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Results for “Fractionation, Field Flow”

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PubMed2000

Separation of different physical forms of plasmid DNA using a combination of low electric field strength and flow in porous media: effect of different field gradients and porosity of the media.

The retention of different physical forms of DNA by an electric field in a chromatography system was studied. We were able to effectively separate the supercoiled and the open circular forms of plasmid DNA using this type of electrochromatography system. Chromatography columns were packed with porous beads, and an axial electric field was applied so that convective buffer flow opposed the direction of electrophoresis of the DNA. A model system composed of approximately equal amounts of the super-coiled and open circular forms of the plasmid pBR 322 (4322 base pairs) was used to test the separation. Chromatography beads (agarose-based) with different porosities were used to determine the effect of the stationary phase on the separation. The porous media did not have a major effect on the separation, but the best separations were obtained using porous chromatography media made with the highest agarose concentration (10% agarose). Selective elution of plasmid DNA with different forms was obtained by either increasing the flow rates or decreasing the electric field strength (by steps or a gradient). In all the separations, the more compact supercoiled form of the plasmid was retained less strongly than either the open circular form (nicked) or the linear form. High molecular weight host genomic DNA was more strongly retained than the plasmid DNA. Increasing the ionic strength of the buffer improved resolution and capacity. The capacity of the separation was determined by injecting increasing amounts of plasmid DNA. Satisfactory separation was obtained at sample loading of up to 360 microg of total DNA on a column with dimensions of 2.5 by 11 cm (bed volume of 54 mL). The retention of DNA depends upon a counter-current flow of electrophoresis and convective flow and could be regarded as a type of field flow fractionation. The retention of the DNA by the electric field and flow is discussed in relation to the diffusion coefficients of the DNA.

Buffers↗

PubMed2005

Fractionation of cell mixtures using acoustic and laminar flow fields.

A fractionation method applicable to different populations of cells in a suspension is reported. The separation was accomplished by subjecting the suspension to a resonant ultrasonic field and a laminar flow field propagating in orthogonal directions within a thin, rectangular chamber. Steady, laminar flow transports the cell suspension along the chamber, while the ultrasonic field causes the suspended cells to migrate to the mid-plane of the chamber at rates related to their size and physical properties. A thin flow splitter positioned near the outlet divides the effluent cell suspension into two product streams, thereby allowing cells that respond faster to the acoustic field to be separated from those cells that respond more slowly. Modeling of the trajectories of individual cells through the chamber shows that by altering the strength of the flow relative to that of the acoustic field, the desired fractionation can be controlled. Proof-of-concept experiments were performed using hybridoma cells and Lactobacillus rhamnosus cells. The two populations of cells could be effectively separated using this technique, resulting in hybridoma/Lactobacillus ratios in the left and right product streams, normalized to the feed ratio, of 6.9 +/- 1.8 and 0.39 +/- 0.01 (vol/vol), respectively. The acoustic method is fast, efficient, and could be operated continuously with a high degree of selectivity and yield and with low power consumption. (c) 2004 Wiley Periodicals, Inc.

Cell Culture Techniques↗

PubMedDate not supplied

Metal content in river suspended particulate matter: data on Po River.

The present study summarizes the last ten years of literature on heavy metal distribution in Suspended Particulate Matter (SPM) and dissolved phase in the Po River (Italy). The work compares different methodologies employed to collect, concentrate and fractionate the samples. The importance of metal speciation as a function of particle size is underlined and two approaches to metal speciation in the colloidal fraction of Po River SPM are presented: Sedimentation Field-Flow Fractionation (SdFFF) and pH-dependent extractions. Finally, emphasis is placed on the need for comparison with a reference "natural background level" of the metal load in Po River particulate matter in order to determine the real human contribution to river pollution. The high values of some trace transition elements, such as Cr and Ni, have been compared with clay sediments around Ferrara and with bricks in historic buildings. The highly comparable natural concentration of these metals in Po fine sedimentary rocks and in historic brick buildings of Ferrara (XII-XVI centuries) can provide information on natural geochemical anomalies.

