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PubMed2001

A data analysis algorithm for programmed field-flow fractionation.

An algorithm that employs numerical integration for analysis of field-flow fractionation (FFF) data is presented. The algorithm utilizes detector response, field strength, and channel flow rate data, monitored at discrete time intervals during sample elution to generate a distribution of sample components according to particle size or molecular weight. The field strength and channel flow rate may either be held constant or programmed as functions of time, and it is not necessary for these programs to follow specific mathematical functions. If experimental conditions are monitored during a run, the algorithm can account for any deviation from nominal set conditions. The algorithm also allows calculation of fractionating power for the actual conditions as monitored during the run. The method provides greatly increased flexibility in the application of the FFF family of techniques. It removes the limitations on experimental conditions incurred by adherence to analytically available solutions to FFF theory, allowing ad hoc variation of field strength and other experimental parameters as necessary to increase sensitivity and specificity of the method. An implementation of the algorithm is described that is independent of the FFF technique (i.e., independent of field type) and mode of operation. To reduce computation time, it uses mathematical techniques to reduce the required number of numerical integrations. This is of particular importance when the perturbations to ideal FFF theory, such as those due to the effects of hydrodynamic lift forces, particle-wall or particle-particle interactions, and secondary relaxation, necessitate relatively lengthy numerical calculations.

Algorithms↗
PubMedDate not supplied

Expression of Vitreoscilla hemoglobin in Escherichia coli enhances ribosome and tRNA levels: a flow field-flow fractionation study.

Asymmetrical flow field-flow fractionation (FFF) was used to separate and quantitate 70S ribosomes, the 30S and 50S subunits, and tRNA in one single analytical procedure. The method was applied to an investigation of the effect of Vitreoscilla hemoglobin (VHb) on the translational machinery of the recombinant Escherichia coli cells. The number of active 70S ribosomes per cell increased dramatically, more than 2-fold, as did also the tRNA levels for the VHb-expressing strain relative to VHb-negative control at the end of a 30-h fed-batch cultivation. This was accompanied by a corresponding 61% increase of a cloned marker enzyme activity. The results clearly indicate that VHb promotes the level of translational components. There should be many other cases in bioengineering where it is important to relate the protein production level in a bioreactor to the ribosome and tRNA levels.

Aerobiosis↗
PubMed2003

Circular asymmetrical flow field-flow fractionation for the semipreparative separation of particles.

A new technique for the separation and characterization of particles and polymers based on asymmetrical flow field-flow fractionation was developed. The new circular asymmetrical flow field-flow fractionation instrument (CAFFFE) resembles a quasi-parallel arrangement of 12 individual flow channels. As compared to the classical asymmetrical flow field-flow fractionation (AF-FFF), which can be used so far only for analytical separation and characterization of particles and polymers, the CAFFFE allows the introduction of higher amounts of sample into the channel in a single run so that semipreparative to preparative separation becomes possible. This was demonstrated by the separation of polymer latex standards.

Journal Article↗
PubMed2004

Effect of channel width on the retention of colloidal particles in polarization, steric, and focusing micro-thermal field-flow fractionation.

The effect of the channel width on the performance of separation by micro-thermal field-flow fractionation (micro-TFFF) of the carboxylated polystyrene latex particles was studied by using the particles in diameter range from 100 nm to 3800 nm. It has been shown that the retention order follows the anticipated polarization, steric, and focusing mechanism in the corresponding size range and under the specific conditions, appropriate to each channel thickness. However, the attractive interactions of the particles with the accumulation wall can complicate the separation as has been proven by the experiments carried out by using the carrier liquids of different ionic strengths. Three channel thicknesses (0.025, 0.100, and 0.250 mm) were tested thus imposing the volumes of micro-channels of roughly 9, 37, and 92 microl. Such an experimental investigation has never been performed with respect to the applicability of the TFFF within an extended range of molar masses or particle sizes. The advantages and drawbacks of different channel widths are discussed with respect to the performance of separation of micro-TFFF but also by taking into account the practical requirements of the construction of the micro-TFFF channel. The principal finding is that very thin channel (w = 0.025 mm) substantially reduces the range of particle sizes or polymer molar masses that can effectively be separated due to the mixed separation mechanism, steric exclusion being effective from smaller particle size. The found dependence of the resolution on the imposed experimental conditions including the channel width has allowed the elucidation of some peculiar results published in the literature, which were contradictory with regard to the known theoretical and experimental findings.

