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PubMed1998

Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy. Effect of ionic strength and pH of carrier solutions.

The effect of ionic strength and pH of carrier solutions on the separation of liposomes by flow field-flow fractionation (flow FFF) has been studied for the determination of accurate vesicle size distribution of liposomes. Retention behaviors of liposomes (PC/PG/cholesterol) are observed in typical buffer solutions (PBS and Tris-HCl) of various ionic strengths as carrier liquids in flow FFF. The average diameters of collected fractions at each flow FFF run are measured by photon correlation spectroscopy (PCS) for the comparison with FFF calculations at corresponding time interval of collected fractions. A reasonable separation of liposomes is observed at I = 0.016 M for both buffer solutions. Retention of liposomes is found to be elongated at ionic strengths higher than an optimum condition found experimentally, but it is shortened at a lower ionic strength due to the electrostatic interaction between the channel wall and the liposomes. Finally, size distributions of liposomes are provided comparing the liposome preparations by flow FFF.

Drug Carriers↗
PubMed1998

Hyphenated thermal field flow fractionation--capillary electrophoresis.

The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered.

Electrophoresis, Capillary↗
PubMed2003

Magnitude and direction of thermal diffusion of colloidal particles measured by thermal field-flow fractionation.

In this paper we provide experimental evidence showing that various types of submicrometer-sized particles (latexes, inorganic, and metallic), suspended in either aqueous or nonaqueous carrier liquids to which a temperature gradient dT/dx is applied, experience a force in the direction opposite to that of dT/dx. This behavior is similar to that of small particles such as soot, aerosols, and small bubbles suspended in stagnant gases across which temperature gradients are applied, a phenomenon known as "thermophoresis in gases." We report the use of a thermal field-flow fractionation (ThFFF) apparatus in two different configurations to establish the direction of particle motion subject to a temperature gradient. The first approach employed the conventional horizontal ThFFF channel orientation. In this case, small electrical potentials were applied across the narrow channel thickness either to augment or to act in opposition to the applied thermal gradient, depending on whether the accumulation wall was maintained at a positive or negative potential relative to the depletion wall. Thus, by observing the changes in the retention behavior of surface-charged latices or silica particles with changes in potential difference across the channel thickness, we were able to ascertain the direction of migration of the particles in the thermal gradient. The second approach involved the use of a ThFFF column oriented vertically in an implementation of a technique known as thermogravitational FFF. In this approach, the convective flow along the channel length (due to density gradients associated with the temperature gradient) couples with the thermal diffusion effect across the channel thickness to result in a combined particle retention mechanism. A retarded upward migration rate is indicative of accumulation of particles at the cold wall, while enhanced upward migration would indicate a hot-wall accumulation. From the results of our investigations, we conclude that submicrometer-sized particles suspended in either aqueous or nonaqueous carrier liquids and subjected to a temperature gradient migrate from the hot wall toward the cold wall of a ThFFF channel.

Journal Article↗
PubMed2000

Combination of asymmetric flow field-flow fractionation (AF4) and total-reflexion X-ray fluorescence analysis (TXRF) for determination of heavy metals associated with colloidal humic substances.

To assess the structural variability of colloidal humic substances and the associated heavy metals an off-line coupling of asymmetric flow field-flow fractionation (AF4) with total-reflection X-ray fluorescence analysis (TXRF) is presented. AF4 allows a rather gentle separation of colloids with a minimum of interference and artifacts as no shear forces, drying, or interactions with a stationary phase are involved. After a calibration with suitable polymer particles of known molecular weight, the molecular weight distribution of colloidal humic substances between 1 and 10(3) kDa can be assessed with AF4. The combination with TXRF permits a simultaneous multielement analysis after preconcentration of samples on the AF4 channel using an optimized buffer. The analysis of seepage and sewage water sample and a sewage sludge sample yielded continuous distributions of the molecular weight and the associated heavy metals. The potential of AF4-TXRF coupling for the study of metal ion exchange equilibria with colloids was demonstrated by spiking seepage water with various heavy metals and subsequent AF4-TXRF analysis of the heavy metals bound to the colloidal fraction (Cu, Cr, Zn, Ni, Co).

Buffers↗
PubMed1993

Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography.

During the downstream processing of monoclonal antibodies (Mab) the formation of dimers and/or oligomers may occur. Today, the most common technique for determination of aggregate content is gel permeation chromatography (GPC) but it has limited resolution and separation speed. Oligomers are particularly difficult to resolve. Asymmetrical flow field-flow fractionation (FFF) is a new analytical method for the separation of Mab aggregates. The monomer and dimer peaks were well resolved and three additional peaks, thought to be trimers, tetramers, and pentamers were partially resolved. The total time for a separation can be kept as short as 6 min. The GPC separation takes about 15 min with less resolution. A study of the reproducibility for the two techniques revealed that the precision was slightly better for GPC. Investigation of the dependence of sample concentration showed that the load limit for FFF was about 4 micrograms of Mab. The GPC technique requires prefiltered samples in order to avoid clogging of the column. For FFF it is possible to inject samples containing precipitated material without any pretreatment. The flow conditions can be adjusted so that the precipitated material elutes with the front, well separated from the monomer peak.

