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PubMed2006

Determination of calibration function in thermal field flow fractionation under thermal field programming.

A new procedure for determining the calibration function able to relate retention and operative parameters to molecular weight of the species in thermal field flow (ThFFF) under thermal field programming (TFP) conditions is presented. The procedure involves determining the average values of retention parameters under TFP and determining a numerical function related to the temperature variations that occur during TFP. The calibration parameters are obtained by a procedure fitting the retention and operative parameters that hold true at the beginning of the TFP. The procedure is closely related to the one previously developed to calibrate the retention time axis under TFP ThFFF and, together, they constitute a full calibration procedure. Experimental validation was performed with reference to polystyrene (PS)-decalin and PS-THF systems. The calibration functions here obtained were compared to those derived by the classical procedure at constant thermal field ThFFF to obtain the calibration function at variable cold wall temperatures. Excellent agreement was found in all cases proving "universality" of the ThFFF calibration concept, i.e. it is independent of the particular system on which it was determined and can thus be extended to ThFFF operating under TFP. The new procedure is simpler than the classical one since it requires less precision in setting the instrumentation and can be obtained with fewer experiments. The potential applications for the method are discussed.

Journal Article↗

PubMed2005

Characterisation of poly(N-isopropylacrylamide) by asymmetrical flow field-flow fractionation, dynamic light scattering, and size exclusion chromatography.

Asymmetrical flow field-flow fractionation (AsFIFFF) was used to determine the hydrodynamic particle sizes, molar masses, and phase transition behaviour of various poly(N-isopropylacrylamide) (PNIPAM) samples synthesised by reversible addition--fragmentation chain transfer (RAFT) and conventional free radical polymerisation processes. The results were compared with corresponding data obtained by dynamic light scattering (DLS) and size exclusion chromatography (SEC). Agreement between the three methods was good except at higher molar masses, where the molar mass averages obtained by SEC were much lower than those obtained by AsFIFFF and light scattering. The aggregation of the polymers, which are thermally sensitive, was studied by DLS and AsFIFFF at various temperatures. In deionised water there was an abrupt change in the particle size due to phase separation at approximately equal to 32-35 degrees C. The critical temperatures determined by AsFIFFF were 3-5 degrees C higher than those obtained by DLS.

Acrylic Resins↗

PubMed2001

TiO2 colloidal suspension polydispersity analysed with sedimentation field flow fractionation and electron microscopy.

Sedimentation field flow fractionation (SdFFF) operated at multi gravitational field is used to analyse a highly polydisperse TiO2 colloidal suspension. From the initial sample, time dependent eluted fractions are collected and submitted to electron microscopy (EM) shape and size analysis. To assess the accuracy of FFF in determining the average size of the different fractions, these are re-introduced into the channel by means of two different procedures, the on-channel concentration of the fractions and the direct re-injection of pre-concentrated fractions (DRI). Both methods appear accurate to determine the average size of every fraction, associated to a lower recovery in the case of DRI. The fractogram band spreading characteristics of the re-introduced fractions are correlated to the particle size distribution measured by EM. After density determination of fractionated particles, the fractogram is calibrated in terms of size and size distribution using data obtained from EM for each fraction. Quantitative analyses, based on particle counting showed high recovery (80-90%) of the eluted species. However, this loss limited the possibility to extend signal information to a quantitative one.

Chemical Fractionation↗

PubMed2003

Lowering the molecular mass limit of thermal field-flow fractionation for polymer separations.

Thermal field-flow fractionation (ThFFF) is capable of separating a wide molecular mass range of polymers by their molecular mass (Mr) and chemical composition. However, retention and resolution decrease significantly for polymers with Mr<20 kDa. Various approaches for increasing the retention of low Mr (<15 kDa) polymers were investigated. Our results showed that temperature conditions and single-component solvents had a limited effect on polymer retention and that certain binary solvent mixtures caused a dramatic increase in retention. The binary solvents approach has enabled the use of a ThFFF system and temperature conditions to separate 2.6 kDa PS from 4.4 kDa PS, thereby extending the applicability of ThFFF to lower molecular masses. The effect of binary solvent mixtures on polymer retention is correlated with the mixture viscosity.

