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PubMed2002

Miniaturized flow fractionation device assisted by a pulsed electric field for nanoparticle separation.

Electric field flow fractionation (EFFF) is a powerful separation technique based on an electrical field perpendicular to a pressure-driven flow. Previous studies of microelectric field flow fractionation (micro-EFFF) indicate that separation performance was limited due to a weak effective electric field caused by polarization layers on the electrode surfaces. In this work, we report on a micro-EFFF device that uses a pulsed voltage scheme to overcome these limitations. The device was fabricated in indium tin oxide (ITO)-coated glass with ITO as electrodes. The effective electric field for pulsed voltage operation was found to be 50-fold stronger when compared with constant voltage operation. A strong influence of pulsed voltage frequency on nanoparticle retention times was observed. Using pulsed voltage, improved separation of polystyrene particles of different surface charge and particle size is demonstrated. Pulsed voltage also offers more parameters compared to the constant voltage mode, e.g., pulse frequency, duty cycle, and waveform to optimize the retention behavior of analytes.

Journal Article↗

PubMed2005

Shifts in polystyrene particle surface charge upon adsorption of the Pluronic F108 surfactant.

Electrical field-flow fractionation (ElFFF) and sedimentation field-flow fractionation (SdFFF) were used in combination to study the adsorption of the triblock polymeric surfactant, Pluronic F108 [(EO)129-(PO)56-(EO)129] to 200 nm polystyrene (PS) latex spheres. The SdFFF technique allowed an accurate determination of the mass of surfactant adsorbed on each particle from a solution of given concentration. To complement this isotherm study, we show that ElFFF can be used to measure fractional coverages of the formed electrically neutral surfactant layers on the charged PS particles. Through a combination of the two techniques it is possible to gain information about the structure of the adsorbate layer. Thus, when Pluronic F108 is taken up by the PS surface from solutions of low concentration, all three blocks appear to adhere to the surface as long as there is free space available. As the solution concentration increases and the fractional coverage reaches approximately 20%, the surface turns crowded enough to let the strongly adsorbing PPO blocks competitively displace the weakly adherent PEO blocks, which gradually rise to extend into the aqueous phase until the surface is fully saturated.

Adsorption↗

PubMed2006

Characterization of protein aggregation: the case of a therapeutic immunoglobulin.

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.

Acetic Acid↗

PubMed2001

Characterization of starch polysaccharides by flow field-flow fractionation-multi-angle laser light scattering-differential refractometer index.

The coupling between flow field-flow fractionation (FFF), multi-angle laser light scattering and differential refractometer index provides a promising technique for fractionation of starch polysaccharides in aqueous conditions. Native starches with different amylose/amylopectin levels (0-70%) as well as a pure amylose sample were characterized. By applying a sudden drop in the cross-flow-rate, clear separation was achieved between amylose (which elutes first) and amylopectin. Flow FFF produced correct relationships between the molecular mass or the gyration radius versus elution volume for the fractionated amylopectin population. The results are also considered in terms of the macromolecular composition of starches.

Amylopectin↗

PubMed2005

Field programming in frit inlet asymmetrical flow field-flow fractionation/multiangle light scattering: application to sodium hyaluronate.

The capability of field-programmed separation in frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been examined for separating a high molecular weight sodium hyaluronate (NaHA) by varying the field programming parameters. Experiments were performed with on-line coupling of the field programming FI-AFlFFF and multiangle light scattering (MALS) detection. Sample relaxation, a pre-requisite step to establish equilibrium states of sample materials prior to the beginning of separation in most forms of FFF techniques, is obtained by hydrodynamically in FI-AFlFFF without stopping the migration flow. Thus, the procedures of sample injection -- hydrodynamic relaxation -- separation in FI-AFlFFF are continuously achieved without halting the sample migration. In this study, field programming in FI-AFlFFF was investigated for the separation of NaHA, water-soluble polysaccharides, by examining the influence of field decay pattern, initial field strength condition, and ionic strength of carrier solution on the successful separation of a degraded NaHA sample. Results were compared with molecular weight calculations of eluting materials among different field programming conditions from multiangle light scattering (MALS) signals. It was found that when the field programming was utilized in FI-AFlFFF, a proper selection of initial cross-flow rate, the field decay pattern, and an appropriate control of final field strength needed to be carefully selected in achieving a successful separation of a broad molecular weight water-soluble polymer sample.

