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Results for “Fractionation, Field Flow”

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PubMed2002

A chip system for size separation of macromolecules and particles by hydrodynamic chromatography.

For the first time, a miniaturized hydrodynamic chromatography chip system has been developed and tested on separation of fluorescent nanospheres and macromolecules. The device can be applied to size characterization of synthetic polymers, biopolymers, and particles, as an attractive alternative to the classical separation methods such as size exclusion chromatography or field-flow fractionation. The main advantages are fast analysis, high separation efficiency, negligible solvent consumption, and easy temperature control. The prototype chip contains a rectangular flat separation channel with dimensions of 1 microm deep and 1000 microm wide, integrated with a 300-pL injector on a silicon substrate. The silicon microtechnology provides precisely defined geometry, high rigidity, and compatibility with organic solvents or high temperature. All flows are pressure driven, and a specific injection system is employed to avoid excessive sample loading times, demonstrating an alternative way of lab-on-a-chip design. Separations obtained in 3 min show the high performance of the device and are also the first demonstration of flat channel hydrodynamic chromatography in practice.

Chromatography, Liquid↗

PubMed2002

Anomalous radial migration of single DNA molecules in capillary electrophoresis.

We report the unexpected radial migration of DNA molecules in capillary electrophoresis (CE) with applied Poiseuille flow. Such movement can contribute to anomalous migration times, peak dispersion, and size and shape selectivity in CE. When Poiseuille flow is applied from the cathode to the anode, DNA molecules move toward the center of the capillary, forming a narrow, highly concentrated zone. Conversely, when the flow is applied from the anode to the cathode, DNA molecules move toward the walls, leaving a DNA-depleted zone around the axis. We showed that the deformation and orientation of DNA molecules under Poiseuille flow was responsible for the radial migration. By analyzing the forces acting on the deformed and oriented DNA molecules, we derived an expression for the radial lift force, which explained our results very well under different conditions with Poiseuille flow only, electrophoresis only, and the combination of Poiseuille flow and electrophoresis. Factors governing the direction and velocity of radial migration were elucidated. Potential applications of this phenomenon include an alternative to sheath flow in flow cytometry, improving precision and reliability of single-molecule detection, reduction of wall adsorption, and size separation with a mechanism akin to field-flow fractionation. On the negative side, nonuniform electroosmotic flow along the capillary or microfluidic channel is common in CE, and radial migration of certain analytes cannot be neglected.

DNA↗

PubMed2003

Mechanism for the separation of large molecules based on radial migration in capillary electrophoresis.

We demonstrate a novel separation mechanism for large molecules based on their radial migration in capillary electrophoresis with applied hydrodynamic flow (HDF). The direction of radial migration depends on the direction of the applied HDF relative to the electric field. The radial migration velocities are size-dependent, which could be attributed to the different degree of deformation under shear flow. Analytical separation was demonstrated on a sample plug containing lambda DNA (48 502 bp) and phiX174 RF DNA (5386 bp) with baseline separation. Alternatively, this separation mode can be performed continuously and is thus applicable to preparative separations. Without the need for gel/polymer or complex instrumentation, this separation technique is complementary to capillary gel electrophoresis and field-flow fractionation. Although large DNA molecules were used to demonstrate the separation mechanism here, these protocols could also be applied to the separation of proteins, cells, or particles based on size, shape, or deformability.

Bacteriophage lambda↗

PubMed2003

On-chip hydrodynamic chromatography separation and detection of nanoparticles and biomolecules.

For the first time, on-chip planar hydrodynamic chromatography is combined with UV absorption detection. This technique is suitable for size characterization of synthetic polymers, biopolymers, and particles. Possible advantages of an on-chip hydrodynamic chromatography system over conventional techniques, such as size exclusion chromatography, and field-flow fractionation are fast analysis, high efficiency, reduced solvent consumption, and easy temperature control. The hydrodynamic separations are performed in a planar configuration realized in fused silica using a mixture of fluorescent and nonfluorescent polystyrene particles with sizes ranging from 26 to 155 nm. The planar chip configuration consists of a 1-microm-high, 0.5-mm-wide, and 69-mm-long channel, an integrated 150-pL injection structure, and a 30-microm-deep and 30-microm-wide detection cell, suitable for UV absorption detection. By combination of the separation data obtained in the new fused-silica chip with those obtained using a previously presented planar hydrodynamic chromatography chip, which was realized using silicon and glass microtechnology, a description of the retention and dispersion behavior of planar hydrodynamic chromatography is obtained. Especially the influence of the sidewalls on the dispersion is investigated. Furthermore a hydrodynamic separation within 70 s of several biopolymers is shown in the glass-silicon chip.