Colloids↗
PubMed1998

Compositional heterogeneity in parenteral lipid emulsions after sedimentation field flow fractionation.

This study examines the size and compositional heterogeneity of particles in a commercial lipid emulsion (Intralipid) before and after equilibration with penclomedine, a highly lipophilic cytotoxic agent. Emulsions were fractionated by sedimentation field-flow fractionation (sedFFF), and particle sizes of the monodisperse fractions were determined by photon correlation spectroscopy. The triglyceride (TG), phosphatidylcholine (PC), and penclomedine (in drug loaded emulsions) contents in each fraction were determined by HPLC. The aqueous-entrapped volume within Intralipid was determined to be approximately 10% by size-exclusion chromatography using [3H]mannitol. Thirteen sedFFF fractions collected from the drug free emulsions yielded particles ranging in size from 154 to 423 nm. Total channel recoveries were 89% and 95% for TG and PC, respectively. Apparent particle densities varied significantly with size, suggesting heterogeneity in composition as confirmed by PC/TG mass ratios which varied dramatically. Computer fits of the distribution profiles suggested populations of phospholipid vesicles and oil droplets containing excess phospholipid in addition to classical emulsion droplets. Drug loading induced a significant shift of the predominant triglyceride containing population to a larger particle size. The penclomedine distribution profile closely mimicked that of the TG rather than the PC fraction. These studies suggest the need to consider not only size distribution but also compositional distribution in characterizing parenteral emulsions.

Antineoplastic Agents↗
PubMed1990

Hydrodynamic relaxation and sample concentration in field-flow fractionation using permeable wall elements.

The advantages of hydrodynamic relaxation in field-flow fractionation, in which an injected sample is driven rapidly toward its equilibrium distribution by flow, are described relative to conventional field-driven relaxation. A new concept for achieving hydrodynamic relaxation, based on the use of permeable wall elements (or frit elements) embedded in the channel walls, is introduced. Here an auxiliary substream of carrier fluid, permeating uniformly into the FFF channel near the inlet, drives the sample, entrained in its own substream, close to its equilibrium configuration. Such frit elements can also be used to enrich the sample at the outlet. Equations are derived and plots are provided for the position of the splitting plane dividing the two substreams; this position defines the strength of the hydrodynamic relaxation. Variations in shear through these frit-modified end regions are also formulated and plotted. The effects of frit elements on band broadening are discussed. It is concluded that permeable wall elements in many configurations may be broadly applicable to FFF and related methods for improved sample introduction, increased separation speed, reduced risk of sample adhesion to the wall, improved flow stability, and sample enrichment.

Chemistry Techniques, Analytical↗
PubMed1988

Sedimentation field flow fractionation of mitochondrial and microsomal membranes from corn roots.

Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.

Cell Fractionation↗
PubMed2004

Influence of ionic strength, sample size, and flow conditions on the retention behavior of pullulan in flow field-flow fractionation.

Polymer molecular parameters such as hydrodynamic size are expected to be invariant regardless of the technique used to measure them, and to vary only, to some extent, with the solvent power and the polymer structure and properties as predicted from polymer chemistry. The hydrodynamic size of five pullulan standards derived from FlFFF in solutions of different ionic strength appears to correlate well to molecular mass as expected for neutral polymers for all fractions except that of lower mass. The correlation also holds for large amounts of injected sample even though with a slope which increases with rising polymer load. The evidence that the same result is obtained also for low sample amounts but with a higher cross-flow rate is interpreted as the manifestation of the presence of hydrodynamic interactions in concentrated polymer systems.

Fractionation, Field Flow↗
PubMed1984

Stability of perfluorocarbon blood substitutes determined by sedimentation field-flow fractionation.

