (SF) into different micronic–submicronic dimensional ranges; and (3) inductively coupled plasma–atomic emission spectrometry (ICP–AES) elemental determination on both the separated fractions and the bulk phase. One specific feature of the UF/SF steps is that they are gentle and thus preserve the complexity of the colloidal features of SPM samples as far as possible. The investigation was performed on a real SPM sample (Po River, Italy). Two SF modes were considered: the so called conventional SPLITT fractionation (CSF) mode and the full feed depletion SPLITT fractionation (FFDSF) mode. These differ in terms of resolution, time (both better in CSF as compared to FFDSF) and operating mode (FFDSF does not require a diluting carrier). Quantitative aspects of the UF step recovery and of the CSF and FFDSF modes were investigated in terms of total mass balance proving that only the FFDSF mode is currently satisfactory for quantitative purposes. Mass balance versus the following elements: Cd, Cr, Cu, Mn, Ni and Pb, was performed using ICP–AES over the 0.2–1.5 and 1.5–20 μm FFDSF SPM fractions, proving that the analytical procedure based on UF/FFDSF/ICP–AES is consistent and useful in the investigation of trace element distribution in different SPM dimensional ranges versus that of the bulk phase. The relevance of aggregation–solubility equilibria concerning colloids of SPM phase is emphasized and further improvement of the procedure is discussed.     

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.     

Fractionation of calcium and magnesium in honeys, juices and tea infusions by ion exchange and flame atomic absorption spectrometry

An analytical procedure was proposed to study the operational fractionation of Ca and Mg in bee honeys, fruit juices and tea infusions. The protocol devised was based on the solid phase extraction of distinct metal fractions on different sorbents, namely strong acidic cation exchanger Dowex 50Wx4, weak acidic cation exchanger Diaion WT01S and strong basic anion exchange resin Dowex 1x4. For the evaluation of the amounts of the metal fractions distinguished, a flame atomic absorption spectrometry was used off-line prior to the determination of Ca and Mg concentrations in the effluents obtained. It was established that Ca and Mg are mostly present in the analysed samples in the form of cationic species (96–100%). The accuracy of the entire fractionation scheme and sample preparation procedures involved was verified by the performance of the recovery tests.