Environmental Monitoring↗

PubMed1998

Submicrometer Particle Sizing by Multiangle Light Scattering following Fractionation

The acid test for any particle sizing technique is its ability to determine the differential number fraction size distribution of a simple, well-defined sample. The very best characterized polystyrene latex sphere standards have been measured extensively using transmission electron microscope (TEM) images of a large subpopulation of such samples or by means of the electrostatic classification method as refined at the National Institute of Standards and Technology. The great success, in the past decade, of on-line multiangle light scattering (MALS) detection combined with size exclusion chromatography for the measurement of polymer mass and size distributions suggested, in the early 1990s, that a similar attack for particle characterization might prove useful as well. At that time, fractionation of particles was achievable by capillary hydrodynamic chromatography (CHDF) and field flow fractionation (FFF) methods. The latter has proven most useful when combined with MALS to provide accurate differential number fraction size distributions for a broad range of particle classes. The MALS/FFF combination provides unique advantages and precision relative to FFF, photon correlation spectroscopy, and CHDF techniques used alone. For many classes of particles, resolution of the MALS/FFF combination far exceeds that of TEM measurements. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗

PubMed2004

Characterization of fluorocarbon-in-water emulsions with added triglyceride.

Fluorocarbon-in-water emulsions are being explored clinically as synthetic oxygen carriers in general surgery. Stabilizing fluorocarbon emulsions against coarsening is critical in maintaining the biocompatibility of the formulation following intravenous administration. It has been purported that the addition of a small percentage of long-chain triglyceride results in stabilization of fluorocarbon emulsions via formation of a three-phase emulsion. In a three-phase emulsion, the triglyceride forms a layer around the dispersed fluorocarbon, thereby improving the adhesion of the phospholipid surfactant to the dispersed phase. In the present study, we examined the effect of triglyceride addition on the physicochemical characteristics of the resulting complex dispersion. In particular, we examined the particle composition and stability of the dispersed particles using a method which first fractionates (classifies) the different particles prior to sizing (i.e., sedimentation field-flow fractionation). It was determined that the addition of a long-chain triglyceride (soybean oil) results in oil demixing and two distinct populations of emulsion droplets. The presence of the two types of emulsion droplets is not observed via light scattering techniques, since the triglyceride droplets dominate the scattering due to a large difference in the refractive index between the particles and the medium as compared to fluorocarbon droplets. The growth of the fractionated fluorocarbon emulsion droplets was followed over time, and it was found that there was no difference in growth rates with and without added triglyceride. In contrast, addition of medium-chain-triglyceride (MCT) oils results in a single population of emulsion droplets (i.e., a three-phase emulsion). These emulsions are not stable to droplet coalescence, however, as significant penetration of MCT into the phospholipid lipid interfacial layer results in a negative increment in the monolayer spontaneous curvature, thereby favoring water-in-oil emulsions and resulting in destabilization of the emulsion to the effects of terminal heat sterilization or mechanical stress.

Chemical Phenomena↗

PubMed2005

DNA separation by EFFF in a microchannel.

This paper theoretically explores the application of electric field flow fractionation (EFFF) for the size-based separation of DNA strands in a microchannel. An axial electric field cannot separate DNA strands in solution because the electrical mobility of the strands is independent of the length. However, lateral electric fields coupled with an axial Poiseuille flow can separate the DNA strands of different sizes. By using regular perturbation analysis, we obtain the effective diffusivity and the mean velocity of the DNA molecules that are undergoing a pressure driven Poiseuille flow in a 2D channel in presence of a lateral electric field. The mean velocities and the dispersion coefficients are then utilized to determine the scaling for length of the channel and the time required for separation of DNA molecules in different parameter regimes. The results show that EFFF can separate DNA strands in the range of 10 kbp that differ in size by about 2.5 kbp in about half an hour in a 1 cm long channel. While DNA strands can be separated by EFFF, the performance of devices based on EFFF seems to be at best comparable to other techniques such as entropic trapping.

DNA↗

PubMed2004

The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L.: identification, linkage analysis and subcellular localization.

The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II.

