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PubMed2004

Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded solid lipid nanoparticles by photon correlation spectroscopy, field-flow fractionation and transmission electron microscopy.

Recently, colloidal dispersions made from mixtures of solid and liquid lipids were described to combine controlled release characteristics with higher drug loading capacities than solid lipid nanoparticles (SLN). It has been proposed that these nanostructured lipid carriers (NLC) are composed of oily droplets which solubilize the drug and which are embedded in a solid lipid matrix. The structures of SLN and NLC based on glyceryl behenate and medium chain triglycerides were characterized by photon correlation spectroscopy (PCS) and laser diffraction (LD), field-flow fractionation (FFF) with multi-angle light scattering detection (MALS), and cryo transmission electron microscopy (cryo TEM). PCS indicates that SLN and NLC differ from a nanoemulsion with respect to Brownian motion due to asymmetric particle shapes. Non-spherical particles, in case of SLN and NLC, lead to higher polydispersity indices compared to the nanoemulsion. In FFF, the nanodroplets elute much earlier than SLN- and NLC-platelets although their PCS and LD data show similar particle sizes. In TEM platelet (for SLN), oil loaded platelet ("nanospoons"; for NLC) and droplet (for nanoemulsion) structures were observed. In contrast to literature reports, the investigated SLN appear as thin platelets. NLC are found to be lipid platelets with oil spots sticking on the surface. Very short diffusion pathways in platelets, increased water-lipid interfaces and low drug incorporation in crystalline lipids are the drawback of SLN and NLC compared to conventional nanoemulsions.

Chemical Phenomena↗

PubMed1998

A micromachined electrical field-flow fractionation (mu-EFFF) system.

In this work, micromachining technologies are employed to develop a miniaturized electrical field-flow fractionation (EFFF) separation system. EFFF systems are used to separate colloidal particles such as cells, liposomes, proteins, or other particulates, and to characterize emulsions and other mixtures according to particle charge density. Macromachining techniques have been used to develop existing EFFF technologies. At the present time, the limiting factor in the development of higher precision EFFF separation systems has been the manufacturing approach. In this paper, the theory behind the operation and resolution of a micron-sized EFFF (mu-EFFF) system is described and the advantages to be gained from application of micromachining technologies are given, thus motivating the need for further miniaturization. A completely fabricated mu-EFFF system is developed, separations are performed, and the mu-EFFF system is compared to the theoretically predicted results as well as the results from current macro EFFF systems.

Biocompatible Materials↗

PubMed1984

Sedimentation field-flow fractionation: a method for studying particulates in cataractous lens.

It is shown that the technique of sedimentation field-flow fractionation (sedimentation [sed] FFF) can be used to determine the particle content and particle size distribution of normal and cataractous lenses. A 31-year-old normal human lens, for example, showed a particle content of 1.5% by weight with diameters ranging from 0.12 micron to 0.9 micron. The urea insoluble material present in the nuclear and cortical fractions from a densely cataractous lens contained particles ranging from 0.12 micron to 1.7 micron, with average sizes of 0.83 micron and 0.82 micron respectively, for the two fractions. These numbers offer a basis for comparison; their actual values may be shifted slightly either up or down depending on the assessment of particle density. These sizes, which correspond to molecular weights of around 2 X 10(9) dalton, are larger than previously reported for lens particulates. The sed FFF method is thus seen to permit fractionation and size analysis of small amounts of lens material in times less than one hour.

Cataract↗

PubMed2006

Pre-apoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation. The effect of channel thickness on sorting performance.

Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.

Apoptosis↗

PubMed1997

Sedimentation field-flow fractionation of cellular species.

More than 15 years ago a pioneering report on the separation of cellular material using sedimentation field-flow fractionation (SdFFF) was published. Surprisingly, only a few reports on SdFFF applications to cell separations are available as yet. The major limitations which seemed to slow the development of SdFFF applications in biology appeared to be related to the development of specific instrumentation. Therefore, guidelines are given for setting up a biocompatible SdFFF apparatus. SdFFF elution of cells with different characteristics was performed to demonstrate the efficiency, completeness, and rapidity of cell separations by this method. Retention data describing the effect of lifting forces on nucleated cells are compared to those for red blood cells (RBC). The effects of flow rates, field intensity, and injection protocol were studied using normal human RBC eluted in isotonic medium as a probe for retention conditions. When possible, data were also compared to previously published reports. Guidelines for elution and separation optimization are given. Using mixtures of nucleated and living red blood cells, viability studies showed a surprisingly high recovery of each type of material.

