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Results for “Fractionation, Field Flow”

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PubMed2001

Optimization of experimental conditions for the separation of small and large starch granules by gravitational field-flow fractionation.

Separation of small and large barley starch granules by gravitational field-flow fractionation was investigated from the point of view of sample pre-treatment, amount of injected sample, and elution conditions. The sample pre-treatment study resulted in the conclusion that it is reasonable to soak the starch granules for at least 24 h prior to separation. The experiments with different amounts of injected sample show that it is possible to increase as well as decrease twofold the sample amount usually used without any change in retention ratios. The implementation of flow-rate gradients for elution of the starch granules reduced total separation time. However, the applied flow-rate gradients did not improve the resolution of peaks A and B compared with the generally used constant flow-rate. Thus, for barley starch granules, the constant flow-rates within the range from 0.8 to 1.0 ml/min seem to provide the best compromise of total separation time, peak resolution and instrumental expense.

Chemical Fractionation↗

PubMedDate not supplied

Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity.

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.

Cell Culture Techniques↗

PubMed2003

Practical implications of ionic strength effects on particle retention in thermal field-flow fractionation.

Modification of ionic strength of an aqueous or non-aqueous carrier solution can have profound effects on the particle retention behavior in thermal field-flow fractionation (ThFFF). These effects can be considered as either advantageous or not depending on the performance criteria under consideration. Aside from the general increase in retention time of particulate material (latexes and silica particles), our experiments indicate improvement in resolution with increases in electrolyte concentration. Absence of an electrolyte in the carrier solution causes deviations from the theoretically expected linear behavior between the retention parameter lambda (a measure of the extent of interaction between the applied field and the particle) and the reciprocal temperature drop across the channel walls. A negative interaction parameter delta(w), of about -0.170 was determined for 0.105- and 0.220-microm polystyrene (PS) latex particles suspended in either a 0.25 or a 1.0 mM TBAP-containing acetonitrile carrier and for 0.220 microm PS in 0.50 and 1.0 mM NaCl-containing aqueous medium. This work also demonstrates that optimum electrolyte concentrations can be chosen to achieve reasonable experimental run-times, good resolution separations, and shifts in the steric inversion points at lower field strengths, and that too high electrolyte concentrations can have deleterious effects such as band broadening and sample loss through adsorption to the channel accumulation surface. The advantages of using ionic strength rather than field strength to effect desired changes are lowered power consumption and possible application of ThFFF to high temperature-labile samples.

Fractionation, Field Flow↗

PubMed1993

Electrical field-flow fractionation in particle separation. 1. Monodisperse standards.

In its original implementation, electrical field-flow fractionation (EFFF) was carried out in membrane-walled channels with the electrodes placed externally to the flow channel. The poor separation efficiency of this system left the technique largely unattended for about two decades. In the present study, we describe a new and simple approach to EFFF, which demonstrates the technique's ability to carry out rapid, high-resolution separations of colloidal samples in aqueous suspensions. The present channels are bounded by the smooth and rigid graphite electrodes which allow for the application of small voltages, typically less than 2 V, across the thin (178 microns) separation space defined by a Mylar spacer. Although this arrangement generates nominal fields of the order of 100 V/cm, polarization of the electrodes considerably reduces the effective field across the bulk of the channel to less than 1% of the nominal value. Nevertheless, under conditions of low ionic strength the system is shown to retain and separate polystyrene (PS) latex standards with sizes ranging from 60 to 10,000 nm. For small particles of comparable zeta-potential, separating in the "normal" mode of EFFF, the size selectivity Sd, is approximately 0.7. As with other FFF techniques, EFFF displays a transition from "normal" to "steric" behavior; the critical diameter for this transition is highly dependent on ionic strength, with values ranging from approximately 500 nm in deionized water to approximately 1200 nm in 133 microM aqueous NaCl under an applied voltage of 1.37 V and a flow of 1 mL/min.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Fractionation↗

PubMed2005

Interface for direct and continuous sample-matrix deposition onto a MALDI probe for polymer analysis by thermal field flow fractionation and off-line MALDI-MS.

A simple interface based on an oscillating capillary nebulizer (OCN) is described for direct deposition of eluate from a thermal field-flow fractionation (ThFFF) system onto a matrix-assisted laser desorption/ionization (MALDI) probe. In this study, the polymer-containing eluent from the ThFFF system was mixed on-line with MALDI matrix solution and deposited directly onto a moving MALDI probe. The result was a continuous sample track representative of the fractionation process. Subsequent off-line MALDI-mass spectrometry analysis was performed in automated and manual modes. Polystyrene samples of broad polydispersity were used to characterize the overall system performance. The OCN interface is easy to build and operate without the use of heaters or high voltages and is compatible with any MALDI probe format.

Fractionation, Field Flow↗

PubMed2005

Capillary magnetic field flow fractionation and analysis of magnetic nanoparticles.