Chromatography, Gel↗

PubMed2004

High-velocity transport of nanoparticles through 1-D nanochannels at very large particle to channel diameter ratios.

We explore the possibility of generating high-velocity flows of nanoparticles through flat-rectangular nanochannels, which are only 50% deeper than the diameter of the particles. Using the shear-driven flow principle, 200-nm particles can, for example, be transported through a 300-nm-deep channel at velocities up to 35 mm/s (upper limit of our current setup). Working under high-pH conditions, the velocity of the carboxylated nanoparticles still respects the small-molecule velocity law, despite the high degree of confinement to which the particles are subjected. The high degree of confinement is also found to lead to a reduced band broadening. When injecting sharply delimited particle plugs, the plate heights observed for the flow of 0.2-microm particles through a 0.3-microm channel (with plate heights of the order of 1-2 microm) are, for example, approximately 1 order of magnitude smaller than for the flow of 1.0-microm particles through a 1.4-microm channel. It is also found that the band broadening is, within its statistical variation, independent of the fluid velocity over a large range of particle velocities (5-35 mm/s). The flow method distinguishes itself from pressure-driven field-flow fractionation and hydrodynamic chromatography in that the mean particle velocity is independent of the particle size over the entire range of possible particle to channel diameter ratios.

Journal Article↗

PubMed2006

Open tubular capillary electrochromatography: technique for oxidation and interaction studies on human low-density lipoproteins.

A novel, open tubular capillary electrochromatographic method was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of approximately 2.5 MDa yielded a stable stationary phase at temperatures 25 and 37 degrees C and at pH values from 3.2 to 7.4. The quality of the coatings was not influenced by variations in the LDL concentration in the coating solutions (within the range of 2-0.015 mg/mL) with the coating procedure used in the study. Radiolabeled LDL stationary phases and scanning electron microscopy, employed to shed light on the location and coating density of LDL particles on the inner surface of the capillary wall, confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 +/- 8%). In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillary coating. Capillaries coated with human LDL particles were submitted to different oxidative conditions by changing the concentration of the oxidant (CuSO4), oxidation time, pH value, and temperature. The oxidation procedure was followed with electroosmotic flow mobility, which served as an indicator of the increase in total negative charges of LDL coatings, and by asymmetrical field flow fractionation, which measured the changes in size of the lipoprotein particles. The results indicated that oxidation of LDL was progressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidation process was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2006

Mechanical degradation and changes in conformation of hydrophobically modified starch.

In this paper, we study the mechanical degradation and changes in conformation of a branched ultrahigh molar mass biomacromolecule, hydrophobically modified starch, as caused by high-pressure homogenization. The characterization was performed with asymmetrical flow field-flow fractionation (AsFlFFF) with multiangle light scattering (MALS) and refractive index detection. The starch which had been chemically modified with octenyl succinate anhydride (OSA) proved to be very large and polydisperse. Upon high-pressure homogenization, the molar mass and rms radius (r(rms)) decreased, and the extent of these changes was related to the turbulent flow conditions during homogenization. The treatment also induced an increase and scaling with size in the apparent density of the macromolecules. To further study the changes in conformation, it was necessary to calculate the hydrodynamic radii (r(h)). This can be determined numerically from the elution times in the analysis and the flow conditions in the AsFlFFF channel. The results showed that the treatment can cause a dramatic decrease in the quotient between r(rms) and r(h), suggesting major conformational changes. These results together could be interpreted as degradation and "crumpling" of the macromolecule, which would give a decrease in r(rms) and an increase in apparent density, together with a "fraying" of more outer parts of the macromolecule, which could give rise to the increase in r(h).

Air↗

PubMedDate not supplied

Effect of NaBH4 concentration and reaction time on physical properties of glutaraldehyde-polymerized hemoglobin.