Cell Fractionation↗

PubMed2001

Ultrahigh molar mass component detected in ethylhydroxyethyl cellulose by asymmetrical flow field-flow fractionation coupled to multiangle light scattering.

Asymmetrical flow field-flow fractionation (flow FFF) was connected to multiangle light scattering (MALS) and refractive index (RI) detectors for characterization of the molar mass distribution and molecular radius of a cellulose derivative, ethylhydroxyethyl cellulose (EHEC). Experimental conditions were optimized to allow study of a wide range of molar mass including even ultrahigh molar mass (UHM) components. The weight-average molar mass was 3.1 x 10(5) g x mol(-1) representing a very broad range (of molar mass) from 4.0 x 10(4) to 10(7) g x mol(-1), which corresponds to from <20 to 200 nm rms radius. The light scattering signal showed the presence of an UHM component, possibly an aggregate of extreme size, i.e., approximately 10(8) g x mol(-1) with a hydrodynamic diameter of 0.35 microm. Careful choice of the pore size in in-line filters is necessary in order to minimize MALS detector noise without removing the UHM component. Flow FFF-MALS-RI was demonstrated to be uniquely suited to detect the presence of UHM components.

Cellulose↗

PubMed1985

Physical characterization of PBCV virus by sedimentation field flow fractionation.

A virus (PBCV) from the symbiotic Chlorella-like algae of Paramecium bursaria has been characterized by sedimentation field flow fractionation (SdFFF). The method yields effective molecular weight M' (molecular weight X buoyancy factor) with a precision of better than 5% under a variety of experimental conditions. The value for M' is used to calculate true molecular weight values through combination with density values; densities were obtained from outside sources, from ultrasonic densitometry, and from sedimentation coefficients determined independently. The actual molecular weight is also determined through SdFFF analysis of the virus in carriers of different densities. Values for the particle diameter are obtained from SdFFF zone broadening studies. The various SdFFF characterization results are compared with molecular weight, particle size, and density values determined by conventional methods.

Animals↗

PubMed1989

Effect of interleukin-1 on the size distribution of cartilage proteoglycans as determined by sedimentation field flow fractionation.

The effect of interleukin-1 (IL-1) on the size distribution of cartilage proteoglycans was studied using sedimentation field flow fractionation (SdFFF), a rapid, high-resolution technique for the separation of proteoglycan monomers and aggregates. During incubation of cartilage in control media, 35S-prelabeled proteoglycan was lost primarily from proteoglycan present in the monomer form; aggregates were conserved. In the presence of IL-1, both 35S-proteoglycan monomers and aggregates were lost, suggesting that IL-1 increases the susceptibility of aggregates to loss from the cartilage matrix. Evaluation of uronic acid as a measure of net change in proteoglycan content indicated that IL-1 causes a net decrease in both monomers and aggregates. Kinetic studies suggested that aggregates are degraded to monomers which then diffuse out of the matrix. Incorporation of [35S]sulfate into cartilage proteoglycans following exposure to IL-1 showed that synthesis of monomers and aggregates is inhibited similarly. SdFFF is a valuable technique for studying proteoglycan metabolism. With its use, changes in proteoglycan monomer and aggregate populations can be detected in response to cytokines such as IL-1.

Animals↗

PubMed1992

Particle size distribution of concentrated perfluorocarbon emulsions by sedimentation field flow fractionation.

Alliance Pharmaceutical Corp's concentrated perflubron (perfluorooctylbromide; PFOB) emulsions are being developed for several medical applications. Sedimentation Field-Flow Fractionation (SdFFF) can provide accurate particle size distribution data when properly calibrated for those emulsions. Since no suitable particle standards are available with the proper density (approximately 1.9 g/cc) and size range (diameter = 0.1-1.0 microns), calibration of the mass distribution was done by comparing the SdFFF detector signal with the actual mass of perflubron extracted from fractions eluted at successive time points and measured by GC. The calibrated mass distribution can then be used to calibrate other instruments such as the photosedimentation instruments used for routine quality assurance measurements.

Blood Substitutes↗

PubMed1994

Theory of field-programmed field-flow fractionation with corrections for steric effects.