This paper reports the purification and analysis of magnetic nanoparticles using capillary magnetic field flow fractionation, which utilizes an applied magnetic field oriented orthogonal to the capillary flow. To validate this approach as a separation method for nanometer-scale particles, samples of magnetic nanoparticles composed of either gamma-Fe2O3 (maghemite) or CoFe2O4 with average diameters ranging from 4 to 13 nm were prepared and characterized by transmission electron microscopy and SQUID magnetometry. Retention of the samples on the capillary was investigated as a function of solvent flow rate and the nanoparticle size and composition; the elution times of the nanoparticles are strongly dependent on their magnetic moments. We demonstrate the use of this method to separate a mixture of nanoparticles into size-monodisperse fractions. The magnetic moments of the particles are calculated based on analysis of the retention parameters and correlate with values obtained in separate SQUID magnetometry measurements.

Journal Article↗

PubMed2006

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.

Amylases↗

PubMed1988

Sedimentation field flow fractionation of fused unilamellar vesicles: comparison with electron microscopy and gel filtration.

Sedimentation field flow fractionation (SF3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-A small unilamellar vesicles to 700-A large unilamellar vesicles (LUVs) and the slower conversion to 950-A LUVs at 4 degrees C was readily monitored by SF3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF3 are discussed and correlated with electron microscopy and gel filtration.

Calorimetry, Differential Scanning↗

PubMed1997

Introducing dielectrophoresis as a new force field for field-flow fractionation.

We present the principle of cell characterization and separation by dielectrophoretic field-flow fractionation and show preliminary experimental results. The operational device takes the form of a thin chamber in which the bottom wall supports an array of microelectrodes. By applying appropriate AC voltage signals to these electrodes, dielectrophoretic forces are generated to levitate cells suspended in the chamber and to affect their equilibrium heights. A laminar flow profile is established in the chamber so that fluid flows faster with increasing distance from the chamber walls. A cell carried in the flow stream will attain an equilibrium height, and a corresponding velocity, based on the balance of dielectrophoretic, gravitational, and hydrodynamic lift forces it experiences. We describe a theoretical model for this system and show that the cell velocity is a function of the mean fluid velocity, the voltage and frequency of the signals applied to the electrodes, and, most significantly, the cell dielectric properties. The validity of the model is demonstrated with human leukemia (HL-60) cells subjected to a parallel electrode array, and application of the device to separating HL-60 cells from peripheral blood mononuclear cells is shown.

Cell Separation↗

PubMed2004

Influence of operating parameters on the retention of chromatographic particles by thermal field-flow fractionation.

We have investigated the retention behavior of chromatographic particles in thermal field-flow fractionation (FFF). Retention time is found to increase with increasing temperature drop across the channel thickness, as expected for species exhibiting a thermophoretic mobility. Experiments have been performed with a vertically oriented channel rather than by using the classical horizontal configuration as this leads to much more reproducible retention data. In acetonitrile, silica-based particles are more retained than octadecyl-bonded silica particles, which confirms our previous finding, by means of a different method, that the thermophoretic mobility of the latter is smaller than that of the former. Whatever the type of particles and the nature of the carrier liquid, the relative retention time is observed to decrease with increasing carrier flow rate. This indicates that a hydrodynamic lift force acts on particles to move them away from the accumulation wall, as is usually observed in all FFF experiments with micrometer-sized particles. However, upward and downward flow directions in the vertical channel lead to similar retention data, indicating that inertial lift forces have a minor influence on retention. In addition, the relative retention time steadily decreases with increasing sample concentration, suggesting that the hydrodynamic lift force increases significantly with sample concentration. Accordingly, we speculate that a new transport phenomenon, called shear-induced hydrodynamic diffusion, not previously accounted for in the modeling of retention in FFF, is controlling the migration of the particles in the FFF channel. Implications of the influence of this phenomenon in other FFF experiments are discussed.

Chemical Fractionation↗

PubMed2005

Flow-based and sieving matrix-free DNA differentiation by a miniaturized field flow fractionation device.

DNA separation is typically done by gel electrophoresis based on its charge property. In our previous work, we reported that dielectrophoresis could be used to manipulate polystyrene nanoparticles' motion by using a miniaturized electrical field flow fractionation device (micro-EFFF) with a segmented electrode operated under a pulsed voltage (PV). In this work, we report the manipulation and separation of DNA molecules using the micro-EFFF. DNA motion was in situ visualized inside the device. Results revealed that dielectrophoresis governed DNA motion, which was strongly correlated with the pulse frequency but not the duty cycle of a PV. A longer retention time of DNA molecules was measured under a PV. The retention time increased with the length of DNA molecules. As the micro-EFFF is flow-based and sieving-matrix-free, it has a potential to be applied to sample preparation in a micrototal analysis system or when fractionated molecules are needed for downstream analysis.