Fractionation, Field Flow↗

PubMed2005

Low-capacity channel designed for particle separation with controlled electric fields and evaluation of involved forces.

Electric field is one of the suitable physical fields applicable to particle separations. Although long rectangular channel is used for particle separation in usual electrical field flow fractionation (FFF), a short low-capacity channel can replace it if the field is precisely controlled. Several separation principles are proposed with this channel. The elution behavior of particles has revealed that the gravitational, diffusion, and hydrodynamic lift force (HLF) play important roles in the determination of the elution behavior of particles. The elution threshold voltage (V(th)) was defined and experimentally determined for various system configurations and particles. The electric force no longer overcomes the other forces, and particles are taken off the wall, when the applied voltage becomes lower than V(th). V(th) values have allowed us not only to estimate surface charge density of a particle but also to evaluate the hydrodynamic lift force against particle.

Electricity↗

PubMedDate not supplied

Effect of glutaraldehyde concentration on the physical properties of polymerized hemoglobin-based oxygen carriers.

Artificial blood substitutes based on glutaraldehyde cross-linked hemoglobin (PolyHb) are currently being developed for use in human subjects needing blood transfusions. Despite the commercial development of PolyHb dispersions, a systematic study of the effect of varying the glutaraldehyde to hemoglobin (G-Hb) molar ratio on the resulting PolyHb physical properties (molecular weight distribution and oxygen binding parameters) has not been conducted to date. The results of this study show that increasing the G-Hb molar ratio elicits a general decrease in the P50 (partial pressure of oxygen at which Hb is half saturated with oxygen) and cooperativity and a simultaneous increase in the weight averaged molecular weight (Mw) of the PolyHb dispersion and methemoglobin (MetHb) level. Three PolyHb dispersions (20:1, 30:1, and 40:1 G-Hb molar ratios) displayed potential as artificial blood substitutes. The 20:1 PolyHb dispersion resulted in the presence of more intramolecularly cross-linked and non-cross-linked tetramers versus cross-linked species that were larger than a tetramer ( approximately 75% tetrameric and approximately 25% higher-order species), lower MetHb level (8%), and P50 (20.1 mmHg) similar in magnitude to that of non-cross-linked Hb. The 30:1 PolyHb dispersion consisted of more higher-order species ( approximately 76%), higher MetHb level (28%), and lower P50 (13.3 mmHg). The 40:1 PolyHb dispersion resulted in a similar P50 of 13.0 mmHg and similar MetHb level (30%); however, this PolyHb dispersion only consisted of species larger than a tetramer. The molecular weight distribution of PolyHb dispersions was determined using asymmetric flow field-flow fractionator (AFFF) coupled with multiangle static light scattering (MASLS). This is the first time that AFFF-MASLS has been used to characterize the molecular weight distribution of PolyHb dispersions.

Biopolymers↗

PubMed2005

Cubosome dispersions as delivery systems for percutaneous administration of indomethacin.

PURPOSE: The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug. METHODS: Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system. RESULTS: Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding. CONCLUSIONS: MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.

Adult↗

PubMed1998

pH-regulated electroretention chromatography: towards a new method for the separation of proteins according to their isoelectric points.

The pH-dependent electroretention behavior of model proteins cytochrome c and ribonuclease A was studied in a hollow fiber arrangement, similar to that used in electrical field-flow fractionation. Field-induced immobilization of the proteins at the inner wall of the fiber was a function of the pH adjusted in the solution surrounding it, indicating that the pH inside the fiber lumen, relevant for protein migration, quickly equilibrates to the regulated value outside. A complete separation of the model proteins was achieved. Advantages of the principle as well as prospects for the development of a technique separating more than two protein species according to their isoelectric points are discussed.

Animals↗

PubMed2005

Field-flow fractionation-inductively coupled plasma mass spectrometry: an alternative approach to investigate metal-humic substances interaction.