This paper deals with the principal perturbation to ideal normal-mode elution of particles in field-flow fractionation (FFF). This perturbation is due to the finite size of particles undergoing migration in the FFF channel. The effects of a first-order correction for particle size are examined. Equations are derived for retention time, fractionating power, and steric inversion diameter for operation at constant field strength, as well as under conditions of both exponential and power programmed field decay. Useful limiting equations for fractionating power are derived and their validity is confirmed for typical experimental conditions. The derived equations are necessary for the future development of a systematic optimization strategy for the selection of operating conditions for particle size analysis by FFF. Calculations confirm our previous conclusion that the fractionating power for exponential field programming varies strongly with particle size; this variation is only slightly reduced by steric perturbations. The uniform fractionating power of power programming is slightly disturbed by steric effects although fractionating power remains much more uniform than for exponential programming. It is shown that a higher uniformity in fractionating power can be gained by manipulating the parameters of power programming but that no improvement is possible with exponential programming. Phenomena giving rise to higher order perturbations and to secondary relaxation are discussed and the conditions identified under which these effects are minimized.

Chemical Fractionation↗

PubMed1995

Separation and characterization of red blood cells with different membrane deformability using steric field-flow fractionation.

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published data from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.

Cell Separation↗

PubMed1990

Particle characterization in centrifugal fields. Comparison between ultracentrifugation and sedimentation field-flow fractionation.

A ten-component mixture of polystyrene latex particles in the 67-1220 nm size range was subjected to analysis by analytical ultracentrifugation (AUC) and sedimentation field-flow fractionation (SdFFF) using programmed and constant fields. The AUC analysis of the mixture yielded diameter values in good agreement with data determined on the separate components; the relative amounts of each component in the mixture were likewise closely reproducing the sample's known composition. Diameters determined by SdFFF, either in a constant- or programmed-field mode, were in good agreement with the AUC for particles smaller than about 500 nm. For the sample's larger components, however, particularly the programmed mode showed diameter values smaller than expected. In addition, field programming resulted in incomplete recoveries of the larger particles, leading to more or less distorted mass distributions for the complex sample. The observed discrepancies, which are thought to result from events at the analytical wall in the FFF channel, suggested a protocol for accurate sizing, as opposed to fingerprinting, of samples with broad size distribution. By tracking sizes and amounts of the different components at different but constant field strengths, and retaining as analytically valid only those data recorded in a retention range from five to about thirty column volumes, it was possible to determine sizes and amounts in good agreement with known parameters for the sample. Unlike the AUC procedure, SdFFF produces fractions of a high degree of uniformity, which lend themselves to a secondary analysis, e.g. by electron microscopy, as shown in the study.

Centrifugation↗

PubMed2003

Coupling thermal field-flow fractionation with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analysis of synthetic polymers.

Thermal field-flow fractionation (ThFFF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have been coupled to yield a powerful combination of techniques for polymer analysis. Thermal FFF's high molecular weight (MW) selectivity and sensitivity to chemical composition are used to separate polydisperse polymers and polymer mixtures into the narrow polydispersity and homogeneous chemical composition fractions essential for MALDI-TOFMS analyses. On the other hand, MALDI-TOFMS's ability to directly measure molecular weight alleviates the need for polymer standards for ThFFF. In this first-time coupling of ThFFF and MALDI-TOFMS, compatibility issues were addressed and optimum conditions and procedures were identified and developed to maximize the capabilities of the combined technique. Depending on the polymer MW and the method of MALDI sample deposition, fractions from 1-10 ThFFF runs were combined for MALDI-TOFMS analysis. Binary solvents were used to enhance ThFFF retention and resolution of low-MW (<15-kDa) polymers, and methods were developed to allow routine MALDI-TOFMS analyses of polystyrene polymers up to 575 kDa. Overall, the MW compatibility of the two techniques was extended from several kilodaltons to several hundred kilodaltons. Polymer fractions were collected after separation by ThFFF and analyzed either by MALDI-TOFMS or reinjection into the ThFFF system. Good agreement was observed between the MW distribution data obtained by MALDI-TOFMS and ThFFF. The application of ThFFF/MALDI-TOFMS to polydisperse polymers and polymer mixtures was demonstrated. This combined technique was also shown to be a viable means for preparing standards from the original polymer sample.