It is shown that the method known as sedimentation field-flow fractionation, which has been applied to the separation and characterization of many industrial and biological particles and recently to emulsions, can be used to obtain high-resolution droplet diameter profiles for perfluorocarbon blood substitutes. Following a description of the methodology, experiments are described for two commercial perfluorocarbon emulsions, Fluosol-DA 20% and Fluosol-43. The droplet diameter profiles for both of these blood substitutes are shown to shift to noticeably higher diameter values in less than 2 months. The diameter at the profile peak for Fluosol-DA 20%, for example, shifts from 0.19 to 0.27 micron in 56 days.

Blood Substitutes↗
PubMed1972

Electrical field-flow fractionation of proteins.

Protein separation has been achieved by electrical field-flow fractionation, a heretofore unrealized separation technique. Some advantages of this method relative to electrophoresis are the low voltage required, the lack of adverse heating and support effects, and the existence of the method as an elution technique. A comparison of theoretical and experimental retention shows good agreement.

Albumins↗
PubMed2002

On void time determination in thermal field-flow fractionation.

Because of the temperature dependence of the carrier liquid density, the mass of carrier which is contained in a thermal field-flow fractionation channel depends on the cold wall temperature and on the temperature difference across the channel thickness. It is observed that the void time of the solvent peak decreases when increasing the average temperature in the channel. The void time is found to be directly proportional to the average carrier density in the channel. The determination of the void time from the knowledge of the channel geometrical volume and the measurement of the volumetric flow-rate leads to significant errors if the thermal expansion of the carrier between the temperature of the measurement and the average channel temperature is not taken into account. Recommendations are given for proper void time determinations in thermal FFF.

Fractionation, Field Flow↗
PubMed2002

Steric-hyperlayer sedimentation field flow fractionation and flow cytometry analysis applied to the study of Saccharomyces cerevisiae.

Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 microm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective "steric-hyperlayer" mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density.

Equipment Design↗
PubMed2006

Estimation of the Hamaker constants by sedimentation field-flow fractionation.

van der Waals forces are one of several forces that control the adhesion between two materials. These forces are important to quantify in adhesion studies because they are always present and are always attractive. The major problem in calculating the van der Waals interaction between colloidal particles is that of evaluating the Hamaker constant. Hence, an accurately determined Hamaker constant for a given material is needed when interfacial phenomena such as adhesion are discussed in terms of the total potential energy between a particle and a substrate. In this paper, a new simple and accurate methodology for the estimation of the Hamaker constant is introduced. The results are in good agreement with those values found in literature.

Adhesiveness↗
PubMed2005

Sedimentation field flow fractionation monitoring of rice starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.

Amylases↗
PubMedDate not supplied

Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins.

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.

Chromatography, Gel↗
PubMed2006

Neural stem cell separation from the embryonic avian olfactory epithelium by sedimentation field-flow fractionation.

The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using "Hyperlayer" elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a "pseudoplacodal" epithelioid cell type from which derived "floating cells". These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.

Animals↗
PubMed2006

Hollow-fiber flow/hyperlayer field-flow fractionation for the size characterization of airborne particle fractions obtained by SPLITT fractionation.

Hollow-fiber flow field-flow fractionation (HF FlFFF) was applied for the separation and size characterization of airborne particles which were collected in a municipal area and prefractionated into four different-diameter intervals >5.0, 2.5-5.0, 1.5-2.5, <1.5 microm) by continuous split-flow thin (SPLIIT) fractionation. Experiments demonstrated the possibility of utilizing a hollow-fiber module for the high-performance separation of supramicron-sized airborne particles at steric/hyperlayer operating mode of HF FlFFF. Eluting particles during HF FlFFF separation were collected at short time intervals (approximately 10 s) for the microscopic examination. It showed that particle size and size distributions of all SPLITT fractions of airborne particles can be readily obtained using a calibration and that HF FlFFF can be utilized for the size confirmation of the sorted particle fraction during SPLITT fractionation.

Journal Article↗