Interaction between metal ions and humic matter was investigated using a hyphenated technique, field-flow fractionation-inductively coupled plasma mass spectrometry (FFF-ICP-MS). Aggregation of a metal-spiked commercial Aldrich humic acid in an aqueous solution of calcium ion or in seawater was examined over time intervals of 0-4320 min. The aggregation was demonstrated by shifts in peak maximum of humic matter from smaller size (2.9 nm) to larger size (5.1 or 5.8 nm in Ca2+ solution or in seawater, respectively) and also by the broadening of size distribution profiles. With FFF, size distribution of humic aggregate was characterized. Further, dominant particle size (2.9 nm), mean particle size (3.8 nm), and diffusion coefficient (1.51 x 10(-6) cm2/s) of humic acid solution were determined. With FFF-ICP-MS, associations of Cd, Cu, and Pb with humic aggregates were examined. The mean diameters of Cd-, Cu-, and Pb-bound humic aggregates in the metal-spiked humic acid were 4.1, 4.5, and 5.8 nm, respectively. These diameters were shifted to 6.0, 6.0, and 6.9 nm, respectively, in the humic acid incubated with calcium solution, whereas they were shifted to 6.5, 5.7, and 7.4 nm, respectively, in the humic acid incubated with seawater for three days. Humic aggregate of small size showed more affinity for Cu than Cd and Pb, whereas the large aggregate showed more affinity for Pb than Cd and Cu, respectively.

Environmental Monitoring↗

PubMed1999

High resolution size determination of 20 nm colloidal gold particles by SedFFF.

PURPOSE: Assessment of lower size limit of Sedimentation Field-Flow Fractionation (SedFFF), specifically to evaluate if the method is suitable to determine the size and size distribution of 20 nm colloidal gold particles with high resolution. METHODS: Sedimentation Field-Flow Fractionation was used to determine the size of the colloidal particles. Due to the high density of gold it was possible to extend the lower size limit of SedFFF well below 20 nm. The size distribution of a gold colloid was obtained from the peak broadening caused by the polydispersity of the sample. The peak broadening due to instrumental imperfections was determined. For comparison purpose the particles were also sized using SEM and PCS. RESULTS: The mean diameter of the particles was determined to be (20.87+/-0.05) nm, the standard deviation in size being 1.04 nm (about 5%). SEM could confirm that the particles are about 20 nm in diameter. A sizing with PCS was not possible. The particles have a strong tendency to aggregate and PCS yields a diameter that is much too large. CONCLUSIONS: At optimized analytical parameters Sedimentation Field-Flow Fractionation is an effective method to measure the size of gold particles as small as 15 nm with an accuracy of about 0.1 nm. The polydispersity of the sample can easily be determined.

Chemical Fractionation↗

PubMed2003

Experimental approaches for size-based metal speciation in rivers.

A review of the different methodologies employed to fractionate and characterize riverine suspended particulate matter is presented. The importance of size-based metal speciation is underlined and the possibility of studying it by the Sedimentation Field Flow Fractionation (SdFFF) technique is illustrated. The studies on the metal load in river Suspended Particulate Matter (SPM) performed over the last ten years are critically reviewed focusing on the different methods employed to collect, concentrate and size-fractionate samples. The fact that there is no homogeneity in methods and data collection in this field is underlined. Among the different fractionation techniques, Field Flow Fractionation (FFF) methodologies have proved to be a good approach to study the role of SPM in metal load and transport. The possibility of studying size-based metal speciation using the SdFFF technique is presented and the importance of metal speciation in rivers is underlined.

Environmental Monitoring↗

PubMed2000

Study of magnetic particles pulse-injected into an annular SPLITT-like channel inside a quadrupole magnetic field.