Journal Article↗

PubMed1996

Isolation and measurement of colloids in human plasma by membrane-selective flow field-flow fractionation: lipoproteins and pharmaceutical colloids.

Using a modified flow field-flow fractionation (flow FFF) technique termed membrane-selective flow FFF, a capability is developed to isolate various colloidal constituents that are naturally present in or may be introduced into blood plasma. Once isolated, the colloids may be subject to quantitative measurement to provide relative amounts of the different constituents and the size distribution curve of each. The potential of the technique has been demonstrated by isolating and measuring (a) the lipoprotein fractions found in human blood plasma, (b) liposyn II pharmaceutical emulsion added at 2 mg/mL of plasma, and (c) amphotericin B colloidal dispersion (a drug delivery agent) added to plasma at 0.5 mg/mL.

Chemical Fractionation↗

PubMed2003

Size analysis of industrial carbon blacks by sedimentation and flow field-flow fractionation.

Carbon black is one of the most useful particulate materials in the industrial field. Among the various physical properties of carbon black, size and size distribution are the most important properties to affect the quality of a final product. However, it is difficult to measure the exact particle size of carbon black since it suffers unavoidable interference from flocculation. In this study, the effects of various factors on the dispersion of industrial carbon blacks were investigated for the determination of size and size distribution of carbon black particles. Sedimentation and flow field-flow fractionations (FIFFF) were used to determine the size of carbon black, and their optimum analytical conditions were tested by changing surfactant, pH, ionic strength, and method of dispersion. The results showed that surfactant structure and its concentration played significant roles in dispersion stability. Carbon black was dispersed well with a nonionic surfactant with a pH of around 8 and an ionic strength of 0.003 M. The mean diameters measured from two types of FFF and photon correlation spectroscopy are in good agreement. This study demonstrates the potential of sedimentation and flow FFF for analyzing highly adsorptive industrial particles and guides for sample preparation.

Journal Article↗

PubMed1982

Sedimentation field flow fractionation of liposomes.

Quantitative analyses of the particle size distribution of liposomes were performed in 30 to 60 minutes by exponential-field sedimentation field flow fractionation. This gentle new separation method exhibits great potential for the high-resolution fractionation and the size or molecular weight analysis of a wide variety of biological macromolecules and colloidal suspensions.

Centrifugation↗

PubMed2004

Working without accumulation membrane in flow field-flow fractionation. Effect of sample loading on retention.

Membraneless hyperlayer flow field-flow fractionation (Hyp FIFFF) has shown improved performance with respect to Hyp FIFFF with membrane. The conditions for high recovery and recovery independent of sample loading in membraneless Hyp FIFFF have been previously determined. The effect of sample loading should be also investigated in order to optimize the form of the peaks for real samples. The effect of sample loading on peak retention parameters is of prime importance in applications such as the conversion of peaks into particle size distributions. In this paper, a systematic experimental work is performed in order to study the effect of sample loading on retention parameters. A procedure to regenerate the frit operating as accumulation wall is described. High reproducibility is obtained with low system conditioning time.

Calibration↗

PubMed1993

Size distribution of liposomes by flow field-flow fractionation.

The applicability of field-low fractionation (FFF) to the characterization of liposomes is discussed and theoretically described. Because of fundamental differences in their driving forces, sedimentation FFF and flow FFF measure different vesicle properties. Sedimentation FFF, although used previously to measure vesicle sizes and size distributions, is fundamentally a technique that measures the effective mass and mass distribution of particles. It is sensitive to small changes in the effective mass of either the biomembrane or its encapsulated load and thus is likely to be useful in characterizing such properties as drug loading, biomembrane volumes and areas, and distributions of these properties. Size characterization by sedimentation FFF can only be done by deducing size from effective mass. Flow FFF, by contrast, provides a direct measurement of vesicle size and size distribution. After demonstrating the high resolution and relative accuracy of size measurement of flow FFF by the separation of polystyrene latex standards, flow FFF was applied to two preparations of DSPC-DSPA liposomes that were sonicated under different temperature conditions. Fractograms and size distributions are reported as a function of sonication time. The rapid elimination of a large diameter tail to the distribution is shown to constitute a major mechanism for distribution narrowing. Finally, results are provided bearing on the reproducibility of size distribution measurements by flow FFF.

Chemical Fractionation↗