The US is about 1.5 days away from exhausting its entire blood supply. Hence, there is an urgent need for the development of universal blood substitutes. One such blood substitute is glutaraldehyde-polymerized bovine hemoglobin. Despite the commercial development of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes, there has been little published work on the effect of reaction conditions on the physical properties of the polymerized hemoglobin dispersion. In this study, the effect of varying the concentration of the quenching agent NaBH(4), glutaraldehyde concentration, and reaction time on the physical properties of PolyHb dispersions was studied by measuring the absolute molecular weight distribution, as well as oxygen-binding properties such as P(50), Hill coefficient, and methemoglobin level (MetHb) of these dispersions. Bovine hemoglobin was polymerized with glutaraldehyde using a parallel synthetic approach. Asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of the PolyHb dispersions. In general, high glutaraldehyde concentrations (20-30 times the molar concentration of hemoglobin) adversely affected the oxygen-binding properties of PolyHb dispersions, while NaBH(4) concentrations (up to 300 times the molar concentration of hemoglobin) and reaction times (up to 24 h) did not appear to have any effect on the oxygen-binding properties of PolyHb dispersions.

Animals↗

PubMedDate not supplied

Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes.

Various oxidized mono/di/tri/poly saccharides were studied as potential hemoglobin (Hb) cross-linkers in order to produce oxygen carriers with high oxygen affinities (low P(50)'s) and high molecular weights (therefore lower macromolecular diffusivities compared to tetrameric Hb). Such physical properties were desired to produce polymerized hemoglobins (PolyHbs) with oxygen release profiles similar to that of human blood, as was demonstrated in work by Winslow (1). In this present study, bovine hemoglobin was cross-linked with a variety of oxidized (ring-opened) saccharides, which resulted in cross-linked Hb species ranging in size from 64 to 6400 kDa (depending on the particular oxidized saccharide used in the reaction) and P(50)'s ranging from 6 to 15 mmHg. A parallel synthetic approach was used to synthesize these carbohydrate-hemoglobin conjugates, and asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of these PolyHb dispersions. Cross-linking reactions were conducted at two pHs (6 and 8), with larger cross-linked Hb species produced at pH 8 (where hydrolysis was most likely to occur between glycosidic bonds linking adjacent saccharide rings) rather than at pH 6. The largest molecular weight species formed from these reactions consisted of Hb cross-linked with ring-opened lactose, maltose, methylglucopyranoside, sucrose, trehalose, and 15 kDa and 71 kDa dextran at high pH (pH 8). The most promising Hb cross-linker was methylglucopyranoside, which resulted in very large cross-linked Hb species, with low P(50)'s and lower methemoglobin (metHb) levels compared to the other Hb cross-linking reagents.

Animals↗

PubMed2002

Influence of natural organic matter on the adsorption of metal ions onto clay minerals.

The influence of natural organic matter (NOM) on the adsorption of Al, Fe, Zn, and Pb onto clay minerals was investigated. Adsorption experiments were carried out at pH = 5 and pH = 7 in the presence and absence of NOM. In general, the presence of NOM decreased the adsorption of metal ions onto the clay particles. Al and Fe were strongly influenced by NOM, whereas Zn and Pb adsorption was only slightly altered. The interaction of the metal ions with the minerals and the influence of NOM on this interaction was investigated by coupling SdFFF with an inductively coupled plasma mass spectrometer (ICPMS) or an inductively coupled plasma atomic emission spectrometer (ICPAES). Quantitative atomization of the clay particles in the ICP was confirmed by comparing elemental content determined by direct injection of the clay into the ICPMS with values from acid digestion. Particle sizes of the clays were found to be between 0.1 and 1 microm by sedimentation field-flow fractionation (SdFFF) with UV detection. Aggregation of particles due to metal adsorption was observed using SdFFF-ICPMS measurements. This aggregation was dependent on the specific metal ion and decreased in the presence of NOM and at higher pH value.

Adsorption↗

PubMed2002

Humic colloid-borne natural polyvalent metal ions: dissociation experiment.