Fractionation, Field Flow↗

PubMed2006

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion.

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.

Chymotrypsin↗

PubMed2004

Laser-induced breakdown detection combined with asymmetrical flow field-flow fractionation: application to iron oxi/hydroxide colloid characterization.

The combination of asymmetrical flow field-flow fractionation (AsFlFFF) with the laser-induced breakdown detection (LIBD) is presented as a powerful tool for the determination of colloid size distribution at trace particle concentrations. Detection limits (D1) of 1, 4, and 20 microg/L have been determined for a mixture of polystyrene reference particles with 20, 50, and 100 nm in size, respectively. This corresponds to injected masses of 1, 4, and 20 pg, which is lower than found in a previous study with the symmetrical FlFFF (SyFlFFF). The improvement is mainly due to the lower colloid background discharged from the AsFlFFF channel. The combined method of AsFlFFF-LIBD is then applied to the analysis of iron oxi/hydroxide colloids being considered as potential carriers for the radionuclide migration from a nuclear waste repository. Our LIBD arrangement is less sensitive for iron colloid detection as compared to reference polystyrene particles which results in a detection limit of approximately 240 microg/L FeOOH for the AsFlFFF-LIBD analysis. This is superior to the detection via UV-Vis absorbance and comparable to ICP-MS detection. Size information (mean size 11-18 nm) for different iron oxi/hydroxide colloids supplied by the present method is comparable to that obtained by sequential ultrafiltration and dynamic light scattering. A combined on-line ICP-MS detection is used to gain insight into the colloid-borne main and trace elements.

Colloids↗

PubMed2004

Field-flow fractionation of cells with chemiluminescence detection.

Field-flow fractionation is a separation technique characterized by a retention mechanism which makes it suitable for sorting cells over a short analysis time, with low sample carry-over and preserving cell viability. Thanks to its high sensitivity, chemiluminescence detection is suitable for the quantification of just a few cells expressing chemiluminescence or bioluminescence. In this work, different formats for coupling gravitational field-flow fractionation and chemiluminescence detection are explored to achieve ultra-sensitive cell detection in the framework of cell sorting. The study is carried out using human red blood cells as model sample. The best performance is obtained with the on-line coupling format, performed in post-column flow-injection mode. Red cells are isolated from diluted whole human blood in just a few minutes and detected using the liquid phase chemiluminescent reaction of luminol catalysed by the red blood cell heme. The limit of detection is a few hundred injected cells. This is lower than the limit of detection usually achieved by means of conventional colorimetric/turbidimetric methods, and it corresponds to a red blood cell concentration in the injected sample of five orders of magnitude lower than in whole blood.

Erythrocytes↗

PubMed2003

Mass overloading in the flow field-flow fractionation channel studied by the behaviour of the ultra-large wheat protein glutenin.

Flow field-flow fractionation (FFF) has previously been used in successful fractionation and characterisation of the ultra-large wheat protein glutenin. The many parameters, which may influence the retention behaviour, especially when analysing extremely high-molecular-mass samples such as glutenin, are here reported. Size determination from the sample retention time, using FFF theory, will as a result have a very low accuracy. The need for direct molecular mass determination, such as by light scattering, in combination with FFF, in order to do accurate size measurements of glutenin is pointed out as well as the importance to minimise the overloading.

Fractionation, Field Flow↗

PubMed2002

Flow field-flow fractionation and characterization of ionic and neutral polysaccharides of vegetable and microbial origin.