Antibodies, Monoclonal↗
PubMed2002

Preferential adsorption of fluorescing fulvic and humic acid components on activated carbon using flow field-flow fractionation analysis.

Activated carbon treatment of drinking water is used to remove natural organic matter (NOM) precursors that lead to the formation of disinfection byproducts. The innate hydrophobic nature and macromolecular size of NOM render it amenable to sorption by activated carbon. Batch equilibrium and minicolumn breakthrough adsorption studies were performed using granular activated carbon to treat NOM-contaminated water. Ultraviolet (UV) absorption spectroscopy and flow field-flow fractionation analysis using tandem diode-array and fluorescence detectors were used to monitor the activated carbon sorption of NOM. Using these techniques, it was possible to study activated carbon adsorption properties of UV absorbing, fluorescing and nonfluorescing, polyelectrolytic macromolecules fractionated from the total macromolecular and nonmacromolecular composition of NOM. Adsorption isotherms were constructed at pH 6 and pH 9. Data were described by the traditional and modified Freundlich models. Activated carbon capacity and adsorbability were compared among fractionated molecular subsets of fulvic and humic acids. Preferential adsorption (or adsorptive fractionation) of polyelectrolytic, fluorescing fulvic and humic macromolecules on activated carbon was observed. The significance of observing preferential adsorption on activated carbon of fluorescing macromolecular components relative to nonfluorescing components is that this phenomenon changes the composition of dissolved organic matter remaining in equilibrium in the aqueous phase relative to the composition that existed in the aqueous phase prior to adsorption. Likewise, it changes the composition of dissolved organic matter remaining in equilibrium in the aqueous phase relative to the adsorbed phase. This research increases our understanding of NOM interactions with activated carbon which may lead to improved methods of potable water production.

Adsorption↗
PubMed2002

Comparison of the molecular mass and optical properties of colored dissolved organic material in two rivers and coastal waters by flow field-flow fractionation.

Colored dissolved organic material (CDOM) is an important sunlight absorbing substance affecting the optical properties of natural waters. However, little is known about its structural and optical properties mainly due to its complex matrix and the limitation of the techniques available. A comparison of two southwestern Florida rivers [the Caloosahatchee River (CR) and the Shark River (SR)] was done in terms of molecular mass (MM) and diffusion coefficients (D). The novel technique Frit inlet/frit outlet-flow field-flow fractionation (FIFO-FIFFF) with absorbance and fluorescence detectors was used to determine these properties. The SR receives organic material from the Everglades. By contrast, the CR arises from Lake Okeechobee in central Florida, receiving anthropogenic inputs, farming runoff, and natural organics. Both rivers discharge to the Gulf of Mexico. Fluorescence identified, for both rivers, two different MM distributions in low salinity water samples: the first was centered at approximately 1.7 kDa (CR) and approximately 2 kDa (SR); the second centered at approximately 13 kDa for both rivers, which disappeared gradually in the river plumes to below detection limit in coastal waters. Absorbance detected only one MM distribution centered at approximately 2 kDa (CR) and 2.2-2.4 kDa (SR). Fluorescence in general peaked at a lower MM than absorbance, suggesting a different size distribution for fluorophores vs chromophores. A photochemical study showed that, after sunlight, irradiated freshwater samples have similar characteristics to more marine waters, including a shift in MM distribution of chromophores. The differences observed between the rivers in the optical characteristics, MM distributions, and D values suggest that the CDOM sources, physical, and photochemical degradation processes are different for these two rivers.

Adsorption↗
PubMed1987

Transport, space, entropy, diffusion and flow. Elements underlying separation by electrophoresis, chromatography, field-flow fractionation and related methods.

We describe here some elements common among separation methods, including the requirement for separative transport, the sensitive utilization of space, and the dissipative (band broadening) roles of entropy, diffusion, and flow. We focus on the need to carefully manage the unceasing competition between separative and dissipative transport in all high resolution methods. We then examine the more specific roles these elements play in influencing separation in several important analytical separation techniques. We begin with electrophoresis and sedimentation, which are conceptually among the simplest of systems because they require no flow and only a single dimension of space. The conflict between separative and dissipative transport is examined closely for these systems and it is shown that the outcome is a theoretical plate number which can be expressed as a ratio of two energies: a structuring energy--delta mu ext, which organizes the separation, and thermal energy RT, which is responsible for its dissipation. It is explained why optimal separation is most often achieved in thin layers or in capillary tubes. Chromatography and field-flow fractionation are then described as two closely related methods in which flow is powerfully coupled with a simple enrichment process occurring at right angles to flow. Unlike electrophoresis, these systems are intrinsically two-dimensional. It is shown how flow and diffusion processes both assume two diametrically opposite roles: those of aiding separation and simultaneously causing its dissipation. With a few equations it is demonstrated that the dissipative role is best contained by reducing the thickness (diameter) of the system or of certain elements within the system.

Chemical Fractionation↗
PubMed2006

Charged species transport, separation, and dispersion in nanoscale channels: autogenous electric field-flow fractionation.