Advantages of the continuous magnetic flow sorting for biomedical applications over current, batch-wise magnetic separations include high throughput and a potential for scale-up operations. A continuous magnetic sorting process has been developed based on the quadrupole magnetic field centered on an annular flow channel. The performance of the sorter has been described using the conceptual framework of split-flow thin (SPLITT) fractionation, a derivative of field-flow fractionation (FFF). To eliminate the variability inherent in working with a heterogenous cell population, we developed a set of monodisperse magnetic microspheres of a characteristic magnetization, and a magnetophoretic mobility, similar to those of the cells labeled with a magnetic colloid. The theory of the magnetic sorting process has been tested by injecting a suspension of the magnetic beads into the carrier fluid flowing through the sorter and by comparing the theoretical and experimental recovery versus total flow-rate profiles. The position of the recovery maxima along the total flow-rate axis was a function of the average bead magnetophoretic mobility and the magnetic field intensity. The theory has correctly predicted the position of the peak maxima on the total flow-rate axis and the dependence on the bead mobility and the field intensity, but has not correctly predicted the peak heights. The differences between the calculated and the measured peak heights were a function of the total flow-rate through the system, indicating a fluid-mechanical origin of the deviations from the theory (such as expected of the lift force effects in the system). The well-controlled elution studies using the monodisperse magnetic beads, and the SPLITT theory, provided us with a firm basis for the future sorter evaluation using cell mixtures.

Magnetics↗

PubMed2006

Onset of non-linearity in zonal elution separators: the concept of effective analyte concentration.

When an analyte injected in a zonal separation method (chromatography, capillary zone electrophoresis, field-flow fractionation) is not highly diluted in the carrier fluid, the retention ratio, R--or ratio of the cross-sectional average migration velocity of the analyte to that of the carrier fluid--depends on the local concentration, c, of the center of mass of the analyte zone, and the zone migration occurs in non-linear conditions. Because the zone broadens as it moves along the separator, R varies continuously from the inlet to the outlet of the separator. That concentration, c(eff), for which R(c(eff)) is equal to the length-averaged apparent retention ratio, R(app), is called effective concentration, and that distance, z(eff), from the separator inlet, for which c(z(eff)) is equal to c(eff), i.e. for which R(z(eff)) is equal to R(app), is called effective position. Assuming that near the onset the non-linear behavior, R(c), is a linear function, values of R(app), c(eff) and z(eff) have been computed in a wide range of operating conditions which are typical of situations encountered in capillary zone electrophoresis, liquid chromatography, or field-flow fractionation. Computations have been performed both in presence and in absence of the dispersion arising from the concentration dependence of the analyte migration rate (called thermodynamic dispersion in chromatography or electromigration dispersion in capillary zone electrophoresis). It is found that, whatever the range of analyte concentration covered from inlet to outlet of the separator, c(eff) is always close to two times the analyte concentration, c(out), at the outlet of the separator, and z(eff) between one-fourth and one-third of the separator length. As c(out) is easily determined from the peak recorded by a concentration-sensitive detector, a simple pragmatic expression is given for the estimation of c(eff). This effective concentration is the appropriate concentration to be used for comparing predictions of theoretical models of R(c) with experimental retention data. This is of particular interest for validating such models in field-flow fractionation.

Chemical Fractionation↗

PubMed2000

Effect of Ionic and Nonionic Solutes on the Transient and Steady States in the Settling of Charged Colloidal Particles.

Centrifugation was applied to study the transport phenomena in various suspensions of charged colloidal silica particles. Isopycnic focusing of colored density marker beads was used to view the evolution of the density gradient and to determine its steady-state shape from the measured positions of the focused zones. The effect of the ionic and nonionic solutes on the transient and steady states was studied. The experimental results are discussed in relation with the published theoretical dependence of the transport coefficients on the volume fraction of the settling particles. The understanding of the transport phenomena and of the interfering particle interactions governing the formation of the density gradient and the subsequent isopycnic focusing under model static conditions is necessary for the optimization of the isoperichoric focusing field-flow fractionation dynamic experiments, but it should allow a more accurate description of the relaxation phenomena and retention in polarization field-flow fractionation as well. Some previous experimental results concerning the study of the transport phenomena of the charged silica particles in an electric field are exploited here to demonstrate the general character of the observed behavior, independent of the field nature. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Fractionation and composition of colloidal and suspended particulate materials in rivers.