The flow field-flow fractionation (FlFFF) analysis of a variety of neutral as well as ionic polysaccharides from plants and micro-organisms shows the generally broad distribution in molecular size of these polymers. This result is also obtained on a commercial sample of pullulan whose size distribution appears much wider than that of any of five standard fractions of the same polymer. Clear evidence of some physico-chemical properties of the polysaccharides is given by the study of the effect of the carrier ionic strength on salep, oxidized salep and konjac, carboxymethylcellulose and hyaluronic acid. While neutral polysaccharides, regardless of their origin, only slightly change size distribution in the presence of a simple electrolyte in solution, charged polymers, either naturally charged or chemically ionized, consistently exhibit very low retention levels in water which dramatically increase even at low salt concentrations. Exclusion mechanisms, including steric effects, are shown to be responsible for the anticipated retention times in water of these species that assume the expected statistical coil behavior only when electric charges are screened by the added electrolyte. Under these conditions, higher retention levels are obtained because the volume adjacent to the accumulation wall becomes more accessible to the sample during relaxation. On the basis of these findings, the elution behavior of a number of polysaccharide samples in-laboratory obtained from the fungus Aureobasidium pullulans under different incubation conditions is attributed to the presence of species varying in physico-chemical properties and molecular size.

Fractionation, Field Flow↗

PubMed2005

Fast "hyperlayer" separation development in sedimentation field flow fractionation.

Specific prototypes of sedimentation field flow fractionation devices (SdFFF) have been developed with relative success for cell sorting. However, no data are available to compare these apparatus with commercial ones. In order to compare with other devices mainly used for non-biological species, biocompatible systems were used for standard particle (latex: 3-10 microm of different size dispersities) separation development. In order to enhance size dependent separations, channels of reduced thickness were used (80 and 100 microm) and channel/carrier-phase equilibration procedures were necessary. For sample injection, the use of inlet tubing linked to the FFF accumulation wall, common for cell sorting, can be extended to latex species when they are eluted in the Steric Hyperlayer elution mode. It avoids any primary relaxation steps (stop flow injection procedure) simplifying series of elution processing. Mixtures composed of four different monodispersed latex beads can be eluted in 6 min with 100 microm channel thickness.

Chemical Fractionation↗

PubMed2006

Programmed cross flow asymmetrical flow field-flow fractionation for the size separation of pullulans and hydroxypropyl cellulose.

Different functions for the programming of the cross flow in asymmetrical flow field-flow fractionation were studied with the aim to find the flow conditions most suitable for the molar mass distribution analysis of high molecular weight polysaccharides. A mixture of four differently sized pullulans covering the molar mass range 5.8 x 10(3)-1.6 x 10(6) g mol(-1) were used as a model sample. Two types of programs were studied, linear and exponential decays, both with and without initial periods of a constant cross flow. For comparison, nonprogrammed runs, i.e. using constant cross flow, were studied. It was found that exponentially decaying cross flow gave the most uniform molar mass selectivity across the fractogram. The programmed cross flow was applied to the molar mass distribution analysis of a technical quality of hydroxypropyl cellulose.

Cellulose↗

PubMed2006

Increasing the sensitivity of asymmetrical flow field-flow fractionation: Slot outlet technique.

A new enrichment approach is described to improve the sensitivity of the asymmetrical flow field-flow fractionation, with and without ICPMS online coupling for elemental detection. For the slot outlet technique, a part of the laminar carrier stream is removed through an additional pump. This allowed an enrichment of the colloidal particles in the separation channel up to a factor of 14. Additional improvements of the separation efficiency permitted us to separate colloids with differences in their molecular masses of only 2 kDa. Different polymer standards, proteins, and synthesized tracer colloids, as well as real samples (wastewater, liquid manure, serums) were used to assess the performance of the new technique.

Colloids↗

PubMed2005

Field-flow fractionation and biotechnology.