DNA, Bacterial↗

PubMed2004

Diosgenin dose-dependent apoptosis and differentiation induction in human erythroleukemia cell line and sedimentation field-flow fractionation monitoring.

To limit or stop cancer spreading, one of the most prevalent strategies is to induce cancer cell death. Differentiation therapy and apoptosis induction are two ways to achieve this goal. Sedimentation field-flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape), we studied the capacity of SdFFF to monitor specific biophysical modifications that occurred during cellular apoptosis or differentiation induction. Then, we used, as an in vitro cellular model of apoptosis and differentiation, diosgenin dose-dependent induction in the polyvalent human erythroleukemia cell line. Two other chemicals were used: phorbol myristate acetate (differentiation inducer) and staurosporine (apoptosis inducer). Our results demonstrated a correlation between SdFFF elution profile changes and induction of effective biological processes. Thus, after acquisition of a reference profile, SdFFF could be used alone to follow chemically induced biological events, suggesting many different applications such as testing series of molecules, evaluation of new cellular/biological models used in different life science fields, or sorting purified populations with the aim of better understanding mechanisms of induced cellular events.

Apoptosis↗

PubMed1991

Isolation of microfilariae from blood by gravitational field-flow fractionation.

Over 100 million persons suffer from diseases caused by filariae infestation, and one billion are at risk. A simple isolation method for both analytical and preparative separation is presented. Based on the simplest field-flow fractionation technique, the gravitational one, effective isolation of microfilariae is achieved. Microfilariae are eluted in the void volume of the channel without pollution by red blood cells. The red blood cell elution peak shows a total absence of microfilariae, as demonstrated after fraction collection and microscopic investigation. The elution mode of microfilariae and red blood cells appears to be a steric one, as confirmed by a reinjection experiment. The simplicity, low cost and the relatively short time required for this separation (10 min) indicate that gravitational field-flow fractionation could become a new separation tool for screening of microfilariae. With both live and dead microfilariae, the high recovery (66-80%) allows preparative fractionation for diagnostic purposes or fundamental research.

Animals↗

PubMed1993

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.

Apoferritins↗

PubMed1990

Evaluation of pinched inlet channel for stopless flow injection in steric field-flow fractionation.

In this article the concept of utilizing a pinched inlet channel for field-flow fractionation (FFF), in which the channel thickness is reduced over a substantial inlet segment to reduce relaxation effects and avoid stopflow, is evaluated for steric FFF using one conventional channel and two pinched inlet channels. It is shown that with the proper adjustment of flow-rate, the stopflow process in FFF can be completely avoided, thus bypassing the flow interruption associated with stopflow and reducing separation time. The maximum flow-rate that can be used for stopless flow operation without incurring zone distortion is shown to agree reasonably well with simple theory; slight departures from theory are attributed to the existence of reduced transport rates of large particles through thin channel structures.

Centrifugation↗

PubMed2002

Hyphenation of sedimentation field flow fractionation with flow cytometry.

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).

Chemical Fractionation↗

PubMed2001

Geometric scaling effects in electrical field flow fractionation. 1. Theoretical analysis.

This work outlines the fundamental scaling laws associated with electrical field flow fractionation channels. Although general FFF theory indicates few advantages from miniaturization, EFFF theory indicates clear advantages to miniaturization of the EFFF channel. Retention, plate heights, resolution, equilibration times, and time constants are examined. The outlined theory predicts scaling advantages in each of these areas after miniaturization. Potential applications, such as the use of these systems for sample preparation in microscale total analysis systems, and improvements associated with these theoretical predictions are also discussed.

Journal Article↗

PubMed2004

Mechanistic investigation of nanoparticle motion in pulsed voltage miniaturized electrical field flow fractionation device by in situ fluorescence imaging.

In our previous study, we reported a miniaturized electrical field flow fractionation device (micro-EFFF) that used a pulsed voltage (PV) to increase the effective electric field and, hence, improved the separation performance. In this work, we developed two micro-EFFFs with planar or segmented electrode design and investigated the particle movement in the flow channels under a PV. Numerical simulation was used to understand the electric field distribution in the micro-EFFFs. When the calculations for the micro-EFFF with a segmented electrode (segmented micro-EFFF) and the micro-EFFF with planar electrodes (planar micro-EFFF) are compared, a stronger electric field at the top electrode segments is found in the segmented micro-EFFF, with the strongest field at the edges of the electrode segments. Nanoparticle motion in both devices was in situ visualized by using a fluorescence microscope equipped with a CCD-camera. Results reveal that electrophoresis governs the nanoparticle movement in the planar micro-EFFF and dielectrophoresis dominates the movement in the segmented micro-EFFF. Two models are postulated to explain the experimental observations of the nanoparticle movement. The mechanistic understanding of controlling nanoparticle motion in a miniaturized environment will help the design and application of micro-EFFF for the separation of charged biomolecules (proteins and DNAs).

Journal Article↗