The association of pollutants (nutrients, heavy metals and organic compounds) with colloidal and suspended particle matter (SPM) plays a dominant role in determining their transport, fate, biogeochemistry, bioavailability and toxicity in natural waters. A scheme for the fractionation and composition of colloidal and SPM from river waters has been tested. All four separation methods, i.e. sieving, continuous flow centrifugation, tangential flow filtration, sedimentation field-flow fractionation, were for the first time used to separate five size particulate fractions from river. Significant (gram) amounts of colloidal material (<1 microm) in three size ranges, nominally 1-0.2, 0.2-0.006 and 0.006-0.003 microm were obtained. The separation scheme was able to process large samples (100 l), within reasonable times (1 day) and the apparatus was portable. The aquatic colloid size was also characterized with high resolution by using sedimentation field-flow fractionation technique. The mass-based particle size distribution for the water sample showed a broad size distribution between 0.05 and 0.4 microm with the maximum around 0.14 microm. There was a systematic increase in the content of organic carbon (estimated by loss on ignition), Mg, Ca, Na, Cu and Zn with decreasing particle size, highlighting the importance of the colloidal (<1 microm) fraction. It was concluded that the colloidal Cu and Zn concentrations in rivers might be much higher than those reported before.

Biological Availability↗

PubMedDate not supplied

Liposome size analysis by dynamic/static light scattering upon size exclusion-/field flow-fractionation.

The aim of the current study was to analyse the particle size distribution of a liposome dispersion, which contained small egg phosphatidylcholine vesicles and had been prepared by high-pressure homogenisation, by various size analysis techniques. Such liposomes were chosen since they can be looked at as a prototype of drug nano-carriers. Three sub-micron particle size analysis techniques were employed: (1) fixed-angle quasi-elastic laser light scattering or photon correlation spectroscopy (PCS), (2) size exclusion chromatographic (SEC) fractionation with subsequent (off-line) PCS size-analysis and quantification of the amount of particles present in the sub-fractions, and (3) field-flow-fractionation coupled on-line with a static light scattering and a refractive index (RI)-detector. When designing liposome-based drug carrier systems, a reliable and reproducible analysis of their size and size distribution is of paramount importance: Not only does liposome size influence the nanocarrier's in-vitro characteristics such as drug loading capacity, aggregation and sedimentation but also it is generally acknowledged that the pharmacokinetic behaviour and biodistribution of the carrier is strongly size-dependent. All three approaches of liposome size analysis used here were found to yield useful results, although they were not fully congruent. PCS indicated either a broad, mono-modal, log-normal size distribution in the range of below 20 to over 200 nm in diameter, or alternatively, a bimodal distribution with two discrete peaks at 30 to 70 nm and 100 to over 200 nm. Which of the two distribution models represented the best fit depended primarily on the data collection times used. In contrast, both fractionating techniques revealed a size distribution with a large, narrow peak well below 50 nm and a minor, broad, overlapping peak or tail extending to over 100 nm in diameter. The observed differences in liposome size distribution may be explained by the inherent limitations of the different size analysis techniques, such as the detection limit and the fact that PCS is overemphasizing bigger particle sizes.

Chemical Fractionation↗

PubMed2003

Turbidimetric detection method in flow-assisted separation of dispersed samples.

Characterization of dispersed samples is an outstanding trend in analytical science. Among flow-assisted separation techniques for dispersed samples, size exclusion chromatography, hydrodynamic chromatography, and field-flow fractionation are the most widely applied. With dispersed analytes separated by these techniques, the UV/vis spectrophotometric detectors work as turbidimeters. To directly convert the analytical signal for quantitative analysis, the extinction properties of the dispersed analyte must be known. A new method is proposed to experimentally obtain-by single-run, flow-assisted separation with UV/vis diode-array detectors-the mass-size (or number-size) distribution function of the analytes when a retention-to-size relationship is either theoretically or empirically available for the chosen separation technique. This approach needs neither standards nor reliance on a method to predict the optical properties of the analytes. Theory and original algorithms are presented. Algorithms are then tested to optimize the numerical routines. Accuracy and robustness of the method are evaluated by simulation, and limitations for the application to experimental data are described. Finally, first application to field-flow fractionation shows validity of the method when applied to a few real cases.

Journal Article↗