The gentle separation mechanism has made field-flow fractionation particularly suited to samples of biotechnological interest, from proteins and nucleic acids to viruses, subcellular units and whole cells. Recent progress in field-flow fractionation technology, as well as the development of coupled techniques combining field-flow fractionation capabilities with the specificity and sensitivity of well-established analytical methods, opens up new biotechnological applications for field-flow fractionation. The most recent appealing applications include: sorting and fingerprinting of bacteria for whole-cell vaccine production; noninvasive and tagless sorting of immature and stem cells; separation of intact proteins and enzymes in top-down proteomics; and the development of flow-assisted, multianalyte immunoassays using nano- and micron-sized particles with immobilized biomolecules.

Animals↗

PubMed2006

Field-flow fractionation of proteins, polysaccharides, synthetic polymers, and supramolecular assemblies.

This review summarizes developments and applications of flow and thermal field-flow fractionation (FFF) in the areas of macromolecules and supramolecular assemblies. In the past 10 years, the use of these FFF techniques has extended beyond determining diffusion coefficients, hydrodynamic diameters, and molecular weights of standards. Complex samples as diverse as polysaccharides, prion particles, and block copolymers have been characterized and processes such as aggregation, stability, and infectivity have been monitored. The open channel design used in FFF makes it a gentle separation technique for high- and ultrahigh-molecular weight macromolecules, aggregates, and self-assembled complexes. Coupling FFF with other techniques such as multiangle light scattering and MS provides additional invaluable information about conformation, branching, and identity.

Fractionation, Field Flow↗

PubMed2006

Size-based speciation of natural colloidal particles by flow field flow fractionation, inductively coupled plasma-mass spectroscopy, and transmission electron microscopy/X-ray energy dispersive spectroscopy: colloids-trace element interaction.

Flow field flow fractionation (FIFFF), inductively coupled plasma-mass spectroscopy (ICP-MS), and transmission electron microscopy (TEM) coupled to X-ray energy dispersive spectrometry (X-EDS) are used in series for the first time to characterize colloids. Results demonstrate the utility of FIFFF-ICP-MS-TEM/X-EDS to relate physical properties (size) of colloids to their chemical properties (chemical composition, surface chemical composition, and colloids-trace elements association). Results suggest that the major part of natural organic matter (NOM) is concentrated in the fraction < 0.01 microm (C2). Aluminum, iron, and manganese are the main colloidal components in the fraction 0.01-0.45 microm (C1). Aluminum occurs as aluminum oxides or aluminosilicates in the whole size range, while iron and manganese occur as individual oxyhydroxides in the size range < 0.20 microm. Within the C2 fraction, Al, Mn, Cu, and Ni elements are complexed to NOM (e.g., humic substances). Iron is complexed to NOM in some samples and probably free in other samples. Lead is totally free in all samples. Within the C1 fraction, Cu and Pb are mostly associated to Fe and Mn oxyhydroxides. Consequently, NOM with Fe and Mn oxyhydroxides are the main colloidal carriers of trace elements in the Loire watershed system.

Colloids↗

PubMed2003

Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

Bacillus cereus↗

PubMed2005

Application of a high-performance liquid chromatography fluorescence detector as a nephelometric turbidity detector following Field-Flow Fractionation to analyse size distributions of environmental colloids.

A new operation mode for HPLC-type fluorescence detectors is presented and evaluated using synthetic and environmental particles in the colloidal size range. By applying identical wavelengths for excitation and emission a nephelometric turbidity or single angle light scattering detector is created which can be easily coupled to flow or sedimentation Field-Flow Fractionation (Flow FFF or Sed FFF) for the analysis of colloidal dispersions. The results are compared with standard UV-vis detection methods. Signals obtained are given as a function of particle size and selected detection wavelength. Conclusions can be drawn which affect the current practice of FFF but also for other techniques as groundwater sampling and laboratory column experiments when turbidity is measured in nephelometric mode and in small sample volumes or at low flow rates.

Chromatography, High Pressure Liquid↗

PubMed2003

Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation.

The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of rhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli. A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A600 of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A600 5.8). AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell(-1), an almost 3-fold increase in the number of tRNAs cell(-1), and up to a 26% increase in the mass fraction of active 70S ribosomes.

Bacterial Proteins↗