Chloroplasts↗

PubMed2005

Characterzation of colloidal and humic-bound Ni and U in the "dissolved" fraction of contaminated sediment extracts.

The dissolved phase of environmental aqueous samples is generally defined by filtration at 0.2 microm or even 0.45 microm. However, it is also acknowledged that colloids <0.2 microm suspended in the aqueous phase can be important for determining contaminant availability and mobility. We have used flow field-flow fractionation (FI FFF) and size exclusion chromatography (SEC) coupled to UV-absorbance (UVA) and inductively coupled plasma mass spectrometry (ICP-MS) to study the dissolved organic matter (DOM) and colloidal binding of U and Ni in water extracts of sediments collected from a contaminated area of the Savannah River Site, a U.S. Department of Energy former nuclear materials production and processing facility, near Aiken, SC. High-performance SEC-UVA-ICP-MS was well-suited to the separation of DOM overthe molecular weight (MW) range of approximately 200-7000 Da. The ICP-MS element specific data indicated that a significant fraction of U was associated with DOM. Uranium exhibited a bimodal distribution and the other fraction was greater than the exclusion limit for the column and coeluted with Al. Flow FFF was used to size this fraction as colloidal with an approximate effective spherical diameter of 0.09-0.12 microm. Element specific ICP-MS data confirmed that U and Al were associated with the colloidal phase. High-field FI FFF was also applicable to sizing DOM but resolution was poorer than SEC. The results of this study suggest that "dissolved" U at this site is predominantly either complexed by DOM or bound to a colloidal fraction while Ni is predominately present as labile complexes or the free cation and, therefore, potentially bioavailable.

Biological Availability↗

PubMed2001

Aggregation of amphiphilic pullulan derivatives evidenced by on-line flow field flow fractionation/multi-angle laser light scattering.

Size-exclusion chromatography (SEC) is a useful steric separation technique for the analysis of water-soluble polysaccharides in aqueous solution. However, in the case of amphiphilic derivatives, the usefulness is limited because of interactions between hydrophobic segments and the stationary phase. Alkyl-bearing pullulans differing from the extent and the length of alkyl groups were characterized using flow-field flow fractionation with on-line coupling multi-angle laser light scattering (F4/MALLS). Comparison of SEC and F4 is presented and the interest of F4 in the field of amphiphilic derivatives is demonstrated.

Glucans↗

PubMed1999

Finite element analysis of the lift on a slightly deformable and freely rotating and translating cylinder in two-dimensional channel flow.

Motivated by the lateral migration phenomena of fresh and glutaraldehyde-fixed red blood cells in a field flow fractionation (FFF) separation system, we studied the transverse hydrodynamic lift on a slightly flexible cylinder in a two-dimensional channel flow. The finite element method was used to analyze the flow field with the cylinder at different transverse locations in the channel. The shape of the cylinder was determined by the pressure on the surface of the cylinder from the flow field solution and by the internal elastic stress. The cylinder deformation and the flow field were solved simultaneously. The transverse lift exerted on the cylinder was then calculated. The axial and angular speed of the cylinder were iterated such that the drag and torque on the cylinder were nulled to represent a freely translating and rotating state. The results showed that the transverse lift on a deformable cylinder increased greatly and the equilibrium position moved closer to the center of the channel compared to a rigid cylinder. Also, with the same elastic modulus but a higher flow rate, a larger deformation and higher equilibrium location were found. The maximum deformation of the cylinder occurred when the cylinder was closest to the wall where a larger shear rate existed. The numerical results and experimental studies are discussed.

Blood Flow Velocity↗

PubMed2003

Syringe filtration methods for examining dissolved and colloidal trace element distributions in remote field locations.