The natural association nature of the humic colloid-borne trace elements is investigated. Rare earth elements (REE) Th and U are chosen as naturally occurring representatives and chemical homologues for actinides of different oxidation states present in nuclear waste. Tri- and tetravalent elements in two investigated Gorleben groundwaters (Gohy-532 and -2227) almost exclusively occur as humic or fulvic colloid-borne species. Their desorption behavior from colloids is examined in the unperturbed groundwater (pH approximately 8) under anaerobic conditions (Ar/1% CO2) by addition of a chelating cation exchanger resin. Particularly, the dissociation process of naturally occurring Eu(III) in the groundwater is compared with the Eu(III) desorption from its humate complex prepared with purified Aldrich humic acid in a buffered aqueous solution at pH approximately 8. The Eu(III) dissociation from the groundwater colloids is found to be considerably slower than found for the humate complex synthesized in the laboratory. This suggests that under natural aquatic conditions the Eu(III) binding in colloids is chemically different from the simple humate complexation as observed in the laboratory experiment. The colloid characterization bythe size exclusion chromatography (SEC) and the flow field-flow fractionation (FFFF) indicates that natural colloid-borne trace elements are found predominantly in colloids of larger size (>15 nm in size), while Eu(III) in its humate complex is found mainly in colloids of hydrodynamic diameters <5 nm. The slower desorption kinetics and the larger colloid size suggest that the polyvalent metal ion binding in natural humic colloids is associated to polynucleation with other co-present trace metal ions. Radiotracer experiments reveal that isotopic equilibria with the naturally colloid-borne trace elements are not attained within a period of more than 100 days, indicating irreversible binding of at least a part of colloid-borne polyvalent trace elements. The different kinetic properties of colloid-bound Eu(III) are discussed taking the aqueous speciation based on thermodynamic data into account.

Chromatography, Gel↗

PubMed2003

Amphiphilic Janus micelles with polystyrene and poly(methacrylic acid) hemispheres.

We describe the synthesis and the solution properties of Janus micelles containing a polybutadiene (PB) core and a compartmentalized corona consisting of a poly(methacrylic acid) (PMAA) and a polystyrene (PS) hemisphere. The Janus micelles were obtained via cross-linking the middle block of a microphase-separated PS-block-PB-block-PMMA triblock copolymer in the bulk state, followed by alkaline hydrolysis of the poly(methyl methacrylate) (PMMA) ester groups. Results of fluorescence correlation spectroscopy, field flow fractionation, light scattering, cryogenic transmission electron microscopy, scanning electron microscopy, and scanning force microscopy indicate that above a critical aggregation concentration of about 0.03 g/L spherical supermicelles are formed from about 30 PS-PMAA micelles in aqueous solution in the presence of NaCl. These supermicelles have radii of 40-60 nm, significantly increasing on ionization (pH >6). In addition, very large spherical objects are observed with radii of 100-250 nm.

Journal Article↗

PubMed2004

Effect of actin concentration on the structure of actin-containing liposomes.

Liposomes encapsulating monomeric actin (G-actin) were produced via extrusion through 400 and 600 nm pore diameter polycarbonate membranes in low ionic strength buffer (G-buffer). After actin-containing liposomes were subjected to high ionic strength polymerization buffer (F-buffer), morphological changes in the structure of actin-containing liposomes were studied using asymmetric flow field-flow fractionation (AFFF) coupled with multiangle static light scattering (MASLS). The general shape of these liposomes was initially determined by fitting three form factors, which describe the angular distribution of scattered light from a spherical thin shell, thin disk, and thin rod, to the experimentally measured light scattering spectra to regress the dimensions of the liposomes corresponding to the proposed geometry. Light scattering spectra that yielded a best fit to the thin disk model were analyzed further and fit to the ellipsoidal of revolution form factor to regress both the major and minor axis dimensions. The results of this analysis showed that actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes in F-buffer, at a low actin concentration (0.1 mg/mL), assumed a spherical shape, which is also the case for plain liposomes (no actin present) in G-buffer and F-buffer. When the actin concentration was increased to 1 mg/mL, the polymerizing actin filaments stretched the initially spherical liposome into a disklike shape. However, when the actin concentration was further increased to 5 mg/mL, the liposomes reverted back to a spherical shape.

Actins↗

PubMed1993

A method for the early evaluation of the effects of storage and additives on the stability of parenteral fat emulsions.

The combination of sedimentation field-flow fractionation (SedFFF) and photon correlation spectroscopy (PCS) is shown to provide a detailed record of the droplet sizes present in fat emulsions commonly used in parenteral nutrition. The technique presented has been used to record size distribution data for a particular emulsion (Liposyn-II), demonstrating its high stability and lot-to-lot uniformity. The technique is also able to demonstrate how additions of small amounts of electrolytes [0.45% (w/v) NaCl, 0.05% (w/v) CaCl2] tend to destabilize the emulsion, suggesting some caution in the use of total parenteral nutrition (TPN) mixtures. In contrast, a 1:1 mixture with human serum caused no sign of instability in the Liposyn-II. Using the emulsion as a carrier for lipophilic drugs necessitates adding solutions of the drug in nonaqueous solvents, such as DMSO (dimethyl sulfoxide). This solvent's destabilizing effect results in a droplet coalescence that becomes severe after 3-5 days following a 10% (v/v) addition, while a 5% (v/v) addition reaches the same level of coalescence in 10 days.