Numerical methods are employed to examine the transport of charged species in pressure-driven and electroosmotic flow along nanoscale channels having an electric double-layer thickness comparable to the channel size. In such channels, the electric field inherent to the double layer produces transverse species distributions that depend on species charge. Flow along the channel thus yields mean axial species speeds that also depend on the species charge, enabling species separation and identification. Here we characterize field-flow separations of this type via the retention and plate height. For pressure-driven flows, we demonstrate that mean species speeds along the channel are uniquely associated with a single species charge, allowing species separation based on charge alone. In contrast, electroosmotic flows generally yield identical speeds for several values of the charge, and these speeds generally depend on both the species charge and electrophoretic mobility. Coefficients of dispersion for charged species in both planar and cylindrical geometries are presented as part of this analysis.

Journal Article↗
PubMed2002

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 FIFFF 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.

Chemical Fractionation↗
PubMed2001

Influence of the carrier composition on thermal field-flow fractionation for the characterisation of sub-micron polystyrene latex particles.

A study on the influence of the carrier composition in a ThFFF system on the retention and thermal diffusion of sub-micron polystyrene latex particles has been carried out. Various factors that may influence retention were studied. These include: the type of electrolyte and surfactant, their respective concentrations, and the addition of an organic modifier. Particle retention is highly sensitive to small changes in the carrier composition. It is demonstrated that under the conditions applied, secondary effects, such as particle-wall and particle-particle interactions, are negligible. Addition of surfactants is required to minimise particle-wall interactions. Generally, retention increases at higher electrolyte concentration. Furthermore, the addition of acetonitrile (ACN) to an aqueous carrier leads also to an increased retention. The type of surfactant as well as its concentration is of influence on the retention time. The three surfactants that were studied, i.e., sodium dodecyl sulfate, Brij 35 and cetyltrimethylammonium bromide, showed significant differences in particle retention behaviour. The observed differences in retention in the carriers can be attributed to actual changes in thermal diffusion. D(T) appears to be mainly determined by the interaction between the particle's surface and the carrier liquid, and is therefore highly sensitive to changes in the chemical composition of the particle surface and the carrier. Strong differences in size selectivity were found for different carrier compositions. This allows a relatively easy optimisation of the separation. On the other hand, it complicates the size and composition analysis of particles.

Chemical Fractionation↗
PubMed2001

Sedimentation field-flow fractionation device cleaning, decontamination and sterilization procedures for cellular analysis.

In Sedimentation FFF (SdFFF) practice, it is known that a large number of cell elutions create aging phenomena of the separator, thereby reducing recovery and modifying elution characteristics. Systematic cleaning procedures are developed to enhance channel lifetime, together with microbial decontamination processes. Cells can be therefore reproducibly eluted for a large number of analyses and collected under sterile conditions, if needed. This is one of the most valuable aspect if further culture or transplantation is required. Decontamination was performed using, as contaminant probe, Staphylococcus aureus, highly adherent pathogenic bacteria that eluted from SdFFF as aggregates.

Bacterial Adhesion↗
PubMed1991

Particle size distribution by sedimentation/steric field-flow fractionation: development of a calibration procedure based on density compensation.

Because of the important but mathematically complex role played by hydrodynamic lift forces in sedimentation/steric FFF, applied generally to particles greater than 1 micron in diameter, retention cannot readily be related to particle diameter on the basis of simple theory. Consequently, empirical calibration is needed. Unfortunately, retention is based on particle density as well as size so that a purely size-based calibration (e.g., with polystyrene latex standards) is not generally valid. By examining the balance between driving and lift forces, it is concluded that equal retention will be observed for equal size particles subject to equal driving forces irrespective of particle density. Therefore by adjusting the rotation rate to exactly compensate for density, retention can be brought in line with that of standards, a conclusion verified by microscopy. Linear calibration plots of log (retention time) versus log (diameter) can then be used. This approach is applied to two glass bead samples (5-30 and 5-50 microns) using both a conventional and a pinched inlet channel. The resulting size distribution curves are self consistent and in good agreement with results obtained independently.

Calibration↗
PubMed1994

Enhancement of performance in sedimentation field-flow fractionation by temperature elevation.

Nonequilibrium theory, combined with the principles of time optimization, show that the time necessary to achieve a given separation in FFF is scaled to eta/T, where eta is viscosity and T is absolute temperature. The eta/T ratio for water (and other common liquids) decreases several-fold for modest temperature gains of approximately 20-60 degrees C, implying a significant advantage for FFF operation at elevated temperatures. This concept was tested by modifying a standard sedimentation FFF apparatus with a heating system. The separation of 0.220-0.742 microns polystyrene latex beads in aqueous carrier liquids was compared at room temperature and at elevated temperatures of 51 and 68 degrees C. Both separation power and speed were improved. In accordance with the predicted eta/T scaling, the separation time of five bead sizes at a given resolution level was reduced by a factor of approximately 2.4 (from 29 to 12 min) in elevating the temperature from 25 to 68 degrees C. Some other potential benefits of temperature elevation in FFF are discussed.

Chemical Fractionation↗