It is well-established that sampling and sample processing can easily introduce contamination into dissolved trace element samples if precautions are not taken. However, work in remote locations sometimes precludes bringing bulky clean lab equipment into the field and likewise may make timely transport of samples to the lab for processing impossible. Straightforward syringe filtration methods are described here for collecting small quantities (15 mL) of 0.45- and 0.02-microm filtered river water in an uncontaminated manner. These filtration methods take advantage of recent advances in analytical capabilities that require only small amounts of waterfor analysis of a suite of dissolved trace elements. Filter clogging and solute rejection artifacts appear to be minimal, although some adsorption of metals and organics does affect the first approximately 10 mL of water passing through the filters. Overall the methods are clean, easy to use, and provide reproducible representations of the dissolved and colloidal fractions of trace elements in river waters. Furthermore, sample processing materials can be prepared well in advance in a clean lab and transported cleanly and compactly to the field. Application of these methods is illustrated with data from remote locations in the Rocky Mountains and along the Yukon River. Evidence from field flow fractionation suggests that the 0.02-microm filters may provide a practical cutoff to distinguish metals associated with small inorganic and organic complexes from those associated with silicate and oxide colloids.

Colloids↗

PubMed2006

Development of an SdFFF-ETAAS hyphenated technique for dimensional and elemental characterization of colloids.

Direct hyphenation of electrothermal atomic-absorption spectroscopy (ETAAS) to sedimentation field-flow fractionation (SdFFF) has been developed to enable elemental characterization of submicron particles as a function of size. This hyphenation is particularly suitable for characterizing colloidal particles of environmental interest, for example water-borne particles. The interface is an automatic capillary injection device (CID) which enables direct introduction of large and variable volumes of colloidal particle suspensions into a hot graphite furnace, thus preconcentrating the colloidal particles on the furnace walls. The method was validated by determination of Fe in certified submicron Fe2O3. The procedure was set up by first optimizing the SdFFF fractionation under programmed field conditions, thus enabling optimum fractionation of particle size. The ETAAS procedure was then tested to determine whether it could be used for direct analysis of Fe2O3 slurries without the need for a mineralization step. CID coupled to ETAAS was subsequently exploited for its ability to enhance the sensitivity, because of the increased injection volume. Statistical tests and data handling were conducted to prove the suitability of the ETAAS-CID module. Finally, off-line and on-line ETAAS-CID-SdFFF hyphenation were investigated. These experiments emphasized the advantages of the on-line coupling, because it enables synchronized sampling, enrichment, and elemental analysis of the flowing eluate. The benefits of the proposed hyphenation are the high specificity of analytical detection, increased sensitivity, reduction of analysis time, and minimum sample handling and contamination.

Journal Article↗

PubMed2002

Microsample preparation by dielectrophoresis: isolation of malaria.

An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.

Animals↗

PubMed2004

Continuous cell washing and mixing driven by an ultrasound standing wave within a microfluidic channel.

Ultrasound standing wave radiation force and laminar flow have been used to transfer yeast cells from one liquid medium to another (washing) by a continuous field-flow fractionation (FFF) approach. Two co-flowing streams, a cell-free suspending phase (flow rate > 50% of the total flow-through volume) and a yeast suspension, were introduced parallel to the nodal plane of a 3 MHz standing wave resonator. The resonator was fabricated to have a single pressure nodal plane at the centre line of the chamber. Laminar flow ensured a stable interface was maintained as the two suspending phases flowed through the sound field. Initiation of the ultrasound transferred cells to the cell-free phase within 0.5 s. This particle transfer procedure circumvents the pellet formation and re-suspension steps of centrifuge based washing procedures. In addition, fluid mixing was demonstrated in the same chamber at higher sound pressures. The channel operates under negligible back-pressure (cross-section, 0.25 [times] 10 mm) and with only one flow convergence and one flow division step, the channel cannot be easily blocked. The force acting on the cells is small; less than that experienced in a centrifuge generating 100g. The acoustically-driven cell transfer and mixing procedures described may be particularly appropriate for the increasingly complex operations required in molecular biology and microbiology and especially for their conversion to continuous flow processes.

Cell Count↗

PubMed2004

Pinched flow fractionation: continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel.