Chemical Fractionation↗

PubMed2003

Dielectrophoresis of microbioparticles in water with planar and capillary quadrupole electrodes.

Dielectrophoresis of single microbioparticles was measured in a planar quadrupole microelectrode (50 mum or 65 mum in working area radius) with a microscope. Carbon and polystyrene microparticles, yeast cells and DNA molecules (about 40 kbp) were adopted as a sample. Their dielectrophoretic mobilities were analysed quantitatively with their intrinsic and surface conductivity, their permittivities and their sizes as well as the conductivity and permittivity of aqueous media. Using the dielectrophoretic mobilities obtained with the planar quadrupole microelectrode, some instances of the separation performance between the microparticles were demonstrated with a fabricated capillary quadrupole microelectrode (82.5 mum in bore radius) under the field flow fractionation regime.

Journal Article↗

PubMed2005

Stability of liposome encapsulated hemoglobin dispersions.

The demand for artificial blood substitutes in cases of elective surgeries, trauma, and civilian mass catastrophes increases every day. However, few studies have been done to characterize the mechanical stability of blood substitutes, especially liposome encapsulated hemoglobin (LEHb) dispersions. In this work, the stability of LEHb dispersions was investigated by fitting Jung et al.'s liposome size distribution model to experimentally measured LEHb size distributions [6] (produced via extrusion) using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering. The effective bending constant (KB) and radius of curvature (R0) of each liposome dispersion were regressed from the size distribution fits. The model was found to be in agreement with the size distributions of LEHbs extruded through 400, 200 and 100 nm pore diameter membranes, but not in agreement with LEHbs extruded through 80 and 50 nm pore diameter membranes. Although the magnitude of KB fluctuated, we deduced a general trend for KB to decrease with decreasing pore diameter, and increasing initial Hb concentration. LEHbs extruded through 400nm pore diameter membranes were stabilized by the spontaneous curvature effect, while those extruded through 80 and 50 nm pore diameter membranes were mostly stabilized by thermal undulations, regardless of the initial Hb concentration. For LEHb dispersions extruded through 200 and 100 nm pore diameter membranes, there was a transition of stabilization mechanism from spontaneous curvature to thermal undulations with increasing initial Hb concentration. Taken together, these results suggest that moderate Hb encapsulation might actually impart better mechanical stability to LEHb dispersions extruded through 200 and 100 nm pore diameter membranes.

Blood Substitutes↗

PubMed2005

Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L.: distinct SHG reside in the cytosol and in the apoplast.

Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (<10 kDa; SHG(S)) and high (>10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using 14CO2 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime.

Arabidopsis↗

PubMed1996

Variations in the fluorescence intensity of intact DAPI-stained bacteria and their implications for rapid bacterial quantification.

As current techniques for the quantification of bacteria are laborious and often imprecise, instrumental approaches such as sedimentation field-flow fractionation (SdFFF) are attractive. In this technique, fluorogenic dyes specific for nucleic acids are used to identify bacterial cells. Bacterial biomass can be quantified directly with SdFFF if the specific fluorescence of bacterial cells is constant. The effect of different growth conditions on the specific fluorescence of one strain each of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus epidermidis stained with 4',6-diamidino-2-phenylindole was examined. Specific fluorescence varied over a 500-fold range, from 0.22 to 103 arbitrary fluorescence units per cell. Specific fluorescence was highest when cells were in log phase, and lowest when cells were in stationary phase. Specific fluorescence decreased when cells harvested in log phase were starved for 7 d in a carbon-free minimal medium, and increased rapidly (within 2 h) after cells were relieved from carbon limitation. Such variations in specific fluorescence must be considered when using gross fluorescence as a direct indicator of bacterial numbers in the SdFFF technique for quantifying bacterial biomass. Moreover, they have serious implications for the application of fluorescence techniques in other instrumental approaches for bacterial enumeration in environmental samples.

Bacteria↗