A concept of "pinched flow fractionation" for the continuous size separation and analysis of particles in microfabricated devices has been proposed and demonstrated. In this method, particles suspended in liquid were continuously introduced into a microchannel having a pinched segment and were aligned to one sidewall in the pinched segment by another liquid flow without particles. The particles were then separated perpendicularly to the flow direction according to their sizes by the spreading flow profile inside the microchannel. Polymer microbeads were successfully separated, and the effects of the flow rate and channel shapes on the separation performance were examined. Also, separated particles were collected independently by making branches at the end of the pinched segment. Since this method utilizes only the laminar flow profile inside a microchannel, complicated outer field control could be eliminated, which is usually required for other kinds of particle separation methods such as field flow fractionation. Also, this method can be applied both for particle size analysis and for preparation of monodispersed particles, since separation can be rapidly and continuously performed.

Journal Article↗

PubMed2004

A microfabricated device for subcellular organelle sorting.

We report a microfabricated field flow fractionation device for continuous separation of subcellular organelles by isoelectric focusing. The microdevice provides fast separation in very small samples while avoiding large voltages and heating effects typically associated with conventional electrophoresis-based devices. The basis of the separation is the presence of membrane proteins that give rise to the effective isoelectric points of the organelles. Simulations of isoelectric focusing of mitochondria in microchannels are used to assess design parameters, such as dimensions and time scales. In addition, a model of Joule heating effects in the microdevice during operation indicates that there is no significant heating, even without active cooling. The device is fabricated using a combination of photolithography, thin-film metal deposition/patterning, and electroplating techniques. We demonstrate that in the microfluidic devices, mitochondria from cultured cells migrate under the influence of an electric field into a focused band in less than 6 min, consistent with model predictions. We also illustrate separation of mitochondria from whole cells and nuclei as well as the separation of two mitochondrial subpopulations. When automated and operated in parallel, these microdevices should facilitate high-throughput analysis in studies requiring separation of organelles.

Cell Fractionation↗

PubMed2003

Room-temperature preparation and characterization of poly (ethylene glycol)-coated silica nanoparticles for biomedical applications.

Monodisperse, spherical, polyethylene glycol (PEG)-coated silica nanoparticles have been prepared at room temperature and characterized for the purpose of biomedical applications. The particles were synthesized by the hydrolysis of tetramethyl orthosilicate (TMOS) in alcohol media under catalysis by ammonia, and their size can range from about 50-350 nm in diameter. We studied the particle size and size distribution using a scanning electron microscope (SEM) and an asymmetric field-flow fractionation (AFFF) multiangle static light-scattering instrument. The chemical and/or physical binding of PEG to the silica nanoparticles was studied by infrared spectroscopy, and the weight percentage of PEG attached to the particles was quantified. The PEG-coated silica nanoparticles showed enhanced colloidal stability when redispersed into aqueous solutions from the dried state as a result of the steric stabilization function of the PEG polymer grafted on the surface of particles. A nonspecific protein-binding test was also carried out to show that the PEG coating can help reduce the protein adsorption onto the surface of the particles, relating to the biocompatibility of these PEG-coated particles. Also, the inclusion of magnetic nanoparticles into the silica particles was shown as an example of the possible applications of PEG-coated silica particles. These silica nanoparticles, as a matrix for encapsulation of certain reagents, have potential for applications to in vivo diagnosis, analysis, and measurements inside intact biologic systems.

Biocompatible Materials↗

PubMed1994

Structural studies of commercial fat emulsions used in parenteral nutrition.

In this paper, we demonstrate that by employing a combination of sedimentation field-flow fractionation (sedFFF) and other characterization techniques, such as photon correlation spectroscopy (PCS) and freeze-fracture electron microscopy (EM), it is possible to show that commercial fat emulsions of similar overall chemical compositions not only may exhibit different size distributions but may have different densities as well. A closer look at the density difference between droplet and suspension medium, on the one hand, and the droplet size, on the other, demonstrates that fat emulsions may have structures other than the traditional oil droplet surrounded by a monolayer of surfactant. From our determined and simulated density differences, we propose that these emulsion droplets may have a multilayered surfactant arrangement as well as an inclusion of water vesicles in the oil phase of the emulsion. Freeze-fracture EM observations provide evidence to confirm the existence of such complex structures. These findings are supported by recent EM work from other laboratories, as well as through chemical verification of elevated water contents in the oil droplets of these emulsions.

Drug Stability↗